Obesity and Factor of Leptin Resistance
Date: Thursday, October 12th, 2000
Title: “The Behavioral and Biological Determinants of Leptin Resistance”
Speaker: Joseph R. Vasselli, Ph.D., St. Luke’s/Roosevelt Hospital, NY, NY
Behavioral Studies of Leptin Resistance
The concept of leptin resistance is almost as old as the discovery of leptin itself. Prompted by the observation of a strong correlation between increasing circulating leptin levels in humans and increasing body fat, Maffei et al. (1995) were the first to propose that leptin may lose its feeding and body weight inhibitory effects as levels of the hormone increase. Campfield et al. (1995) were the first to experimentally demonstrate leptin resistance in a lean strain of mice (AKR/J) made obese with a high fat diet (HF), noting that administration of 3 times more exogenous leptin to the obese mice was required to provoke significant feeding and body weight inhibition, in comparison with chow-fed control mice. The concept that leptin resistance may reflect functional alterations of the leptin response mechanism is supported by observations that the phenomenon can be observed in dietary obese rodents even when leptin is administered centrally (Lin et al., 2000; Widdowson et al., 1997).
Leptin resistance is not difficult to demonstrate experimentally, as done in our laboratory using adult male S-D rats made obese on 60% HF. In response to 1.0 mg rMuLep/kg body weight i.p., obese rats eating HF showed no feeding or body weight inhibitory effects of leptin over a 24-hr test period, in comparison with chow-fed lean control rats, which displayed significant decreases of 3.5 g (-15%) in 24-hr food intake, and 5.6 g (-1.0%) in body weight. For testing, the response of each rat to i.p. leptin is compared to its response to a previous i.p. saline injection under identical test conditions. Despite this striking difference in responsiveness to injected leptin, we noted that levels of plasma leptin of the groups differed by only 5-6 ng/ml, with the leptin-resistant HF group having levels slightly above 20 ng/ml. Leptin levels were measured using the Linco RIA kit for rat leptin.
However, in the above testing, as in virtually all demonstrations of leptin resistance to date (El-Haschimi et al., 2000; Widdowson et al., 1997), the responsiveness of an obese group maintained on a HF diet is compared to that of a lean group fed a chow diet. This leaves the results open to the criticism that potential differences of palatability, rather than an underlying biological impairment of leptin action, is responsible for loss of the leptin effect observed in the obese group. According to the “palatability” hypothesis of leptin resistance, the inhibitory effects of leptin may remain intact in dietary obese animals and in most obese humans, but may simply be overridden by the continuous availability of more attractive diets (Arch et al., 1998).
To investigate this possibility, groups of young, male, S-D rats were fed either chow or 45% HF, on which the groups spontaneously maintained equal body weights throughout the growth period. For saline- and leptin-injection testing only, half of each group was switched to the alternate diet. In support of the palatability hypothesis, we observed that the feeding-inhibitory effect of leptin was abolished when chow-maintained rats were switched to the HF diet for testing. However, this was not true for the group maintained and tested on the HF diet, which responded with significant feeding and body weight reductions to injected leptin. For the group switched from HF to the chow diet for testing, the feeding and body weight inhibitory effects of injected leptin were even more potent (p<0.05) than those observed in the chow-maintained and tested group. Thus, palatability can alter the feeding-inhibitory effects of leptin, but is not the sole basis for leptin resistance. Leptin levels of the groups, although significantly different, were well below the 20 ng/ml level that was associated with leptin resistance in our demonstration study cited above.
Two more studies were conducted to examine the relationship between obesity and leptin level. The first study showed that, independent of maintenance diet, obesity per se and elevated leptin levels (> 20 ng/ml) associated with it, correlate with the expression of leptin resistance in DR (diet-resistant) and DIO (diet-induced obese) rats. In this study, DIO rats made obese on HF but switched to a maintenance diet of laboratory chow for an extended period, remained significantly resistant to i.p. leptin (1.0 mg/kg BW). The second study showed that male Zucker obese and lean rats are both resistant to the effects of injected leptin (2.0 mg/kg i.p.). Both the lean and obese groups had leptin levels of at least 20 ng/ml (in this case leptin levels were measured by the Amgen rodent leptin assay, which uses a polyclonal antibody to murine leptin).
To summarize, a palatable, high fat diet can diminish the feeding-inhibitory effect of leptin, but alone does not account for the phenomenon of leptin resistance. This is borne out by observations in our laboratory that maintenance of a group of rats on HF but at normal body weight for an extended period does not abolish leptin responsiveness. Reciprocally, maintenance of a group of obese rats on a laboratory chow diet for an extended period, during which the rats maintained obese body weights, does not restore leptin responsiveness, i.e., the group remains leptin resistant. Our data also indicate that in rats, circulating leptin levels above approximately 20 ng/ml appear to be required for the expression of leptin resistance. This would suggest that obesity induces leptin resistance as a result of its effect on leptin levels.
Biological Mechanisms of Leptin Resistance
Biological processes hypothesized to be involved in leptin resistance were reviewed, including leptin binding to blood borne proteins (one of which is the soluble leptin receptor), active and/or passive transport of leptin into the brain, leptin receptor expression levels, and alterations of leptin receptor-second messenger responsiveness (see Ahima and Flyer, 2000 for a review). Binding of leptin to proteins in the blood actually decreases with increasing obesity, rendering this possibility unlikely (Housenecht et al., 1996). Several studies have now documented relatively reduced levels of CSF leptin in obese humans, however, in comparison to levels in lean humans (Schwartz et al., 1996), and two studies showing decreased blood-brain capillary leptin transport in HF obese rat models have been published (Banks et al., 1999; Burguera et al., 2000). Leptin receptor binding and second messenger activation have been extensively studied, and evidence for leptin receptor down-regulation in vitro in the presence of high leptin concentrations, (Uotani et al., 1999), and in vivo in a dietary obese rat model (Madiehe et al., 2000) has been found. Also the Flier group, although not being able to show decreased mRNA for long form leptin receptor in the hypothalamus of obese HF rats, was able to show reduced binding of STAT-3 (receptor second messenger) to nuclear elements in these animals in response to both peripheral and central leptin administration (El-Haschimi et al., 2000).
Because leptin has several peripheral actions also mediated by long-form receptors, we chose to examine the ability of i.p. leptin to stimulate lipolysis in the adipocytes of chow-fed (leptin-responsive) and HF (leptin-resistant) groups of rats. We found no impairment of leptin-induced adipose tissue lipolysis in either group of rats, demonstrating that the long form of the leptin receptor functions normally in rat adipocytes, in spite of a state of central leptin resistance induced by HF. We concluded that leptin resistance in obese HF rats may be limited to long-form receptors in the CNS. We also examined the phosphorylation (activation) of STAT-3 in the hypothalamus of these groups. Results showed elevated levels of phosphorylated STAT-3 in the brains of HF rats, which could not be further stimulated when the rats were injected with leptin. These results are consistent with the maximal stimulation of hypothalamic leptin receptors by elevated leptin concentrations in HF rats, and offer a potential explanation for the association between elevated circulating leptin levels and the expression of leptin resistance observed in our behavioral testing.
Brief results of a pilot study on aging-induced leptin resistance were presented, which suggested that this form of leptin resistance may not be dependent upon increased adiposity or on circulating leptin levels above 20 ng/ml. Finally, the question of the availability of human data on the issue of leptin resistance was addressed. Virtually no data exist, save for several correlational studies of metabolic rate vs. leptin levels in men and women (overall non-significant findings). On a more positive note, however, a highly significant correlation between palatability ratings of a test meal and fasting leptin levels in a sample of men and women with a wide range of BMI was recently reported, although control procedures in this study were noted to be inadequate (Raynaud et al., 1999). Obviously, a great deal more will be known when investigators are able to administer leptin to humans experimentally.
In summary, although several biological mechanisms have been identified which may participate in leptin resistance, the sequence of events which initiate and maintain this state remains to be determined, and more information regarding potential treatment issues, (i.e., role of diet composition in onset, potential changes in perceived palatability, reversibility of the effect) is required.
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Q. Your experiment with the HF diet showed that this diet doesn’t induce leptin resistance, but why do you feel strongly that you have ruled out any role of palatability in mediating leptin resistance?
A. I believe that diet palatability can in general modulate the strength of leptin’s feeding inhibitory effect, as it can that of many other feeding-inhibitory agents such as CCK, due to different degrees of diet acceptability which accompany diets with different sensory, textural, and/or nutrient qualities. In the case of leptin resistance, diet palatability can indirectly create a state of leptin resistance as a result of its effects on caloric intake and nutrient partitioning, if these effects result in obesity and elevated leptin levels. Also, diet palatability can alter the expression of already established leptin resistance when animals are tested on more than one diet. There is currently no evidence, however, that diet palatability contributes directly to the biological mechanisms which underlie leptin resistance.
Q. What was the caloric level of the chow-like diet used in your studies, and what was the source of the carbohydrate in the diet?
A. The chow-like diet was designed to mimic the composition and caloric content of standard laboratory chow (Purina). The carbohydrate content came from starch, and the caloric content matched that of Purina chow almost exactly (3.5 Kcal/g).
Q. If the carbohydrate source had been sugar, the rats would have become obese more quickly…and differing rat strains could have different responses to this type of dietary manipulation.
A. Yes, a high sugar content in the diet may have induced obesity more rapidly, and it is well established that differential responsiveness to diet composition can be observed among different rat and mouse strains. The issue you are driving at, I believe, is whether
the high fat content of the diet is required to induce leptin resistance, or whether carbohydrate-induced obesity can also result in leptin resistance. I have data (not shown here tonight) which indicates that carbohydrate overconsumption which leads to obesity
can also result in leptin resistance. I.e., leptin resistance is not high fat diet specific.
Q. Did you look at dose-response measures of leptin action, and whether the response profile (i.e. the slope or threshold) changes with age or with the palatability of the diet?
A. We did examine dose-response curves for leptin feeding inhibition, and found that the shape of the curve depends on both the strain and age of the rats. There are large differences in responsiveness to leptin by rat strain. In general, all strains become less responsive to the feeding inhibitory effects of leptin as they age. We have not systematically investigated the effect of diet type on leptin responsiveness, however.
Q. What would happen if you looked at the effect of a HF diet after the animals became leptin resistant on carbohydrates?
A. Good question. We have not yet looked at responsiveness to a HF diet in rats made leptin resistant on a carbohydrate diet. I assume that the rats would be resistant to the feeding inhibitory effects of leptin on a HF diet as well.