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Discussion
Moderator, Phillip James, M.D.
Circulating Leptin Levels and Their Signi cance James: There are a number of issues to deal with as a result of these lectures so let us progress logically and rst consider the emphasis given to leptin being a good index of total body fat once we have standardized some of the modulating in uences. Prentice: I do not like the phrase calorie counter, but if you tease out the other factors that modulate leptin, e.g., the pulsatility, diurnal effects, gender relationships, cortisol effects, etc., there is still an underlying, very strong relationship with total body fat that we should not neglect. Friedman: There are intrinsic factors to the fat cell that are coordinated with leptin production and these have to do with the lipid content or adipocyte cell volume as Rudi Leibel set out. There is a lot of evidence that the lipid content per cell is highly correlated with the amount of leptin produced. The issue is how the lipid content per cell can be counted. Steve ORahilly suggested that there might be a stretch receptor within the cell membrane. I like that idea because how would you detect an incremental increase in lipid superimposed upon a large background value? In view of the work on cholesterol by Brown and Goldstein,1 we need to think whether the same argument could be made for leptin. The solution identi ed by Brown and Goldstein is of the importance of a sub-compartment that equilibrates with a small amount of cholesterol, so theoretically there may be a mechanism involving a cellular sub-compartment controlling leptin production. It is hard to get at this on an in vitro basis because no cell culture model replicates the sort of dynamic changes in leptin production seen in vivo. On top of the unknown primary determinant of leptin production, there are extrinsic factors that we have heard about such as the cytokines, insulin, cortisol, and so on, all of which have been reported to alter leptin production. Nevertheless, there is a much more profound intrinsic determinant relating to the lipid content that we do not understand. Leibel: I agree the primary factor appears to be the size of adipocytes, so if one looks at the different depots in
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animal tissues, there is a very tight correlation between the cell size and its leptin production. In humans the total fat mass is a rst order determinant of circulating leptin. Then the distribution of fat makes a difference with subcutaneous fat, probably by virtue of its cell size, producing more leptin per cell than, for example, intraabdominal depots. This is probably why the distribution of fat enters into a multivariate regression equation when you try to account for the level of circulating leptin. Then the next effect relates to gonadal steroids that also have an effect on lipid distribution. If you then remove this distribution effect, the androgens overall tend to lower circulating leptin levels, whereas estrogens tend to raise them. That is the reason why, in postmenopausal women, you see a slight decrease in leptin per unit of body fat. This effect is not nearly as important, however, as the impact of puberty in children where one sees the androgen effect in boys having a much more powerful effect in lowering leptin per unit of body fat than the estrogen effect which then becomes apparent in girls. Chehab: There is a problem assessing leptin in pregnancy because of the coexisting release of circulating leptin receptor that competes with the radioimmunoassay for leptin. Thus, if you take ob/ob mice and make them pregnant, maintaining their leptin levels arti cially with leptin throughout pregnancy, you nd their apparent leptin is sky-high, re ecting leptin receptor release. In humans, the soluble binding activity is not enhanced so you can measure leptin levels in pregnant women. Prentice: The difference between species is important. I understand that there is a threefold increase in leptin in pregnant rats and the same is true in humans. This seems odd in that you would expect, in late gestation, a drive to increase the supply of energy and nutrients. The short form leptin receptor, which binds most of the leptin is, however, also shed in pregnancy, so this will alter the binding of leptin. Drevon: We have measured leptin levels in weeks 17 and 35 of pregnancy in women and nd about a 20% increase in plasma leptin, which is a modest rise. Intriguingly we nd that women with female [fetuses] have higher levels than women with male fetuses.
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Yajnik: We have measured leptin levels in cord blood in both Indian babies and in Caucasian babies born at the Whittington Hospital, London. The Indian babies are only 2.7 kg weight on average compared with the white babies being 3.5 kg. Nevertheless, the cord leptin levels are essentially identical in the two groups, i.e., 5.5 ng/mL. In practice, the leptin levels are related to all aspects of anthropometry at birth except length and the babies with the highest leptin concentrations have the highest subscapular fat measurements and the smallest triceps values. This suggests to me that truncal adiposity may be more important in determining leptin concentrations. Friedman: Let us ask the questions why leptin levels differ between people. The leptin level itself is not necessarily predictive of whether you are going to be lean or obese. One can have a low leptin level and be obese and a high leptin level and be lean. The way I think about leptin levels is as follows: how much we weigh to some extent is a re ection of how much leptin we make per unit fat mass and then how responsive to it we are. So someone who is obese with a high leptin level is likely to have no problem making leptin, but is insensitive to its effects. Someone who is lean with a high leptin level is also relatively insensitive, but they make a lot of it per unit fat mass. So we may, in the end, be talking about genetic differences in the pathways controlling how much leptin we make per unit fat cell and how responsive we are to the generated leptin. Olsen: In my mind, the effects of glucocorticoids on leptin levels seem to relate to a pharmacologic rather than physiologic effect. ORahilly: Our data show a very clear physiologic, diurnal rhythm in leptin levels that is entrained by meals. The meal effect dominates any possible effect of glucocorticoids and, although pharmacologically doses will increase leptin levels, their impact physiologically is minor. Markus: We have measured over 24 hours cortisol and leptin levels in healthy volunteers with a very narrow body mass index (BMI, kg/m2) range. We nd a very good correlation between cortisol and leptin, so we think that this may be part of an entrained, endogenous variation and a possible direct effect of cortisol on leptin production. I am not so much concerned with the diurnal variations as such, but with the mean cortisol levels seen on a 24-hour basis; these show the best correlations with leptin. Chehab: We nd, in transgenic mice, which we have generated with essentially no lipid stores in the adipocytes, that these cells still keep secreting leptin, so how does this work?
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Friedman: I think each fat cell makes a certain amount of leptin because it is an adipocyte, so if you have a large number of tiny fat cells, you will produce appreciable leptin. Greenburg: If one considers the unique data from Leibel and Hirsch at the Rockefeller, one hears that there is a limit to fat cell volume before hyperplasia sets in with a replication of fat cell numberso presumably this can also affect leptin production. ORahilly: We have been looking at isolated adipocytes and considering the relationship between peroxisome proliferatoractivated receptor (PPAR ) and fat cell size. The bigger a fat cell gets, the less PPAR there is and this seems to me, potentially, an attractive auto-regulatory mechanism for controlling leptin production because the PPAR effect is upstream of a whole range of genes that are important in maintaining the adipocytes phenotype. So as a fat cell becomes larger, it reduces its PPAR expression and thereby controls a number of mechanisms concerned with fatty acid and glucose uptake. This could therefore provide a neat potential mechanism for regulating fat cell size. Groop: I would like to throw in a component of genetic control here because if you look at genomic-wide scans to assess the relationship between particular genomic expressions and leptin levels or degrees of fat mass, then one actually fails to see any speci c relationships in most studies. This emphasizes the fact that if we are trying to look at genetic controls, we are going to have to be much more speci c and also take account of non-genomic modulators of leptin production and fat mass determinants. Drevon: Perhaps we should not simply think about the total lipid within an adipocyte but also the impact of speci c fatty acids, particularly the long-chain n-3 fatty acids that we think could be important determinants of leptin expression. We have recently published our ndings that very long-chain n-3 fatty acids down-regulate leptin expression.2 We have also looked at the effects of conjugated linoleic acid. There is a small effect in vitro, but it is not as big a modulator of leptin expression as the long-chain polyunsaturated fatty acids (PUFAs). Korotkova: We have also found an effect of essential fatty acids but, in this case, through feeding pregnant and lactating rats a low essential fatty acid containing diet. This reduces leptin levels in the offspring throughout the rst 3 weeks of life. Angelin: I am intrigued by the n-3 fatty acid effect because that implies you are feeding a diet that is physiologically re ecting a reduction in fat cell volume. Is this a direct effect of the speci c fatty acids or is there
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a change in the adipocyte that then leads to a change in leptin transcription? The question is if the dietary component is having a real physiologic effect. Drevon: When assessing the effects of PUFA diets, e.g., in the pregnancy studies being conducted by Korotkova and Strandvik3 one needs to be sure that one is not looking at the complicating effect of changes in energy intake, so pair feeding studies are essential. We have looked at the expression of leptin in response to different kinds of fatty acids and we have been able to narrow the responsive component of the promoter region for leptin production down to 200 bases. I therefore conclude that we are looking at a direct effect of the fatty acids on the expression of the messenger for leptin production. Leibel: We have been looking at leptin in 10-day-old suckling rats. The vast majority of circulating leptin in these animals is derived from brown, not white adipose tissue. The control of leptin expression in white and brown adipose tissue is, however, very different, with the sympathetic nervous system suppressing the generation of leptin in brown adipose tissue. In white adipose tissue, however, the rate of leptin production seems to relate to the caloric ux through the organ itself. In humans there is very little brown fat beyond the rst year of life, but the potential role of the sympathetic system may be important. The distinctions between species in the proportion of brown and white adipose tissue need to be considered before we extrapolate our understanding of leptin control from one species to another. Astrup: We have undertaken a study where we have compared the meal-induced increases in leptin. We were unable to discern any difference in the response to high-fat and high-carbohydrate diets, i.e., there was the same acute meal-induced increase in leptin in humans. The fatty acid composition of the diet may modulate leptin production, but the macronutrient composition of meals does not seem to be important in relation to the early response. James: Why does the leptin level fall when we fast? Astrup: This is unknown but I think that insulin is one of the acute mediators [that] could affect the fall in leptin secretion on fasting. Prentice: I still dont understand what sets the tonic level of leptin output, [or] what uxes leptin is responding to. I showed some years ago, with James Stubbs, that very simple covert dietary manipulations can completely fool the short-term regulation of appetite control in human volunteers. Erlanson-Albertsson: Do we know if leptin is only able to regulate energy-dilute foods that are, for example, starch based, whereas leptin fails to inhibit the impact of
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high-fat high-sucrose foods that are also very tasty? Perhaps leptin is primarily involved in energy balance but cannot modulate the cerebral systems involved in the rewards-related behavioral response to eating tasty foods. Martin: Animals deprived of food will do everything in their power to reduce further weight loss. They reduce their physical activity, metabolic rate, and heat output, and therefore do everything to conserve energy. The problem with the so-called set-point concept is that they dont take into account the way in which there are indeed, for example, annual weight cycles, as Andrew Prentice showed in his annual weight cycling data from the Gambia. Before the Green Revolution and other changes in this last century, ad libitum food was unusual in humans so both animals and humans displayed annual cycles of body weight. Thus the modern environment is extremely unusual. Friedman: The cycling in body weight of hibernating animals is clearly an example of the systems plasticity, but these animals display well-regulated cycling. In human terms we also need to recognize the very different experiences of different groups over the millennia. With the advent of agriculture and animal domestication in the Middle East and its spread to Europe, the environmental and selective pressures became very different from the traditional hunter-gatherer lifestyle. James: Andrew Prentice gave a marvelous overview at the recent Vienna obesity conference of the pandemic recurrent famines through the last few centuries implying that European populations, anyway, have not been free of famines. Prentice: Yes, but as Jeff Friedman indicates, different environments do provide very different evolutionary backgrounds. We are currently thinking about the simple factors leading to an obesity epidemic but in the developing world we still have an enormous problem of trying to overcome the sub-optimal growth of most of the worlds children. Despite 25 years of effort with tests of different interventions, we are making poor headway, so the environmental factors may be complex. Erlanson-Albertsson: The distinctions between leptin and insulin are valuable. Insulin resistance is good for an obese individual because it allows fatty acids to be released from stores, whereas leptins lipolytic action implies that it would not be particularly valuable physiologically to have leptin resistance. Norum: What is the turnover time of the messenger for leptin and for leptin in the blood? Leibel: The turnover time in the blood is 90 to 120 minutes and the changes in expression of messenger
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RNA for leptin are fairly rapid, at least in rodent experiments, with depressions in leptin expression within 8 to 10 hours. So obviously an acute plasma effect is due to the release of leptin from stored pools. It has been shown that both insulin and corticosteroids affect this secretion. Prentice: Flier proposes that there is a pulsatile release of leptin with a pulsatility of about 17 minutes that implies that there must be a central driver that could in turn relate to pulsatile corticosteroid release. Martin: I am very skeptical when people describe pulses in hormonal systems. The gonadotrophin-releasing hormone (GNRH) pulsatility concept has permeated the literature and in uenced our thinking too much. We have looked very hard for leptin pulses in sheep using our own immunoassay. We nd absolutely no evidence for leptin pulses even with 5 and 10 minutes sampling throughout a 24-hour period. Cortisol, however, shows extreme pulsatility. I think if we were a bit more skeptical about the pulsatility question and put in random numbers in a model analytic program, we would then conclude that there were pulses that in practice simply re ected random errors and variations. ORahilly: I am very happy to hear that there may not be pulsatility because it seems intrinsically implausible that the entire human fat mass would be controlled, presumably neuronally, to generate short-term pulses in leptin. Plasma Leptin Binding Fruhbeck: It is very clear that there are both free and bound fractions of plasma leptin, but as far as I know we understand little about the relative potency or functions of these two fractions. It is not clear whether it is the active or bound leptin that interacts with the transport apparatus within the central nervous system (CNS) or elsewhere. Prentice: If leptin has multiple effects, then there is a host of different ways to control the different effects of leptin. The hormone could be degraded at different rates or bound to change its activity in relation to selective receptors so that the partitioning of leptin in different environments, e.g., the cerebrospinal uid, could become very important. Lord: Of the 1000 or so papers published on leptin, most have been general epidemiologic studies on blood leptin without regard to its binding. We need to know what it is bound to. I understand that lean people may have predominantly unbound leptin that ampli es our concern about the active component. ORahilly: There was a paper presented at the European Society for Endocrinology last week that looked at the binding activity in human serum. If you make a monoNutrition Reviews , Vol. 60, No. 10

clonal antibody against the extracellular portion of the leptin receptor you can effectively remove all the leptin binding activity in serum, so there is not much doubt that the vast amount of leptin binding activity is determined by the soluble form of the leptin receptor. What is not clear to me from any of the literature is whether the amount of circulating leptin receptor, responsible for leptins binding, is in any way regulated physiologically. In our patients with genetic leptin de ciency we can give plenty of leptin during treatment, but the level of circulating soluble leptin receptor does not change at all under these circumstances, so this seems to prove that leptin itself does not affect the amount of circulating leptin receptor. James: What happens when you eat or starve? ORahilly: I do not believe there are any signi cant changes in the circulating amount of leptin receptor. Clement: Our collaborator in Paris has recently published an analysis of free and bound leptin in patients with mutations of the leptin receptor. Even the homozygous leptin receptor de cient subjects, with complete leptin resistance, still have leptin gene expression correlated with the amount of fat within adipocytes. Most of the circulating leptin is bound by the short-form of the mutated receptor but the remaining free leptin, i.e., 10 to 20% of circulating leptin is still correlated with the amount of body fat. Leptin Signaling to the Brain James: There has been a suggestion that leptin is re ecting overall body fat but that insulin levels are signaling to the brain the selective accumulation of abdominal fat. Could this be true? Leibel: We have been looking at the regional distribution of body fat in patients using magnetic resonance analyses. Insulin does seem to relate very well to intraabdominal fat and could in turn be in uencing the net production of leptin. Markus: I am not clear about the differential effects of insulin in the brain compared with other metabolic organs. If there is peripheral insulin resistance in obesity, is the brain still showing insulin sensitivity? Porte: This has not been assessed very well but Jessie Roth, a number of years ago,4 suggested that brain insulin sensitivity was not different in obese animals with peripheral insulin resistance. This needs to be reassessed. We do know that the transport of insulin into the brain across the endothelial cells is reduced in insulin resistance so the insulin receptor levels in hyperinsulinemic animals are reduced in muscle, in adipose tissue, and in the endothelial capillary beds of the brain. NevertheS71

less, the insulin receptor levels in the neurones themselves are not reduced. The biologic activity of the insulin at this neuronal level has not been determined under these conditions but it is at least possible that the insulin sensitivity itself is maintained. Astrup: If we look in humans at the ad libitum intake of a lunch following a xed standardized breakfast, then we nd that 70% of the variation in the amount eaten at lunch is explained by the insulin response to the standardized breakfast. We have shown this in two different studies in normal weight adults and consider that the insulin response may be responsible for the satiety effect and the cessation of food intake. In the obese subjects, however, we have found a blunted relationship. This, of course, does not tell us about insulin resistance within the brain. Prentice: I am sure that many are familiar with Christos Mantzoross5 work in anorectics where they have found a very distinct relationship between plasma and cerebrospinal uid (CSF) levels of leptin. The system seems to maintain CSF levels of leptin and attens out the variations in blood leptin. Thus when blood levels are low, one actually nds a higher CSF level. Is this another means whereby the brain is dampening the impact of peripheral inputs? Do we nd the same for insulin in anorectics? Porte: It is very dif cult to be very interventionist in human studies. We have data on the steady-state relationships between plasma and CSF insulin. We nd a curvilinear relationship and, from our animal studies with insulin infusions, conclude that the transport of insulin into the brain is the limit on the brain input of insulin. At relatively low plasma insulin levels, transport is saturated but uptake re ects both transport and insulin diffusion. Within the physiologic range, transport plays the major role in determining leptin uptake into the CSF. In humans we nd a curvilinear relationship between plasma and CSF levels of both insulin and leptin so we surmise that the transport mechanism is the key to the control of CSF levels for both hormones. Friedman: I agreethere are now a number of reports about the importance of the transport system. There are probably selective transport mechanisms through the ventricles at least into the arcuate nucleus. The issue, however, is what the relationship of this transport is to the amount of leptin at the synapse at the nerve cell and in the CSF. There is at least 100 times more leptin in plasma than you nd in CSF. An important unknown is the turnover rate of leptin in the CSF. So the Mantzoros concept is attractive but the amount of transport does vary and the degree of saturation of the transport system could be an important control mechanism but it is hard to
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reach a rm conclusion on this based simply on measurements of CSF leptin. Leibel: I agreelooking at spinal uid concentrations of leptin and attempting to infer its fundamental control is like monitoring the exhaust of a car and trying to gure out what is happening at the level of the pistons. What is going on at the level of the synapses is probably very different from events measured in terms of spinal leptin. In crude terms, however, if we have animals with no functional leptin receptor, we still nd leptin in the spinal uid because the blood levels are much higher than normal. This reinforces, I think, the importance of active transport with much higher levels then inducing other entry mechanisms. James: What is the speed at which leptin traverses the receptor transport system? Friedman: Uptake is demonstrably rapid with neuronal biochemical changes observed within 15 minutes of giving a single peripheral dose of leptin. Martin: I am astonished by the report of 100-fold differences between plasma and CSF leptin. When we measure the leptin in sheep and measuring third ventricular leptin levels, we nd that the plasma level is only double that in the CSF. There is a nice close relationship between the two systems as shown by our 24-hour monitoring of samples taken every 20 minutes. James: How is the turnover of leptin explained? Once bound to receptors, how is it then processed? Is there an extra dimension to leptin metabolism? Friedman: The big surprise is the amount of leptin coming out in the urine. ORahilly: We do know that very high plasma leptin levels occur in renal failure. Most insulin disappears through receptor-mediated endocytosis with only modest amounts excreted in the urine. With leptin, however, the kidneys seem to have a bigger role. We, of course, in giving leptin to children are providing physiologic rather than pharmacologic amounts, but unfortunately we have not measured urinary leptin in these children. James: When there is renal failure, is the anorexia explained by high leptin levels? ORahilly: That has been suggested as a primary driving force in suppressing appetite in renal failure but there are so many other uremic factors involved. Leibel: The renal effect is rapid because if you clamp the renal artery of an animal, the plasma leptin levels will rise within 30 minutes. Lord: Michael Kaiser showed from radiolabeled studies that [upon] giving leptin, about 80% comes out in the
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urine with about 15% being metabolized in the liver in these rat studies. These turnover studies have not been conducted in renal failure patients but there is a big issue about their anorexia and malnutrition. Rat studies, however, show that a non-leptin fraction is primarily responsible for the anorexia of uremic animals. Central Interactions of Leptin Bjorntorp: We know that the serotoninergic system is very much involved in the regulation of energy intake, so what is the relationship between this system and leptin? Friedman: Serotonin comes from the raphe nucleus and there are clear projections of this serotonergic system to the hypothalamus, but the precise cells receiving this input are still being explored. A drug that inhibits serotonin reuptake and potentiates the serotonin (5HT) responses may involve a variety of selective receptors. Serotonin is downstream of leptin so if you give the reuptake inhibitor to stimulate a 5HT effect, the weight goes down and therefore leptin levels fall in response to the reduced body fat, which then means that the lower leptin is stimulating food intake and blunting the impact of a serotonin reuptake inhibitor such as sibutramine. Experimentally one can limit this blunting by maintaining leptin. It is unclear what all these control pathways are. Martin: We have undertaken infusions with leptin to assess the impact on the GNRH system. As leptin is infused into the 3rd ventricle at 14 micrograms/hour, one can show that there is a signi cant but delayed reduction in the frequency but not the amplitude of the leutinizing hormone (LH) pulse. This delayed effect is, however, explained by the leptin having inhibited feed intake so this in turn alters LH pulse frequency. The impact of orexin is very different since a single injection induces a remarkable increase in LH pulse frequency. These were mature males so we are past any effects relating to puberty. James: What is the mysterious property in food that modulates the LH pulse frequency in these circumstances? Porte: This could be an insulin response to fasting but we do not know. Insulin and leptin both come into the CSF and have some similar properties. We now focus on leptin but we need to recognize our long-standing knowledge on insulin that again relates to reproduction. We have to remember that when giving insulin into the third ventricle, it is still a long distance from its site of action. If insulin is injected into the hypothalamus directly then only extremely small amounts are needed to induce its effects. The response to these minute amounts, we believe, re ects the ready transport of insulin from plasma.
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James: Dan Porte had a marvelous diagram on the relationship of leptin to the neural pathways presented in last years Nature special Insight supplement.6 I understand that the concept is that the proopiomelanocortin (POMC) and cocaine-amphetamineregulated transcript (CART) system is the catabolic pathway with the neuropeptide Y (NPY) and the Agouti-related protein (AgRP) system being anabolic. The concept is of a balance between these two systems with effects of both leptin and insulin on this balancing mechanism. Porte: This is a simpli ed view of the mechanism, but it is a beginning. Friedman: I agree with Dan Portes view on this. There are lots of inputs from a number of factors and the same neurone seems to respond to multiple inputs. The evidence suggests that perhaps insulin and leptin are sensed by the same neurones. Fatty acid signaling may be involved but this proposition is based on knockout models that induce changes in lipid metabolism, which in turn seems to affect the central sensitivity to leptin and other hormones. The last 5 to 10 years of work has suggested that the two pathways interact at multiple levels so NPY neurones are linked to POMC neurones that interact downstream at the Agouti-related protein interactions with the MC4 receptor. In addition, the NPY neurones connect to the melanocortin-concentrating hormone (MCH) neurones that project downstream. Leibel: I agree that Dans proposal is reasonable. The insulin and leptin system seem to be able to sense not only the overall storage of body energy but also the rates of movement of substrate through various metabolic systems. The time axis of physiologic relevance of insulin and leptin may turn out to be somewhat different. James: How does this system relate to the shift in overall balance in body weight or, what some people would term, set points? We know, for example, of seasonal adjustments in animals [that] are responsive to photoperiod changes, and [that] are linked to seasonal variations in body weight. These seasonally appropriate weights are defended if there is an unusual change in input or energy expenditure. What central mechanisms are responsible for this readjustment? Leibel: My perception is that animals and humans are set to maintain relatively speci c levels of body fatness. If this is perturbed, for example by semi-starvation, then a set of metabolic changes occur even though the individual is now maintained in energy balance at a lower body weight, which implies that the individual is behaving as if in a semi-fasted state. From a teleologic point of view, the organism is trying to restore itself to its original state in terms of body fat. Insulin and leptin may be playing subtlety different roles but somehow the signals to the
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central nervous system are specifying the reduction in energy stores. Porte: One way of readjusting the supposed set point is by making a brain lesion. One can induce changes in the balance point of body weight with brain lesions and have animals readjusting themselves to attain a different weight. Even Zucker rats end up with a balance once obese so that overfeeding then induces a reduction in intake to bring the body weight back to its obese value. ORahilly: I think we should recognize that the study of genetic abnormalities provides a more physiologic insight than brain lesions. Humans who have lost one copy of their melanocortin 4 receptor expand their fat mass until they are at a higher weight. We know that this receptor system operates downstream of leptin and this single gene cause of obesity is probably the commonest. I think we have been too generous in giving equal weight to insulin and leptin. The simple fact is that if you lack leptin you become enormously obese, whereas if you lack insulin you lose weight and become thin. Obviously there is a peripheral catabolic effect of insulin deprivation with secondary hyperphagia in the insulin de ciency state. Nevertheless, it has been shown that if you replace leptin in an animal with a low insulin level, you restore appetite to normal, so the hyperphagia of insulin de ciency is mediated, at least in part, through leptin. The second point is that if you consider carefully reports on the knockout studies relating to the brain insulin receptor, there is a rather unimpressive increase in food intake and body mass. The effect is not comparable with the extraordinary impact of the loss of the brain leptin receptor, so I conclude that leptin is of far greater signi cance than insulin in controlling body weight. Porte: We have to be careful before concluding that knockout experiments truly knock out a system completely: we do not know how obese an animal would become if it had truly lost all its insulin receptors. The second point relates to the interpretation of experiments. If you treat a diabetic animal with centrally administered leptin you can reduce its food intake, but that does not mean that leptin de ciency was the cause of the animals overeating. One can also treat with insulin to reduce the food intake and the low leptin level is maintained. If one considers the development of this system of control from an evolutionary point of view, we know that the insulin system is present in C elegans, Drosophila, and other very primitive forms of life, and this system has an important role in the control of both reproduction and lifespan. In this insulin-signaling pathway it is possible to knockout a neuronal component and increase the lifespan and fat storage capacity of the organism. This system is older in evolutionary terms than the leptin
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system so, Steve, the insulin system could be more important! Friedman: I think it is clear now that leptin is a major player in weight regulation with the leptin system as a catabolic process having important cross talks with the insulin signaling system that is largely anabolic. When one considers the impact of different knockout experiments, one can see that a knockout of POMC or the MC4 receptor leads to a phenotypic change and it is in this catabolic pathway that a single knockout will induce increases in body fat. If, however, one develops an NPY knockout mouse you get far more subtle changes arising from knockouts in the anabolic pathway. This is simply telling us that the anabolic pathway is in some ways indispensable so there are more redundant mechanisms in this pathway. There is probably less redundancy in the catabolic pathway, which is why single-gene mutations alter the phenotype. Then when it comes to the issue of set points, some consider the whole scheme hard-wired in the sense that adiposity will be permanently in uenced by a melanocortin receptor allele, whereas others take a more plastic view of this balancing mechanism. Markus: As a clinician working with obese patients I have the impression that the plasticity in terms of body weight control is only unidirectional. Thus if individuals gain weight and then restabilizes 10 kg heavier it is terribly dif cult to get that weight down again. When obese individuals nally succeed in lowering their weight and then maintaining it for up to 10 years, they still highlight the need to be constantly on their guard against overeating. So the plasticity is only in terms of an upward movement in body weight. Leibel: I agree with the observation, but cannot explain it. Obese people have enormous and persisting problems in maintaining a reduced body weight. The resistance to maintaining a lower body weight seems to be much stronger than the resistance to weight gain. Biologically the anabolic pathway seems, therefore, much more effective than the catabolic processes. Putting an animal into what it perceives to be a negative energy state induces anabolic responses that are so powerful that they seem to continue to act almost inde nitely. Garza: I am wondering about the timeframe for these weight disturbances. If somebody is force-fed over a short period of time, then they tend to return spontaneously to their previous weight. If, however, you look at the epidemiology of obesity, e.g., in the United States, the same accumulation of energy over a longer period with a progressive gain in weight leads to a major but unsuccessful effort of so many people to return to their previous lower body weight. The recidivism seen after weight loss programs is exceptionally common. Why
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then do we see these differences between the acute and chronic increases in body weight? Leibel: I think one reason why people have dif culty in reducing their weight is that the environmental factors conducive to their weight gain remain the same, so all the genetic and molecular pathways that we have been discussing determine where an individual will be located in the spectrum of responses to a speci c environment. Thus if people are in an environment that promotes weight gain, the body weights of most of the population will rise. Conversely, in changed circumstances the body weights of a population can fall, but the relative order of the individuals within the populations weight spectrum will tend to stay the same. ORahilly: We have to remember that in modern societies the secular changes in obesity may not re ect eating more energy but the inability to down-regulate appetite in the face of a marked reduction in physical activity. It may well be that our biologic regulatory systems work better at a high-energy throughput but I do not know of animal experiments that have looked explicitly at this. Bjorntorp: If we look at the rate of weight gain in the population, we are merely talking about an energy gain amounting to an immeasurable 50 kilocalories a day or so and the challenge is to relate this to the neuroendocrine background affecting the propensity to weight gain. When treating obese subjects, we always want them to lose the weight within a year, not 10 years. Theoretically it might be better to very slowly decrease weight and therefore not challenge a slowly adapting set point. James: The only data that I know relating to this progressive decline in weight are the as yet unpublished national data sets on body weight in Japan where sequential surveys have shown that women below the age of about 50 have been becoming progressively thinner for the last 10 years. Although this is often considered a behavioral adaptation to cosmetic concerns, I doubt this explanation because there is a uniform shift in the whole distribution of percentile BMI curves in these women with a uniform slow decline. I have not heard Japanese investigators commenting on Japanese women becoming much more hungry now so it might be that very slow environmental shifts with, for example, increased physical activity could work. Chehab: Might this not simply re ect that Japanese birth rates have gone down so that there is less pregnancyassociated weight gain? Markus: Was it truly a reduction in weight on a secular basis or a cohort effect?
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Angelin: What about the smoking habits? I wonder whether nicotine and/or smoking can help in changing the balance or set point? James: The birth rates are going down slowly, but this is in insuf cient to account for the observed secular changes which I think are probably genuine and not simply a cohort effect given the age groups presented. Smoking in Japanese women is relatively uncommon. Reseland: We have looked at the effect of nicotine on leptin expression. In practice we nd leptin levels are lower in smokers than nonsmokers and when we provided nicotine chewing gum or cigarettes to smokers, we observed a decline in plasma leptin levels. In vitro cultures of primary adipocytes with physiologic concentrations of nicotine, however, led to no observable changes in the production and secretion of leptin. Friedman: If we observe a low leptin level in someone who is losing weight, the simplest explanation is that they are becoming more sensitive to leptin for some reason. We know of a report on acetylcholine receptor M3 knockouts lead to obesity. So acetylcholine and endocanabinoids could relate to a nicotine response. Changes in leptin levels may simply re ect other changes and not imply direct effects. Groop: I wonder if anybody has undertaken meta-analyses of leptin receptor mutation changes in terms of body fat and taken account of smoking in these studies? Could genetic subtypes respond therefore to nicotine much more than others? ORahilly: I know little about a nicotine effect, but it does raise the issue of peripherally active and effective mechanisms in energy balance including the activation of brown adipose tissue and uncoupling proteins. Sven Enerback has found a winged helical transcription factor (FOXC2) in adipose tissue that binds the [lipoprotein lipase] promoter and when transgenically over-expressed in adipose tissue converts white into brown fat.7 The beta-3 adrenergic receptor and a whole range of genes typically found expressed in brown fat are activated so this transcription factor may prove to be one of the integrating mechanisms that underlies leptins effect in upregulating thermogenesis. This factor counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance Bjorntorp: We selected women who were lean from our surveys and looked for this gene but found no relationship to the body weight, insulin resistance, and other metabolic features of these women. To our surprise, however, we found a relationship to psychiatric disorders that Leif Groop has also studied.
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Groop: The molecular expression in visceral fat is about ve times higher than in subcutaneous fat and there is a strikingly strong inverse correlation with measures of insulin sensitivity, not only in visceral fat but in skeletal muscle also. We need more studies before we can extend our conclusions. Cortical Effects on the Neural Leptin Network James: We have been discussing the central control of intake and reproduction as though we are dealing with a hard-wired scheme of neuronal interactions with receptor and neurotransmitter changes. However, Per Bjorntorp would like us to think in broader terms and consider cortical in uences. Bjorntorp: I, like many colleagues, was terri ed of moving away from the precision of genetic analyses and radioactive counters when considering the issue of stress-related eating and the general impact of stress. A reading of the literature is worrying because the measurements seem very inexact and often based on questions as to whether individuals feel stressed or not and whether they eat more or less as a result. We have, however, known for a long time that patients with hypercortisolemia in Cushings disease eat a lot and conversely those with Addisons disease eat less. When we then go to Jean Renauds experiments on adrenalectomy in rodents he nds a decrease in food intake linked to what appears to be excessive leptin sensitivity. A stepwise increase in corticosterone as the replacement hormone leads to a progressive reduction in leptin sensitivity and increasing food intake. If too much corticosterone is given, the animals eat more and become fat with high leptin levels analogous to the human leptin resistance of obesity. A number of studies have been undertaken in humans where glucocorticoids given to both normal and obese individuals increase leptin levels. A recent paper by Kelly Brownell8 involved a laboratory-based stress for individuals with a monitoring of the neuroendocrine responses in terms of cortisol effects. The individuals are then provided with snacks in a room supposedly at the end of the study but when in practice eating was being monitored. Those who eat more snacks were the individuals with the greater neuroendocrine and cortisol response. Therefore, although the obesity epidemic relates to physical inactivity and inappropriate foods, stress may be an additional factor. Greenberg: We have to recognize that there is a minuteby-minute monitoring of plasma events as shown by Camp elds studies on the changes in plasma glucose and fatty acid levels in relation to the onset of eating. There is no reason why stress should not have equivalent short-term effects.
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Leptin and Reproduction ORahilly: The data that Farid Chehab showed on the genetic modi cation of reproduction in ob/ob mice are interesting because some animals went into puberty with little or no leptin. This ts with observations in humans with congenital lipodystrophy and vanishingly low plasma leptin concentrations. These children enter puberty relatively normally so I wonder whether, because of the hyperinsulinemia in these conditions, there is genuine cross talk between leptin and insulin in terms of its reproductive effects? Insulin might actually substitute for leptin if present at high levels. Friedman: I have been told of women with lipodystrophy who were very hyperphagic before leptin treatment. When given leptin their food intake dropped and for the rst time they became interested in sexual relationshipssuddenly they started using make-up and behaving differently, so leptin may have much broader effects. Given that, in evolutionary terms, animals were reproducing before the emergence of leptin, it is hard to argue strongly that one of leptins principal actions is the control of reproductive capacity. Chehab: We may need to distinguish between invertebrates and the different levels of vertebrate evolution. Higher order animals may, in fact, have leptin locked in to their control processes even though this is not apparent in C elegans or Drosophila. Lord: Leptin seems to have evolved at the same time as the emergence of backbones in species. Since leptin is then conserved throughout the later evolution of animals, it implies a strong role. Friedman: I agree that there is a huge selective advantage in having a system for interpreting the nutritional state of an animal correctly and this is probably important for every physiologic system. So it is quite logical that leptin evolved to maintain an animal in a robust nutritional state but we need to have a clearer understanding of leptins many roles. James: If we look at the secular reduction in the age of puberty, do we believe that there could have been a progressive increase in leptin at particular ages, thereby in uencing the age of puberty? ORahilly: I think it is a very attractive hypothesis. On a global basis, if a proportion of children are particularly sensitive to leptin they will shift the whole distribution pattern of pubertal timing, but it is dif cult to know how this could be tested. James: I wonder whether we can link Chehabs and Prentices presentations with Leibels comments about
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the interactions between the sex hormonal changes in puberty and leptin. I am also impressed by Andrew Prentices analysis of weight cycling and fertility in Gambian women where the implication is that there is an early fall off in fertility with weight loss. Martin: I think we need to distinguish metabolic determinants of the onset of puberty from metabolic issues in the reproducing mature animal or human. I think we are going to see very different relationships between leptin and its effects during puberty and reproduction. Chehab: It is well recognized that women who lose too much body fat stop menstruating so there are similarities in that a minimum amount of fat will also delay the onset of puberty. Prentice: The Gambian data unfortunately cannot distinguish a biologic effect of weight loss from a social effect relating to coital frequency at a time of year when everybody is working extremely hard. I suspect, however, that it could be a biologic effect with leptin involved. I am not convinced there is a xed threshold for body fat because other environmental factors will affect it, so we need to recognize the exibility of the system. Martin: The Frisch work 9 ignored the many decades of animal research showing that puberty is not simply determined by nutritional factors so one can rear pigs, for example, to reach puberty at a lower level of body fat, but they still enter puberty before another group who have had a more acute manipulation. The time relationship is important one is not going to induce puberty in very early childhood simply by making babies very fat. Prentice: If we consider the issue of lactation as part of the reproductive process, we have to recognize that lactation presents a major metabolic demand for the woman. She either has to increase her intake, or markedly enhance the nutrient uxes for milk production. In small animals with large, fast-growing litters, the demand for milk production can only be met by a big increase in food intake. In humans we seem to be able to rely greatly on fat and other nutrient stores but can eat more. Leptin could be an important candidate molecule for orchestrating the metabolic changes in lactation, but we must recognize the enormous species differences in the demand. In animals, plasma leptin levels plummet during lactation, [which is] consistent with a signal to the animal to eat a lot more. The human work from the Childrens Nutrition Center in Baylor, Houston, where there were careful measurements of both fat mass and leptin suggest no signi cant differences in leptin levels between the non-lactating and lactating women. So species differences are crucial.
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Immunology Lord: I am very intrigued by Chehabs analysis on the rescuing of the sterility of the ob/ob mouse on a Black 6 background, by backcrossing it onto a Balb C background. There is good evidence in rodents that successful pregnancy relies on having a TH2 response within the placenta and the Balb C mice are the paradigm for looking at TH2 responses, whereas the Black C background highlights the TH1 model of immune responses. Have you looked at the immunologic responses of pregnancy in your experiments? Chehab: No, not at all. We have found, however, that backcrossing to different backgrounds other than the Balb C67 background also allowed the heterozygous ob mice to become fertile. So the responses are not exclusively relating to the Balb C strain. Hanson: I would like to add to Graham [Lord]s analysis of the immune issue. It has become clear that in some way we select mates who are different in their human leukocyte antigen (HLA) constitution, this determining their smell which may be how we choose immunologically different mates. The difference in HLA between parents determines the strength of the immune response to the fetus. The presentation of CD1 antigens in the placenta to the natural killer cells of the mother may be part of this relationship. The surprise is that the cytokines from the mothers immune response to the fetus will rst direct the ovum; secondly it will prepare the deciduum for the implantation of the egg. A number of cytokines are important, especially the leukin inhibiting factor. Then the growth of the placenta is determined by IL3, granulocyte-macrophage colonystimulating factor, and by granulocyte-stimulating factor coming from the mothers immune response. Several of the hormones generated by the placenta to protect pregnancy are also induced by these cytokines. Finally, the prostaglandin that induces delivery is also dependant on this interaction. These analyses derived from animal studies have some support in humans. In our studies in Pakistan looking at almost 3000 children and their parents, 48% of the parents were related by the marriage of cousins and this is evident over several generations. The children of related parents died 20% more frequently than those of unrelated parents and those born alive had signi cantly lower birth weights. These ndings could relate to a weaker stimulus to the mothers immune response. If one considers the issue of leptin, one could conclude in crude terms that leptin is certainly needed for the function of the immune system, but it would appear that leptin may have more selective effects. The T1 helper cells providing protection against intracellular pathogens seems particularly to need leptin, but if some of these clones were directed against their own tissues, it would enhance
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autoimmune disease. So the system needs to be balanced. We should also not conclude that low immune responses are ineffective. I have seen several girls with severe anorexia nervosa and I always ask them about the problems that they might have with infections, but there do not seem to be any, so perhaps there is an adaptation to different leptin levels. Then I would like to come to the Korotkova model where one group is given an ordinary diet and the other an essential fatty acid (EFA)restricted diet during pregnancy and/or lactation. If the dams are then exposed to ovalbumin in the food, then they will transfer tolerance to the offspring but there is a reduction in the response in the offspring if the mothers have been on an EFA-restricted diet. So it is not just a question of leptin levels when we are dealing with immunologic responses. Lord: The existence of the TH1/TH2 dichotomy is very clear in animal models but not quite as clear in humans. We found, and other groups subsequently con rmed, that in the absence of leptin the TH1 immune response is completely de cient, but this is not as clear-cut in humans. Hanson: There are a couple of papers from the Gambia looking at growth in newborns. The supposed dominance of the TH2 system in newborns that we inferred from animals does not seem to hold true for children. Lord: Looking at cord blood of fetuses that have not been infected in utero, there is no selective channeling to either TH1 or TH2 dominance. We are essentially dealing with undifferentiated, nave T-cells in newborns that have never been exposed to antigens, so this issue of the balance of TH1 and TH2 in the newborns cells does not, I think, come into it. Groop: Graham Lord suggested that leptin-de cient animals are protected from some T-cell-mediated hepatitis and also from experimental encephalitis, so are there any data on the rescuing of diabetes in the nonobese diabetic (NOD) mice by actually crossing them with the ob/ob mice? Lord: We are breeding these crosses at the moment but there is also a paper in press on the NOD mice where the provision of recombinant leptin worsens the pancreatic islet in ltration with an early onset of Type 1 diabetes and greater leptin receptor expression in the islets. If we can rescue the diabetes in the NOD mouse by crossing it with the ob mouse and then re-expressing the diabetes by leptin injections, this would be interesting. James: Let us stay with the immunologic issues in man, if we can. I am reminded of the Gambian data published by Andrew Prentices group four years ago10 setting out the unusual premature deaths from infections in adults
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who were born small during the hungry season. Is there the remotest possibility that this re ects the entraining of the immune system and that in some way the leptin fetal signaling system is involved? Prentice: This is perfectly feasible, but at present we know very little about this area and are looking into it. It is perhaps important to recognize that the thymus is a very responsive organ to dietary changes and there is a remarkable reduction in size during starvation, which Lars Hanson has suggested may re ect the need to shut down the cognate immune system and rely on the innate immune system. This gamble may have been particularly appropriate in groups of humans living in small huntergatherer communities where they might not be exposed to a host of novel infections but one might still ask why we should shut down our immune system under these conditions. We have found that the size of the thymus is affected by seasonality but we cannot nd any correlation between thymic size adjusted for body weight and circulating leptin levels adjusted for body weight. Hanson: The two most expensive systems in terms of energy are the central nervous system and the immune system. The two may even be linked. If one has knockouts of the CC3 receptor, one of the essential cytokine receptors, the cerebellum does not develop normally. We know that the immune system is very wasteful because it is producing a lot of cells that are being eliminated through apoptosis. This turnover is understandable in the immune system because there are real advantages in the apoptosis of some of these cell lines, but the very fast turnover of cells in the CNS is dif cult to understand. James: Not only does the immune system require energy, of course, but a whole array of different nutrients. Golden, about 30 years ago, showed the importance of the zinc in the recovery of thymus size and immune function in malnourished children;11 vitamin A and other micronutrients may also be involved. Lord: I should have emphasized that starvation involves a complex neurohumoral response with changes in insulin, free fatty acids, and micronutrient de ciencies including vitamin A, zinc, and selenium. The response to malnutrition is interesting because it seems to be predominantly a cell-mediated immune de cit. We are also looking at thymic size and leptin levels in immature babies to see if there is any correlation. We are using magnetic resonance imaging as a very sensitive measure of thymic volume and measuring leptin levels in cord blood because the thymus goes through a major phase of development from week 26 onwards. Again we have found no correlation between leptin levels and thymic size, but we are dealing with very complex systems. Garza: Have animal models been tested to assess the
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thymic response to nutrition? If one uses a starvation model but supplies adequate vitamins and minerals, does one still see the same thymic involution? Lord: As far as I know, this has not been done. Hanson: In previous meetings we have highlighted the important contribution of malnutrition to deaths from infection in children from poor countries, but we dont understand it at all well. The lethal impact of severe protein-energy malnutrition is easy to understand, but with vitamin A, which is so crucial, there is still confusion because we know we can prevent 20 to 30% of deaths but when we look more speci cally at diarrhea- or pneumonia-related deaths, the impact is not as clear-cut. We know that zinc will shorten the time course of diarrhea, but we do not seem to see remarkable effects in terms of death rates. We have a relatively poor understanding of the different components of the immune system that are affected by different micronutrients. Garza: We need to be very careful before concluding that energy supply as such is the critical factor in explaining the impact of malnutrition on growth, survival, and immune function in young children. The supposed lack of energy is really a proxy for poor dietary quality and after the age of about 8 months, only 12% of energy intake is needed for growth. So we must not underestimate the importance of micronutrients. Leibel: Let us think about the therapeutic implications of using leptin in relation to immunology. If we are now generating compounds that are either centrally or peripherally acting through the leptin mechanism, do we open up the possibility that we will alter the immune responses and perhaps highlight autoimmune phenomena in patients being successfully treated to reduce their weight? Lord: We have been trying to think about this in a number of ways. First one can do some simple mathematic modeling of leptin binding to its receptors in terms of pharmacokinetics, but it does depend on what you consider to be the KD of the leptin receptor. One would perhaps expect 50% of the leptin receptors to be occupied and the fatter you get, the more rapidly you would move to 100% occupancy. Trying to increase leptin levels above this occupancy level would not necessarily have any effects on autoimmunity, so we should be thinking in physiologic terms. The second point is that in most studies of very large numbers of obese patients, we do not nd a preponderance of autoimmune disease unless you classify cardiovascular disease as an autoimmune phenomenon. I recognize, of course, the growing evidence about the role of in ammatory responses in cardiovascular disease. Thirdly, in experimental models with leptin given to normal mice, the evidence is somewhat contradictory.
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Norum: It seems a paradox that when infection occurs, there is an increase in leptin with a reduction in food intake but in infections, we also have a greater energy expenditure as a result of temperature changes and inammatory responses, so this does not seem to be an appropriate set of responses for survival. Lord: We have seen experimentally that as cytokines are produced, there is a very rapid wave of leptin induction. If, however, the infection persists for long enough to induce a loss of body weight or body fat, then this tends to produce much lower leptin levels, so one could argue that the early large surge in leptin at the start of an infection is important to help the immune response kick in. Hanson: The human race has been through an extraordinary period of evolution in terms of combating infection and coping with insuf cient food. We know that with an infection interleukin 1 (IL1) and tumor necrosis factor (TNF ) will increase rst. IL6 levels will then rise and tend to block somewhat the effect of IL1 at the IL1 receptor level, which in turn will reduce the subsequent production of TNF . There is also the induction of soluble TNF receptors [and] then leptin levels will rise with the stimulation of IL10. IL10 will dampen the cytokine receptor activities and the leukocytes and the in ammatory cells will also try to suppress this reactivity. The dif culty comes with the multiple sequential infections which may disrupt this sequence and we know from our earlier studies in Pakistan nearly 40 years ago that there are very large groups of children with manifest infection for 100 to 150 days per year, so the immune system is being hit very hard. Groop: I believe that there is evidence that the immune system may be related to the development of insulin resistance and that there is a surprisingly good inverse correlation between C-reactive protein levels in the blood and measures of insulin sensitivity. Do we have analogous data on the relationship between serum leptin levels and C-reactive protein? Janssen: There is a good correlation between IL6, which is supposed to regulate C-reactive protein induction, and leptin levels in patients in Gothenburg. Both IL6 and serum leptin, of course, are related to BMI and fat mass, both being produced by adipose tissue. Friedman: About 20% of HIV patients, I hear, develop lipodystrophy that can be quite severe, so one would expect this to suppress leptin levels. I wonder, therefore, whether there are particularly severe infections relating to immune de ciencies in this subgroup of patients.
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Leptin, Blood Pressure, and Cardiovascular Disease Bjorntorp: I would like to take up Fru hbecks point on the relationship between leptin and blood pressure. Leptin apparently activates the sympathetic nervous system, not only in terms of its thermogenic pathways, but also those branches regulating blood pressure. We know that obese animals and humans tend to be both hyperleptinemic and hypertensive and experiments with leptin infusions given either systemically or intra-thecally induce increases in sympathetic nervous system (SNS) activity and blood pressure. Obese animals with mutations in the leptin receptor, e.g., the db/db mice or fa/fa Zucker rats, are normotensive, implying a fundamental relationship. We therefore looked at polymorphisms in the leptin receptor gene in 300 men aged 52 years and found that men with mutations in the second exon have lower blood pressures. We then looked at the 64 hypertensive men who in practice had a somewhat increased BMI as well as elevated leptin levels. Only one of the 64 had an unusual allele implying that this polymorphism protects individuals from hypertension. Polymorphisms in this exon seem to relate to clinically signi cant differences in blood pressure. We therefore infer that, with this abnormal polymorphism, one signal from the leptin receptor relates to the SNS and the modulation of blood pressure, but that another signal to appetite regulation may operate normally. James: What is the proportion of people with these abnormal alleles in your population group? Bjorntorp: Twenty- ve percent of the general popula tion have an abnormal allele in exon 8, but only 6% have an abnormal allele in exon 4. Astrup: Can you distinguish between the effects of these alleles on BMI and on blood pressure? Soderberg: In the northern part of Sweden we have a couple of case-control studies on atherosclerosis in males and in both studies there is a very strong interaction between high leptin levels and high blood pressure. James: What is the prevalence across the world of these exon changes? Leibel: The polymorphisms described in the leptin receptor have been described in perhaps half a dozen population groups. I think the prevalence of abnormalities in exon 4 in some population groups is higher than that found in Sweden. Angelin: I think we need to be careful about these polymorphic analyses and coupling them with functional variations. One needs at least 700 to 800 people for these analyses to ensure that one has a reasonable assessment.
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Groop: I agree there are problems with case-control studies. Given the known environmental effects on blood pressure, it may be important to have family-based controls before one can begin to infer anything from the polymorphism. We need meta-analyses of the relationship between polymorphisms in the leptin receptor and the different indices of risk in relation to blood pressure, BMI, etc. The study of morbidly obese individuals may give very different results, so caution is needed before the meta-analyses are available. Fruhbeck: We have studied 400 morbidly obese French adults and found no relationship between these polymorphisms and either an obesity-related phenotype or hypertension. However, I believe there is a San Antonio study that did nd an increase in systolic blood pressure associated with polymorphisms of the leptin receptor gene. Soderberg: Four independent groups have shown a relationship between high leptin levels and cardiovascular disease. Two studies have shown a relationship to the onset of clinical stroke and we have found, in a prospective study, that those in the highest quartile of leptin levels had greater rates of myocardial infarction, even after adjusting for BMI and other risk factors such as cigarette smoking, serum cholesterol levels, apo A and apo B, and blood pressure. There are also new studies showing a close association between the stenosis and sclerosis of the arterial wall and high leptin levels. Friedman: I think we need to be sure that we adjust for body fat rather than BMI in these relationships because the leptin/BMI correlation is about 0.5 to 0.6, whereas the leptin/body fat correlation is 0.9 or better. Without this direct correction for fat mass itself, it may be that leptin is simply a surrogate for body fat or for visceral fat. Groop: I think ideally one also needs to be sure that one is not simply linking to cytokines such as IL6 and TNF otherwise we may just be looking at a nonspeci c effect. Prentice: One really should consider whether excess leptin is switching on a proin ammatory response. It might be interesting to study those taking aspirin because if the relationship with IL6 and C-reactive protein disappeared, then a persisting relationship with leptin would become more persuasive. Friedman: We may be dealing with different doseresponse thresholds for different systems with insensitivity to leptin in terms of some cellular types, whereas others are responding. Soderberg: It would be valuable to have selective mea sures of body fat rather than BMI but the epidemiologic studies I mentioned are very large. With all the earlier
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discussions on the importance of high levels of insulin and pro-insulin in relation to cardiovascular disease, one might infer that insulin was mechanistically involved, but we have found that once one adjusts for leptin, these associations with insulin disappear and so far leptin is proving to be the strongest predictor. In our prospective studies we will certainly be able to look at the aspirin effect. Lord: We do know that fat people tend to get more TH2 type diseases such as asthma so in looking at different clinical problems, we need to think about the impact of changes in TH1/TH2 balance. Yajnik: Paul Zimmet some years ago looked at his analyses on the metabolic syndrome and found that, if he introduced leptin-related analyses in place of insulin, he obtained much stronger predictors of risk. Prentice: The dif culty in these multi-regressional analyses is that it requires good measures of the real levels of leptin and insulin. Since insulin is more variable over a short timeframe, a single measure of leptin is likely to give a better prediction of overall leptin levels than analogous measures of insulin. Porte: One issue that is never taken into account and has not been set out clearly in publications is that the previous days food intake makes a big difference to the overnight fasting insulin level. One therefore has to be careful and one also needs to recognize that prior nutrition affects insulin sensitivity rather quickly. James: Let us now come to the issues in relation to the developing world and the remarkable acceleration in diabetes and cardiovascular disease rates that are becoming apparent. Dr. Yajnik has remarkable data showing that Indian babies, although born small, have excess body fat and that if they accelerate in growth after birth, then they have a marked insulin resistance and elevated blood pressure in childhood.12 They therefore seem sensitized to quite modest changes in diet and tend to accumulate body fat selectively in the abdomen with an ampli ed risk of diabetes and heart disease at very modest levels of body weight. Dr. Yajnik, have you yet been able to bring leptin into this picture? Yajnik: We are currently awaiting the results on leptin but we may well still not know whether it just represents body fat. In India we already have 21 million diabetics and expect by 2025 to have 60 million! We already see a vefold difference between diabetes rates between rural and urban Indians. In our studies in six Indian cities we have found that 15% of adults over 20 years of age have diabetes with a further 15% having impaired glucose tolerance. In those who have transferred into the city for less than 5 years, the body fat increase is from
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19% in a rural environment to 21 to 22% in the urban slums, but the diabetes prevalence rates have already increased from about 0 to 5% with impaired glucose tolerance going up from 10 to 15%. In the middle class living in the cities for more than one generation, the diabetes prevalence rate is 15%, with 20% affected by glucose intolerance. We nd in our preliminary measurements a progressive increase in leptin levels as we go from the rural to urban slum to the middle-class environment. These leptin levels predict all the adverse outcomes but we still do not know if the relationship is independent of body fat. We have standardized the blood measurements by not only recording previous dietary intake for three days, but in addition have provided everybody with a standardized meal for dinner the night before they were studied with a xed 10-hour fasting period and three separate samples of fasting blood ve minutes apart. Porte: What about issues relating to differential lifespan? Are we dealing simply with an age relationship? Yajnik: Lifespan 50 years ago was about 40 years in the rural environment and 45 years in urban communities, but now rural lifespans are about 58 years and in an urban environment they are about 65 years with big differences in the causes of death. I am not sure, however, about the data where one has excluded differential infant and childhood mortality rates. Rosenberg: As you begin to look at the Indian data, how are we going to distinguish issues of receptor-based resistance from circulating levels of leptin as well as insulin? Do we have population-based evidence on agerelated changes in insulin resistance and should we be thinking of how to assess this in terms of leptin. Friedman: We have a problem in trying to standardize this for leptin. Whereas we now have reasonable cellbased assays for insulin action in terms of glucose transport and in vivo studies on glucose uptake with clamp techniques, we have no in vivo assays for functional leptin sensitivity. We will therefore need to progress and see whether we can develop these techniques but there is a real challenge in humans because we are often looking at central nervous system responses. Prentice: Even in clamp studies we ignore the interactions of insulin with fatty acid metabolism as we monitor glucose uptake, but we will have even more problems with leptin. We know with insulin we have an agerelated decline in pancreatic output, but I do not know whether we have any evidence that the adipocyte wears out in terms of leptin production. ORahilly: In type 2 diabetes the decompensation of the pancreatic beta cells is clearly accompanied by histologic
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changes in the islets and the amyloid deposits seem to play a contributory role to the fall in insulin production. There does not seem to be anything like this happening with the adipocyte in terms of leptin production. As far as I know, there is no fall in plasma leptin with age. The only odd phenomenon is the extraordinarily low leptin levels seen in those individuals who are heterozygote mutation carriers. The individuals with exceptionally low leptin levels are over the age of 60, but we only have a few cases as yet. Friedman: Perhaps I could be provocative and suggest that as we become obese, extra lipid is deposited in many cells including the blood vessel wall, muscle, heart, and liver. The accumulation of intracellular pools of fat perhaps occurs because we are less sensitive to leptin so we reset the signaling for leptin despite having higher levels of cellular fat. This intracellular lipid then inhibits insulin signaling and this cellular fat is also involved in the development of cardiovascular disease. Normally one would expect that to reduce this tissue lipid would involve a greater response to leptin, but we have already become leptin insensitive. Cancer Olsson: There have been a number of studies that have related features of the metabolic syndrome, where high circulating leptin will be found, to the development of certain cancers such as prostate, breast, and colon. We decided, therefore, to look at prostatic cancer in a little more detail by studying in situ cancer within the prostate, this being extremely common in men. We found that there was a substantial over-expression of the long leptin receptor in these cancer sites when compared with the expression in normal tissue. We have therefore continued with a nested case-control prospective study that has been running from 1986. We have compared the features of 120 cases of prostate cancer that emerged since then with 240 controls, controlling for age and other background factors. We nd what seems to be a critical level of leptin corresponding to a BMI of about 25 to 26, which links to a substantial increased risk of the later development of prostate cancer. One could argue that this is really crude epidemiology, but although we did not control for more selective measures of adiposity, we did adjust for levels of insulin-like growth factor 1 (IGF1), testosterone, and other hormones. Leptin did seem to be an independent predictor with much higher BMI levels being less hazardous than expected because of the counteracting effect of the greater estrogen production in those with a higher BMI; estrogens then limit the progression of prostatic cancer.13 James: Are you implying that the local presence of
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leptin is inducing angiogenic changes and the development of the metastatic spread? Olsson: YesI think we are dealing with the development of the cancer from an in situ state. James: More generally, Bergstrom et al.14 is about to announce its recalculation of the relationship in the European Union between excess body weight and prostate cancer. I believe that they are nding a much stronger relationship than we found in the World Cancer Research Fund report on Diet and Cancer.15 Tommy Olsson is now beginning to unravel the mechanisms involved. Leptin and Bone Holmang: During fetal growth and development the hormonal and metabolic environment provided by the mother is important for ensuring adequate growth and development. There is now a major interest in the fetal programming of adult chronic diseases but bone has not been looked at as carefully. We therefore wanted to assess the impact of increased maternal leptin levels on the fetus and whether this modi es the eventual adult body composition. We therefore gave pregnant rats intraperitoneal injections of human recombinant leptin at days 8, 10, and 12 of gestation, i.e., in early pregnancy. We found that leptin-exposed male offspring had a signi cantly reduced body weight with a more modest reduction seen in the female rats. By 10 weeks of age, the leptin-exposed females had a signi cantly reduced total body weight with reductions in the fat depots, e.g., parametrial. Bone effects were also apparent with spinal, tibia, and femoral lengths reduced in both the leptinexposed male and female animals. The cortical bone thickness was signi cantly reduced but no effects were seen on the trabecular bone mass. These leptin-exposed groups also had a reduced intra-abdominal adipose tissue. This suggests that leptin could be an important mediator of the fetal development of bone and adipose tissue. We checked on the food intake of these animals and although there was a reduction in intake of about 16% after the rst injection, the second and third injections induced no signi cant decrease in intake. The weights of the dams, with or without extra leptin were also the same. Reseland: I would like to deal with human rather than rat data. We were surprised to discover that primary cultures of human osteoblasts expressed messenger RNA for leptin and we found expression levels roughly equivalent to those seen in the placenta and white adipose tissue. In our primary cultures the leptin mRNA was present in differentiated but not in undifferentiated osteoblasts, nor was it present in immortalized cells of osteosarcoma. The long form of the leptin receptor was
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also present in all the cell types. These data excluded contamination with adipocytes and on immunostaining we found leptin itself in the differentiated cells with the leptin being secreted into the medium. Leptin expression developed in the mineralization phase of differentiation. On giving leptin to a cell culture we observed that leptin protected the cells from apoptosis induced by retinoic acid. We also found that leptin increased the proliferation of the osteoblasts to the same extent as that observed with classic hormones and vitamin D3. We found that bone mineralization and collagen deposition were also increased, as were the expressions of IL6 and transforming growth factor . We also have preliminary data indicating that leptin decreases the osteoclastic activity. Astrup: This seems to con ict with the recent Cell paper,16 which found exactly the opposite, i.e., that leptin reduces bone mineral content. Holmang: That paper did not deal with the characteris tics of the osteoblast and they claim to have found no traces of leptin receptor on the osteoblasts. There is a recent paper suggesting that leptin induces an increase in bone mineral content. Lord: We have also found that the reduction in osteocalcin observed in vivo in starvation was prevented by giving leptin. Prentice: I think it makes sense to have an integrating signal such as leptin, which allows the body and bone to speed or slow growth during periods of nutritional abundance and deprivation. We already know, however, that leptin is not essential for bone growth and development. Thus we have heard that in the ob/ob and db/db mice both grow well in terms of bone mass with a good robust skeleton being available to support their extra size. The same seems to be true in the children studied by Steve ORahilly with either leptin de ciency or the MC4 mutation and massive obesity. The leptin-de cient pregnant animals also seem to produce normal offspring at deliver in terms of bone size. Leptin may be involved in the ne-tuning of bone responses, as suggested by epidemiologic studies, which are now showing a relationship between serum leptin and either bone mass or bone mineral density measured by dual energy X-ray absorptiometry in humans at all ages. One has to be careful, however, in these studies because both leptin and general bone status are intimately linked with the size of the individual, their fatness, their lean body mass and their state of maturation, so it is dif cult to tease out the particular role of leptin. Everything that I have heard suggests that leptin could work at many different levels. The main osteogenic stimuli in practice are weight bearing, gravity, the strain imposed on bones by muscle and at least from
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puberty onwards, the effect of sex hormones. We know that leptin affects estrogenic status by modulating aromatase activity in adipocytes. Recently a randomized trial with estrogen replacement therapy in women has shown an increased leptin level so there is a dialogue between leptin and estrogens. We also know that leptin interacts with growth hormone, IGF1, and IGF binding protein 3. These are closely linked and it would appear that in post-menopausal women, both growth hormone and IGF1 levels appear to be important in maintaining the bone health of women into their eighties. Many anabolic hormones that affect the skeleton are also cosecreted with insulin, e.g., amylin, calcitonin, gene-related peptide, and these are signals of food intake. The calcium parathormone set point may also be affected by food intake so unraveling any effect of leptin will be dif cult. It has always fascinated me that the peak in leptin levels during the circadian cycle comes just after midnight and precedes the parathormone and bone resorption peaks of activity by about one hour. One therefore wonders whether leptin is part of the signaling circadian rhythm process. We have already heard that there can be direct effects of leptin on bone and that the absence of leptin appears to produce a more osteopetrotic phenotype. Cortical bone seems not to be affected, but trabecular bone volume is much increased, which implies that the volume of other tissues within the marrow space is reduced. The in vitro data that we have heard about, and the controversy about the net effect of leptin, suggest we may well be looking at cross talk between the osteoblasts and osteoclasts that is so important for the regulation of bone remodeling. If leptin also regulates apoptosis in the osteoblast, then we have another potentially important regulatory process. We also know that there are inverse relationships in ageing bone between osteoblasts and adipocyte numbers, so again leptin may be involved in the adipocyte transformation. Whatever the local factors and regulation, it is important to always keep in mind the overall in vivo effects. For many years I have been studying poorly growing children in the Gambia who can lose bone mass very rapidly under adverse conditions when they stop eating. The Gambian children have a low bone mineral content for their size and this is the complete reverse of what we see in extreme conditions of leptin de ciency. Last weeks international meeting in Madrid on bone and mineral metabolism also reported studies on recovered anorectics who, 10 years after their original diagnosis, still have a low bone status and a propensity to fracture. Given the contrasts in Gambian children and anorectics with the enhanced bone masses observed in truly leptinde cient children, it is dif cult to consider leptin de S83

ciency per se as responsible for the major impact on bone of natural nutritional deprivation. Bjorntorp: As I understand it, we seem to be getting different effects of leptin if given centrally or peripherally. Friedman: In agouti mice there is increased linear skeletal growth at a time when we know there is an inhibition of melanocortin signaling. This implies that there is a relationship between de cient leptin signaling, at least through the melanocortin pathway and increased skeletal growth. Steve ORahillys children with MC4 receptor abnormalities also have increased skeletal growth, so at this integrated level there does seem to be a relationship, but we do not know really the nature of the output of melanocortin signaling in terms of the control of bone mass and there could be peripheral effects. I think we also need to be cautious about drawing too many conclusions from studies on ob/ob mice, because they are so complex and one gets a host of secondary abnormalities as a result of their obese state which may overwhelm the direct effects of leptin de ciency. James: Thank you everybodywe have had a remarkable discussion, not only about the control of insulin signaling in different physiologic states, but also an extraordinary range of effects of insulin at a cellular level with implications for reproduction, early fetal programming, immunologic control, the development and control of body weight and body fat, an impact on bone and its responsiveness to food and, nally, to emerging issues in relation to Type 2 diabetes, hypertension, and the development of cardiovascular disease. It is clear from this symposium that we are now in a phase of research where the challenge is to see how leptin acts as an integrating signal for the bodys response to so many different environments.
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