Effect of insulin therapy on endothelial function and insulin-stimulated endothelial function in patients with type 2 diabetes and ischemic heart disease. PhD thesis Christian Rask Madsen University of Copenhagen, February 2001. Christian Rask Madsen: PhD thesis. Page i. Abbreviations AGE advanced glycation end-products ADMA assymmetrical dimethylarginine BH4 tetrahydrobiopterin BMI body mass index DAG diacylglycerol DCCT The Diabetes Control and Complications Trial DDAH dimethylarginine dimethylaminohydrolase DIGAMI Diabetes Mellitus, Insulin Glucose Infusion in Acute Myocardial Infarction EDHF endothelium-derived hyperpolarising factor eNOS endothelial nitric oxide synthase ET endothelin FFA free fatty acids HDL high density lipoprotein IR insulin receptor IRS insulin receptor substrate LDL low density lipoprotein L-NAME NG-nitro-L-arginine methyl ester L-NMMA NG-monomethyl-L-arginine L-NNA NG-nitro-L-arginine MAPK mitogen activated protein kinase NADH nicotinamide adenine dinucleotide (in its reduced form) NADPH nicotinamide adenine dinucleotide phosphate (in its reduced form) NO nitric oxide PI3K phosphatidyl inositol-3 kinase PKC protein kinase C UKPDS United Kingdom Prospective Diabetes Study VSMC vascular smooth muscle cell Christian Rask Madsen: PhD thesis. Page ii. Preface The Finnish researcher, Professor Hannele Yki- now that it was established at the department. Järvinen, often begins her lectures by We thus planned to measure insulin-stimulated proposing that “Diabetes is a vascular disease glucose uptake and endothelial function in diagnosed by measuring blood glucose”. When patients at the same examination day. Lars Dr. Christian Torp-Pedersen asked me, in said that I had not described the aim clearly 1997, to start the work which has resulted in enough in the protocol, and I realized it was the thesis you are about to read, the only because I had not expressed a well-defined aspect of the project which seemed decided hypothesis of whether insulin resistance on from the start was that it should somehow causes endothelial dysfunction, or the other embrace type 2 diabetes and cardiovascular way around. I suggested that we could study disease. Early plans for the study was an intervention in our patients that would be discussed with Drs. Per Hildebrandt, Thomas expected to improve endothelial function but Melchior, and Lars Køber and with Christian’s did not by other known or proposed wife, Dr. Marie Seibæk. Before acknowledging mechanisms influence insulin-stimulated those that contributed to the study, I would glucose uptake: antioxidants, for example. like to describe the preparations that went Such a study has since been made in patients ahead of the work presented in the thesis with essential hypertension, a condition proper. associated with insulin resistance, and it did not support that endothelial dysfunction Per had then accepted a position in the causes insulin resistance . In the end, steering comittee of the DIGAMI-2 trial and measuring insulin-stimulated glucose uptake thought we should attempt to explain a was abandoned at a meeting in Christian and mechanism for the improved survival seen Marie's house, when Marie, momentarily with insulin therapy of patients with diabetes recovering from absent-mindedness during the and acute myocardial infarction. We had clever children’s bed-time manoeuvres, said (with a ideas that we were not perseverant enough to very concerned look): “You want to make 60 pursue. To identify abnormalities of insulin clamps? That’s a lot of work”. We restricted effects in the myocardium which could be a ourselves to measuring endothelial function. target for insulin therapy, Thomas wrote a protocol draft which described experiments Christian said that there would be plenty of aimed at measuring insulin-stimulated glucose patients to recruit. I first screened patients uptake during coronary catheterisation and who had been examined by coronary thus to measure directly whether the human arteriography in our department. It is true that heart is insulin resistant in patients with type 2 at least 10% of the more than 2000 patients diabetes. This idea was abandoned because that receives this procedure each year at our we agreed that it would not be possible to institution are expected to have type 2 measure the supposedly quite small coronary diabetes. But the inclusion criterium of arterio-venous glucose differences. But it glycemic dysregulation and the exclusion seems that given a sound hypothesis one criterium of left ventricular dysfunction should not readily accept that a study cannot severely decimated my regimen of potential be done for technical reasons. Measuring the research subjects. Not being able to find the arterio-venous glucose difference in the patients was the most frustrating part of my human coronary circulation is indeed PhD work. feasible . My first thanks go to Christian. He has a large Marie, in her PhD work, had measured whole- capacity for learning new things and an body insulin sensitivity in patients with admirable ability to organize, in his mind, ischemic heart disease at Gentofte University complicated and contradictory facts, and make Hospital. Insulin resistance, like endothelial arbitrary decisions when it is necessary. I dysfunction, may be one of the earliest beleive that when he respects a collegue it is changes leading to vascular disease, and regardless of age and accreditation. And he is Christian was eager to preserve the very good at making us remember, in his own euglycemic hyperinsulinemic clamp techique belief, that we work because it is enjoyable. Christian Rask Madsen: PhD thesis. Page iii. My supervisor and I like the idea that this Drs. Thure Krarup and Erik Christiansen made research will ultimatively benefit the patients, important contributions to the protocol and to but his and my primary motivation are selfish. the article manuscript and were thorough and Realizing this makes me even more amazed of succesful at initiating and maintaining insulin the attitude of the patients I have adressed. therapy in the treatment group of patients. Very few of them have denied to participate, Anne Marie Råby-Magle kindly helped with and those that did went through dietary advice to patients. I was fortunate to uncomfortable study days and accepted be able to consult Dr. Philip Hougaard about thorough changes to their life-styles during the statistical analysis. intervention period without second thoughts. I Ulla Jørgensen, Anne-Mette Budde, Kirstine admire and respect their motivation and I Serup-Hansen, and Berit Thomsen all worked thank both those that participated and those hard as study nurses in clinical trials that that wanted to, but could not. partly financed the project. Dr. Jens Otto Lund Two years ago, Dr. Nikolaj Ihlemann started to provided us with office and laboratory space. expand the work in this PhD thesis by Trine Schnor, pharmacist at the Central establishing a model of local forearm Hospital Pharmacy of Copenhagen County, hyperglycemia and studying the effect of helped with procedures for producing drugs hyperglycemia on the acetylcholine vasodilator for infusion. Niels Jensen, engineer at Gentofte response and the insulin-stimulated Hospital, was of assistance many times when acetylcholine response in the human forearm. there was technical problems with the It has been wonderful to have someone with laboratory equipment. whom I could always discuss methods and Per Hilderbrandt, Thomas Melchior, Marie data and who could help to digest the Seibæk, and Professor Niels-Henrik Holstein- literature. Nikolaj has a wonderful ironic sense Rathlou gave important intellectual input at of humour, and at some point he will not be various stages of the study. able to not make fun of this preface. He is the best collegue I have had. Finally, I would like to thank University of Copenhagen for my salary, the Danish Heart I thank Lars for saying “I’m not sure that I Foundation and Emil Torp-Pedersen's understand this” at the right times, thus Foundation for financial contributions, and pointing out weaknesses in design or data Bayer Denmark for donating blood glucose interpretation. I am grateful to Lene Eskildsen, analyzers for home-monitoring of blood who screened hundreds of patient records and glucose. The Danish Medical Research Council was my first technical assistance, and to and the Danish Diabetes Foundation have Dorthe Baunbjerg Nielsen, who has been a financially supported the projects described in very skilful technician and data organizer. the section on perspectives. References 1. McNulty PH, Pfau S, and Deckelbaum LI. Effect of plasma insulin level on myocardial blood flow and its mechanism of action. Am.J Cardiol. 2000;85(2):161-5. 2. Natali A, Sironi AM, Toschi E, Camastra S, Sanna G, Perissinotto A, Taddei S, and Ferrannini E. Effect of vitamin C on forearm blood flow and glucose metabolism in essential hypertension. Arterioscler.Thromb.Vasc.Biol. 2000;20(11):2401-6. Christian Rask Madsen: PhD thesis. Page iv. Abstract Background. Decreased endothelial function affected by insulin infusion in any group. In and blunted insulin-stimulated endothelial healthy controls, insulin infusion increased the function may be mechanisms responsible for ACh response by ∼ 120%, but had no effect in development of atherothrombotic disease in patients. Surprisingly, both the ACh and SNP type 2 diabetes, but it is unknown whether response had decreased after 3 days of insulin hypoglycemic drug therapy can modulate therapy in the treatment group of patients. these abnormalities. The aim of this study was After 2 months, hemoglobin A1c in the to examine whether long-term intensive insulin treatment group had decreased by 2.5 therapy can improve endothelium-dependent ± 0.4%. In this group, the ACh and SNP vasodilation as well as the acute effect of responses had not changed significantly, but insulin to stimulate stimulate endothelium- insulin stimulation now increased the ACh dependent vasodilation. response by ∼ 80%. In the time control group, vascular responses were unchanged. The Patients and methods. Patients with type 2 inhibitory effect of L-NMMA on the insulin- diabetes and stable ischemic heart disease and stimulated ACh response was similar in lean, healthy control subjects were studied. patients and healthy controls and had not Forearm blood flow was measured by venous changed after 2 months in the treatment occlusion plethysmography during dose- group of patients. response studies of acetylcholine (ACh) and sodium nitroprusside (SNP) infused into the Conclusions. The patients with type 2 brachial artery. ACh was repeated during diabetes and ischemic heart disease in this intrabrachial infusion of insulin, resulting in study had decreased endothelium-dependent high physiological serum insulin concentrations and endothelium-independent vasodilation. in the forearm with minor elevation of Their vascular dysfunction may be improved systemic serum insulin. Co-infusion of insulin by BH4, suggesting a relative deficiency of this an ACh was repeated during infusion of L- cofactor in the vasculature, but further studies NMMA, an inhibitor of nitric oxide (NO) are needed to confirm this preliminary finding. production. Finally, ACh infusion was repeated In lean, healthy subjects, insulin increased during co-infusion of tetrahydrobiopterin endothelium-dependent vasodilation, likely by (BH4), a cofactor or NO synthase. Patients a local effect on the vasculature. This effect were re-studied after 3 days and after 2 was absent in patients with poor glycemic months of therapy with 4 daily subcutaneous control, but present after 2 month’s insulin insulin injections (treatment group, n = 19) or therapy. The mediator of this effect was not after 2 months without hypoglycemic drug established. Surprisingly, after 3 days of therapy (time control group, n = 9). Certain insulin therapy, endothelium-dependent and - dose-response studies were only performed in independent vasodilation was paradoxically subgroups of patients. Thus, studies with decreased. Planned research include studies to insulin and L-NMMA was not performed in all determine whether tumor necrosis factor-α patients and studies with SNP were not has a role in mediating endothelial insulin performed in all healthy controls. The BH4 resistance. Future research may show whether studies were only performed in a subgroup of therapy directed against endothelial insulin patients. resistance can prevent atherothrombotic complications in type 2 diabetes. Results. The ACh and SNP responses were lower in patients compared to healthy controls. BH4 infusion increased the ACh response in patients. Basal blood flow was not Christian Rask Madsen: PhD thesis. Page v. Resumé Baggrund. Nedsat insulin-stimuleret virkning hos patienter. Basal endotelfunktion er muligvis en årsag til blodgennemstrømning blev ikke påvirket af udvikling af hjertekarsygdom ved type 2 insulin-infusion i nogen gruppe. Hos raske diabetes, men det vides ikke om blodsukker- kontrolpersoner øgede insulin-infusion AK- nedsættende behandling kan bedre denne virkning med ∼ 120%, men påvirkede ikke AK- tilstand. Formålet med det nærværende studie virkning hos patienterne. AK- og NNP-virkning var at undersøge hvorvidt intensiv var overraskende nedsat efter 3 dages insulin- insulinbehandling kan bedre endotel-afhængig behandling i behandlingsgruppen. Efter 2 vasodilatation samt den akutte virkning af måneders var hemoglobin A1c faldet 2.5 insulin på endothel-afhængig vasodilatation. ± 0.4% i behandlingsgruppen. I denne gruppe havde AK- og NNP-virkninger ikke ændret sig, Patienter og metoder. I studiet undersøgtes men insulin-stimulation øgede nu AK-virkning patienter med type 2 diabetes og stabil med ∼ 80%. I tidskontrolgruppen var alle iskæmisk hjertesygdom og slanke, raske vaskulære effekter uændrede. Den forsøgspersoner. Underarms-gennemblødning hæmmende effekt af L-NMMA på AK-virkning blev målt med venøs okklusions-pletysmografi var lige stor hos patienter og raske under dosis-virknings-undersøgelser af kontrolpersoner og ændrede sig ikke efter 2 acetylkolin (AK) og natrium nitroprussid (NNP), måneder i behandlingsgruppen. infunderet i a. brachialis. AK blev gentaget Konklusioner. Patienterne med type 2 under intra-arteriel infusion af insulin, som diabetes og iskæmisk hjertesygdom i dette øgede serum-koncentrationer af insulin i studie havde både nedsat endothel-afhængig underarmen til høje fysiologiske værdier med og endothel-uafhængig vasodilatation. Deres en kun lille stigning i systemisk serum-insulin. vaskulære dysfunktion blev muligvis bedret af Samtidig infusion af insulin og AK blev BH4, hvilket antyder en relativ mangel på gentaget med infusion af L-NMMA, en denne kofaktor i vaskulært væv, men hæmmer af nitrogenoxid (NO) produktion. yderligere studier er nødvendige for at Desuden blev den samtidige infusion of insulin bekræfte dette præliminære fund. Hos slanke, og AK gentaget med infusion af raske forsøgspersoner øgede insulin endothel- tetrahydrobiopterin (BH4), en kofaktor til NO afhængig vasodilatation, formentlig via en syntase. Patienterne blev genundersøgt efter 3 lokal effekt på karrene. Denne effekt dage og efter 2 måneder i behandling med fraværende hos patienter med dårlig insulin, givet som 4 daglige subkutane glykæmisk kontrol, men var til stede efter 2 injektioner (behandlingsgruppe, n = 19) eller måneders insulinbehandling. Den medierende efter 2 måneder uden antidiabetisk behandling faktor af denne effekt blev ikke påvist. Efter 3 (tidskontrolgruppe, n = 9). Visse dosis- dages insulinbehandling var både endothel- virknings-undersøgelser blev kun udført i afhængig og -uafhængig vasodilatation undergrupper af deltagere. Således blev overraskende nedsat. Egne planlagte projekter undersøgelser med insulin og L-NMMA ikke vil søge at vise om TNF-α har betydning ved udført hos alle patienter og studier med NNP endotelial insulinresistens. Fremtidige studier ikke udført hos alle raske kontrolpersoner. vil måske kunne vise om behandling rettet Resultater. AK- og NNP-virkninger var mindre mod endotelial insulinresistens kan forebygge hos patienter sammelignet med raske hjertekarsygdom ved type 2 diabetes. kontrolpersoner. BH4-infusion øgede AK- Christian Rask Madsen: PhD thesis, table of contents. Page vi. Table of contents page Abbreviations i. Preface ii. Abstract iv. Resumé (abstract in Danish) v. Introduction...................................................................................................................... 1 Background....................................................................................................................... 1 Endothelial function and dysfunction ................................................................................. 1 Endothelium-derived vasorelaxing or vasoconstricting factors .............................................. 2 Insulin sensitivity and insulin resistance............................................................................. 2 Hyperglycemia, insulin resistance, and cardiovascular disease. ............................................ 3 Hypotheses ....................................................................................................................... 3 Methods ............................................................................................................................ 4 Applied methods ..................................................................................................................... 4 Subjects ................................................................................................................................. 4 Patients .......................................................................................................................... 4 Healthy controls .............................................................................................................. 4 Experimental protocol.............................................................................................................. 4 Preparations and measurement of blood flow..................................................................... 4 Acetylcholine and sodium nitroprusside response ............................................................... 5 Insulin-stimulated acetylcholine response .......................................................................... 5 L-NMMA-inhibited insulin-stimulated acetylcholine response................................................. 5 Acute effect of BH4 on the acetylcholine response.............................................................. 5 Intervention design ................................................................................................................. 5 Patient allocation ............................................................................................................. 5 Early and late repeat examination ..................................................................................... 6 Acetylcholine reproducibility study..................................................................................... 6 Biochemical analyses. ...................................................................................................... 6 Calculations and statistical analyses .......................................................................................... 6 Ethical considerations .............................................................................................................. 7 Choice of methods: alternatives and criticism ............................................................................ 8 Endothelium-dependent stimulus and vascular bed............................................................. 8 Measurement of blood flow and determinants of variability related to experimental design .... 9 Acetylcholine................................................................................................................. 10 Sodium nitroprusside ..................................................................................................... 11 L-NMMA ........................................................................................................................ 11 Insulin-stimulated acetylcholine response ........................................................................ 11 Christian Rask Madsen: PhD thesis, table of contents. Page vii. Experiments to control for the insulin-stimulated acetylcholine response ............................ 11 Local insulin stimulation ................................................................................................. 12 Serial pharmacological studies on each experimental day .................................................. 12 Design of clinical intervention study ................................................................................ 13 Patient selection ............................................................................................................ 13 Choice of healthy control group ...................................................................................... 13 Concommitant medication and competing risk factors for endothelial dysfunction................ 14 Statistical analyses......................................................................................................... 14 Results ............................................................................................................................ 15 Insulin stimulation ......................................................................................................... 15 Metabolic control ........................................................................................................... 16 Blood flow data ............................................................................................................. 16 Acetylcholine response................................................................................................... 16 Sodium nitroprusside response ....................................................................................... 16 Insulin-stimulated acetylcholine response ........................................................................ 16 L-NMMA inhibition of insulin-stimulated acetylcholine response .......................................... 16 Effect of BH4 on the acetylcholine response..................................................................... 17 Effect of short-term insulin therapy on acetylcholine and sodium nitroprusside responses .... 17 Effect of long-term insulin therapy on vascular responses ................................................. 17 Acetylcholine and sodium nitroprusside responses and insulin-stimulated responses in the time control group ................................................................................................................ 17 Acetylcholine reproducibility ........................................................................................... 17 Systemic circulatory responses........................................................................................ 17 Discussion....................................................................................................................... 17 Interpretation of own results.................................................................................................. 17 Acetylcholine responses before treatment ........................................................................ 17 Local insulin stimulation ................................................................................................. 18 Metabolic effects of insulin treatment .............................................................................. 18 Insulin-stimulated acetylcholine response ........................................................................ 18 Effect of BH4 on the acetylcholine response..................................................................... 19 Effect of short-term insulin therapy on acetylcholine and sodium nitroprusside responses .... 19 Effect of long-term insulin therapy on acetylcholine and sodium nitroprusside responses ..... 19 Effect of long-term insulin therapy on insulin-stimulated acetylcholine response.................. 19 L-NMMA inhibition of the insulin-stimulated acetylcholine response .................................... 20 Discussion of related previous literature .................................................................................. 20 High glucose concentrations and endothelial function ....................................................... 20 Effects of insulin on vasodilation in vivo in animals and healthy humans ............................. 21 Effects of insulin on vasodilation in insulin resistant subjects ............................................. 22 Mediators of insulin-stimulated vasodilation ..................................................................... 22 Christian Rask Madsen: PhD thesis, table of contents. Page viii. Insulin-stimulated PI3K-Akt-eNOS activation .................................................................... 23 Effects of insulin on eNOS expression .............................................................................. 24 Mechanisms of endothelial insulin resistance .................................................................... 24 Effects of insulin on vascular smooth muscle cell function in vitro ...................................... 24 Effects of insulin on endothelium-independent vasorelaxation ex vivo or vasodilation in vivo 26 Hypoglycemic or insulin sentisizing interventions and endothelial function in vivo ................ 26 Hypothetical mechanism of improved insulin-stimulated acetylcholine response .................. 26 Endothelial dysfunction as a determinant of peripheral insulin resistance ............................ 27 Conclusions..................................................................................................................... 27 Implications.................................................................................................................... 27 Implications for improved endothelial function and clinical outcomes.................................. 27 Implications for the concept of endothelial insulin resistance ............................................. 28 Perspectives.................................................................................................................... 29 BH4.............................................................................................................................. 29 TNF-α........................................................................................................................... 29 Birth weight .................................................................................................................. 29 FFAs and PKC................................................................................................................ 30 References ...................................................................................................................... 31 Table of contents ..............................................................................................................vi Tables 1-5 Figures 1-18 Article manuscript Christian Rask Madsen: PhD thesis, Page 1 of 40. Background Introduction The world-wide prevalence of type 2 diabetes Endothelial function and dysfunction is increasing because sedentary lifestyles and Functions of the vascular endothelium are obesity are becoming more frequent, and it is numerous. In short, these functions include estimated that 221 million peple will have type vasomotor control, regulation of thrombosis 2 diabetes in 2010 (1). The most important and fibrinolysis, influence on inflammatory complication of type 2 diabetes is responses, and regulation of vascular cardiovascular disease. At my institution, remodelling. For example, the endothelium almost half of patients referred for coronary synthesizes vasorelaxing factors acting locally arteriography are suffering from type 2 in an autocrine or paracrine fashion and diabetes or impaired glucose tolerance (2). controls mononuclear cell adhesion to its own Endothelial dysfunction is regarded as the surface by regulating the expression of adhesion molecules. Endothelium-dependent earliest detectable causal factor in the vasorelaxation and mononuclear cell adhesion development of atherothrombotic disease (3). This abnormality is found in patients with type have been the endothelial functions most 2 diabetes and in subjects with insulin commonly examined in vitro or in vivo. resistance (4), a central factor in the The most thoroughly studied endothelium- pathogenesis of type 2 diabetes. Furthermore, derived vasodilating factor is NO, which has the degree of endothelial dysfunction is been shown to be a mediator of all the associated with the severity of insulin endothelial functions mentioned above. In resistance (5-7), and hyperglycemia is vascular tissue, it is synthesized by the isoform associated with endothelial dysfunction (see of NO synthase called endothelial NO synthase page 20). As insulin resistance and (eNOS). Apart from the synthesis of NO, it has hyperglycemia, the two most important extra- been suggested that, during physiological pancreatic abnormalities in type 2 diabetes, conditions, the enzyme catalyzes the are both independent risk factors for production of superoxide, which may regulate cardiovascular disease, they may determine gene expression (14) or be a precursor for a the development of atherothrombosis through putative hyperpolarizing and vasodilating detrimental effects on the vascular factor, hydrogen peroxide (15). endothelium. There are several lines of evidence to suggest Insulin stimulates endothelial function, but that endothelial dysfuntion, especially NO- individuals with insulin resistance associated mediated endothelial function, has a causal with obesity (8) or type 2 diabetes (9) are role in atherothrombotic disease. As an resistant to effects of insulin on the example, a single reference to a review or endothelium. Accordingly, insulin resistance original article is mentioned with each item. may exist at the level of the endothelium, which is supported by studies in vitro (10, 1. NO inhibits cellular mechanisms thought to 11). be early determinants of atherosclerosis Insulin therapy improves both glycemic control and thrombosis (16). and insulin sensitivity in patients with type 2 2. Endothelial dysfunction is temporally diabetes (12). In addition, insulin therapy of related to the development and regression patients with type 2 diabetes, started during of atheroma in animal models of admission for acute myocardial infarction, atherosclerosis (17). improve their long-term mortality (13). The 3. Inhibition of vascular NO production aim of the present study was to examine accelerates atheroma formation in the whether insulin therapy improved endothelial abscence of increased blood pressure in function and insulin-stimulated endothelial animal models of atherosclerosis (18). function in patients with type 2 diabetes and ischemic heart disease. 4. Gene transfer of NO synthase inhibits atheroma formation in animal models of atherosclerosis (19). 5. Endothelium-dependent vasodilatation of coronary and limb conduit and resistance Christian Rask Madsen: PhD thesis, Page 2 of 40. vessels is decreased in subjects with risk experimental conditions (30, 31). Among factors for cardiovascular disease (20). endothelium-derived vasoconstrictors are prostaglandins (e.g. thromboxane A2) and 6. Endothelial dysfunction is associated with endothelins (ETs) (e.g. ET-1). future risk for cardiovascular events in patients with established coronary artery Insulin sensitivity and insulin resistance disease in small selected populations (21- 23). Insulin has effects on carbohydrate, lipid, and amino acid metabolism. In glucose metabolism 7. In subjects with risk factors for vascular alone, several intermediary reactions are disease, therapy known to prevent regulated by insulin. However, insulin cardiovascular events also improve sensitivity is frequently synonymous with the endothelial function (24). ability of insulin to stimulate glucose uptake. 8. Certain human eNOS polymorphisms are Insulin-stimulated glucose uptake can be associated with impaired endothelium- measured in vivo by the hyperinsulinemic dependent vasodilatation as well as euglycemic clamp (32), which was used in cardiovascular disease in case-control many of the studies referred to in this thesis. studies. Such polymorphisms include a The principle of this test is to infuse insulin G894T mutation in exon 7 (with the intravenously at a constant dose, often corresponding change in amino acid sufficient to cause complete inhibition of sequence Glu298Asp) (25); a mutation in hepatic glucose output. Hyperinsulinemia intron 4 (4a/b); and a T786C mutation in induced this way would quickly lead to the 5’-end of the eNOS promotor region. profound hypoglycemia, but during the test, glucose is infused at a variable rate to 9. Treatment that likely has an effect maintain plasma glucose at a constant specifically on endothelial dysfunction concentration in the normal range. Whole-body improves clinical end-points of ischemic insulin sensitivity can thus be expressed as the disease. There is very sparce information rate of glucose infusion necessary to maintain to support this point, but an example is a euglycemia (32). study where oral supplements of L- arginine, the substrate for NO synthase, Insulin resistance is the result of a decreased improved walking distance in patients with effect of insulin to stimulate glucose uptake, or intermittent claudication and peripheral the result of requirements for increased insulin arterial occlusive disease, supposedly by concentrations to stimulate the same glucose restoring vascular NO formation and uptake. Skeletal muscle is quantitatively the vasodilator function (26). most important tissue responsible for insulin resistance (33). Endothelium-derived vasorelaxing or Insulin resistance is probably the earliest vasoconstricting factors abnormality in the pathogenesis of type 2 Several factors synthesized in and released diabetes (34). Transition from the insulin- from the vascular endothelium has effects on resistant nondiabetic phenotype to the state of local vasomotor function. NO, which is a lipid- diabetes, however, also requires development soluble gas, diffuses to VSMC and is of a relative β-cell insufficiency (35). The responsible for relaxetion in response to cGMP sequence of development of insulin resistance produced after activation of soluble guanylate and deficient insulin production, respectively, cyclase when this enzyme binds NO. The in the natural history of type 2 diabetes is vasorelaxing pathway distal to cGMP formation controversial (36). Insulin resistance may in VSMC is incompletely understood (27). Apart result from genetic causes as well as acquired from NO availability, NO-cGMP-mediated factors, primarily obesity (37). Circulating vasodilation is regulated by guanylate cyclase molecules secreted by adipocytes represent a activity (28). major focus to understand the association between obesity and insulin resistance. Other endothelium-dependent vasodilators are Furthermore, such substances are particularly prostacyclin and endothelium-derived important for the discussion in the present hyperpolarizing factor (EDHF). EDHF is study because they may explain the probably representative of several factors not simultaneous occurence of derangements in yet completety characterized. Endothelium- insulin-dependent glucose transport and derived vasodilating factors may substitute for metabolism and insulin-dependent endothelial each other during pathological (29) or Christian Rask Madsen: PhD thesis, Page 3 of 40. function (4). Such adipocyte-derived signals (DIGAMI) study, which included patients with include free fatty acids (FFA) (see page 24), diabetes (> 80% type 2) admitted for acute TNF-α (see page 29) and leptin (37), but in the myocardial infarction, showed a relative risk near future, newly discovered factors (38) may reduction of 28% for total mortality (13) prove to be important. extended to over 3 years after the infarction (46). One purpose for the on-going The molecular causes of insulin resistance are DIGAMI-2 trial is to determine whether this incompletely understood. Since the rate- effect is due to insulin-glucose-potassium limiting step in insulin-stimulated glucose infusion during the first few days of the acute metabolism is thought to be glucose phase of myocardial infarction or due to long- uptake (39), much effort is directed at term effects. identifying the molecular causes of alterations in the insulin signaling transduction Insulin resistance has been shown in several pathway (40) and in the translocation of the studies to be associated with an increased risk glucose transproter protein 4 (GLUT4) from of cardiovascular disease (47), and a intracellular vesicles to the cell prospective study of the role of insulin membrane (41). The insulin signaling pathway, resistance is on-going (48). The results of the which will be discussed in several sections of Diabetes Control and Complications Trial this thesis (see page 23) is activated by the (DCCT) (44) and the UKPDS (45) have refuted binding of insulin to the insulin receptor (IR). previous concerns for the safety of insulin This stimulates tyrosine autophosporylation of therapy, based on observations in certain the IR and tyrosine phosporylation and animal studies that insulin had pro-atherogenic activation of insulin receptor substate (IRS). properties. Although fasting circulating insulin IRS acts as a docking protein for phosphatidyl concentratrations have been shown to be an inositol-3 kinase (PI3K), which it activates. The independent risk factor for cardiovascular primary substrate for PI3K is disease (49), the current prevailing view is that phosphatidylinositol (4,5)-phosphate. Its elevated basal insulin levels in patients with product phosphatidylinositol (3,4,5)-phosphate type 2 diabetes is a marker of insulin interacts with Akt (protein kinase B) (42) which resistance, resulting from a compensatory is recruited to the cell membrane and activated increase of insulin secretion to maintain by serine/threonine phosphorylation by a insulin-stimulated glucose uptake (47). kinase not yet identified (41). The final Several mechanisms linking hyperglycemia and coupling between Akt and GLUT4 translocation insulin resistance to endothelial dysfunction has not been identified. There may be parallel have been described (see page 20). signaling pathways than the one outlined here, including activation of protein kinase C (PKC) ζ and PKC λ (41). Decreased expression or Hypotheses impaired activity of the IR, IRS, and Akt may The study aimed at testing the following each play a role in insulin resistance (41). hypotheses, pertaining to patients with type 2 Hyperglycemia, insulin resistance, and diabetes in poor glycemic control and cardiovascular disease. complicated with ischemic heart disease, versus age-matched, lean, healthy control Several prospective observational studies have subjects: shown hyperglycemia to be an independent risk factor for cardiovascular disease (43). 1. In patients compared to healthy controls, Disappointingly, insulin therapy to patients endothelium-dependent vasodilation is with type 1 diabetes (44), or and intensive depressed whereas endothelium- treatment policy with insulin or oral independent vasodilation is unaffected. sulphonylureas to patients with type 2 2. In healthy controls, local forearm diabetes (45) did not improve cardiovascular hyperinsulinemia acutely increases events or total mortality, although the The endothelium-dependent vasodilation. United Kingdom Prospective Diabetes Study (UKPDS) study (45) did show a non-significant 3. In patients, the effect of local forarm 16% reduction of the relative risk of hyperinsulinemia on endothelium- myocardial infarction in patients managed with dependent vasodilation is decreased an intensive hypoglycemic treatment policy. compared to healthy controls. However, the Diabetes Mellitus, Insulin Glucose Infusion in Acute Myocardial Infarction Christian Rask Madsen: PhD thesis, Page 4 of 40. 4. In patients, insulin therapy administered last few years were screened and potential with the goal of optimizing glycemic control participants contacted by telephone. A decision improve endothelium-dependent to include or exclude each patient was made vasodilation; and this effect is dependent after an out-patient visit. All patients who were on long-term therapy (2 months) rather found eligible and who accepted participation than short-term therapy (3 days). were included. In patients, insulin-stimulated endothelium- Details of clinical characteristics of patients are dependent vasodilation is improved by long- listed in tables 1 and 2. All patients except two term insulin therapy; and this improvement is had a history of hypertension, and ACEI due to increased nitric oxide (NO)-mediated therapy was originally initiated because of vasodilation. hypertension in all cases. Healthy controls Methods Healthy volunteers were recruited after Applied methods newspaper advertisements. They were non- SUBJECTS smoking and did not take high-dose vitamin supplements. All were lean (BMI < 25 kg m-2) Patients and all had normal fasting blood glucose (< 5.6 mmol l-1) on two seperate occasions, normal Patients with type 2 diabetes and ischemic plasma cholesterol (< 6.0 mmol l-1) and a heart disease were included. Ischemic heart normal electrocardiogram. disease was documented by prior myocardial infarction or coronary atherosclerosis Details of clinical characteristics of healthy demonstrated during angiography. Thus, this controls are listed in table 1. diagnosis was confirmed by prior acute EXPERIMENTAL PROTOCOL myocardial infaction in 10 patients, and because of examinations made in relation to Preparations and measurement of blood flow coronary artery by-pass grafting in 15 patients and percutaneous transluminal coronary plasty In patients, oral hypoglycemic drugs were in 3. To ensure inclusion of patients with a withdrawn 2 weeks before the study. All typical type 2 diabetes phenotype and a high medicine was interrupted for 24 hours probability of remaining clinically stable preceeding any of the examinations, and the 4 without their usual hypoglycemic medicine, patients who were smokers abstained from inclusion was restricted to obese patients smoking for 8 hours before each study. Both (body mass index (BMI) > 27 kg m−1) with patients and healthy controls arrived at the diabetes onset at age 50 years or older and laboratory at 8 AM after 8 hours of fasting. An with a diabetes duration of > 2 years without arterial cannula with an external diameter of 1 periods of ketoacidosis. The mean diabetes mm (Ohmeda, Swindon, UK) was inserted into duration was 7.6 ± 1.2 years. In addition, the the brachial artery of the non-dominant arm. patients were required to have a hemoglobin During the whole experiment, isotonic saline A1c (HbA1c) ≥ 8.0%. was infused through the cannula with intermittent co-infusion of solutions of Patients were only included if they had been vasoactive drugs, keeping the total infused receiving a statin (atorvastatin 10-20 mg or volume constant at a rate of 1 ml min−1. A simvastatin 10-20 mg) for > 3 weeks if venous cannula was placed in the medial clinically indicated (total cholesterol without cubital vein of both arms for aspiration of cholesterol-lowering therapy ≥ 5.0 mM). blood samples. In the perfused arm, this Patients with systolic dysfunction (left cannula was inserted retrogradely into a deep ventricular ejection fraction > 50%) or vein where the tip could not be palpated, to decreased renal function (plasma creatinine > allow aspiration of blood from metabolically 130 mmol/l) were excluded. active muscle tissue, rather than from Recruitment of patients took place at a skin (50). department of cardiology and at two The study subjects were examined in the departments of endocrinology at hospitals in supine position with both forearms at Copenhagen, Denmark (the teaching hospitals horizontal level with the right atrium. Forearm of Gentofte, Frederiksberg, and Hvidovre). blood flow was measured by an electrically Patient records from hospitalizations within the calibrated strain gauge venous occlusion Christian Rask Madsen: PhD thesis, Page 5 of 40. plethysmograph (D.E. Hokanson, Bellevue, Insulin-stimulated acetylcholine response WA, USA) (51). Upper arm cuffs were inflated Following the sodium nitroprusside dose- to 40 mm Hg for ~ 15 seconds with ~ 15 response study, the acetylcholine dose- second intervals. Measurements were made response study was repeated during co- simultaneously in both arms immediately infusion of insulin (Actrapid, Novo Nordisk before the start of any drug infusion and after Scandinavia, Malmö, Sweden) 0.05 mU kg−1 completion of the periods described for each min−1 started 20 minutes before and drug infusion below. Any infusion period was maintained during the dose-response study. extended by the duration of blood flow This study was performed in all patients and in measurement, which lasted approximately 2 the same 10 consecutively included healthy minutes. Generally, four recordings were made controls mentioned above (see figure 2a). with the plethysmograph. The recordings are sensitive to mechanical noise, i.e. artifacts due L-NMMA-inhibited insulin-stimulated to voluntary or involuntary muscle contractions acetylcholine response in the forearm, movement of the arm because of breathing, and untowards contact between Following the insulin-stimulated dose-response the strain gauge and, for examle, the bed study, the combined insulin and acetylcholine cover or infusion lines. Furthermore, large study was repeated during co-infusion of NG- variations in blood flow could sometimes monomethyl-L-arginine acetate (L-NMMA) readily be indentified during recordings. When (Clinalfa) 1.6 and 3.3 mg min-1 for 5 minutes such artifacts or large variations were at each dose preceeding acetylcholine infusion recognized during recordings, an extra and maintained at 3.3 mg min-1 during recording or extra recordings were made until acetylcholine infusion. This study was carried at least three stable recordings had been out in the last 17 consecutively included obtained. After the examination, the tangent to patients and in the same 10 healthy controls each curve was marked with pencil on the as mentioned above (see figure 2c). printed recording using a ruler, and the Acute effect of BH4 on the acetylcholine inclination of the tangent measured on a response digitizing board connected to a personal computer with custom-made software. The As a pilot study for a planned study (see the blood flow was calculated as the mean of all section on perspectives), the effect of stable recordings. tetrahydrobiopterin (BH4) (Clinalfa) on the acetylcholine response was examined as the The protocol for the pharmacological dose- last of 4 dose-reponse studies in that days response studies are shown in figures 2a-2c. infusion protocol (see figure 2a). This study Acetylcholine and sodium nitroprusside was carried out in the first 7 consecutively response included patients and was thus preceeded by dose-response studies of acetylcholine, sodium Accumulated dose-response studies were nitroprusside, and acetylcholine during insulin made with acetylcholine chloride (Clinalfa, stimulation. To avoid oxidation, BH4 was Läufelfingen, Switzerland) 7.5, 15, 30, and 60 brought into solution immediately before use µg min−1 for 5 minutes at each dose (total in saline bubbled with nitrogen. BH4 500 µg infusion time at each dose appoximately 7 min−1 was infused for 5 minutes preceeding a minutes including duration of blood flow repeated acetylcholine dose-response study. measurement). Subsequently, a dose-response As for sodium nitroprusside, BH4 was study with sodium nitroprusside dihydrate administered in opaque syringes and infusion (Roche, Basel, Switzerland) 1, 3, and 10 µg lines. min−1 were made with the same duration of each dose as for acetylcholine. Because of its INTERVENTION DESIGN sensitivity to light, sodium nitroprusside was Patient allocation administered in opaque syringes and infusion lines. A flow diagram of the intervention design is shown in figure 1. The first 18 patients The acetylcholine response was examined in consecutively included were randomised 1:1 to all patients and all healthy controls, and the a treatment group and time control group. To sodium nitroprusside response was examined obtain a more precise estimate of the changes in all patients and in 10 consecutively included in insulin-stimulated endothelial function, 10 healthy controls. additional patients were allocated to the Christian Rask Madsen: PhD thesis, Page 6 of 40. treatment group. Clinical characteristics of patient at the late repeat examination day due patients, according to treatment group, are to technical reasons in both cases. Therefore, listed in table 2 and 3. the L-NMMA studies in the treatment group only included 11 paired observations. Patients in the treatment group started insulin therapy in the evening of the initial Acetylcholine reproducibility study examination, following a similar administration of insulin as the one used for long-term An acetylcholine reproducibility study was treatment in the DIGAMI trial (13). Thus, they performed at the early repeat examination in 9 injected themselves subcutaneously with fast- patients and at the late repeat examination in acting insulin (Actrapid Pen, Novo Nordisk) 3 4 patients by a series of 3 dose-response times daily at meals and intermediate-acting studies with acetylcholine, sodium insulin (Insulatard Pen, Novo Nordisk) at bed- nitroprusside, and acetylcholine, respectively time. Patients were given instructions (see figure 2b). regarding insulin dose and diet several times Biochemical analyses. daily in the beginning of the treatment period and at least once a week in the later stages of HbA1c was measured by latex enhanced the treatment period. turbidimetric immunoassay and expressed relative to hemoglobin measured by the The patients in the time control group colorimetric cyanide free alkaline hematin continued without hypoglycemic drugs while method (COBAS MIRA, F. Hoffmann-La Roche, monitoring and reporting their own blood Basel, Swizterland). glucose as a safety measure. Blood samples were drawn immediately before Early and late repeat examination measurements of blood flow. Blood glucose In the first 9 consecutively included patients in was analysed in full blood by enzymatic the treatment group, acetylcholine and sodium colometry (COBAS MIRA) and insulin was nitroprusside responses were re-examined analysed in serum by double antibody after 3 days (“early repeat examination”) radioimmunoassay (Insulin RIA 100, according to the protocol illustrated in figure Pharmacia & Upjohn Diagnostics, Uppsala, 2b. In all patients, the acetylcholine and Sweden). Blood pressure and heart rate were sodium nitroprusside responses were re- read from a recording of the intra-arterial examined after 2 months (“late repeat pressure and an electrocardiogram examination”). At the early and late repeat immediately after each measurement of blood examination, patients in the treatment group flow. took no insulin, the last insulin dose being the CALCULATIONS AND STATISTICAL ANALYSES intermediate-acting insulin at bed-time the night before the study. Medical therapy Analyses of blood flow during acetylcholine or additional to hypoglycemic drugs was sodium nitroprusside does-response studies unchanged throughout the study, except in are described below. Comparisons of one patient in the time control group who catagorical variables of patients characteristics stopped atorvastatin due to side effects. One were made with tests of proportions as well as patient in the time control group was taken out exact binomial testing (Fisher’s exact test). All of the study before the late repeat examination other comparisons were analyzed with two because of symptomatic hyperglycemia, but sample or paired t tests as appropriate. data from the initial examination were included Assumptions of Normality was tested by visual in analyses. inspection of Normal plots and assumptions of equal variances in two sample t tests were The insulin-stimulated acetylcholine response tested with the F test (variance ratio test) was re-examined at the late repeat (Microsoft Excel 97 software). examination in the last 17 consecutively included patients (13 in the treatment group, 4 Blood flow responses during dose-response in the time control group) (see figure 2c). The studies were compaired by mixed models using L-NMMA inhibited insulin-stimulated the mixed procedure in the Statistical Analysis acetylcholine response was re-examined at the Software, version 8 (SAS Institute, Cary, NC, late repeat examination in the same 17 USA). Blood flow was log transformed because patients. However, the L-NMMA study had not the residual variation increased for increasing been accomplished in one patient at the initial values of predicted blood flow (the examination day and was not completed in one untransformed plot of residuals as a function Christian Rask Madsen: PhD thesis, Page 7 of 40. of predicted blood flow was “<” shaped), Ethical considerations whereas the residual variation based on The study was carried out according to the transformed data was independent of Declaration of Helsinki II. It was approved by predicted blood flow (the residual plot had an the local ethics comittee and by the Danish even scatter). Medicinal Agency. All subjects gave written Pharmacological study (e.g., the acetylcholine informed consent. dose-response study or insulin-stimulated The primary ethical considerations of the study acetylcholine dose-response study) entered the pertained to the risk of arterial cannulation. In model as a fixed effect, thus testing for a a review of 8208 catheterisations (the majority systematic difference of overall effect between being left-sided heart catheterisations, most two dose-response studies. The interaction frequently with cannulation of the femoral between pharmacological study and dose of artery), the incidence of complications vasodilator (i.e., the dose-dependent requiring operation was 0.28% (52). These progression of either the acetylcholine or complications include hemorrhage, sodium nitroprusside response) also entered pseudoaneurism, or thrombosis. The arterial the model as a fixed effect, thus testing for a cannulas used in the experiments in the systematic difference of dose-dependent present study have a narrower outside progression of the vasodilator response in each diameter (1.0 mm), are shorter (4.5 cm) and dose-response study. With a linear dose- have less contact with the arterial wall than response curve, this would translate into a catheters used for heart catheterisation; different inclination of the acetylcholine dose- therefore, the risk for complications may be response curve and the insulin-stimulated smaller than that referred to above. acetylcholine dose-esponse curve, independent Accordingly, an British research group has of any difference in overall effect). Other fixed carried out cannulation of the brachial artery effects were group allocation (i.e., patients to perform experiments similar to those in the versus healthy controls and treatment group present study (51). Furthermore, a group at versus time control group); and experimental the University of Pisa, Italy, has performed day. Study subject and the interaction between approximately 2000 similar experiments study subject and dose of vasodilator (i.e., the without observing serious complications dose-dependent progression of the vasodilator (Stefano Taddei, personal communication). response in each study subject) entered the model as random effects, thus taking into Another concern was the withdrawel of oral account the supposedly random variation of hypoglycemic drug therapy from the patients both the overall individual response and the in the time control group. Generally, there is dose-dependent progression of the vasodilator no reason to beleive that the long-term response, both with a mean of zero. P-values prognosis for patients such as those in this from tests of the interactions mentioned will study is affected by withdrawing such therapy not be quoted. The statistical models used are for a period of 8 weeks. In fact, at the time of further described on page 14. initiation of the study there was no evidence from clinical trials indicating that hypoglycemic Statistical significance was defined as a two- drug therapy improved the prognosis in sided p < 0.05. Values (including geometric patients with type 2 diabetes. Thus, symbols with error bars in the figures) are withdrawel of oral hypoglycemic drug therapy presented as mean and standard error of the according to protocol was found to be safe, mean, unless otherwise specified. given sufficient monitoring of metabolic Differences between values for plasma lipids, control. Symptomatic hyperglycemia was a HbA1c, insulin, and glucose were calculated as predefined secondary exclusion criteria. the mean of individual differences and may Finally, the risk of hypoglycemia in the patients therefore not be identical to differences in the treatment group had to be considered. between mean values of groups (as read in the In patients with type 2 diabetes, the risk of tables). severe hypoglycemia is relatively small (45). Please note that in the cases where graphs Attempts to prevent complications as the ones show paired data, observations were included discussed were made by adequate training of only for patients with complete data. However, the person that performed the arterial statistical analyses considered all observations. cannulations (the author of this thesis); by cannulating the non-dominant arm with the Christian Rask Madsen: PhD thesis, Page 8 of 40. purpose of limiting short- or long-term endothelium-dependent agonists. As in the functional loss if complications to cannulation coronary circulation, acetylcholine is usually should occur; by instructions to the subjects to administered as intra-arterial infusion to avoid avoid physical exercise for 24 hours after systemic effects, i.e. hypotension and arterial cannulation; by teaching self- secondary reflex vasoconstriction (51). monitoring of blood glucose to patients in both Increased forearm blood flow during treatment groups; by frequent telephone acetylcholine infusion in the human forearm consultations by the author to monitor model is determined by vasodilation of metabolic control of the patients in both resistance vessels, i.e. arterioles and small treatment groups; and by thorough arteries. In contrast, flow-mediated explanation of the symptoms of hypo- and vasodilation (61, 62) has been used as a hyperglycemia, and the actions to be taken if measure of endothelium-dependent such symptoms were to occur. vasodilation of a conduit arteries (usually the Patients with diabetes and ischemic heart brachial artery) (63). Here, the stimulus is disease have an adverse prognosis (13). As shear stress, i.e. the tangential force on the the study was expected to yield information vessel wall exerted by the flowing blood. Flow- with potential importance for the future mediated vasodilation is non-invasive, both by improvement of prevention and treatment of virtue of the experimental stimulus, post- cardiovascular disease in such patients, the ischemic hyperemia, as well as the fact that risks described above were found to be in vessel diameter can be measured by proportion to the possible benefits of study. ultrasound; it has been shown to be endothelium-dependent (in animals (64)) and Choice of methods: alternatives and mediated by NO (in humans (65)); it is criticism probably the most important physiological Endothelium-dependent stimulus and vascular stimulus for endothelium-derived NO (66); and bed it correlates with coronary endothelium- dependent vasodilation (67). In contrast, Although a plethora of important vascular agonist-stimulated vasodilator response in the endothelial functions exists, only a few human forearm model has never been different strategies have been used to measure compared with coronary endothelium- it in humans in vivo. Investigators have dependent vasodilation or brachial artery flow- studied plasma concentrations of markers of mediated vasodilation. The relative importance endothelial activation, such as von Willebrandt of the different mediators of the acetylcholine factor and circulating adhesion molecules (53); vasodilator response of forearm resistance concentrations of nitrate (NO3−) and nitrite vessels are not identical with those in conduit (NO2−), the stable catabolites of NO, in plasma vessels where atherosclerosis occur (68)). The or urine (54); or whole-body 15N-arginine-15N- primary reason that the acetylcholine response citrulline kinetics (55). However, most or the is perceived as a measure of endothelial literature in this field describe endothelium- function with implications for the pathogenesis dependent vasodilation. It is directly involved of atherothrombotic disease is the well- in normal vasomotor control (56, 57) and is established fact that the classical risk factors probably responsible for symptoms because of for atherosclerosis - hypercholesterolemia, insufficient circulation (58). In addition, as hypertension, and diabetes - are associated several endothelial functions are commonly with abnormal endothelium-dependent affected simultaneously, endothelium- vasodilation in both the coronary arteries (69- dependent vasodilation is frequently viewed as 71), the brachial artery (63) (72) (73), and a measure of general endothelial function (20). forearm resistance vessels (74-76) (references Endothelium-dependent vasodilation was first only made to the first published studies in studied in man by visualizing coronary artery each catagory). diameter by arteriography during intra- Helmuth Drexler and co-workers have coronary infusion of acetlycholine (59). Since combined advantages of the forearm model the introduction of measurement of and flow-mediated vasodilation by using one- endothelium-dependent vasodilation in the dimensional ultrasound (resembling A-mode forearm (in the following referred to as “the echocardiography, no longer in clinical use) to human forearm model”) (60), this method has image flow-mediated vasodilation of the radial become the most popular in studying artery during pharmacological manipulation in vasodilator responses to acetylcholine or other the forearm by drug infusion into the brachial Christian Rask Madsen: PhD thesis, Page 9 of 40. artery. Endothelium-dependent vasodilation transducer or flow probe, for example, may be has also been measured as the response of applied or placed), but excluding blood supply forearm skin perfusion to acetylcholine applied that does not result in distension of the by iontophoresis (77, 78), or as maximum forearm during venous return, for example hyperaemic response to local heating of the part of the blood supply to bone. Thus, foot skin (79). different methods may estimate blood flow to various tissues (muscle, skin, bone) differently, We chose to use the technique of intra- and furthermore, hyperemic stimuli (exercise, brachial infusion of acetylcholine because it heating, drugs) may differentially increase can be combined with local insulin stimulation blood flow in certain tissues. (see below), does not require extensive training to operate adequately (as does one- In 8 patients with valvular or ischemic heart dimensional ultrasound, Helmuth Drexler, disease, measurements with plethysmography personal communication), and does not have of blood flow at rest or stimulated by isotonic limitations in obese subjects (as with two- hand grip correlated well with measurements dimensional ultrasound of the brachial artery using an electromagnetic flow-meter (85). The because of impaired image quality, Keld flow-meter was applied to the surgically Sørensen, personal communication). isolated brachial artery and calibrated against controlled blood flow in canine femoral In the present study, acetylcholine (and artery (85). The correlation was best at blood sodium nitroprusside, see page 10) was flows below 15-20 ml (100 ml)−1 min−1 (the examined in a cumulative dose-response flow range relevant to the present study), and study. The method makes it possible to detect plethysmography overestimated blood flow differences in effects without prior knowledge relative to that measured by the flow-meter at of whether they exist at low or high doses. In rest and during hyperemia (85). In contrast, addition, the comparison of dose-response plethysmography was shown to underestimate studes, when a difference is present at several leg blood flow at rest compared to the dye- doses, has a statistical power which exceeds dilution method (86). comparison at a single dose (see page 14). In small populations, the mean acetylcholine Measurement of blood flow and determinants response has been shown to be reproducible of variability related to experimental design on the same day, and the sodium nitroprusside Forearm blood flow was measured by mercury- response on the same day as well as on in-silastic strain gauge venous occlusion different days (see the sections below for each plethysmography (51, 80, 81). Very few drug). Such presentation of reproducibility can studies have employed methods other than only suggest the presence or the absence of a plethysmography for measurement of systematic error, for instance due to endothelium-dependent forearm vasodilation, tachyphylaxia. Reports on intra-individual but methods, probably more accurate, exist variability of basal or stimulated blood flow are wich involve invasive manipulation (e.g. rare in the literature. The intra-individual measurement with doppler flow wire in the coefficient of variation for basal flow was 13 ± brachial artery (82-84) or demand great 2% (87) or 15.5 ± 2.3% (88) on the same day ressources (e.g. magnetic resonance but 31% on different days (89). For the imaging (83)). Venous blood flow as a vasodilator response to unilateral forearm substitute for limb blood flow cannot be exercise it was 17%. The interquartile range of measured by the thermodilution method as in the difference between vasoconstrictor the leg (8), because a vein collecting the responses to intra-brachial infusion of majority of venous return, as the fermoral vein angiotensin II or noradrenaline from one day in the leg, is not accessible. to the other was ≤ 27% (89). Forearm or leg strain gauge plethysmography Because blood flow through arterio-venous have been validated against other methods of anastomoses in the hand is more variable than blood flow measurement. Discrepancies flow to forearm skeletal muscle and between flow measurements with different determined, among other factors, by techniques may be caused by differences in temperature regulation, the standard what they measure: plethysmography procedure is to occlude the hand circulation by measures total limb blood flow, including that a wrist cuff inflated to suprasystolic pressure through skin collaterals branching off before during measurement of forearm blood the distal brachial artery (where a doppler flow flow (51). However, a wrist cuff may not be Christian Rask Madsen: PhD thesis, Page 10 of 40. important when studying pharmacological vasodilating factors accounting for the vasodilator responses (90). vasodilating effect of acetylcholine have not been identified as only approximately 40% A resting period seperating the time of arterial (dependent upon species and vascular bed) of cannulation from the first blood flow vasodilation can be blocked by measurements, as in the present study, is pharmacological inhibition of NO and standard in the literature, although arterial or prostacyclin production. venous cannulation have been shown to not alter basal blood flow (86). Acetylcholine has a half-life in plasma of a few seconds because it is rapidly catabolised by Blood flow has been shown not to be plasma cholinesterase. This may an explain dependent on forearm circumference (90), but why vasodilatation to acetylcholine may (93) a large part of the interindividual variability of (or may not (90)) depend on forearm length. insulin-stimulated vasodilation in healthy Plasma cholinesterase activity is variable, in subjects seems to be explained by relative limb part atributtable to genetic variation of its muscle content (87) and density of muscle encoding gene (94). To avoid variability of the capillaries (91). acetylcholine response due to rapid and Comparison of vasodilator (to acetylcholine or unpredictable catabolism of acetylcholine, sodium nitroprusside) or vasoconstrictor (to L- some authors have used the synthetic NMMA) responses between groups have cholinergic agonist metacholine (see table 5c). limited value because the pharmacological However, the use of metacholine has been response is dependent on factors independent critisized, because although it activates the of the pharmacodynamics properties, namely same receptor as acetylcholine (95), the vessel wall tension (due to blood pressure and response has not been found to be inhibited by preconstriction) and vessel wall geometry L-NMMA (96). (wall-lumen ratio) (51). For the same reasons, Acetylcholine is considered a physiological and because absolute and relative increments stimulus for the endothelium, as endothelial of blood flow during drug infusion are cells may be stimulated by cholinergic dependent on basal blood flow (51), it is perivascular axons or by acetylcholine impossible to determine if comparisons synthesized and released by endothelium in an between groups of absolute or blood flow autocrine or paracrine fashion (97). However, relative to base-line are most valid; this is true it is unknown whether intra-brachial infusion, for comparisons in the same group during as in the present study, produces dose-response studies on the same day or physiologically relevant concentrations of different days. acetylcholine in plasma or at the luminal or Acetylcholine abluminal surface of the endothelium. Although acetylcholine is often called a Other endothelium-dependent agonists have receptor-dependent agonist of eNOS, the been used in the human forearm model. French researcher Paul Vanhoutte has Serotonin has the potential advantage that its suggested that such an agent is merely vasodilating effect can be completely inhibited eliciting a “response to a vasodilator that by L-NMMA (98), but it is a disadvantage causes endothelium-dependent relaxation in when comparing with one’s own results that it vitro” (92). In humans, the primary argument has only been used in few human studies. for acetylcholine being an endothelium- Bradykinin (99) and substance P (99, 100) dependent vasodilator is that its effect can be have also been used, but have no obvious partly inhibited by L-NMMA (see page 11). advantages over acetylcholine. Acetylcholine stimulates endothelial NO The vasodilator response to acetylcholine production after binding to endothelial reach a plateau at 3-14 minutes after the muscarinic G-protein coupled receptors, thus adminstration of a new dose in a dose- stimulating phospholipase C which mediates response study similar to that in the present Ca2+ influx through the cell membrane and study (101). Thus, the infusion period of 5 activation of eNOS after increased binding of minutes was chosen to allow measurement of Ca2+ to eNOS-associated calmodulin. Increased blood flow during a steady state of intracellular Ca2+ also stimulates production of vasodilation. arachidonic acid metabolites, e.g. prostacyclin, The mean acetylcholine-stimulated through activation of phospholipase A2. All vasodilatation in the leg of patients with Christian Rask Madsen: PhD thesis, Page 11 of 40. ischemic heart disease was reproducible when streptozotocin-treated rats, a model of the dose-reponse study was repeated on the diabetes (109). same day (102). Increased inhibition by L-NMMA of basal blood Doses of acetylcholine were chosen to elicit flow or a pharmacological vasodilator response expected vasodilator responses from one close does not prove the existance of increased NO to the smallest detectable to one as large as production. Rather, it suggests increased NO possible whilst still avoiding systemic effects, available for signaling (because of increased guided by previous literature. NO production or because of decreased NO break-down, e.g. due to increased vascular Sodium nitroprusside superoxide production) or an increase of NO The mechanism of sodium nitroprusside as a signaling (e.g. because of decreased activity of NO donor in vascular tissue is unknown. The guanylate cyclase, the “NO receptor” in NO generating activity has been localized to vascular smooth muscle cells responsible for membrane fractions of VSCMCs (103), and production of NO’s second messenger, cGMP). there is evidence that NO is released on the The IC50 of L-NMMA for eNOS activity is 3-7 extracellular VSMC surface by reduction of µmol l−1 (110). The dose of L-NMMA chosen in sodium nitroprusside through activity of the present study is similar to high doses in membrane-bound NADH oxidase (104). On the previous studies. It produces, calculated by the other hand, it has been suggested that sodium formular in the appendix, a plasma nitroprusside actually donates NO+ concentration of approximately 1.4 mmol l−1. intracellularly (105). The intracellular L-NMMA concentration in The only frequently used alternative NO donor endothelium is unpredictable. There are no in human studies is nitroglycerin. It is reports on unspecific effects of L-NMMA dependent on adequate intracellular administered in similar doses. concentrations of cysteine. It does not have Insulin-stimulated acetylcholine response any clear advantages or disadvantages compared to sodium nitroprusside. The infusion of insulin was first planned with the intention of studying the very early effects The mean sodium nitroprusside response in 10 of insulin on endothelium-dependent and - healthy controls was different by ≤ 6.8% when independent vasodilation, as opposed to the repeated after 2 hours or after 3 weeks (90). effects after 3 days and after 2 months, and Doses of sodium nitroprusside were chosen to the insulin-stimulated acetylcholine response make vasodilator responses match the was not a pre-specified end-point in the expected acetylcholine responses, guided by original protocol. This pharmacokinetic previous literature. understanding was superseded by the concept of the insulin-stimulated acetylcholine L-NMMA response as a test for endothelial insulin At the time of starting the present study, the sensitivity, in the sense that is used in this only eNOS inhibitor available for human use thesis. Therefore, only 13 patients in the was L-NMMA. As a methylated analogue of L- treatment group and 4 patients in the time arginine, L-NMMA is a competitive inhibitor of control group had this response tested at the NO synthase. Physiologically, a low late repeat examination day. endogenous production of L-NMMA Experiments to control for the insulin- exists (106). L-NMMA inhibits NO synthase stimulated acetylcholine response activity, but not the reduction of O2, leaving intrinsic superoxide-generating activity The studies of the L-NMMA inhibited insulin- unaffected (14, 107). On the other hand, NG- stimulated acetylcholine response was nitro-L-arginine (L-NNA), which has recently designed to detect whether any change of the been used for humans by infusing its inactive insulin-stimulated acetylcholine response was precursor NG-nitro-L-arginine methyl ester (L- endothelium-dependent and mediated by NO. NAME) (108), inhibits both NO production and An alternative to the L-NMMA control would be O2 reduction. Preliminary evidence suggest to measure the insulin-stimulated sodium that such actions of L-arginine analogue nitroprusside response. An increase in this inhibitors may be relevant physiologically, response after clinical intervention would because L-NNA inhibits the upregulated suggest that an increase of the insulin- superoxide production in aorta from stimulated acetylcholine response was Christian Rask Madsen: PhD thesis, Page 12 of 40. endothelium-dependent only if the former was coupling of local glucose concentrations and unchanged or had increased; and vice versa. local blood flow, because co-infusion of L- An increase of the insulin-stimulated sodium glucose (which is not recognised by glucose nitroprusside response would not preclude that transporter proteins) did not have the same an increase in the insulin-stimulated effect (116). acetylcholine response was also endothelium- A link between glucose transport and blood dependent. Furthermore, it would not suggest flow is further substantiated by the observation which factor (e.g. NO, prostacyclin, EDFH) was that during experiments where whole-body mediating the change of the insulin-stimulated and leg glucose uptake were constant in acethylcholine response. subjects with a wide variety of insulin Local insulin stimulation sensetivity, namely in lean, healthy subjects, obese subjects, and patients with type 2 The method of local insulin stimulation in the diabetes, leg blood flow increased by the same forearm has been employed in for metabolic amount (from ∼ 0.2 to ∼ 0.4 l min−1) (118). studies for decades since it was first These conditions were achieved during described (111). In subjects with a history of euglycemic hyperinsulinemic clamps with step- regional sympathectomy, vasodilation to wise increments of systemic insulin infusion systemic insulin infusion is delayed in the (40-1200 mU m−2 min−1) and hyperglycemic innervated calf and abolished in the hyperinsulinemia with step-wise increments of denervated forearm by intravenous infusion of systemic glucose infusion (resulting in blood L-NMMA, suggesting a direct NO-mediated glucose concentrations of 4-20 mmol effect of insulin which is modulated by l−1) (118). Furthermore, vasodilation to sympathetic activity (112). This finding is systemic insulin infusion is intact in patients explained by the well-established ability of with type 2 diabetes if blood glucose is insulin to stimulate central nervous system clamped at each patient’s prevailing fasting sympathetic activity (113). Insulin-stimulated blood glucose (119). In contrast, insulin’s sympathetic effects on peripheral vasomotor vasodilatory effect does not seem to be function, probably vasoconstrictor effects, may dependent on carbohydrate metabolism (120). be different in insulin sensitive and insulin Glucose infusion, but not fructose infusion at resistant individuals because sympathetic the same molar dose, caused an increase in activity in obese (114) and elderly (115) circulating concentrations of endogenously subjects is increased in the fasting, basal state, produced insulin. Indirect calometry showed but reduced during insulin stimulation that carbohydrate oxidation was similar during compared to lean controls. the two infusions, but only glucose infusion Intra-arterial infusion of insulin without increased calf blood flow; and co-infusion of concommitant infusion of glucose has been insulin and glucose further elevated circulating critisized for being unphysiological because it insulin concentrations as well as blood results in a reduction of local glucose flow (120). concentrations, whereas physiological increase Studying vasodilation to intra-arterial infusion in circulating insulin levels in any vascular bed of insulin in the forearm rather than the leg is usually accompanied by an increase in blood has the advantage that a larger rise of glucose (116). Such experimental conditions systemic insulin levels can be expected during during intra-brachial insulin infusion have also intra-femoral infusion, because the basal blood been made responsible for the fact that many flow to the leg is approximately 10 times studies have not found an effect of higher than in the forearm, and because the intrabrachial insulin infusion to increase basal dose necessary to raise the concentration of blood flow (116). Notably, one study found insulin is directly proportional to forearm or that similar insulin concentrations in the blood flow. forearm caused increased basal forearm blood flow in healthy individuals during systemic Serial pharmacological studies on each hyperinsulinemia, but not during local experimental day hyperinsulinemia (117). On the other hand, It is generally acknowledged that it is a big intra-arterial co-infusion of D-glucose to advantage to compare pharmacological maintain local euglycemia augmented the vasomotor responses on the same day in a vasodilatory effect (from 20 to 47%) of single subject to avoid the inter-individual insulin (116). The authors speculated that the variability related to day-to-day variation (51). local rate of glucose uptake may explain this Christian Rask Madsen: PhD thesis, Page 13 of 40. However, this design could be flawed by carry- prior infusion of any of the other drug over effects, tachyphylaxia or time effects. combinations was found more important than There are no reports in the literature of carry- controlling for systematic effects of serial over effects or tachyphylaxia of acetylcholine infusions. Therefore, the infusion protocol was or sodium nitroprusside responses (see the planned with a fixed sequence. sections on each drug above). Design of clinical intervention study There may have been a positive carry-over effect of insulin from the first infusion The intervention design used was an open (together with acetylcholine) to the second parallel group intervention. A cross-over design (together with sodium nitroprusside or with would have had the benefit of paired acetylcholine and L-NMMA). There is reason to observations between insulin treatment and suspect this because protein expression and withdrawal of pharmacological treatment. This activity of eNOS is already apparant after one would be less suitable considering the potential hour in cell culture experiments (121). carry-over effects of insulin treatment, depend Accordingly, the increase in basal limb blood to an unpredictable degree on the duration of flow during local or systemic insulin stimulation wash-out period. Furhtermore, insulin- in humans is time-dependent within a few treatment is expected to result in weight gain hours (122). (as was indeed the case), which may in itself unfavorably influence insulin sensitivity and A circadian rhytm has been described for basal insulin dose requirements, and possibly and stimulated forearm blood flow with a endothelial function. lower basal forearm blood flow and a larger vasodilation to the α-adrenergic antagonist Patient selection phentolamine in the morning than in the The patient selection was primarily motivated afternoon or evening (123). Incidentally, there by the observations made in the DIGAMI trial was no diurnal variation of vasodilation to as described in the introductory section (see sodium nitroprusside in this study (123). page 1). By including patients with manifest The pharmacological vasomotor response has ischemic heart disease we selected patients been found to be dependent on basal blood individually prone to the vascular complications flow in some (51, 93), but not all (90), studies. of type 2 diabetes. This population can be If wash-out periods between pharmacological expected to have more severe insulin studies do not allow for a complete return of resistance than patients with type 2 diabetes blood flow to the initial basal level, a larger without clinical signs of vascular disease response can be expected. (124). A final consideration is that the duration of the Choice of healthy control group vasoconstrictor effects of L-NMMA is several hours. Therefore, L-NMMA can only be used as The most obvious alternatives to the control the last of a series of infusions. group the healthy controls included in the present study would be healthy volunteers One pharmacological study on each of several matched with patients for BMI or another experimental days in the same individual index of obesity; or patients with ischemic would not be feasible because of the time heart disease and normal glucose tolerance, invested by the study subject, the invasive matched with diabetic patients for BMI. It nature of the methods, and ressources. One would have been an attempt to answer if the pharmacological study on a single day in vascular function studied was dependent on different individuals would require large groups the hyperglycemic phenotype or obesity, for sufficient statistical power. If untoward perhaps obesity-related insulin-resistance; or if effects of serial infusions has to be accepted, it was dependent on the manifestation of randomisation of the sequence of infusion may ischemic heart disease. Other authors have ensure that these effects are not systematically suceeded, in human studies of endothelial favoring a certain result. But sequence function, to show that cardiovascular risk randomisation in a small sample size may factors are additive with respect to their easily confound results unpredictably. The association with endothelial dysfunction (125). main focus was the acetylcholine response and However, matching patient groups would the insulin-stimulated acetylcholine response. probably not be feasible because insulin Obtaining an estimate of the acetylcholine sensitivity would likely not be similar in obese response without the possible influence of a and type 2 diabetic patients, respectively; and Christian Rask Madsen: PhD thesis, Page 14 of 40. matching for additional factors that would Statistical analyses compete for an influence on endothelial The analytical strength of the dose-response function (medication and plasma lipoproteins study is that if the response is different in two (see page 14), blood pressure, situations, most often the difference is present microalbuminuria, and smoking status) would at several doses, thus increasing the be very difficult. Therefore, the more modest confidence of the observation made at one ambition was chosen of simply studying dose. To take advantage of this fact, an whether the diabetic patients, with their analysis comparing the difference at each combined characteristics, had a vascular dose, i.e. in seperate two-sample tests, is function different from that of lean, healthy inadequate (130). The analysis of dose- volunteers. response studies cannot be studied with very Concommitant medication and competing risk simple methods such as those available in factors for endothelial dysfunction standard spreadsheets. The general problem is that all such methods require that observations The 3-hydroxy-3-methylglutaryl-CoA reductase are independent – which statistically implies inhibitors (statins) may improve endothelial that the observations are not correlated function independent of their effect on (covariance equal to zero). We considered cholesterol metabolism, for example by using simple data reduction (average or slope increasing endothelial NO synthase (eNOS) of observations in one individual). We were expression (126), and statin therapy of reluctant to use such methods because they subjects with desirable cholesterol levels use data reduction, the consequence being increases their endothelium-dependent that more experiments are required to study a vasodilatation (127). In a recent study, phenomenon than without data reduction. cholesterol levels, even in the range considered desirable according to current We therefore sought professional help, and Dr. guidelines (< 5.0 mmol l−1), was shown to be Philip Hougaard accepted to advise us. The negatively correlated with endothelium- choices we had were analysis of variance with dependent vasodilatation (128). According to repeated measures; and mixed models. The this study, a difference in plasma cholesterol of clear recommendation was mixed models. In 1.0 mmol l−1 would be associated with a addition to the help of Dr. Hougaard I difference in maximal endothelium-dependent consulted a textbook on the subject (131). vasodilatation of 64% (read from figure 4 in An outline understanding of mixed models this article (128)). Thus, plasma cholesterol must take an outset in the more familiar concentrations and statin therapy status were analysis of variance. In a standard analysis of the characteristics that were most likely to variance a model is formed to explain the influence endothelial function independent of overall variance (sum of squares of all the characteristics we wanted to modify, differences between the overall mean and namely hyperglycemia and insulin resistance. each observation) of a variable (blood flow) as With the purpose of avoiding composed of variance caused by variables of hypercholesterolemia and establishing a interest (e.g. treatment) and unspecified homogeneous patient group with regard to variability. The effects of a variable is termed a cholesterol levels and statin medication, fixed effect. In the general framework of my patients were only included if they were study a fixed effect will imply that the dose treated with a statin when clinically indicated. response curve of a individual is parallel Treatment with aspirin (102) and angiotensin shifted according to each influential effect. converting enzyme inhibitors (24) may have Repeated measures (each dose response improved the endothelium-dependent curve) can be handled with analysis of responses in the patients, and smoking may variance with repeated measures, but there have impaired it (129). Medication in the are limitations. The only effects that can be patients and differences in cholesterol levels studied are fixed effects (see below) and data and blood pressure between the patients and must be complete. If one flow is missing, all healthy controls makes it impossible to observations for that individual are excluded conclude which factors are responsible for any from the analysis. This last problem can be differences in acetylcholine responses, but solved by various methods of interpolation, but does not limit the interpretation of the effect of the analysis then addresses real as well as insulin therapy on vascular responses in the virtual data. For the present study, the patients. repeated measures would also not be handled Christian Rask Madsen: PhD thesis, Page 15 of 40. completely correctly. Models can be formulated other effects (and interactions) were specified which take into account that each dose as fixed effects. response curve are repeated measures, but it As for model assumptions we received the is not possible to formulate models which also advice to study in detail plots of predicted take into account that several dose response values versus residuals (differences between curve are from the same individual. the model and the original observations). The Mixed models is a general term for a large assumptions of the model require that variety of analysis of continuous and residuals are normally distributed and not categorical data. Within this framework, only dependent on the predicted value. Our initial random effect models of continuous data were models of untransformed data did indicate used. The basic difference between mixed normal distribution of residuals, but the models and a standard analysis of variance is distribution was heavily dependent on the that the models include covariance terms value of the predicted value. After logarithmic (correlation) and can therefore fit correlated transformation of blood flow data the model data. In contrast to analysis of variance, the assumptions appeared fulfilled. models can handle interaction effects including The acutal data handling was performed by several variables. Thus, in the present study, myself in cooperation with Christian Torp- effects of interest (and interactions), most Pedersen (who has extensive experience with importantly long-term therapy, were entered SAS programming). SAS programs were as fixed effects. Other effects were entered as checked by Philip Hougaard. random effects. Random effects are a particular strength of mixed models. As The biologist reading this account may worry described above, a fixed effect implies a that the results of the experiments depended parrallel shift of a dose response curve. on the use of complex models, or even that Furthermore, it implies that for the important the complex models were used to obtain a interaction between dose and individual, each positive results. Fortunately the models were individual is associated with a particular slope robust. Omission of interactions terms or of a dose response curve. It is more likely that omission of transformation gave the same the slopes of a dose response curve represent overall results (i.e. significant results for the random variation around a mean slope. By same experiments, but with different p- entering the interaction between dose of values). acetylcholine and individual as a random effect With this description of mixed models I do not is thereby assumed that the slopes vary pretend to be able to account for the models normally with a mean of zero. This appears used in a mathematical sense. The biologist reasonable, except for the fact that the slope using mathematical methods need to is obviously not zero. This problem is handled understand the use, the limitations and model by adding interaction between individual and checking of complex methods – and to ensure treatment as a fixed effect, thus assuming that by professional advice that the methods are each treatment is associated with a mean correctly used. My understanding of mixed slope, and that individuals receiving that models is limited to this approach. treatment have slopes that vary from this average slope. In this account I have used the term slope to make the subject intuitively Results understandable. It is also possible not to assume any particular relationship between Insulin stimulation dose and response. This was actually the During insulin infusion, local serum insulin was method used because the large variation of raised to 133 ± 14.6 mU l−1 and 105 ± 16 in the results make strict assumption of the the total patient group at the initial relationship between dose and response examination and in the healthy controls, uncertain. respectively (p = 0.3). Local serum insulin was Model formulation and model assumptions are not different during insulin stimulation critical in any use of models. The final model between any group of subjects or any used for the data specified all variables of examination day (p > 0.2). interest and all interactions. Individual and the Local serum insulin was not statistically interaction between individual and dose different in healthy controls (105 ± 16 mU l−1, (without assuming a linear relationship) was p = 0.3) or during insulin stimulation with or the only random effect variable included; all Christian Rask Madsen: PhD thesis, Page 16 of 40. without L-NMMA in the different groups of blood pressure did not change in the treatment subjects or on different examination days in or the time control group (table 2, p > 0.5). the two groups of patients. Blood flow data Systemic insulin was raised by 1.3 ± 0.6 mU l−1 during insulin infusion in healthy controls and All data were missing at the late repeat by ≤ 3.7 mU l−1 in patients in either group at examination for one patient who was any day. Local and systemic blood glucose withdrawn from the time control group decreased by ≤ 0.8 mmol l−1 at any day in any because of symptomatic hyperglycemia. Data group (table 1). from dose-response studies with L-NMMA were missing in one patient at the initial All local insulin data were missing, due to examination and in another patient at the late failure of placing a venous cannula, in 2 repeat examination for technical reasons. patients at the initial examination, in 3 patients Blood flow data from measurements at certain at the late repeat examination, and in one doses (between one and three doses in a healthy control subject. single dose-response study) were missing in Please note that the fasting blood glucose, five patients. measured immediately after venous Acetylcholine response cannulation in the morning, was generally somewhat higher than the levels measured Basal blood flow was not different in healthy immediately before insulin stimulation (table 1- controls and in the total patient group at the 4). initial examination (2.1 ± 0.2 and 1.9 ± 0.2 ml (100 ml)−1 min−1, respectively, p = 0.5) The Metabolic control acetylcholine response was lower in the total Fasting serum insulin was more than twice as patient group compared with healthy controls high in patients compared with healthy (figure 3, p = 0.03). controls (16.2 ± 7.8 versus 6.6 ± 0.5 mU l−1) Sodium nitroprusside response which, considering their elevated fasting blood glucose, indicates that they were insulin The sodium nitroprusside response was lower resistant (132). In the treatment group, fasting in the total patient group at the initial systemic serum insulin increased significantly examination than in healthy controls (figure 4, from the initiale examination to the late repeat p = 0.03). examination (p = 0.01), which was probably a result of insulin administration. There was no Insulin-stimulated acetylcholine response hypoglycemic events that required other Twenty minutes of insulin infusion had no management than patient-initiated effect on basal forearm blood flow in healthy carbohydrate intake. controls (before insulin stimulation 2.5 ± 0.5; Plasma cholesterol was not different in the during insulin stimulation 2.9 ± 0.6 ml (100 total patient group (4.6 ± 0.2 mmol l−1) ml)−1 min−1; n = 10, p = 0.2) or in the total compared with the total group of healthy patient group at the initial examination (2.1 ± controls (4.9 ± 0.1 mmol l−1, p = 0.1), but 0.2 ml (100 ml)−1 min−1 without change, n = plasma triglycerides were higher (table 2, p < 28). 0.0001). In healthy controls, insulin had a large In the treatment group, fasting blood glucose stimulatory effect on the acetylcholine decreased after 2 months of insulin therapy to response (149 ± 47, 110 ± 33, 100 ± 45, and about half the value at the initial examination 106 ± 44% increase of blood flow during the 4 (table 3) and HbA1c decreased from 10.0 ± 0.4 doses of acetylcholine, figure 5, p < 0.0001). to 7.5 ± 0.2%. In the time control group, In contrast, such an effect was absent in the fasting blood glucose was unchanged from the total patient group at the initial examination initial to the late repeat examination (table 2), (figure 6, p = 0.3). and although HbA1c deteriorated slightly, this L-NMMA inhibition of insulin-stimulated was not statistically significant (table 2, p = acetylcholine response 0.07). Plasma triglycerides decreased in the treatment group (table 2, p = 0.0004), but In healthy controls, L-NMMA decreased blood were unchanged in the time control group. flow in response to co-infusion of insulin and Total plasma cholesterol, HDL cholesterol and acetylcholine by 46 ± 9, 43 ± 9, 30 ± 15 and 21 ± 14% during the 4 doses of acetylcholine Christian Rask Madsen: PhD thesis, Page 17 of 40. (figure 5, p < 0.0001). In patients at the initial no effect of insulin on the acetylcholine examination, the corresponding relative response (figure 17, p = 0.7). vasoconstriction to L-NMMA was 41 ± 6, 25 ± 15, 40 ± 5 and 33 ± 8% (figure 19, p < Acetylcholine reproducibility 0.0001). The vasoconstriction to L-NMMA was The acetylcholine response was reproducible not different between these subgroups of when repeated after the sodium nitroprusside patients and healthy controls (p = 0.5). infusion (2.3 ± 0.2, 3.6 ± 0.5, 6.2 ± 1.1, and There was no difference between decrements 9.3 ± 1.7 ml (100 ml)−1 min−1 at each dose of of insulin- and acetylcholine-stimulated blood acetylcholine during the first dose-response flow during L-NMMA infusion in healthy study; 2.8 ± 0.4, 4.9 ± 0.8, and 7.6 ± 1.4 ml, controls or in patients at the initial examination and 10.6 ± 1.8 (100 ml)−1 min−1 at each dose day (figure 7). during the repeated study; n = 13, p = 0.2). Effect of BH4 on the acetylcholine response Systemic circulatory responses After 5 minutes of BH4 infusion, blood flow Blood flow in the arm contralateral to the was unchanged compared to basal (3.6 ± 1.2 perfused arm were unchanged throughout the versus 3.8 ± 1.0, p = 0.9). The acetylcholine studies. As an example, blood flow in the response during BH4 infusion was larger than control arm is displayed in figure 20. In the during acetylcholine alone (figure 8, p < subgroup of healthy controls who were studied 0.0001). during insulin stimulation and in the total patient group at any examination day, systolic Effect of short-term insulin therapy on blood pressure was unchanged during acetylcholine and sodium nitroprusside acetylcholine infusion, but decreased by 7-12 responses (range at highest dose) mmHg during sodium nitroprusside infusion and increased by 4-10 At the early repeat examination in patients in mmHg during L-NMMA infusion (p ≤ 0.01). the treatment group, both the acetylcholine (figure 9) and the sodium nitroprusside (figure 10) response had decreased (p = 0.007 and Discussion 0.009, respectively). Interpretation of own results Effect of long-term insulin therapy on vascular Acetylcholine responses before treatment responses Like the present study, all previous studies, After 2 months of insulin therapy, the except one (133), have demonstrated acetylcholine (figure 11) and sodium decreased endothelium-dependent nitroprusside responses (figure 10) were vasodilatation in patients with type 2 diabetes unchanged (p = 0.09 for both). with similar methods (6, 24, 76, 134-142). In contrast, insulin-stimulation now had a Indeed, several studies have also found significant effect in patients in the treatment impaired flow-mediated vasodilation (73, 143, group (58 ± 25, 84 ± 66, 120 ± 93, and 69 ± 144). Most of these studies included patients 36% increase of blood flow during the 4 doses without clinical signs of cardiovascular disease. of acetylcholine, figure 12 and 13, p = 0.0002). In the majority of previous studies of forearm This result was similar when analyzed by responses to nitrovasodilators, their effect repeated measurements ANOVA (p = 0.02). have been shown to be similar in patients with The decrements of insulin- and acetylcholine- type 2 diabetes and healthy controls, although stimulated blood flow during L-NMMA infusion some have found an impaired response (76, were unchanged by insulin therapy (figure 14, 136). Given that such an impairment of p = 0.9). endothelium-independent vasodilation is Acetylcholine and sodium nitroprusside representative of the general pathophysiology responses and insulin-stimulated responses in of type 2 diabetes - or other risk factor for the time control group atheroscleroses - it is probably smaller than the decreased endothelium-dependent In the time control group, acetylcholine (figure vasodilation, and some studies may not have 15) and sodium nitroprusside responses (figure had the statistical power to detect it. Thus, 16) did not change after 2 months (p = 0.09 decreased brachial artery vasodilation to and 0.6, respectively). At this time, there was sublingual nitroglycerin was associated with Christian Rask Madsen: PhD thesis, Page 18 of 40. risk factors for cardiovascular disease in a and more efficient therapy. For example, in the study with a large sample size (n = 800) (145) DIGAMI trial HbA1c decreased 1.1 ± 0.1% in and preliminary data from a study of 90 the intervention group and 0.4 ± 0.1% in the patients with type 1 diabetes showed control group after 3 months (46). decreased forearm vasodilation to nitroglycerin After a new steady state of glycemic control, infusion (146). A decreased response to HbA1c changes to a new level after at least one exogenous NO may be explained by some of month, so HbA1c at the initial study day the mechanisms that have also been probably overestimated glycemic control after implicated in the endogenous NO production, 2 weeks without hypoglycemic medication, and e.g. breakdown of NO by reactive oxygen the small (and statistically insignificant) species before reaching targets for NO increase in HbA1c most likely did not reflect a signaling, or dysregulation of the signal real change in glycemic control. pathway distal to NO, e.g. decreased guanylate cyclase activity (see page 24). The decreasing plasma concentrations of triglycerides was probably associated with a Local insulin stimulation decrease of FFA, which, unfortunatley, were By design, local serum insulin during insulin not measured. FFA may directly influence stimulation was not different in patients and in insulin sensitivity (149) and both endothelial healthy controls. A stimulating effect of insulin function and insulin-stimulated endothelial on endothelium-dependent vasodilatation may function (see page 24). exist at a higher concentration of insulin. This Insulin-stimulated acetylcholine response consideration was taken into account in a study where endothelium-dependent In the healthy controls, insulin had no effect vasodilation was measured after more than 3 when infused alone, but was a relative potent hours of systemic insulin infusion and the stimulus for the acetylcholine response. This lacking effect of insulin was present despite confirms previous findings in lean, healthy insulin levels 38 times higher than levels subjects (150, 151). An explanation of this sufficient for an effect in lean, healthy synergistic effect of insulin and acetylcholine controls (6). has recently been suggested at the molecular level (see page 23). In a study from our institution (2) of patients with type 2 diabetes and ischemic heart The present study is the first to examine disease, mean systemic insulin concentrations insulin-stimulated vasodilator responses in 2 hours after a glucose load during a standard patients with type 2 diabetes and manifest oral glucose tolerance test were 106 mU l−1 atherosclerotic disease. (measured by ELISA, which may be expected The decreased insulin-stimulated acetylcholine to yield lower results than the RIA used in the response in patients compared to controls may present study). The local increase of insulin be explained by changes in the insulin concentrations during insulin stimulation in the signalling pathway in endothelial cells (see present study is thus comparable to a page 24). physiological increase of insulin concentrations in a similar patients group. The comparisons of seperate and combined effects of insulin and acetycholine has the The increases in systemic serum insulin were limitation that the experimental protocol only small, but they are not trivial. Circulating allows direct comparison of the acetylcholine insulin concentrations 2-3 times the fasting responses with or without insulin infusion. The level stimulates central nervous system question of whether insulin had any effect on sympathetic activity occur with similar potency its own would be answered correctly if the to high physiological levels (114, 147, 148). It protocol had included an infusion of insulin is unknown if a relative increase of systemic alone during a period of the same length as serum insulin as small as 28 ± 12%, as in our sum of the insulin (20 minutes) and healthy controls, stimulates sympathetic acetylcholine (∼ 28 minutes) infusions. activity. Previous studies of insulin’s effect on the Metabolic effects of insulin treatment response to an endothelium-dependent The 2.5 ± 0.4% decrease of HbA1c was larger vasodilator in patients with type 2 diabetes than that seen in large clinical trials, which have all used systemic hyperinsulinemia (see may both be explained by a high initial value table 5c). The present study is the first to Christian Rask Madsen: PhD thesis, Page 19 of 40. examine the effect of locally elevated insulin have occured in the present study, when concentrations on this response in such chronic hyperglycemia was quickly patients. This is important because systemic ameliorated. mechanisms (117) or glucose Insulin-stimulated vasodilation may be coupled administration (116) has been suggested to be to glucose uptake (see page 12). If this is also necessary elements in insulin-stimulated true for NO-mediated vasodilation (to vasodilation. Furthermore, there has previously endogenously produced or exogenously been concerns that impaired insulin-stimulated provided NO), and if basal glucose uptake after vasodilation in subjects with obesity or type 2 3 days of insulin therapy had decreased due to diabetes are due to test conditions of the sudden lowering of blood glucose, such a euglycemia (119). This does not seem to be mechanism could also explain the result from relevant in the patient group of the present the early examination day. Again, this study, where the potentiation by insulin of the explanation is not supported by direct vasodilator response to an endothelium- experimental evidence. dependent agonist exist during during spontaneous hyperglycemia. Effect of long-term insulin therapy on acetylcholine and sodium nitroprusside Effect of BH4 on the acetylcholine response responses The effect of BH4 on the acetylcholine A recent study showed that adding response was large. However, it should be intermediate-acting bed-time insulin to existing interpreted with hesitation claimed by the fact treatment with metformin in patients with type that control experiments were not made to 2 diabetes, but no clinical signs of demonstate that the response was cardiovascular disease, increased the forearm endothelium-dependent and by the vasodilator response to acetylcholine, whereas circumstance that the study design was not the sodium nitroprusside response was optimal for comparing the acetylcholine unchanged (153). Apart from patient selection, response and the combined BH4 and the discrepancy between results from that acetylcholine response, divided as they were study and the present study may be that by 3 dose-response studies with infusion of although the effect on glycemic control was acetylcholine, insulin, and sodium less (HbA1c decreased from 9.0 ± 0.3% to 7.6 nitroprusside. ± 0.1%), the treatment duration was longer (6 Effect of short-term insulin therapy on months). acetylcholine and sodium nitroprusside The fact that the sodium nitroprusside responses response was unchanged after 2 months in the There is no previous literature that offers an treatment group shows that insulin therapy did explanation for the decreased acetylcholine not result in any apparant change of vascular and sodium nitroprusside responses after 3 sensitivity to NO. days of insulin therapy. However, there is Effect of long-term insulin therapy on insulin- evidedence that hyperglycemia at short term stimulated acetylcholine response may increase endothelium-dependent and - independet vasodilation. The acetylcholine- The present study is the first to show that the induced vasodilation of arterioles of the local effect of insulin on peripheral vasodilation supraspinatus muscle in rats, studied by to an endothelium-dependent agonist is intravital microscopy, was unchanged during impaired in patients with type 2 diabetes one hour of superfusion with 200 mg (100 compared to lean, healthy controls. It is also ml)−1 glucose but increased by superfusion of the first to show that this effect of insulin can 300 mg (100 ml)−1 glucose (152). This increase be improved by glycemic control. could not be bloced by L-NAME or In previous studies, long-term treatment with meclofenamate (a cyclooxygenase inhibitor) the insulin sentisizer troglitazone improved and was accompanied by a decreased brachial artery flow-mediated vasodilation in production of NO, measured directly with an patients with impaired glucose tolerance (154), NO-sensitive microelectrode, but an increased but did not change forearm acetylcholine vasodilator response to sodium nitroprusside. responses or vasodilation to systemic Thus, hyperglycemia apparantly increase hyperinsulinemia in patients with obesity- vascular NO sensitivity acutely in vivo. associated insulin resistance (155). In these Speculatively, the opposite mechanism might Christian Rask Madsen: PhD thesis, Page 20 of 40. studies, glycemic control was unchanged by High glucose concentrations and endothelial troglitazone therapy. function L-NMMA inhibition of the insulin-stimulated Hyperglycemia is the most conspicuous trait of acetylcholine response the diabetic phenotype, and high glucose concentrations seem to directly affect The acetylcholine vasodilator response is only endothelial function. In a study in vitro, partly mediated by NO. One study, endothelium-dependent vasorelaxation of representative of several, showed inhibition of rabbit aorta decreased after incubation for 6 39% of vasodilation to acetylcholine by L- hours in media with high glucose NMMA (at an acetylcholine dose of 30 µg concentrations (157), and in another study ex min−1) (156). In healthy controls, we observed vivo, 72 hours of intravenous glucose infusion a similar relative L-NMMA inhibition of the far in rats decreased acetylcholine-stimulated greater combined insulin and acetylcholine vasorelaxation (158). Glucose-induced response. This is consistent with a substantial endothelial dysfunction seems mediated by part of the insulin potentiation of the reactive oxygen species, which can degrade acetylcholine response being mediated by NO. NO, because it was prevented by treatment in There is only one published human study in vivo with probucol, a scavenger of reactive which the effect of L-NMMA on insulin- oxygen species, and by antioxidant enzymes stimulated vasodilation to an endothelium- ex vivo (157). Such antioxidant protection may dependent agonist was examined (151). The L- occur physiologically, as high glucose NMMA experiments in this study suffer from concentrations induced endothelial antioxidant the same limitations of interpretation, namely enzymes in cell culture (159). It was also that definite proof of involvement of NO in the achieved by in vitro transfection of the Mn2+ insulin-stimulated acetylcholine response is SOD gene (160). In healthy humans, local only acheived if L-NMMA causes a decrement forearm hyperglycemia for 6 hours decreased of the insulin-stimulated acetylcholine endothelium-dependent vasodilatation (161). response larger than the L-NMMA-induced NO production decreases when endothelial decrement of the acetylcholine response alone. cells are cultured in high levels of glucose for In fact, these decrements were similar (based 5-7 days (162, 163). Conversely, the in vitro on incomplete numerical data and read from transfection of eNOS gene to aorta (164) and figure 4) (151). carotid artery (165) of alloxan-treated rabbits The L-NMMA response was not different in prevent impaired endothelium-dependent patients and healthy controls, suggesting that vasorelaxation. The mechanisms responsible the NO-mediated part of insulin-stimulated for decreased NO production are unclear, as acetylcholine response in healthy controls there are reports of downregulation of eNOS (where insulin potentiated the acetylcholine protein (163) as well as eNOS mRNA and response) is similar to that in patients (where protein upregulation accompanied by increased insulin had no effect). superoxide production which can degrade NO (162). A source of superoxide may be Taken together, the L-NMMA experiments are eNOS itself. Thus, preliminary results show compatible with a role of NO in the insulin that elevated superoxide production in aorta potentiation of the acetylcholine response in from streptozotocin-treated rats can be healthy controls and do not exclude a role for inhibited by L-NNA, a NO synthase inhibitor NO in improving the insulin-stimulated (see page 11) (109). However, the quantitively acetylcholine response after insulin therapy. most important source of superoxide is However, these experiments do not prove a thought to be an NAD(P)H oxidase which role for NO in the insulin potentiation of the resembles the phagocytic NADPH oxidase, but acetylcholine response, and the most straight- favors NADH as a substrate (166). Activity of forward interpretation is that NO does not such an enzyme has been demonstrated in mediate the improvement of the insulin- endothelial cells and VSMCs cultured in high stimulated acetylcholine response after insulin glucose concentrations (167) and human therapy. saphenous veins ex vivo (168), and may be Discussion of related previous literature caused by activation of PKC (167). The discussion is limited to studies relevant to Oxidative stress, i.e. an imbalance between the regulation of endothelium-derived reactive oxygen species and cellular vasomotor substances. antioxidant defense systems (169) may also be Christian Rask Madsen: PhD thesis, Page 21 of 40. generated through alterations of glucose proteins, resulting in formation of advanced metabolism. Increased cellular glucose uptake glycation end-products (AGEs). They may increases the flux of glucose through the directly catabolize NO (182), decrease sorbitol pathway (also known as the pyolol prostacyclin production (183), and reduce the pathway), which consumes NADPH by the expression of eNOS (184). aldose reductase reaction and reduces NAD+ Another mechanism for hyperglycemia-induced by the sorbitol reductase reaction (170). endothelial dysfunction may be through Among the consequences of an overactive upregulation of assymmetrical dimethyl sorbitol pathway is depletion of cytosolic arginine (ADMA). ADMA is an endogenously NADPH, a cofactor for eNOS and necessary to produced L-arginine analogue and an inhibitor maintain the primary intracellular antioxidant, of NO synthase (like L-NMMA, another glutathion; and an increase of the cytosolic methylated L-arginine analogue) (106). NADH/ NAD+ ratio, which is likely to promote Preliminary result showed that in the production of reactive oxygen species, in streptozotocin-treated rats, hyperglycemia was part through stimulating superoxide production correlated with plasma ADMA levels, and by vascular NAD(P)H oxidase (171). These activity of DDAH, the enzyme that catabolizes metabolic changes may be the reason that ADMA, was decreased in aorta although DDAH aldose reductase antagonism was able to expression was unchanged (185). Similarly, prevent impaired endothelium-dependent DDAH activity, but not expression, was vasorelaxation of aorta from alloxan-treated decreased in cultured VSMC after incubation diabetic rabbits (172). By a different for 2 weeks at high concentrations of glucose mechanism, increased glucose concentrations compared to physiological may inhibit glucose 6-phosphate concentrations (185). dehydrogenase, which catalyzes the first intermediary reaction in the pentose Finally, eNOS function and endothelium- phosphate pahtway, the primary source of dependent vasodilation associated with insulin NADPH (173, 174). High glucose resistance or hyperglycemia may be impaired concentrations may also lead to oxidative because of a relative vascular deficiency of a stress through glucose autoxidation or lipid cofactor for eNOS, BH4 (see page 29). peroxidation (169). Effects of insulin on vasodilation in vivo in Hyperglycemia is associated with activation of animals and healthy humans certain isoforms of PKC, and with increased intracellular concentrations of diacylglycerol A vasodilating effect of insulin has been (DAG), a physiological activator of PKC, in demonstrated in several vascular beds in vascular tissues (175). Impaired endothelium- humans in vivo. Studies with inhibition of NO dependent relaxation during high glucose production with L-NMMA and studies in isolated concentrations was prevented by a PKC vessels in vitro suggest that the effect is direct inhibitor (176). Furthermore, an inhibitor of rather than dependent on circulating factors or the PKC β isoform prevented impaired the autonomic nervous system. Insulin- endothelium-dependent vasodilation in stimulated vasodilation in the arm or the leg is mesenteric or intestinal arterioles in vivo, both for the major part dependent on vasodilation after oral administration to streptozotocin- of resistance vessels in skeletal muscle and treated diabetic rats (177) and after independent of vasodilation in skin (87, 186). administration during 45 minutes of Systemic hyperinsulinemia for ≥ 1 hour or local superfusion with high glucose forearm or leg hyperinsulinemia for ≥ 20 concentrations (178). Specifically, the latter minutes with insulin levels in the high study showed that decreased NO production, physiological or low supraphysiological range measured directly with a NO-sensitive increased blood flow in many studies by ∼ 50% electrode, was prevented by PKC (see table 5a). inhibition (178). The same PKC β inhibitor increased insulin-induced eNOS Systemic euglycemic hyperinsulinemia also expression (179). Interestingly, vitamin E may caused vasodilation of the human internal inhibit DAG-PKC activation (180) independent carotid artery (measured by 2-dimensional of its antioxidant effects (181). ultrasound) after 15-60 minutes; arterial diameter had regained baseline diameter 15 Moreover, chronic hyperglycemia may impair minutes after the termination of intravenous endothelial function by irreversibly modifying insulin-glucose-potassium infusion (187). Christian Rask Madsen: PhD thesis, Page 22 of 40. All studies of local limb hyperinsulinemia, Effects of insulin on vasodilation in insulin except two (150, 188), did not demonstrate resistant subjects any effect of insulin in healthy, lean subjects, A decreased or even abolished effect of insulin regardless of dose and duration of intra- in subjects with obesity or type 2 diabetes arterial insulin infusion (see table 5b). have been demonstrated in several studies However, intracoronary insulin infusion in non- using systemic hyperinsulinemia (8, 114, 155). diabetic patients increased coronary blood flow In one study of subjects with obesity- by 22% after 60 minutes (189). Furthermore, associated insulin resistance, only local insulin stimulation caused vasodilation of pharmacological doses of insulin had a hand veins with (190) or without (191) vasodilator effect (8). precontraction during infusion of norepinephrine. Vasodilation to local limb hyperinsulinemia has not been studied in obese or diabetic subjects. The present study examined the effect of However, the vasodilator response in hand insulin on the acetylcholine vasodilator veins of subjects with low-grade obesity and response. Previous studies from two borderline hypertension was blunted compared laboratories showed that insulin potentiated to lean, healthy controls (191). vasodilation to endothelium-dependent agonists: local forearm hyperinsulinemia Mediators of insulin-stimulated vasodilation increased forearm blood flow during intrabrachial acetylcholine infusion by 41% at It is beleived that NO is an important mediator the highest dose (151); and systemic of insulin-stimulated vasodilation. The hyperinsulinemia increased leg blood flow to vasodilating effect of systemic (150, 196) and intra-femoral metacholine at the highest dose local (151) hyperinsulinemia could be inhibited by 20% (150). by L-NNMA. The results in the former studies (150, 196) actually suggest that the In keeping with human studies, in vitro studies majority of insulin’s effect is mediated by NO. have shown a vasodilating effect of insulin on Furthermore, sodium nitroprusside vasodilator isolated skeletal muscle arterioles (192, responses were not potentiated by 193)and conduit arteries (194) from animals. systemic (150) or local (151) insulin Interestingly, in rat gastrocnemius arterioles stimulation (see page 21). the vasodilating effect was present during insulin concentrations of 10 mU l−1, i.e. the Apart from stimulation of endothelium- order of magnitude of basal insulin dependent vasodilators (e.g. NO or concentrations (192). prostacyclin) or vasoconstrictors (e.g. thromboxane A2 or ET-1), insulin may exert In humans studies, a dependency on both time effects on vascular tone by modulating and dose has been described (122), although sympathetic nervous system activity (see page it is not clear in this presentation (122) 12) or modulating the effects of circulating or whether data of time and dose are based on endothelium-independent vasoconstrictors. sequential and accumulative doses of insulin. A This hampers the interpretation of in vivo direct comparison of the time-dependency of responses to insulin. insulin-stimulated vasodilation and insulin- potentiated vasodilation to an endothelium- In canine conduit coronary arteries, insulin- dependent agonist cannot be made because stimulated relaxation was completely inhibited infusion of acetylcholine or metacholine only by combined pharmacological inhibition of lasted < 30 minutes. However, it is interesting eNOS (with L-NNA) and cyclooxygenase (with that insulin-stimulated vasodilation is minimal indomethacin) or removal of the endothelium; within the first 30 minutes (195) and shows a but there was no effect of pharmcalological gradual increase within the first 3 hours (8), inhibition in coronary microvessels (0.1-100 whereas insulin's effect on vasodilation to ng/ml) (197). Unfortunately, results of acetylcholine or metacholine (during local inhibition with either L-NNA or indomethacin stimulation) is potent within 20 minutes (in the were not reported (197). In isolated arterioles present study and a previous study (151)). from rat cremaster (192) or Therefore, it is possible that the preferential gastrocnemius (193) muscle, a vasorelaxing mechanisms in these two situations are in fact effect of insulin (10-104 mU l−1) was completely very different, e.g. by induction of eNOS and absent after exposure to L-NNA or removal of stimulation of immediate eNOS activation, endothelium. respectively (see page 23). Christian Rask Madsen: PhD thesis, Page 23 of 40. The vasodilator effects of insulin has been Certain studies suggest that insulin-stimulated suspected to modify vascular responses to vasodilation is dependent of glucose uptake or autonomic nervous system stimulation or metabolism (see pages 12 and 24). Perhaps hormones. Thus, insulin inhibited the the most interesting mechanism of insulin- vasoconstrictor response of norepinephrine stimulated vasodilation, discussed in the next and angiotension II in the rabbit femoral section, is the recently discovered insulin signal artery (198) and in the human forearm (188). transduction pathway in endothelium, which is Adrenergic innervation of VSMC is primarily similar to that in skeletal muscle and other relayed through α1 (contraction) and β2 tissues where the insulin signal is primarily (relaxation) receptors. However, insulin’s affecting glucose metabolism. vasodilating effect does not seem to be mediated by adrenergic or cholinergic Insulin-stimulated PI3K-Akt-eNOS activation mechanisms in humans, as the increase of calf A newly described Ca2+-independent blood flow during systemic hyperinsulinemia mechanism of eNOS activation is could not be blocked by propranolol (a non- phosporylation of eNOS on serine 1177 by Akt, selective β-adrenoceptor blocker) (at a high which is itself activated by phosporylation dose, 1 mg kg−1 min−1), prazosin (an α1- following PI3K activation (207). The PI3K-Akt selective adrenoceptor blocker), or pathway of eNOS activation is discussed in the atropin (199). This study, however, is a manuscript article (208). Until know, stimuli for contradiction of an earlier report (200), where this pathway has been shown to include VEGF, propranolol blocked the vasodilating effect of insulin, fluid shear stress, and estrogen (207). intra-brachial insulin infusion, even at a dose Akt-dependent activation of endothelial NO of insulin (0.1 mU kg−1 min−1) which did not production exists in vivo (209). Interestingly, affect systemic blood glucose; phentolamine (a the acetylcholine response, a Ca2+-dependent non-selective α-adrenoceptor blocker) did not stimulus, was shown to be dependent on Akt: affect insulin’s effect (200). in vivo transfection of dominant-negative Akt In a study of healthy, lean volunteers, insulin abolished acetylcholine-induced vasodilation of was infused intrabrachially at a dose of 0.1 mU rabbit femoral artery in vivo, and in vitro kg−1 min−1 for 2-4 hours with intravenous transfection of dominant-negative Akt inhibited infusion of glucose to maintain local glucose acetylcholine-induced vasorelaxation of mouse levels. Neither infusion of insulin alone nor aorta (209). The transduction of the intrabrachial infusion of ET-1 antagonists alone mechanical stimulus of shear stress to the changed forearm blood flow. However, ET-1 PI3K-Akt-eNOS pathway is not known, but antagonists increased blood flow during insulin preliminary results in cultured endothelial cells infusion, and in the presence of ET-1 suggest that PI3K activation induced by fluid antagonism, vasoconstriction to L-NMMA was shear stress may involve phosporylation of larger with insulin infusion than without (201). platelet-endothelial cell adhesion molecule-1, a Thus, the study did not identify a vasodilating constituent of endothelial cell junctions (210), effect of insulin, but suggested an insulin- or IRS-1 (211). induced change in the balance of endothelium- A suggestion of how a Ca2+-dependent derived vasodilating and vasoconstricting stimulus (e.g. acetylcholine (212)) and a Ca2+- factors. independent stimulus (Akt phosporylation) may Insulin stimulates NO production from cultured interact was recently given by a study of cell- rat aortic endothelial cells (202) and human free function of bovine eNOS with a serine- umbilical vein endothelial cells (10, 203) within aspartate mutation at the serine residue that is a few minutes (10) and with an ED50 of 500- phosphorylated by Akt (213). This mutation, 700 nM (∼ 72 U l−1, an order of magnitude 103 through the negative charge of aspartate, was larger than physiological concentrations). designed to mimick phosphorylation. The Insulin also stimulates the production of ET-1 mutated enzyme exhibited an increase of from cultured endothelial cells (204, 205) and catalytic activity at any concentration of Ca2+, VSMC (206). There are no studies of the effect perhaps by increasing the electron flux of insulin-stimulation on the release of other through the reductase domain of the enzyme endothelium-derived vasodilating or - and by inhibiting the Ca2+-binding cofactor constricting factors from isolated endothelial calmodulin from eNOS (213). cells. Christian Rask Madsen: PhD thesis, Page 24 of 40. Effects of insulin on eNOS expression concentration-dependently inhibited PI3K activity, Akt phosphorylation and eNOS Insulin may not only affect eNOS expression activity (221). The molecular mechanism by through a glucose-lowering effect. Insulin which high glucose inhibits insulin signaling in increased the expression of eNOS mRNA in rat endothelial cells is not known, but in a cultured epididymal microvessels (121) and eNOS fibroblast cell line, high glucose activates PKC protein in endothelial cells cultured from and inhibits IR-dependent tyrosine bovine aorta (121), human umbilical phosphorylation as well as dephosphorylation vein (214), human aorta (214), and human of focal adhesion kinase, whereas TNF-α coronary arteries (163) with a maximum after inhibits insulin signaling through activation of 12 hours of insulin stimulation (121). This phosphotyrosine phosphatase and inhibition of effect may be mediated by PI3K (121), which tyrosine phosphorylation, but not through may thus be a mediator of both immidiate (10, inhibition of dephosporylation of focal adhesion 11, 203) (see the previous section) and late kinase (222). effects of insulin on NO production. Incubation with a high glucose concentration inhibited Some studies have found elevated circulating insulin induction of eNOS protein after 24 concentrations of ET-1 in patients with type 2 hours (163). diabetes, and ET receptor expression and ET-1 effects are altered during insulin resistance Mechanisms of endothelial insulin resistance and hyperglycemia (223). ET-1 has compound Insulin resistance of glucose uptake in obesity effects on glucose transport. Thus, in may be mediated by elevated FFA (149). adipocytes ET-1 stimulated GLUT4 Endothelial NO producition may also be translocation and glucose uptake through influenced by FFA, which inhibit eNOS activity activation of the ETA receptor (224, 225), but in vitro (215). Moreover, in lean healthy inhibited insulin-stimulated glucose subjects, leg metacholine vasodilator response uptake (226). In rats, ET-1 impaired indirect (but not sodium nitroprusside response) was measures of insulin sensitivity (227), and in impaired during intravenous infusion of FFA or healthy humans, ET-1 inhibited leg glucose during elevated FFA due to inhibition of uptake during hyperinsulinemic euglycemic endogenous insulin production by somatostatin clamp without altering leg blood flow (and infusion (216). Furthermore, leg vasodilation inhibited whole-body glucose uptake, although induced by systemic hyperinsulinemia was not examined during steady-state) (228). In inhibited by 4 hours of intravenous infusion of cultured VSMCs ET-1 inhibits insulin-stimulated FFA (217). Leg blood flow during association of PI3K with IRS-2 as well as PI3K hyperinsulinemia was similar with and without activity, apparently through PKC FFA infusion, so the decreased insulin activation (229). Although speculative, it is vasodilator response was only present as a thus possible that ET-1 can inhibit insulin- smaller increase relative to baseline blood flow stimulated PI3K-Akt-eNOS activation in (which was increased during FFA infusion endothelium. alone, before the hyperinsulinemic clamp was Effects of insulin on vascular smooth muscle started) (217). Apart from being small, this cell function in vitro effect was delayed, as infusion for 2 hours did not affect insulin-stimulated vasodilation (218). Insulin may have vasodilatory effects by a The impairment of insulin-stimulated direct effect on VSMCs. Two groups in vasodilation was apparantly due to a decrease particular have pursued studies of the effects of NO-mediated vasodilation, as of insulin on VSMCs, either primary cultures of vasoconstriction to L-NMMA was lower with vascular smooth muscle cells (on rat tail FFA infusion than without (217). collagen gels, a situation where they do not proliferate) from canine femoral artery grown Both TNF-α (see page 29) and high glucose for 1-2 weeks (230-235), or cultures, after concentrations may inhibit the PI3K-Akt-eNOS several passages, of vascular smooth muscle signaling pathway. TNF-α inhibits Akt cells from human abdominal tissue (236-238). phosphorylation in endothelial cell culture (219) and preliminary results show that One study showed that incubation with insulin TNF-α inhibit IRS-1 by serine (10-40 mU l−1 for 20 minutes) inhibited VSMC phosphorylation (220). Other preliminary contraction as well as the increase in results showed that in endothelial cell culture, intracellular Ca2+ concentration after incubation with glucose for 12 hours stimulation with serotonin or angiotensin Christian Rask Madsen: PhD thesis, Page 25 of 40. II (230). These effects were similar after VSMCs has been shown to constitutively preincubation with insulin 40 mU l−1 for 1 express NO synthase, but there is no week (230). These effects were apparantly agreement of which isoforms are present, mediated by voltage-operated Ca2+-channels, which may be dependent on species and cell because it could be completely inhibited by culture procedures. Primary cultured verapamil. The authors speculated that the nonproliferated canine VSMC expressed iNOS, mechanism could be inhibition of Ca2+-influx by but no eNOS, by Western blot (233). In changing the sensitivity of voltage-operated contrast, cultured human VSMC expressed Ca2+-channels to more depolarized values eNOS mRNA, detected by Northern blot, through stimulation of the Na+-K+ pump, whereas iNOS expression was not because the effects of insulin were only minor examined (238). Neither study examined nNOS during high extracellular K+ concentrations and expression. These findings seem irreconcilable, completely inhibited by oubain (an inhibitor of regardless of the fact that iNOS expression the the Na+-K+ pump) (230). could be an artifact of the cell isolation procedure which takes several hours and Removing glucose from the medium or although there may be a discrepancy of inhibiting glucose uptake with phloridzin protein and mRNA expression. It is probably completely inhibited the effects of insulin (143 not a cell culture artifact, since it was also mU l−1 for 30 minutes) on Ca2+ influx and cell detected in freshly dispersed VSMC (234). contraction (231). A later study showed that Conversely, nNOS mRNA and protein was pharmacological inbibition of glycolysis or demonstrated in rat carotid artery (241). abscence of glucose in the medium prevented insulin-stimulated elevation of the Regardless of the source, cultured lactate/pyruvate ratio (an index of increased nonproliferated canine VSMC had a basal cytosolic NADH/NAD+ ratio) (170) and cGMP production of NO, which was inhibited by L- production (234). Incubation with β- NMMA (232, 234). Serotonin, insulin, or their hydroxybutyrate, which reduces NAD+ in the β- combination did not increase NO hydroxybutyrate dehydrogenase reaction, production (234). Insulin increased the elevated the lactate/pyruvate ratio and production of cGMP (232, 236) as opposed to mimicked insulin-stimulated cGMP inhibit its catabolism (236)) and such production (234). It follows that these effects stimulation could be blocked by genistein (a of insulin on VSMC are dependent on tyrosine kinase inhibitor, and thereby an IR glucolysis, possibly by a change to a more kinase inhbibitor) (236). Furthermore, L-NMMA electropositive standard reduction potential. (232, 236) and methylene blue (an inhibitor of Although speculative, this mechanism could guanylate cyclase) (236) inhibited basal cGMP involve the redox balance of heme of production, and L-NMMA inhibited insulin- guanylate cyclase, which is inactivated in its stimulated cGMP production after serotonin oxidized, ferric (Fe3+) form (239). Keeping the pretreatment (232, 234). L-NMMA also blocked heme of guanylate cyclase in its active ferrous the inhibiting effect of insulin (143 mU l−1 for (Fe2+) form may be controlled by a flavoprotein 30 minutes) on serotonin-induced oxidoreductase (171, 239) that uses NADPH as contraction (232). Insulin and the NO donor a cofactor (generated, for example, by the SNAP had additive effects on cGMP production, pentose phosphate pathway (171), see page and insulin-stimulated cGMP production was 20). On the other hand, superoxide production not inhibited by L-NMMA if pretreatment with from vascular NAD(P)H oxidase (which favors SNAP had restored cGMP to control NADH as a cofactor (166)) may oxidize values (234). Thus, insulin potentiated NO- ferreous heme. This can be counteracted by mediated cGMP production, but this effect was promoting lactate production in the lactate not mediated through NO formation. dehydrogenase reaction, thereby decreasing A later study showed that insulin had effects the NADH/NAD+ ratio (240) (Pyruvate + NADH on VSMC independent of Ca2+ transients (233). + H+ ↔ L-lactate + NAD+). Thus, glycolysis and Intracellular Ca2+ was clamped by adding Ca2+ the lactate/pyruvate ratio may influence ionophores to a medium with very high guanylate cyclase sensitivity to NO depending calcium concentration (1 µM-1.8 mM). This on whether cellular enzymatic activity promote had a constrictor effect, but precluded oxidation or reduction of guanylate cyclase intracellular Ca2+-dependent signaling. Insulin heme from NADPH or NADH. inhibited ionophore-induced constriction, and this effect was abolished by a guanylate Christian Rask Madsen: PhD thesis, Page 26 of 40. cyclase inhibitor or L-NMMA (233). Inhibition of insulin for 45 minutes did not have any Ca2+ influx as well as cGMP production and effect (242). conctraction during “clamped” intracellular Ca2+ Only one published study has examined the concentrations may be mediated by effect of a blood glucose-lowering intervention stimulating PKC activity (235). on endothelium-dependent vasodilation. It Insulin also stimulated cAMP production in showed that the forearm vasodilator response VSMC (236), and NO-cGMP production in to intrabrachially infused acetylcholine VSMC potentiated the catecholamine-induced increased after adding bed-time intermediate- increase in VSMC cAMP concentrations (237). acting insulin to established therapy with metformin in patients with type 2 diabetes Effects of insulin on endothelium-independent without manifest cardiovascular disease (153). vasorelaxation ex vivo or vasodilation in vivo In previous reports of studies with long-term Taken together, cell culture studies show that treatment with the insulin sentisizer insulin potentiate responses to NO formed in troglitazone, glycemic control was or supplied to VSMC. This could account for an unchanged (154, 155). They showed that such unknown part of the insulin-stimulated treatment improved brachial artery flow- acetylcholine response in vivo or increase the mediated vasodilation in patients with impaired apparant vascular smooth muscle sensitivity glucose tolerance (154), but did not change for NO. This is not supported by studies in forearm acetylcholine responses or vivo. As described above (page 22), insulin vasodilation to systemic hyperinsulinemia in had no vasodilating effect on rat cremaster patients with obesity-associated insulin muscle arterioles when endothelium was resistance (155). removed (192). In isolated rat gastrocnemius Hypothetical mechanism of improved insulin- muscle arterioles, removal of endothelium or L- stimulated acetylcholine response NNA changed the vasodilator response to a vasoconstrictor response (193). On the other Insulin therapy in the present study may have hand, insulin has been shown to inhibit improved endothelial function by increasing phenylephrine-induced vascular contraction in the expression of eNOS (121, 163) or by rat aorta with endothelium removed (194). In increasing NO production through upregulation lean, healthy humans, systemic of endothelial BH4 concentrations (243). Such hyperinsulinemia increased blood flow by 32% mechanisms may be relevant even though an (lowest dose) and 19% (two highest doses) in effect insulin therapy could not be lean, healthy volunteers, a difference which demonstrated on the acetylcholine response was not statistically significant (150). In a later itself. It could also have increased NO study from the same laboratory, the sodium production by limiting any of the effects nitroprusside response in lean, healthy mentioned above of hyperglycemia or elevated volunteers and patients with type 2 diabetes FFA on endothelial function. was neither larger nor statistically different Finally, insulin therapy could have improved when repeated during systemic endothelial signaling through the endothelial hyperinsulinemia (6), but the sample size for PI3K-Akt-eNOS pathway (see page 28). The the insulin-stimulated sodium nitroprusside last putative mechanism is certain to earn study in patients with type 2 diabetes was very inspiration from the intense research interest small (n = 3). The sodium nitroprusside in the similar signaling abnormalities in skeletal response in a control group of lean, healthy muscle and fat (244). This signaling pathway is volunteers (n = 6) in another study (151) was inhibited by the pro-inflammatory cytokine neither larger nor statistically different during TNF-α (see page 29). In patients with type 2 intra-brachial insulin infusion at the same dose diabetes, cytokine levels in plasma (245) and as in the present study. cytokine secretion from monocytes (246) Hypoglycemic or insulin sentisizing decreases with increased glycemic control. interventions and endothelial function in vivo Similarly, thiazolidinediones, a new class of insulin sentisizing drugs, may improve insulin Insulin treatment from 48 hours following signaling through modulation of cytokine streptozotozin treatment prevented impaired activation, as they prevent the inhibitory endothelium-dependent relaxation of aorta effects of TNF-α on IR kinase, IRS-1, and PI3K from diabetic rats, whereas incubation with activity in fat cells (247), and decrease Christian Rask Madsen: PhD thesis, Page 27 of 40. cytokine levels in plasma in patients with type 5. In patients, a potentiation of the 2 diabetes (245). vasodilator response by insulin was present after 2 months of insulin treatment in a Endothelial dysfunction as a determinant of condition of improved glycemic control. The peripheral insulin resistance mediator of this vasodilator effect of insulin The ability of insulin to increase NO-dependent was not identified. basal blood flow and a blunted vasodilator There are tree limitations, in particular, to the response to insulin in insulin reistance and study. One is that the treatment allocation was type 2 diabetes has been taken as evidence only partly randomized. Another is that that endothelium-dependent vasodilatation pharmacological control experiments were not may determine skeletal muscle glucose uptake able to identify whether the insulin-stimulated by regulating glucose supply to peripheral acetylcholine response, and its changes, were tissues (248). This issue has been the subject NO mediated; and, indeed, whether changes of a particularly unrelenting debate (249, 250) were endothelium-dependent. The third is that and is outside the scope of this discussion. follow-up of the insulin-stimulated acetylcholine response in the time control Conclusions group after 2 months was only performed for the last 4 patients, resulting in little statistical The present study examined peripheral power to detect any changes unrelated to vasodilation of resistance vessels in patients insulin therapy. with type 2 diabetes and ischemic heart disease as well as in lean, healthy volunteers. The following conclusions are listed in the Implications sequence of the hypothesis (see page 3). Implications for improved endothelial function 1. The previous well-established findings of and clinical outcomes decreased endothelium-dependent vasodilation Restoration of the normal response with in patients with type 2 diabetes was confirmed respect to endothelium-dependent in the present group of patients with type 2 vasodilatation has been demonstrated with diabetes and ischemic heart disease, compared numerous clinical interventions known to to age-matched, lean, healthy controls. improve the prognosis of people with risk Endothelium-independent NO-mediated factors from cardiovascular disease. Thus, vasodilation was also found to be impaired. decreased endothelium-dependent This was also the result of certain previous vasodilatation has been improved by exercise studies of patients with diabetes or other training in heart failure, by ACEI treatment in conditions associated with endothelial heart failure, type 1, and type 2 diabetes, by dysfunction, but is in disagreement with the statin therapy in patients with majority of studies of patients with hypercholesterolemia or by antihypertensive uncomplicated type 2 diabetes. therapy in arterial hypertension. Improvement 2. The results confirmed previous findings of abnormal endothelial function has also been in healthy non-obese people regarding the linked to therapy for which the evidence for a ability of locally elevated insulin levels to beneficial clinical outcome is still missing or stimulate vasodilation to an endothelium- controversial, i.e. hypoglycemic therapy in dependent agonist. patients with type 2 diabetes, orally administered L-arginine for coronary or 3. In patients compared to healthy peripheral vascular disease, or orally controls, insulin-stimulated potentiation of the administered vitamin C for patients with vasodilator response to an endothelium- ischemic heart disease. Given the evidence for dependent agonist was absent. the role of endothelial dysfunction in the 4. Despite of a considerable improvement pathogenesis of vascular disease outlined in of glycemic control, insulin therapy did not the background section, it is likely that change either endothelium-dependent or - restoration of insulin-stimulated endothelial independent vasodilation after 2 months. function towards the normal response is Surprisingly, both responses were depressed beneficial. It may be very difficult to obtain after 3 days of insulin treatment, where a evidence for this without means to selectively considerable improvement of fasting blood improve or inhibit insulin-stimulated glucose was already acheived. endothelial function in models of Christian Rask Madsen: PhD thesis, Page 28 of 40. atherosclerosis, which will more likely be improved glucose tolerance and normalized acheived by molecular biology techniques than skeletal muscle Akt activity and glucose pharmacological approaches. uptake (253). Furthermore, exercise training of normal rats increase skeletal muscle PI3K There are important theoretical reservations activity and Akt phosphorylation (254) and towards insulin therapy in this context. Insulin pancreatic islet cell transplantation in signaling pathways may be differentially streptozotocin-treated diabetic rats improved affected in type 2 diabetes and insulin IR kinase, IRS, and PI3K activity (255) and Akt resistance. The insulin signal transmitted by activity (256) in skeletal muscle. The present IRS may be viewed as diverging into the PI3K study supports the suggestion of a common pathway described above (see page 23) and pathway of insulin signaling in muscle, fat, and into the mitogen activated protein kinase endothelium. Furthermore, it supports a (MAPK) pathway, which relays insulin’s effects shared defect of insulin-stimulated glucose as an anabolic and mitogenic hormone. In uptake and insulin-stimulated NO production in insulin resistance, the PI3K pathway of insulin insulin resistance associated with obesity and signaling seems selectively impaired, whereas type 2 diabetes, because insulin treatment and the MAPK pathway has been shown to be improved glycemic control improved a unaffected, both in endothelium (11) and in functional measure of insulin-stimulated skeletal muscle (251). Accordingly, endothelial NO production. administration of insulin may beneficially upregulate insulin’s effects on glucose Insulin resistance secondary to hyperglycemia transport and metabolism and endothelial NO has been interpreted as an adaptive response production, but adversely upregulate insulin’s to maintain a normal cellular glucose uptake effects on vascular proliferation, perhaps during a chronic state of hyperglycemia (257). leading to diabetic complications other than Thus, even though patients with type 2 atherosclerosis, for example retinopathy. diabetes experience hyperglycemia after a Hyperinsulinemia may also alter vascular meal, primarily due to decreased inhibition of homeostasis in a pro-atherogenic fashion by hepatic glucose output, postprandial glucose signaling through other than the classical uptake is normal (258). It has been suggested insulin-receptor dependent pathways. For that the hexosamine pathway is a metabolic example, insulin stimulates NF-κB in vascular sensor of glucose levels that mediates insulin smooth muscle cells (252). This nuclear factor resistance in response to hyperglycemia (259). is thought to be important for several early This notion that insulin resistance may protect events in atherogenesis, for example by insulin sensitive tissues from activation of the genes for TNF-α and VCAM-1. complications (257) has recently been Obviously then, the overall vascular effects of challenged (175). Thus, vascular disease is a insulin administration may be complex and major complication in type 2 diabetes even paradoxical. Understanding vascular insulin though vascular tissues are insulin sensitive by signaling will be important because in the virtue of eNOS activation through the insulin- future, pharmacological agents with selective PI3K-Akt pathway. Glucose uptake in effects on insulin signaling will become endothelium is primarily dependent on insulin- available, one example being the insensitive GLUT1. In contrast to vascular thiazolidinediones (see page 26). smooth muscle cells, GLUT1 is not down- regulated in endothelial cells during Implications for the concept of endothelial hyperglycemia (260). In this context, insulin resistance endothelial insulin resistance may be viewed as It is well-established that improved glycemic an untoward result of an adaptive response control in patients with type 2 diabetes that does not protect endothelium from increases insulin sensitivity, measued as increased cellular glucose uptake but adversely whole-body insulin-stimulated glucose affects NO production, which is dependent on uptake (12). It is unknown whether clinical the same signal transduction pathway as insulin therapy improves expression or activity glucose uptake. Such a concept is, of course, of the IR, IRS, PI3K, or Akt in any tissue. entirely speculative. However, treatment of Goko-Kakizaki rats (a non-obese models of type 2 diabetes) with phloridzin (which improve glucose levels by inhibition of renal glucose reabsorption) Christian Rask Madsen: PhD thesis, Page 29 of 40. Perspectives endothelium-independent vasodilatation, i.e. the sodium nitroprusside response, will be The study presented in this thesis has made in each subject on a seperate study day. prompted new questions regarding the natural history of endothelial dysfunction of insulin TNF-α resistance and type 2 diabetes and the Acute subclinical inflammation in healthy mechanisms of the decreased effect of insulin humans leads to a substantial temporary to stimulate endothelial function. Therefore, reduction of endothelial function (265), which work has been planned in co-operation with may explain the increased risk for several collegues, with central efforts delivered cardiovascular events shortly after infectious by Drs. Christian Torp-Pedersen, Nikolaj disease (266). On the other hand, Ihlemann, Helena Domínguez, and Thomas atherogenesis has been described as a chronic Hermann, all at Gentofte University Hospital. inflammatory condition (267). The pro- The project on FFAs described below is the inflammatory cytokine TNF-α probably plays a focus of my current work at the Joslin Diabetes role in the development of insulin Center, Boston, MA, USA, supervised by Dr. resistance (268) which is an important risk George L. King. factor for cardiovascular disease (47). Thus, BH4 abnormal expression of TNF-α may be an explanation for the association between One planned study will continue the work with vascular inflammation, metabolic insulin BH4. Paradoxically, conditions as different as resistance, endothelial insulin signaling, and hypercholesterolemia, hypertension, diabetes, atherogenesis. and smoking may all lead to the same disease, atherosclerosis. It is an exciting prospect that A planned study will examine the acute effect they may all cause a common of TNF-α on either the acetylcholine response pathophysiological condition which lead to or the insulin-stimulated acetylcholine endothelial dysfunction and ultimatively to response in young, healthy male volunteers. atherosclerosis (261). Such a condition is Birth weight beleived to be represented by increased vascular production of superoxide anion or In the first descriptions of the insulin other reactive oxygen species (261). Oxidative resistance syndrome (269), one of the central stress may inhibit NO-mediated endothelial observations was that the development of type function by degrading NO (262). Alternatively, 2 diabetes and cardiovascular disease often it may lead to decreased intracellular occurs simultaneously in individuals with concentrations of the reduced, active form of insulin resistance. One of the most interesting BH4, a cofactor for eNOS. Suboptimal BH4 theories that attempts to explain this “common concentrations lead to “uncoupling” of the soil” theory is that intrauterine growth oxidase and reductase domain of NO retardation may predispose to insulin synthase (263) which results in oxidation of resistance, type 2 diabetes and cardiovascular molecular oxygen instead of L-arginine and disease decades later in adult life (270). synthesis of superoxide instead of NO. Thus, Genetic factors cannot adequately explain a BH4 deficiency may be both an effect and higher frequence of a history of low cause of vascular oxidative stress (264). birthweight in patients with type 2 diabetes (271). However, growth retardation The forearm vasodilator response to co- due to malnutrition or placental insufficiency infusion of BH4 and acetylcholine will be may adversely affect several organ systems compared to the acetylcholine response in permanently. Such changes are termed “fetal patients with insulin resistance. BH4 is an programming” (270). As metabolic insulin antioxidant, although much less potent than resistance is one of the earliest changes for example vitamin C. Experiments in the detectable in individuals at risk for type 2 planned study controlling for an anti-oxidant diabetes (272), insulin-stimulated endothelial effect will be made on seperate study days by function may be an early vascular change in infusing a stereoisomer (6S-BH4) without individuals prone to the development of type 2 cofactor capacities. In experiments in vitro we diabetes. have shown that 6R-BH4 (the active stereoisomer) and 6S-BH4 are equipotent anti- The planned study will examine endothelial oxidants (figure 18). Additional experiments function and insulin-stimulated endothelial will control for the effect of 6R-BH4 on function in young men born in 1980 and Christian Rask Madsen: PhD thesis, Page 30 of 40. randomly selected from the Danish Medical Birth register and Central Personal Register into two groups of birth weight in the lowest 10%-percentile or the highest 75%-percentile of their year, respectively. All had normal gestational age. These subjects were recently characterized with regard to a host of metabolic characteristics, including data from an euglycemic hyperinsulinemic clamp. FFAs and PKC The metabolic dysregulation in type 2 diabetes includes increases in plasma concentrations of both glucose and FFAs. Circulating FFAs are thought to inhibit insulin-stimulated glucose uptake, and this effect may be caused by a direct interaction with insulin signaling (273) rather than a previously proposed mechanism of allosteric inhibition of glycolytic enzymes. As FFAs activate PKC in non-vascular cells (274), and since vascular PKC is activated in animal models of diabetes, we plan to study the effect of FFAs on endothelial PKC activation. Furthermore, as insulin signaling through the pathway involving PI3K and Akt is decreased in the vasculature of rat models of type 2 diabetes, whereas the pathway involving mitogen-aktivated protein kinase (MAPK) is intact (11), we hypothesize that FFA-induced PKC activation may inhibit the PI3K/Akt pathway and simultaneously activate the MAPK pathway, leading to decreased nitric oxide (NO) expression and increased endothelin-1 (ET-1) expression, respectively. 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Fasting blood glucose [mmol l−1] 14.2 ± 3.3 5.1 ± 0.1 5.3 ± 0.1 Fasting serum insulin [mU l−1] 16.2 ± 7.8 5.4 ± 0.3 6.6 ± 0.5 Systolic blood pressure [mm Hg] 142 ± 5 117 ± 4 117 ± 5 Diastolic blood pressure [mm Hg] 71 ± 2 63 ± 1 61 ± 2 Plasma cholesterol [mmol l−1] 4.6 ± 0.1 4.9 ± 0.1 5.3 ± 0.2 Plasma triglycerides [mmol l−1] 2.1 ± 0.2 0.8 ± 0.1 1.0 ± 0.1 *in whom the insulin-stimulated acetylcholine response was studied. Table 2. Clinical characteristics of patients in the two treatment groups. Treatment group Time control p group n 19 9 . Age [years] 63 ± 2 63 ± 3 n.s. Sex (male/female) 17/2 10/0 n.s. Body mass index [kg m−2] 31.4 ± 1.2 33.4 ± 2.2 n.s. Smokers 3 (11%) 1 (11%) n.s. Microalbuminuria 4/19 (21%) 4/9 (44%) n.s. Oral hypoglycemic drugs 16/19 (84%) 5/9 (56%) n.s. Statin therapy 15/19 (79%) 9/9 (100%) n.s. ACEI therapy 4/19 (21%) 1/9 (11%) n.s. Long-acting nitrates 3/19 (16%) 1/9 (11%) n.s. Calcium blockers 6/19 (32%) 2/9 (22%) n.s. Abbreviations: ACEI: angiotensin converting enzyme inhibitor Table 3. Serum insulin and blood glucose during insulin stimulation in healthy controls and in patients at the initial examination. Treatment group Time control group Healthy control group Before During Before During Before During Local insulin 13.3 ± 1.4 121.2 ± 19.5 13.9 ± 2.3 154.2 ± 20.4 5.4 ± 0.5 105 ± 16 Systemic insulin 13.2 ± 1.44 16.2 ± 1.4 14.0 ± 2.4 17.7 ± 2.3 5.5 ± 0.4 6.8 ± 0.5 Local glucose 13.2 ± 0.9 12.7 ± 0.8 11.5 ± 0.8 10.7 ± 0.8 4.9 ± 0.1 4.4 ± 0.1 Systemic glucose 12.8 ± 0.8 12.5 ± 0.8 11.4 ± 0.8 10.9 ± 0.8 5.1 ± 0.2 4.6 ± 0.2 The heading “Insulin” represents the concentration of insulin in serum, expressed in units of mU l−1. The heading “Glucose” represents the concentration of glucose in blood, expressed in units of mmol l−1. The heading “Local” represents the arm where insulin is infused. The heading “Before” represents concentrations just before insulin infusion. The heading “During” represents concentrations 20 minutes after the start of insulin infusion. Table 4. Clinical parameters for patients according to treatment group and for healthy controls. Treatment group Time control group Examination day: Examination day: Healthy control Early Late Late group Initial Initial repeat repeat repeat n 19 9 19 9 8 31 Body mass [kg] 94 ± 4 . 98 ± 5 104 ± 8 104 ± 8 74 ± 2 Fasting blood glucose [mmol l−1] 14.7 ± 0.9 8.3 ± 0.6 7.5 ± 0.2 13.1 ± 0.9 13.0 ± 1.0 5.1 ± 0.1 Hemoglobin A1c [%] 10.0 ± 0.4 . 7.5 ± 0.2 9.5 ± 0.5 10.0 ± 0.6 . Fasting serum insulin [mU l−1] 15.4 ± 1.4 17.1 ± 3.1 19.4 ± 2.4 16.2 ± 2.9 14.7 ± 3.3 5.5 ± 0.3 Systolic blood pressure [mm Hg] 142 ± 5 138 ± 6 148 ± 6 143 ± 11 145 ± 10 117 ± 5 Diastolic blood pressure [mm Hg] 71 ± 2 64 ± 3 76 ± 3 71 ± 3 75 ± 3 63 ± 1 Plasma cholesterol [mmol l−1] 4.6 ± 0.2 . 4.5 ± 0.2 4.6 ± 0.3 4.8 ± 0.4 4.9 ± 0.1 Plasma HDL cholesterol [mmol l−1] 1.1 ± 0.1 . 1.3 ± 0.1 1.0 ± 0.1 1.1 ± 0.1 1.3 ± 0.1 Plasma triglycerides [mmol l−1] 2.0 ± 0.3 . 1.4 ± 0.2 2.4 ± 0.5 1.6 ± 0.5 0.8 ± 0.1 Abbreviations: Hg: mercury; HDL: high density lipoprotein. Table 5a. Previous studies of vasodilation during systemic hyperinsulinemia in lean, healthy subjects. First author Year See Age BMI Vascular Blood flow Insulin level [mU l−1] Duration [minutes] BF increase [%] table bed measurement technique Laakso 1990 5a 33 ± 2 (weight 68 ± 2 kg) leg thermodilution 21, 49, 3492* 180-550 −4, +37, +65a (Baron)  Laakso 1990 5a 35 ± 3 (weight 67 ± 2 kg) leg thermodilution 524* 60, 120, 180 +36, +44, +72 (Baron)  Kelley  1990 . 54 ± 2 27.1± 1.0 leg impedance 77 240 no plethysmography Anderson  1991 . 20.7 ± 0.3 25.1 ± 0.1 forearm plethysmography 72, 144 60, 120 +55, 65b Bak  1992 49 ± 1 27.3 ± 1.0 forearm plethysmography 231 180 +4 (NS) Bonadonna 1993 . 23 ± 1 lean forearm indocyanine dye 30, 70, 498, 1587*c 130* (not available, NS), 16 (NS), 23, (Ferrannini)  dilution 25 Vollenweider 1993 . 28± 4 21.8 ± 0.8 calf plethysmography 63 120 35 (Scherrer)  Utriainen (Yki- 1995 . 24 ± 1 22.2 ± 0.7 forearm plethysmography 64-484 120−360 +15-115 Jarvinen)  Dela  1995 . 59 ± 1 25.7 ± 0.8 leg thermodilution 416,770, 15936 120-360 19, 26, 50d Abbreviations: not statistically significant (NS). Blood flow increase at highest dose of agonist or NO donor (∆BF). Certain studies have different protocols for insulin infusion that fits the catagories of more than one of the tables 5a-c. In those cases, cross-reference to additional notes have been made in the column “See table”. * Value selected from several similar or calculated from data presented in the article. a Read from figure 2. b Read from figure 5. c These doses administered on different days. d Read from figure 1. Table 5b. Previous studies of vasodilation during local hyperinsulinemia in lean, healthy subjects. First author Year See table Age BMI Vascular Insulin level Duration ∆BF [%] bed [mU l−1] [min] Creager  1985 . forearm .a 15 + 55 Natali (Ferrannini)  1990 . 23 ± 3 22.4 ± 1.5 forearm 125 100 0b (NS) Lembo  1993 . 28 ± 3 lean forearm 63* 30 + 5 (NS) Steinberg (Baron)  1994 5c 36 ± 3 25.9 ± 1.4 leg 50 20 + 49 Jern  1994 . 28 (range: 22.7 (range: 19.6- forearm 98 90 + 81 (+ 61 in 22-36) 25.5) control arm) Taddei (Ferrannini)  1995 5c 47 ± 6 23.1 ± 1.4 forearm 48 20 + 3 (NS) Cardillo  1998 . 52 ± 2 24.6 ± 0.2 forearm 231 120 − 8c (NS) Present study 2001 5c 56 ± 2 23.7 ± 0.6 forearm 133 20 + 16 (NS) Abbreviations: not statistically significant (NS). Blood flow increase at highest dose of agonist or NO donor (∆BF). Certain studies have different protocols for insulin infusion that fits the catagories of more than one of the tables 5a-c. In those cases, cross-reference to additional notes have been made in the column “See table”. * Value selected from several similar or calculated from data presented in the article. a Dose 0.1−1 mU kg−1 min−1 b Read from figure 1. c Read from figure 1. Table 5c. Previous studies of vasodilation to endothelium-dependent and -independet agonists during local or systemic hyperinsulinemia. First author Year See Age Phenotype BMI Vascular Insulin Insulin Duration Agonist & dose ∆BF [%] NO donor & ∆BF [%] table bed administration level pre-agonist [µg min−1] dose [µg [mU l−1] [min] min−1] Steinberg 1994 5b 38 ± 1a healthy 28.2 ± 1.4a leg systemic 43a 180 MCh 2.5-10 + 20 SNP 1.8-7.0 + 19 (Baron)  (NS) Taddei 1995 5b 47 ± 6 healthy 23.1 ± 1.4 forearm Intra-brachial 48 20 ACh 1.6 -165* + 41 SNP 11-44 − 2 (NS) (Ferrannini)  Present study 2001 5b & c 56 ± 2 healthy 23.7 ± 0.6 forearm Intra-brachial 105 20 ACh 3.8-60 + 106 . . Steinberg 1996 . 34 ± 2a obese 33.8 ± 1.6a leg systemic 257a 200 MCh 2.5-12.5 − 26b SNP 1.8-7.0 − 60c (Baron)  (NS) (NS) Steinberg 1996 . 40 ± 3a type 2 36.0 ± 3.4a leg systemic 1023a 200 MCh 2.5-12.5 − 33b SNP 1.8-7.0 − 44c (Baron)  diabetes (NS) (NS) Present study 2001 5b & c 56 ± 2 type 2 32.0 ± 1.1 forearm Intra-brachial 133 20 ACh 3.8-60 + 15 . . diabetes (NS) Abbreviations: metacholine (MCh), acetylcholine (ACh), not statistically significant (NS). Blood flow increase at highest dose of agonist or NO donor (∆BF). ”Agonist” designates endothelium-depent agonist. For aid in identifying studies from the same laboratory, the name of a key researcher (often the senior researcher) is mentioned in parenthesis after the first author. Certain studies have different protocols for insulin infusion that fits the catagories of more than one of the tables 5a-c. In those cases, cross-reference to additional notes have been made in the column “See table”. a Values are from studies with metacholine, but are similar to values for sodium nitroprusside studies. b Read from figure 1 and 2. c Read from figure 3. * Value selected from several similar or calculated from data presented in the article. References to table 5a-c. 1. Laakso M, Edelman SV, Brechtel G, and Baron AD. Decreased effect of insulin to stimulate skeletal muscle blood flow in obese man. A novel mechanism for insulin resistance. J.Clin.Invest. 1990;85(6):1844-52. 2. Kelley DE and Mandarino LJ. Hyperglycemia normalizes insulin-stimulated skeletal muscle glucose oxidation and storage in noninsulin-dependent diabetes mellitus. J.Clin.Invest 1990;86(6):1999-2007. 3. Anderson EA, Hoffman RP, Balon TW, Sinkey CA, and Mark AL. Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans. J.Clin.Invest. 1991;87(6):2246-52. 4. Bak JF, Moller N, Schmitz O, Saaek A, and Pedersen O. In vivo insulin action and muscle glycogen synthase activity in type 2 (non-insulin-dependent) diabetes mellitus: effects of diet treatment. Diabetologia 1992;35(8):777-84. 5. Bonadonna RC, Saccomani MP, Seely L, Zych KS, Ferrannini E, Cobelli C, and DeFronzo RA. Glucose transport in human skeletal muscle. The in vivo response to insulin. Diabetes 1993;42(1):191-8. 6. Vollenweider P, Tappy L, Randin D, Schneiter P, Jequier E, Nicod P, and Scherrer U. Differential effects of hyperinsulinemia and carbohydrate metabolism on sympathetic nerve activity and muscle blood flow in humans. J.Clin.Invest. 1993;92(1):147-54. 7. Utriainen T, Malmstrom R, Makimattila S, and Yki-Jarvinen H. Methodological aspects, dose-response characteristics and causes of interindividual variation in insulin stimulation of limb blood flow in normal subjects. Diabetologia 1995;38(5):555-64. 8. Dela F, Larsen JJ, Mikines KJ, and Galbo H. Normal effect of insulin to stimulate leg blood flow in NIDDM. Diabetes 1995;44(2):221-6. 9. Creager MA, Liang CS, and Coffman JD. Beta adrenergic-mediated vasodilator response to insulin in the human forearm. J Pharmacol Exp Ther 1985;235(3):709-14. 10. Natali A, Buzzigoli G, Taddei S, Santoro D, Cerri M, Pedrinelli R, and Ferrannini E. Effects of insulin on hemodynamics and metabolism in human forearm. Diabetes 1990;39:490-500. 11. Lembo G, Rendina V, Iaccarino G, Lamenza F, Volpe M, and Trimarco B. Insulin reduces reflex forearm sympathetic vasoconstriction in healthy humans. Hypertension 1993;21(6 Pt 2):1015-9. 12. Jern S. Effects of insulin on vascular responses to mental stress and norepinephrine in human forearm. Hypertension 1994;24(6):686-94. 13. Taddei S, Virdis A, Mattei P, Natali A, Ferrannini E, and Salvetti A. Effect of insulin on acetylcholine-induced vasodilation in normotensive subjects and patients with essential hypertension. Circulation 1995;92(10):2911- 8. 14. Cardillo C, Kilcoyne CM, Nambi SS, Cannon RO, III, Quon MJ, and Panza JA. Vasodilator response to systemic but not to local hyperinsulinemia in the human forearm. Hypertension 1998;32(4):740-5. 15. Steinberg HO, Brechtel G, Johnson A, Fineberg N, and Baron AD. Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent. A novel action of insulin to increase nitric oxide release. J.Clin.Invest. 1994;94(3):1172-9. 16. Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G, and Baron AD. Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance. J.Clin.Invest. 1996;97(11):2601-10. Patients Controls n = 28 n = 31 Initial n = 19 n=9 n = 31 examination Treatment Insulin No Early repeat n=9 examination Late repeat n = 19 n=8 examination Treatment Time control group group . Figure 1. Flow diagram for clinical interventions and examinations of vascular function. All 19 patients in the treatment group participated in the late repeat examination after 2 months; the first 9 consecutively included patients in the treatment group also participated in the early repeat examination after 3 days. Hypoglycemic drugs were withdrawn in all patients 2 weeks prior to the initial examination. Treatment consisted of insulin 3 times daily at meals and intermediate-acting insulin at bed-time. Hypoglycemic therapy was witheld in patients in the time control group until the late repeat examination day. One patient in the time control group was taken out of the study before the late examination because of symptomatic hyperglycemia. . Insulin BH4 log(ACh dose) [µg min ] log(SNP dose) [µg min ] −1 −1 60 30 10 15 7.5 3 1 32 60 82 3 5 7 4 6 8 5 3 10 12 14 17 19 21 22 25 Time after arterial cannulation [min] Figure 2a. Infusion protocol at the initial examination day. The narrow, black graphs representsacetylcholine dose-response studies (doses at left ordinate), the bold, gray graph represents sodium nitroprusside infusion (doses at right ordinate). Insulin and BH4 was given in doses of 50 µU kg−1 min−1 and 500 µg min−1, respectively. This protocol was followed for the first 10 consecutively included patients. Only 7 of these participated in the BH4 study at the end of the day. Wash-out periods between dose-response studies were of 20 minutes duration (as shown) or more, until blood flow had returned to basal. Abbreviations: acetylcholine (ACh); tetrahydrobiopterin (BH4); sodium nitroprusside (SNP). . log(ACh dose) [µg min ] log(SNP dose) [µg min ] −1 −1 60 30 10 15 7.5 3 1 32 60 82 3 5 3 10 12 15 Time after arterial cannulation [min] Figure 2b. Infusion protocol followed for 9 patients at the early repeat examination day and for 4 patients at the late repeat examination day. Abbreviations as in figure 2a. . L-NMMA Insulin Insulin log(ACh dose) [µg min ] log(SNP dose) [mg min ] −1 −1 60 30 10 15 7.5 3 1 32 60 82 3 5 7 5 7 2 9 7 10 12 14 17 19 21 21 24 Time after arterial cannulation [min] Figure 2c. Infusion protocol followed for 10 healthy controls and for 17 patients at the initial examination day and for 15 patients at the late repeat examination day. Abbreviations: L-NMMA (NG-monomethyl-L- arginine), otherwise as in figure 2a. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 7.5 15 30 60 Acetylcholine dose [µg min−1] Figure 3. Forearm blood flow during intra-brachial infusion of acetylcholine in 28 patients with type 2 diabetes and ischemic heart disease (closed circles ●) and in 31 lean, healthy control subjects (open circles ○). The blood flow response to acetylcholine was lower in patients than in controls (p = 0.03). Error bars represent standard error of the mean. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 1 3 10 −1 Sodium nitroprusside dose [µg min ] Figure 4. Forearm blood flow during intra-brachial infusion of sodium nitroprusside in 28 patients with type 2 diabetes and ischemic heart disease (closed circles ●) and in 10 lean, healthy control subjects (open circles ○). The blood flow response to sodium nitroprusside was lower in patients than in controls (p = 0.0008). Error bars represent standard error of the mean. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 7.5 15 30 60 Acetylcholine dose [µg min−1] Figure 5. Forearm blood flow during intra-brachial infusion of acetylcholine (closed circles ●) and during combined infusion of acetylcholine and insulin (0.05 mU kg−1 min−1, started 20 minutes before, and maintained during, acetylcholine infusion), with (open triangles ▽) or without (open circles ○) infusion of NG-monomethyl-L-arginine (L-NMMA) in 10 lean, healthy control subjects. Blood flow at dose = 0 µg min−1 was recorded as base-line before start of the insulin infusion. The blood flow response to actylcholine was higher during insulin stimulation and L-NMMA inhibited the combined insulin and acetylcholine response (p < 0.0001). Error bars represent standard error of the mean. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 7.5 15 30 60 −1 Acetylcholine dose [µg min ] Figure 6. Forearm blood flow during intra-brachial infusion of acetylcholine (closed circles ●) and during combined infusion of acetylcholine and insulin (0.05 mU kg−1 min−1, started 20 minutes before, and maintained during, acetylcholine infusion) (open circles ○) before long-term intervention in 28 patients with type 2 diabetes and ischemic heart disease. Blood flow at dose = 0 µg min−1 was recorded as base-line before start of the insulin infusion. The blood flow response to actylcholine was unchanged during insulin stimulation (p = 0.3). Error bars represent standard error of the mean. 14 12 10 [ml (100 ml) min ] −1 8 ∆ blood flow 6 −1 4 2 0 -2 -4 0 7.5 15 30 60 Acetylcholine dose [µg min−1] Figure 7. Decrements of forearm blood flow during intrabrachial infusion of NG-monomethyl-L- arginine (L-NMMA) as a co-infusion with acetylcholine and insulin (0.05 mU kg−1 min−1, started 20 minutes before, and maintained during, acetylcholine infusion) compared with combined infusion of acetylcholine and insulin in 10 lean, healthy control subjects (open triangles ▽) and in 17 patients with type 2 diabetes and ischemic heart disease (closed triangles ▼). Blood flow at dose = 0 µg min−1 was recorded as base-line before start of the insulin infusion. Blood flow decrements were not different in patients and controls. Error bars represent standard error of the mean. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 7.5 15 30 60 −1 Acetylcholine dose [µg min ] Figure 8. Forearm blood flow during intra-brachial infusion of acetylcholine (closed circles ●) and during combined infusion of acetylcholine and tetrahydrobiopterin (BH4) (500 µg min−1, started 5 minutes before, and maintained during, acetylcholine infusion) (open circles ○), a cofactor for endothelial nitric oxide synthase (eNOS), in 7 patients with type 2 diabetes and ischemic heart disease. Blood flow at dose = 0 µg min−1 was recorded as base-line before start of the insulin infusion. The blood flow response to acetylcholine was higher during BH4 infusion (p < 0.0001). Error bars represent standard error of the mean. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 7.5 15 30 60 Acetylcholine dose [µg min−1] Figure 9. Forearm blood flow during intra-brachial infusion of acetylcholine in 9 patients with type 2 diabetes and ischemic heart disease before (closed circles ●) and after (closed squares ■) 3 days of insulin therapy. The blood flow response to acetylcholine was lower after 3 days of insulin therapy (p = 0.007). Error bars represent standard error of the mean. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 1 3 10 −1 Sodium nitroprusside dose [µg min ] Figure 10. Forearm blood flow during intra-brachial infusion of sodium nitroprusside in 9 patients with type 2 diabetes and ischemic heart disease before (closed circles ●) and after 3 days (closed squares ■) and 2 months (closed triangles ▲) of insulin therapy. Compared to the situation before insulin therapy, the blood flow response to sodium nitroprusside was lower after 3 days of insulin therapy (p = 0.009), but unchanged after 2 months (p = 0.09). Error bars represent standard error of the mean. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 7.5 15 30 60 Acetylcholine dose [µg min−1] Figure 11. Forearm blood flow during intra-brachial infusion of acetylcholine before (closed circles ●) and after (closed triangles ▲) 2 months of insulin therapy in 19 patients with type 2 diabetes and ischemic heart disease. The blood flow response to acetylcholine was unchanged by insulin therapy (p = 0.09). Error bars represent standard error of the mean. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 7.5 15 30 60 Acetylcholine dose [µg min−1] Figure 12. Forearm blood flow during intra-brachial infusion of acetylcholine (closed circles ●) and during combined infusion of acetylcholine and insulin (0.05 mU kg−1 min−1, started 20 minutes before, and maintained during, acetylcholine infusion) (open circles ○) before insulin therapy in 13 patients with type 2 diabetes and ischemic heart disease. Blood flow at dose = 0 µg min−1 was recorded as base-line before start of the insulin infusion. The blood flow response to actylcholine was unchanged during insulin stimulation (p = 0.5). Error bars represent standard error of the mean. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 7.5 15 30 60 −1 Acetylcholine dose [µg min ] Figure 13. Forearm blood flow during intra-brachial infusion of acetylcholine (closed circles ●) and during combined infusion of acetylcholine and insulin (0.05 mU kg−1 min−1, started 20 minutes before, and maintained during, acetylcholine infusion) (open circles ○) after 2 month’s insulin therapy in the same 13 patients as in figure 12. Blood flow at dose = 0 µg min−1 was recorded as base-line before start of the insulin infusion. The blood flow response to actylcholine was higher during insulin stimulation (p = 0.0002). Error bars represent standard error of the mean. 14 12 10 [ml (100 ml) min ] −1 8 ∆ blood flow 6 −1 4 2 0 -2 -4 0 7.5 15 30 60 Acetylcholine dose [µg min−1] Figure 14. Decrements of forearm blood flow during intrabrachial infusion of NG-monomethyl-L- arginine (L-NMMA) as a co-infusion with acetylcholine and insulin (0.05 mU kg−1 min−1, started 20 minutes before, and maintained during, acetylcholine infusion) compared with combined infusion of acetylcholine and insulin in 11 patients with type 2 diabetes and ischemic heart disease before (closed triangles ▼) and after (open triangles ▽) 2 months of insulin therapy. Blood flow at dose = 0 µg min−1 was recorded as base-line before start of the insulin infusion. Blood flow decrements were not different before and after insulin therapy (p = 0.9). Error bars represent standard error of the mean. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 7.5 15 30 60 Acetylcholine dose [µg min−1] Figure 15. Forearm blood flow during intra-brachial infusion of acetylcholine before (closed circles ●) and after (open circles ○) 2 months without hypoglycemic drug therapy in 8 patients with type 2 diabetes and ischemic heart disease. The blood flow response to actylcholine was unchanged during this period (p = 0.09). Error bars represent standard error of the mean. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 1 3 10 −1 Sodium nitroprusside dose [µg min ] Figure 16. Forearm blood flow during intra-brachial infusion of sodium nitroprusside before (closed circles ●) and after (open circles ○) 2 months without hypoglycemic drug therpay in 8 patients with type 2 diabetes and ischemic heart disease. The blood flow response to sodium nitroprusside was unchanged during this period (p = 0.6). 20 18 [ml (100 ml)−1 min−1] 16 14 12 Blood flow 10 8 6 4 2 0 0 7.5 15 30 60 −1 Acetylcholine dose [µg min ] Figure 17. Forearm blood flow during intra-brachial infusion of acetylcholine (closed circles ●) and during combined infusion of acetylcholine and insulin (0.05 mU kg−1 min−1, started 20 minutes before, and maintained during, acetylcholine infusion) (open circles ○) in 8 patients with type 2 diabetes and ishcemic heart disease. Furthermore, blood flow during acetylcholine infusion (closed triangles △) and combined insulin and acetylcholine infusion (closed triangles ) in the same patients after 2 months without hypoglycemic therapy. Blood flow at dose = 0 µg min−1 was recorded as base-line before start of the insulin infusion. Insulin did not have an effect on the acetylcholine response before or after this period. (p > 0.7). Error bars represent standard error of the mean. 9 8 BH4 0 µM Light emission [10 counts s ] 7 −1 6 BH4 1 µM 5 3 4 3 BH4 2.5 µM 2 BH4 5 µM 1 0 BH4 100 µM 1 2 3 4 Time from adding xanthine oxidase [minutes] Figure 18: anti-oxidant effect of 6R- and 6S-BH4 in vitro. Luminol-enhanced chemiluminescence of the superoxide producing reaction between xanthine 400 nM and xanthine oxidase 2 mU ml−1 in the presence of 6R-BH4 (closed circles ●), 6S-BH4 (open circles ○), or superoxide dismutase (closed triangles ▼). 6R- and 6S-BH4 are equipotent anti-oxidants in the dose range examined. 20 18 16 [ml (100 ml)−1 min−1] 14 12 Blood flow 10 8 6 4 2 0 0 7.5 15 30 60 Acetylcholine dose [µg min−1] Figure 19. Forearm blood flow during intra-brachial infusion of acetylcholine (closed circles ●) and during combined infusion of acetylcholine and insulin (0.05 mU kg−1 min−1, started 20 minutes before, and maintained during, acetylcholine infusion), with (open triangles ▽) or without (open circles ○) infusion of NG-monomethyl-L-arginine (L-NMMA) in 17 patients with type 2 diabetes and ischemic heart disease before long-term intervention. Blood flow at dose = 0 µg min−1 was recorded as base-line before start of the insulin infusion. The blood flow response to actylcholine was higher during insulin stimulation and L-NMMA inhibited the combined insulin and acetylcholine response (p < 0.0001). Error bars represent standard error of the mean. 20 15 [ml (100 ml)−1 min−1] Blood flow 10 5 0 7.5 15 30 60 −1 Acetylcholine dose [µg min ] Figure 20. Forearm blood flow in the forearm contralateral to the perfused forearm during intra- brachial infusion of acetylcholine (closed circles ●) and during combined infusion of acetylcholine and insulin (0.05 mU kg−1 min−1, started 20 minutes before, and maintained during, acetylcholine infusion) in 10 lean, healthy control subjects. Blood flow at dose = 0 µg min−1 was recorded as base-line before start of the insulin infusion. The blood flow response to actylcholine in this arm was unchanged during acetylcholine infusion with or without co-infusion of insulin (p = 0.8). Error bars represent standard error of the mean.