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CLA: BACKGROUND AND CLINICAL STUDIES ON BODY FAT REDUCTION Michael Pariza University of Wisconsin DR. ANDERSON: Our next speaker is Dr. Michael Pariza, and he is the Director of the Food Research Institute of Wisconsin and distinguished professor and chairman of the Department of Food Microbiology and Toxicology. He holds several affiliated appointments, and I am not going to try to read the whole thing because I want to give Mike as much time as we can up here at the podium Michael Pariza? DR. PARIZA: Thank you. Let me say something for the record regarding the relationship between cachexia and the immune response. Tumor necrosis factor alpha (TNF-α) was first isolated about 10 years ago. It was originally called "cachexin" because it is such a powerful mediator of cachexia. TNF-α is a cytokine, a product of the immune system. So, if we were at an immunology seminar, the relationship between immune response and cachexia, mediated via TNF-α, would not be disputed. The research I will now discuss actually began 20 years ago. At the time there was considerable concern about mutagens forming in hamburger during frying. We got involved in this along with several other groups and verified that yes, indeed, this happens. A lot of work was conducted to isolate and identify the structures of the mutagens, and determine the potential risk from consuming them in food. In general these mutagens are present at quite low levels. In the course of our work we discovered there was a mutagenesis-inhibitory factor in the extracts too. Now, finding an inhibitor of mutagenesis in the Ames test is not in itself striking. There have been many such factors isolated over the years. Generally this work does not come to much because there are so many ways that mutagenesis could appear to be inhibited in a bacterial test system, and then there is the biochemical gap between the bacterial system and the development of cancer in an animal. However, there were special properties associated with this inhibitor that made me think there might be something more to it. At the time I thought we might be dealing with a novel cytochrome P-450 inhibitor. So, we partially purified the stuff and decided that the time had come to either paint or get off the ladder. We tested it for anticancer activity in a mouse model. The model that we used was the two- stage carcinogenesis model in mouse skin. Dr. Shubik, in fact, was one of the pioneers in establishing this system. These are the positive control mice. I am sure you, and certainly Dr. Shubik, have all seen this many times. These mice were all treated with a single low dose of 7,12-dimethylbenzanthracene, and then they were given twice weekly applications of the tumor promoter "TPA." Fourteen weeks later this is what the positive controls looked like. The red wart-like protrusions are, of course, papillomas, an early stage of skin carcinogenesis. Many of these will progress to carcinomas. Another group was treated exactly the same way except that 5 minutes before we gave them the carcinogen we treated them with a single low dose of what we were then calling the "mutagenesis modulator" that had been partially purified from hamburger. This is what those mice looked like at 14 weeks. Most of them had no tumors at all, and even those that had tumors had only very small 233 tumors. We convinced ourselves that this was not an artifact; one could not replicate the effect with corn oil or the like. We repeated the experiment three times before publishing because we wanted to be sure that the effect was real. The study was published in 1983. We then set to isolate and identify the active compound responsible for this inhibition. I am not an analytical chemist. Fortunately we have people on campus with whom we can collaborate, but I tell you it was a chore. It was 3 years of misery. There have to be 11 million things in a hamburger, and I know because I counted them all. And we were lucky. You cannot use this kind of a test system. You have to develop an assay that is much easier and quicker to use. We developed such a system, and eventually succeeded in isolating the active compound which was then synthesized and tested directly. The active compound was conjugated linoleic acid (CLA). This is the cis-9, trans-11 isomer of CLA, a C-18 fatty acid with a cis double bond between carbons 9 and 10, and a trans double bond between carbons 11 and 12. By comparison, linoleic acid is a C-18 fatty acid with cis double bonds between carbons 9 and 10, and 12 and 13. There are other CLA isomers as well, but cis-9, trans-11 CLA is the major CLA isomer in dairy products. You will hear more about this later from Dr. Yurawecz. When we were conducting this research virtually no one else anywhere was paying attention to CLA. Our publication (Carcinogenesis 8:1881-1887, 1987) announcing that CLA exhibited biological activity (i.e., anticarcinogenic activity) was the first instance of an association of a mixture of conjugated dienoic isomers of linoleic acid with a potentially positive physiological effect. For the next five years or so this paper lay largely unnoticed in the scientific literature. Today, of course, everything has changed. At last count this paper had been cited more than 100 times. Moreover, new publications on CLA appear weekly. Our web site, http://www.wisc.edu/fri/clarefs.htm, catalogues the world's literature on CLA since 1987; it is updated weekly. Mark Cook and I began our collaboration in 1990, and soon after we conducted the work that he was describing. I was trying to discover the mechanism whereby CLA inhibited cancer, but what we accomplished was to open up a number of new areas of inquiry. At least part of the reason that we found multiple effects of CLA is that there were two CLA principal isomers in our preparation. We think these two isomers are doing different things, so one might expect multiple effects. Early on, we found that CLA is a growth factor for rats. This is interesting because, as Dr. Cook told you, CLA also enhances the immune system. In fact, CLA is the only food substance of which we are aware that simultaneously enhances growth and the immune system. In this experiment we began feeding CLA to female rats when they were impregnated, so the developing pups were exposed throughout embryonic development. CLA feeding continued after birth, and we found that CLA was actually concentrated in the mother's milk over that found in other maternal tissues. At day 10 of lactation, we observed a reproducible, statistically significant increase in body size for the pups from the dams that had been fed CLA. This increased body size continued after the animals were weaned and maintained on diet supplemented with 0.25% or 0.5% CLA. The same result was seen for the male and female offspring. CLA was clearly acting as a growth enhancer. The question, then, was what is causing the growth enhancement: are they simply getting fat? After all CLA is a lipid. Is it just that simple? We are not feeding very much, you realize, a very low level, but is that all it is? The answer is no. The increased body size is due to increased lean body mass. Body fat is very substantially reduced. Weanling mice fed CLA-supplemented diet do not exhibit enhanced body size, but neither do they lose weight. In fact body weight is unchanged. Nonetheless, the animals fed CLA eat less food. How 234 can that be? It because there of changes in body composition and carcass energy. In these mice, body fat was reduced from 14 percent (controls) to 4 percent in the CLA-fed animals. By contrast, lean body mass increased from 18 percent (controls) to 20 percent (CLA-fed). Body water is increased in the CLA-fed animals because water is associated with muscle. That, of course, is the key to the feed efficiency effect: CLA-fed mice weigh the same as controls because the increase in body water compensates for fat loss, and because they have more body water and less body fat, there is less retained energy in the carcass. We have, also, done a fair amount of work on the biochemical mechanism for this body composition effect. You should realize that the adipocyte is the major site for storing fat, whereas skeletal muscle is the principal site for burning fat. CLA affects both. Adipocyte lipoprotein lipase, the enzyme involved in bringing fat from the blood into adipocytes, is substantially reduced by CLA. Conversely, carnitine palmitoyltransferase, the rate limiting enzyme in fatty acid beta-oxidation, is enhanced by CLA feeding. The effect is especially evident when mice are fasting. The enhancement of lean body mass may be mediated via changes in the way cytokines, especially TNF-α), effect muscle (Lipids 32:853-858, 1997). CLA produces similar effects in pigs. Within 2 weeks of feeding CLA-supplemented diet, there was a difference in back fat accumulation detectable by ultrasound measurements. The effect increased with time. At 14 weeks, the time of slaughter, the CLA-fed pigs exhibited more than 25% reduction in fat coupled with a significant enhancement in lean. The effect is every bit as dramatic as you would get with growth hormone except that the quality of the meat is enhanced in this case. The problem with growth hormone is it results in less juicy meat. That does not happen here. There is actually a retention of fat within the muscle, whereas fat outside the muscle is reduced. This shows a comparison of three completely independent studies. The first is our study, which I just described. The next is a study conducted at Iowa State University. The third is a study conducted at Agriculture Canada. In all cases body fat was reduced and feed efficiency enhanced. In one case growth was also significantly enhanced. There is no argument any longer as to the effectiveness of CLA in swine. There are numerous other studies from around the world now that verify this. We also did extensive toxicological studies, comparing our CLA-fed pigs to control pigs. We could spend an hour talking about the data, but the bottom line was that the pathologist that was involved with this concluded, "No histomorphologic tissue alterations attributable to the addition of CLA to the feed of pigs at dosages of .48% or .95% were observed." That brings us to the clinical trial, which began last December in Madison. 80 clinically obese subjects that were enrolled into this double-blind study. The average age was 40. Forty subjects were given a product called "Tonalin CLA 90," a very high grade of CLA which is totally characterized and could be approved as a drug. They took 3 grams of this product per day, for a total daily dose of 2.7 grams of CLA per day. The 40 control subjects were given 3 grams of vegetable oil per day. In addition, all subjects were advised to reduce their calorie intake and exercise. As a result, most subjects lost weight. We detected no adverse effects. Moreover, some of these subjects are on drugs for diabetes and the like. This indicates that there was no indication of adverse CLA-drug interactions, either. Most subjects lost body weight. The subjects taking CLA lost slightly more weight but the difference 235 was not statistically significant. So these human subjects taking CLA reacted similarly to the experimental animals, where CLA does not induce weight loss per se. We identified a subgroup-- about 30 percent of the CLA subjects and a smaller number of placebo subjects-- who gained lean body mass while on the trial. The CLA subjects who gained lean body mass lost fat mass and a small amount of body weight. These subjects were actually experiencing a change in body composition. By contrast the placebo subjects who gained lean body mass, gained much more fat mass and body weight. From this subgroup we identified a six subjects on CLA, and 4 subjects on placebo, who gained both lean body mass and body weight. The control subjects gained most of their weight as fat mass, whereas the subjects taking CLA gained about the same amount of lean body mass as fat mass. The results are highly significant, p < 0.02. We conclude that CLA is probably not going to be, at least under the conditions that we have tested, effective at causing a fat person to lose a lot of fat mass. However, the indication is that CLA may be very effective in helping to prevent obesity. People who have lost weight who take CLA may regain a lot less body fat in comparison with controls. That is where we think CLA will be most helpful. So, I will stop at that point. Thank you. References: Food Research Institute web page www.wisc.edu/fri/clarefs.htm [References on Conjugated Linoleic Acid (CLA)] DISCUSSION DR. ANDERSON: Thank you, Dr. Pariza. The paper is now open for discussion. DR. BIGELOW: Sandy Bigelow, Ross Products. Did you look for immune parameters in your clinical trial? DR. PARIZA: We will. We have got measurements. We just haven't worked it all in yet. We have got that. DR. SHUBIK: Shubik. Mike, how long were those studies, the time frame? DR. PARIZA: It was in there, I just went so fast. The double-blind phase was 6 months. DR. SHUBIK: It was 6 months from beginning to end? DR. PARIZA: Yes. DR. SHUBIK: Thank you. DR. GORI: Gio Gori. What effect, if any did this have on life span, expectancy and what effect on end pathology? 236 DR. PARIZA: That is a very, very important question, a very interesting question. Dr. Kritchevsky has been trying to get us to do that for some time. We just haven't done it yet, but it is not easy to do. We could have done it since we got started at one point with it, but we didn't follow through. DR. GORI: Do you think that the controls, do you think that fat would be more resistant to subsequent starvation? DR. PARIZA: No, quite the opposite. Obese people who are traumatized are much more susceptible to various complications than people who are not obese. PROF. SOMOGYI: Arpad Somogyi, Berlin. As you know, there are two major groups of anabolic agents being used in animal husbandry, steroid type of compounds and these currently cause quite a bit of problems between Europe and the United States at the level of the WTO and the other group is the antibiotics, of course, the anxiety that useful antibiotics in human medicine might fail as a consequence of increased resistance. Would you care to speculate what is the potential of CLA to move into the vacuum? Would the use of the two other groups of compounds be banned or restricted? DR. PARIZA: We think CLA is going to be very beneficial in this area. We think it may very well reduce the need for antibiotics. We need to study this of course, but the indications are that CLA will be useful in reducing antibiotic use in agriculture. I think it may, also, be very useful in reducing the use of some of these other things you are talking about as well. Does that answer the question? Yes? Okay, good. DR. MC GUIRE: McGuire, University of Idaho. As far as that goes there are very few steroids involved in the pig industry. So, I don't think that this is going to have any impact on steroid use. Antibiotic use it may very well have. DR. BEHLING: Alison Behling, Eastman Chemical Company. I noticed that a lot of the animal studies were done in male animals. DR. PARIZA: No, they have been done in males and females. DR. BEHLING: Okay, what about the clinical study? DR. PARIZA: Equal division of men and women. DR. BEHLING: Okay, thank you. DR. PARIZA: In fact, I think there were slightly more women in that trial. DR. ANDERSON: You showed the slide of the reduction in the epidermal fat pad in the animals. It looked to me from where I was sitting that the intestine looked rather large in the CLA-fed animals compared to the control. Was that just an artifact that I was seeing in the slide? DR. PARIZA: Yes, I suspect it has to do with the time of day they were killed. I don't think it is a real phenomenon. DR. LONGFELLOW: Dave Longfellow, NCI. Mike, it might be interesting to speculate in terms of what about obese humans who have high body fat depots of other dioxins, whatever, PCBs, what about mobilization through CLA of some of those if it gets into that stream? 237 DR. PARIZA: I don't think CLA is causing a big mobilization of fat. The problem is if you accumulate that body fat, you are going to have to get rid of it the old-fashioned way. If you go on a diet, that will of course cause fat mobilization. Where I think CLA is going to be most really effective is keeping the yo-yo syndrome from occurring, once you have gone through all the trouble of losing that body weight. You know, you go to Weight Watchers. You get all excited. You lose 50 pounds. You look great, and a year later you are right back to where you were because people just cannot keep it up. What people don't realize is that obesity is a chronic condition. You don't get over it. You can't be cured of obesity. So, you have got to have ways of preventing it, and that is where we think CLA is going to be helpful. DR. SCIMECA: Joe Scimeca, Kraft Foods. Mike, why do you think there is such a big difference in the growth and feed intake in the various pig studies? DR. PARIZA: Oh, well, you are dealing with different pig strains. I am amazed that it is as reproducible as it is, given that all kinds of strains of pigs being used. Also, there may be differences in feeding and husbandry practices. DR. ANDERSON: One more quick question, up here? DR. REED: Don Reed, at Oregon State. You mentioned the chance that when you lost weight and gained it back, if you took CLA you would increase the body mass that is more protein. How about those individuals who would elect to use ketosis to lose body weight? What would CLA do during that process and afterwards? DR. PARIZA: I have no idea. Mark, do you want to talk about that? DR. COOK: People who go ketotic during weight loss? DR. REED: Yes, they reduce their carbohydrate intake to the point that they mobilize fat and cause ketosis. DR. COOK: In theory CLA should cause a mild ketotic effect, but we have no handle. We have not seen any evidence of that, but there is a lot of fat that is coming out and it has been perhaps because of the increased carnitine palmitoyltransferase we are not seeing as much ketosis. I don't know. DR. ANDERSON: We can have one more quick question. DR. GORI: Gio Gori. These effects of CLA may not be unique. There may be other substances or other fatty acids that they do something similar. Would it be reasonable to have a screening procedure to see whether -- DR. PARIZA: Sure. We have looked at a lot of other things, and there doesn't seem to be something else that is mimicking it, but sure. I mean if we hadn't done the experiments we did back in the eighties this probably never would have been discovered. Why would it be? DR. ANDERSON: In answer to your question, Gio, a number of years ago I fasted animals that had been fed various fatty acids, and what I was looking for, I was feeding an odd chain fatty acid. So, I would get gluconeogenesis and one of my control groups was a high linoleic acid group because there was an old theory of gamma oxidation giving propionate and the linoleic-fed group did lose more fat, the high linoleic acid. 238 I had one more quick question, and then we will get on. What percent fat do you use in these diets? DR. PARIZA: It varies. In the mouse diets we have used 5 percent to 30 percent fat, and we always see the effects of CLA on body composition. I think Dr. Ip is going to talk more about that. DR. ANDERSON: Okay, thank you. GRAPHICS NOT AVAILABLE 239
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