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Fasting Regulates Hypothalamic Neuropeptide Y, Agouti-Related Peptide, and Proopiomelanocortin in Diabetic Mice Independent of Changes in Leptin or Insulin¹

Fishberg Center for Neurobiology (T.M.M., H.M., J.S., J.L.R., T.L.,C.V.M.), Neurobiology of Aging Laboratories (T.M.M., H.M., C.V.M.), Department of Anesthesiology (J.S.), and Department of Geriatrics and Adult Development (T.M.M., H.M., C.V.M.), Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Charles V. Mobbs, Neurobiology of Aging Laboratories, Box 1639, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, New York 10029-6574. E-mail: mobbsc{at}alum.mit.edu

	Abstract

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Fasting increases hypothalamic neuropeptide Y (NPY) and agouti-related peptide (AGRP) messenger RNA (mRNA) and reduces hypothalamic POMC mRNA, and is also characterized by a reduction in plasma leptin, insulin, and glucose, each of which has been implicated in the regulation of hypothalamic gene expression. To further evaluate the roles of leptin, insulin, and glucose in mediating effects of fasting, we examined hypothalamic gene expression in nondiabetic and streptozotocin (STZ)-induced diabetic mice both under ad lib fed and 48-h fasted conditions. In both diabetic and nondiabetic mice, fasting stimulated hypothalamic NPY and AGRP mRNA and inhibited hypothalamic POMC mRNA and adipose leptin mRNA. However, in diabetic mice fasting had no effect on plasma leptin and insulin while decreasing plasma glucose, whereas in nondiabetic mice fasting decreased plasma leptin, insulin, and glucose. Furthermore, in nondiabetic fasted mice, NPY and AGRP mRNA were higher, and POMC mRNA and plasma glucose were lower, than in diabetic ad lib fed mice, even though insulin and leptin were similar in these two groups. These data are consistent with the hypothesis that although leptin and insulin regulate hypothalamic gene expression, glucose or other factors may have independent effects on hypothalamic and adipose gene expression under conditions of low insulin and leptin.

	Introduction

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FASTING regulates hypothalamic gene expression, and in turn hypothalamic gene expression appears to mediate many effects of fasting (1); however, the mechanisms by which fasting regulates hypothalamic gene expression are not fully elucidated. Fasting produces an elevation of hypothalamic neuropeptide Y (NPY) messenger RNA (mRNA) (2, 3, 4, 5, 6) and agouti-related peptide (AGRP) mRNA (7, 8) and a decrease of hypothalamic POMC mRNA (6, 9, 10). Plasma leptin, insulin, and glucose also decrease with fasting (11, 12, 13). Because Ahima et al. (14) demonstrated that leptin injection can reverse many neuroendocrine effects of fasting, many studies have corroborated that many effects of fasting appear to be mediated by the decrease in leptin that accompanies fasting (15, 16, 17, 18). However, because leptin, glucose, and insulin comprise a mutually regulatory loop (12) and tend to co-vary, it is difficult to determine the independent role of each factor in the regulation of hypothalamic gene expression. Leptin appears to independently regulate hypothalamic gene expression because leptin-deficient mice exhibit elevated NPY mRNA (6, 19, 20); elevated AGRP mRNA (8, 21), and decreased POMC mRNA (6, 10, 22), even though leptin-deficient mice also exhibit elevated plasma insulin and glucose (20). Conversely, infusion of leptin can reverse the effect of fasting on NPY mRNA (14) even when leptin is infused directly into the brain (23). Furthermore, fasting appears to act only on those NPY neurons that express leptin receptors (24). These studies suggest the hypothesis that the effects of fasting on hypothalamic gene expression are largely, if not entirely, mediated by the fall in circulating leptin that occurs during fasting. On the other hand, some effects of fasting on hypothalamic gene expression may be independent of leptin because insulin infused directly into the hypothalamus blocks the effects of fasting on hypothalamic NPY mRNA (4) and because effects of fasting on NPY mRNA are observed before a decrease in plasma leptin (25). Furthermore, in db/db mice that are insensitive to leptin, fasting further elevates hypothalamic NPY mRNA and decreases hypothalamic POMC mRNA (6) and further elevates AGRP mRNA (8). In addition, impairment in glucose metabolism increases hypothalamic NPY mRNA (26), consistent with direct effects of glucose on hypothalamic slices that are enhanced by simultaneous application of insulin (27). Therefore, some effects of fasting could be mediated by direct effects of glucose on hypothalamic gene expression or enhancement of these effects by insulin, presumably acting on leptin-sensitive neurons. To assess potential independent effects of leptin, insulin, and glucose on hypothalamic gene expression, we examined the effect of fasting on hypothalamic NPY, AGRP, and POMC gene expression in normal and moderately insulin-deficient mice, in which we hypothesized that fasting would fail to regulate insulin and leptin.

	Materials and Methods

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Animals and treatment
Retired breeder male C57Bl/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). We had previously observed that moderate insulin deficiency could be produced more reliably, with less mortality, in retired breeder than in younger mice (28). Mice were individually housed with free access to feed and water under 12-h light, 12-h dark cycle (lights on at 0700 h). All studies had been approved by the appropriate Institutional Animal Review Board. Mice (n = 30) were injected ip with streptozotocin (STZ, freshly purchased from Sigma, St. Louis, MO, dissolved in PBS, pH 7.6, at a concentration of 15 mg/ml) at a dose of 150 µg/g BW, or PBS (n = 16), and insulin-deficient diabetes was inferred by a plasma glucose level of >300 mg/dl, one week after STZ injection. Blood glucose from tail vein was measured by a GLUCOMETER ELITE (Bayer Corp. Co., Elkhart, IN). STZ-injected diabetic or saline-injected nondiabetic mice were either fed ad libitum, or fasted for 48 h (n = 8 per group). STZ-injected nondiabetic mice (plasma glucose, <200 mg/dl) were fed ad libitum (n = 5; this number was determined by including all STZ-injected mice that did not develop diabetes). To directly assess effects of leptin on hypothalamic gene expression, a group of STZ-induced diabetic mice was also injected ip with leptin (R&D Systems, Minneapolis, MN) at a dose of 0.5 µg/g BW twice daily (at 1000 h and 1500 h) for 2 days, following the protocol of Ahima et al. (14) (n = 9). Mice were killed between 1 and 3 weeks after STZ injection at the end of light period (between 1700 and 1900 h) by exposure to carbon dioxide for about 5 min, followed by cardiac puncture and decapitation. This method was necessary to obtain sufficient plasma to analyze insulin and leptin, but produces stress-related elevations in corticosterone in some mice, so glucocorticoid measurements would have been difficult to interpret and were not performed. Brains were quickly removed and hypothalamus was dissected out, frozen on dry ice, and stored at -70 C until use. Epididymal white adipose tissue was also removed, frozen on dry ice and stored at -70 C until use. Glucose was measured by a Lifescan One-Touch ll glucose meter (Johnson & Johnson, Mountain View, CA), and insulin and leptin were assayed by ELISA with commercial kits (Crystal Chem, Inc., Chicago, IL). All but two samples were within the linear range of the standard curves, and these two samples were eliminated from analysis.

RNA analysis
Total RNA was extracted in TRIzol (Life Technologies, Inc., Gaithersburg, MD) and 3 µg of total RNA from hypothalamus, estimated by spectrophotometer, was subjected to Northern blot analysis to detect POMC, NPY, and AGRP mRNA. POMC mRNA was also assessed by RNase protection assay with 3 µg of total RNA from hypothalamus. Seven micrograms of total RNA from adipose tissue was also subjected to Northern blot analysis to detect leptin mRNA. Northern blot analysis was performed by using single-stranded internally labeled DNA probes as described previously (6, 12). To monitor RNA loading, membranes were reprobed and hybridized with ³²P-labeled probe encoding 18S ribosomal RNA. RNase protection assay for POMC mRNA was also performed as described previously with the standard ranging from 0.5 to 10 pg. The total integrated densities of hybridization signals were determined by phosphoimager (STORM 860, Molecular Dynamics, Inc., Sunnyvale, CA). Data were analyzed and presented per unit microgram total RNA loaded onto the gels, but the results were essentially the same if normalized to the 18S rRNA band.

Statistical analysis:
Statistical analysis entailed a two-way (STZ-diabetic/nonSTZ-nondiabeticXfed/fasting) ANOVA followed, when indicated by appropriate P values (P < 0.05), by Tukey-Kramer posthoc test. In those analyses that included the streptozotocin-treated nondiabetic mice and the diabetic leptin-treated mice, a one-way ANOVA was carried out (each group representing a level of the single overall experimental variable), followed, when appropriate, by a Tukey-Kramer posthoc test. A P value of less than 0.05 was considered significant.

	Results

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Plasma glucose, insulin, and leptin levels
One week after STZ treatment, mice exhibiting plasma glucose greater than 300 mg/dl (determined in the ad lib fed state in the late afternoon) were defined as diabetic. By this criteria, 84% of the STZ-injected mice became diabetic, and the remainder were classified as STZ-injected nondiabetic mice, and no mice died due to STZ treatment. Plasma glucose was significantly influenced by both diabetes [F (1, 28) = 152.0; P < 0.0001] and fasting [F (1, 28) = 115.7; P < 0.0001], and there was a significant interaction [F (1, 28) = 27.6; P < 0.001] on plasma glucose by two-way ANOVA. Diabetic mice exhibited a significantly higher nonfasted plasma glucose (416.1 ± 15.5 mg/dl, P < 0.05) than nonSTZ-injected controls (171.0 ± 9.2 mg/dl) or STZ-injected nondiabetic mice (156.8 ± 11.5 mg/dl) (Fig. 1A; P < 0.05, Tukey-Kramer). Fasting significantly reduced plasma glucose in nonSTZ-injected controls (to 94.3 ± 11.7 mg/dl) and in diabetic mice (to 192.9 ± 20.6 mg/dl). Leptin treatment for 2 days in a group of diabetic mice did not significantly alter plasma glucose.

View larger version (22K): [in this window] [in a new window]   Figure 1. Effects of STZ-induced diabetes and fasting on plasma glucose (A), insulin (B), and leptin (C) levels (mean ± SE, n = 5–9/group). Groups with different letters are statistically different (P < 0.05, ANOVA followed by Tukey-Kramer test).

Plasma leptin was significantly influenced by both diabetes [F (1, 28) = 17.8; P < 0.001] and fasting [F (1, 28) = 14.0; P < 0.001], and there was a significant interaction [F (1, 28) = 10.7; P < 0.01] on plasma leptin. Plasma leptin was significantly lower in STZ diabetic mice (1.06 ± 0.36 ng/ml, P < 0.05) than in ad lib fed nonSTZ-injected controls (7.85 ± 1.84 ng/ml) (Fig. 1C). Fasting significantly reduced plasma leptin in nondiabetic controls to levels exhibited by ad lib fed diabetic mice. However, fasting did not further reduce plasma leptin in diabetic mice. Leptin treatment increased plasma leptin levels in STZ diabetic mice.

Body weight
There was no significant difference in initial body weight between groups (Table 1). Body weight significantly decreased as the diabetic state developed in STZ-injected mice (Table 1). A two-day fast caused significant decrease in body weight in control and STZ diabetic mice to a similar extent (-5.95 ± 0.44 g/2 days and -5.70 ± 0.23 g/2 days, respectively). Leptin replacement caused significantly greater loss of body weight (-1.26 ± 0.14 g/2 days) in ad lib fed diabetic mice compared with the change in body weight of ad lib fed diabetic mice not injected with leptin (-0.58 ± 0.18 g/2 days, P < 0.05). Note that although the combined effect of diabetes and fasting was to decrease total body weight by about 7.2 g, because these mice were retired breeders (approximately 9 months old) with initial body weights of about 30.6 g, their final body weights were approximately equal to the body weights of nondiabetic fasted 5-month-old mice (12). At the time mice were killed all mice in the study appeared healthy, and no mice died during the study.

Adipose leptin mRNA
Adipose leptin mRNA was significantly influenced by both diabetes [F (1, 28) = 20.3; P < 0.001] and fasting [F (1, 28) = 14.3; P < 0.001], but there was not significant interaction [F (1, 28) = 3.1; P = 0.09]. Leptin mRNA in epididymal white adipose tissue was significantly lower (by about 70%, P < 0.05) in diabetic ad lib fed mice than in nondiabetic controls (Fig. 2). Fasting significantly decreased leptin mRNA in both nondiabetic control (by about 58%, P < 0.05) and in diabetic mice (by about 60%, P < 0.05). Exogenous leptin did not change endogenous expression of leptin mRNA in diabetic mice, even though this treatment reduced body weight and increased plasma leptin levels (Fig. 1).

View larger version (28K): [in this window] [in a new window]   Figure 2. Effects of STZ-induced diabetes and fasting on leptin mRNA in the epididymal adipose tissue (mean ± SE, n = 5–9/group). Groups with different letters are statistically different (P < 0.05, ANOVA followed by Tukey-Kramer test).

View larger version (57K): [in this window] [in a new window]   Figure 3. Effects of STZ-induced diabetes and fasting on hypothalamic NPY (A), AGRP (B), and POMC (C) mRNAs, and 18S rRNA (D), as assessed by Northern blot analysis. Total RNA (3 µg) from individual animal was loaded in each lane. Lanes 1–4, Fed control mice. Lanes 5–8, Fasted control mice. Lanes 9–12, Fed STZ-induced diabetic mice. Lanes 13–16, Fasted STZ-induced diabetic mice. Blots represent only a representative subset (n = 4) of all samples quantified (n = 8 for control and diabetic fed and fasted, 5 for STZ-injected nondiabetic, and 9 for leptin-injected). Quantification of NPY (E), AGRP (F), and POMC (G) mRNA signals, and 18S rRNA signal (H), mean ± SE, n = 5–9/group, expressed as percentage of fed, nondiabetic controls. Groups with different letters are statistically different (P < 0.05, ANOVA followed by Tukey-Kramer test). Although the presented data reflect band density per unit microgram total RNA loaded into each lane, the mRNA results were essentially the same if normalized to the 18S rRNA band.

POMC mRNA was significantly influenced by fasting [F (1, 28) = 17.8; P < 0.001], but the effect of diabetes was not significant [F (1, 28) = 1.4; P = 0.25] nor was there a significant interaction [F (1, 28) = 1.6; P = 0.22] (Fig. 3, C and G). Hypothalamic POMC mRNA was slightly lower (by 21%) in diabetic ad lib fed mice than in nondiabetic ad lib fed controls, but this effect did not achieve statistical significance (Fig. 3, C and G; P = 0.16). Fasting significantly reduced POMC mRNA both in nondiabetic mice (by about 47%, P < 0.05) and in diabetic mice (by about 32%, P < 0.05). Furthermore, POMC mRNA was significantly lower in nondiabetic fasted mice than in diabetic ad lib fed controls (P < 0.05). Leptin injection did not significantly increase POMC mRNA in diabetic mice. Assessment of POMC mRNA by Northern blot analysis was corroborated by RNase protection assay of the same samples, in which POMC mRNA levels were quantified as follows (numbers represent absolute picograms POMC mRNA per microgram hypothalamic RNA, mean ± SEM): nondiabetic ad lib fed: 0.82 ± 0.13 pg; nondiabetic fasted: 0.43 ± 0.07 pg; diabetic ad lib fed: 0.63 ± 0.10 pg; diabetic fasted: 0.33 ± 0.05 pg; diabetic, leptin-treated: 0.72 ± 0.20 pg.

	Discussion

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The main purpose of these studies was to assess the extent to which effects of fasting on hypothalamic gene expression may be mediated by factors other than fasting-associated reduction in leptin and insulin, as suggested by studies in adrenalectomized rats (25). Consistent with earlier studies, in normal nondiabetic mice, fasting stimulated hypothalamic NPY (6) and AGRP (8) mRNAs, and reduced hypothalamic POMC mRNA (6). Fasting in normal mice also reduced plasma insulin, leptin, and glucose, and, as described in the introduction, evidence suggests each of these might produce independent effects on hypothalamic gene expression. In diabetic mice, fasting also stimulated hypothalamic NPY and AGRP mRNAs, and reduced hypothalamic POMC mRNA. However, in diabetic mice fasting had no significant effect on plasma insulin and leptin, which were already at low levels similar to levels observed in fasting nondiabetic mice. Although low, leptin, and insulin levels in diabetic mice were well within the linear range of the assays, and although a nonsignificant trend toward a decrease was observed in fasting diabetic mice, the magnitude of this trend was far smaller than the significant effect of fasting in nondiabetic controls, whereas the effect of fasting on hypothalamic gene expression was similar in controls and diabetic mice. Furthermore, hypothalamic NPY and AGRP mRNAs were higher, and hypothalamic POMC mRNA was lower, in nondiabetic fasted mice than in diabetic ad lib fed mice, although plasma insulin and leptin were similar in these two groups. Similarly, hypothalamic NPY and AGRP mRNA were higher in diabetic fasted mice than in nondiabetic fasted mice, although plasma insulin and leptin were similar in these two groups. Finally, leptin injection in diabetic mice, whereas reducing body weight and roughly normalizing plasma leptin, did not significantly influence NPY, AGRP, or POMC mRNAs in diabetic mice (though the injection paradigm did not completely mimic normal leptin secretion patterns). Taken together, these studies indicate factors other than leptin and/or insulin can independently regulate hypothalamic gene expression. It is of some interest that all three hypothalamic genes examined exhibited similar dependencies on leptin, insulin, and other factors, suggesting that these three genes are regulated through common mechanisms.

Leptin mRNA exhibited a pattern of expression similar to, but slightly different from, the pattern of expression of POMC mRNA. As with POMC mRNA, leptin mRNA was inhibited by fasting in both nondiabetic and diabetic mice, even though, in diabetic mice, fasting did not influence plasma insulin or leptin. This observation suggests that fasting can reduce leptin mRNA independent of any change in plasma insulin (or plasma leptin), possibly through a reduction in plasma glucose, as previously suggested (12). However, because fasting did not influence plasma leptin in diabetic mice, the effects of fasting on plasma leptin levels, as opposed to leptin mRNA, may be completely dependent on insulin.

It is remarkable that the effects of fasting on hypothalamic gene were quantitatively very similar in diabetic and nondiabetic mice, despite the large differences in the regulation of plasma leptin and insulin. For example, fasting induced NPY mRNA by 346% in nondiabetic mice and induced NPY mRNA by 375% in diabetic mice, even though in the diabetic mice fasting entailed no change in leptin or insulin. This observation is consistent with the study of Hanson et al. (25), which showed that after 15 h of fasting, hypothalamic NPY mRNA was similarly elevated in intact and adrenalectomized/steroid-replaced rats, even though in the former group fasting had not yet reduced plasma leptin at that time point. These results suggest that, at least under 48-h fasted conditions, effects of leptin and/or insulin on hypothalamic gene expression are additive with, rather than multiplicative with (e.g. synergistic with) other factors such as glucose. The lack of such synergy is statistically corroborated by the lack of a significant statistical interaction between fasting and diabetic state (e.g. statistically, the effect of fasting was the same in diabetic and nondiabetic mice on expression of each gene examined).

One factor that plausibly contributes to many of the effects observed in the present study, including effects of both diabetes and fasting, would be changes in glucocorticoid secretion. In diabetic, adrenalectomized rats, glucocorticoid injection directly stimulates NPY mRNA (29). Furthermore, glucocorticoids are elevated by fasting (25) and in streptozotocin-induced diabetes (30). While the elevation of glucocorticoids are not necessary for fasting-induced NPY mRNA elevation (25), they do appear to be important for the reduction of CRF mRNA by diabetes (30).

Because interference with glucose metabolism can induce hypothalamic NPY mRNA (26), plasma glucose also constitutes a plausible candidate mediating effects of fasting independent of changes in insulin and leptin. In particular, the reduction in plasma glucose by fasting in diabetic mice could plausibly account for effects of fasting on hypothalamic gene expression and leptin mRNA in diabetic mice. Furthermore, NPY and AGRP mRNA was not as elevated, and POMC mRNA was not as reduced, in diabetic ad lib fed mice as in nondiabetic fasted mice, even though these two groups exhibited similar plasma leptin and insulin. This result could be explained by an effect of elevated plasma glucose in diabetic mice to partially compensate for the effects of low leptin and insulin, thus attenuating effects of low leptin and insulin in diabetic ad lib fed mice. Similarly, the observation that leptin mRNA was further inhibited by fasting in diabetic mice despite no change in insulin is consistent with our previous observations that leptin mRNA is independently related to plasma glucose levels (12, 31) and is regulated by glucose metabolism in adipocytes (32).

Nevertheless, differences in plasma glucose do not explain all aspects of gene expression observed in the present study. In particular, NPY and AGRP mRNA were elevated (e.g. showed a fasting-like profile) in diabetic ad lib fed mice compared with nondiabetic ad lib fed mice. Because plasma glucose is higher in the diabetic mice than in the nondiabetic mice, the fasting-like profile of diabetic mice cannot be explained by reduction in glucose but may be mediated by reduction in insulin and/or leptin. Consistent with previous reports (4, 14, 20), these data suggest that insulin and/or leptin can act independently of plasma glucose to regulate hypothalamic gene expression. A final observation suggests that another factor in addition to glucose, insulin, and leptin, may be involved in the regulation of gene expression by fasting. Hypothalamic NPY and AGRP mRNA was higher, and leptin mRNA was lower, in diabetic fasted mice than in nondiabetic-fasted mice (POMC mRNA exhibited the same trend as leptin mRNA in this respect, though the effect did not achieve statistical significance). Thus diabetic fasted mice exhibited an exaggerated fasting-like phenotype compared with fasted nondiabetic mice even though plasma insulin and leptin were similar, and glucose was actually higher (and thus should oppose the fasting-like profile), in diabetic fasted than in nondiabetic fasted mice. These results suggest that some factors other than insulin, leptin, or glucose, may account for this exaggerated fasting-like profile in diabetic mice. In addition to a probable role for glucocorticoids, as discussed above, other factors, including gut hormones such as cholecystokinin (33), could also be involved in mediating leptin- and insulin-independent effects of fasting.

In the present studies, insulin and leptin covaried except when leptin was injected into diabetic mice. Leptin injection into diabetic mice reduced body weight and increased leptin (at least at the time the mice were killed), but did not normalize hypothalamic gene expression or leptin mRNA. These results suggest that the paradigm of leptin replacement used, while having some biological effect, was insufficient to influence hypothalamic gene expression. This observation suggests that the effect of insulin deficiency on hypothalamic gene expression is not entirely mediated by a reduction in leptin, but may be due in part to reduced leptin-independent effects of insulin on hypothalamic function, consistent with previous reports (4). Nevertheless, in the present studies leptin injections may not have achieved physiological concentrations throughout the day, so it is possible that a more physiological or constant replacement protocol would have had a greater effect on hypothalamic gene expression.

Although the results of the present study were described in terms of the effects of fasting, it is equally logical to view the results in terms of the effects of feeding. Thus, because effects of feeding are opposite to the effects of fasting, the present study and previous studies indicate that feeding reduces hypothalamic NPY and AGRP mRNA, and stimulates hypothalamic POMC mRNA. Furthermore, the present study suggests that some effects of feeding on hypothalamic gene expression are independent of leptin and insulin, and plausibly mediated by glucose. Consistent with this conclusion, glucose infused directly into the carotid artery induces hypothalamic c-fos (presumably with no change in insulin or leptin) (34), and we have directly demonstrated that feeding induces hypothalamic immediate-early gene expression in leptin-deficient ob/ob mice as well as in normal mice before leptin increases (Mizuno et al., unpublished observations). Electrophysiological evidence suggests that glucose, leptin, and insulin act on the same hypothalamic neurons in vitro (27, 35, 36). Because feeding-sensitive neurons seem to be primarily or exclusively leptin-sensitive (24), these data suggest that feeding, glucose, leptin, and insulin may primarily on a common set of neurons. Furthermore, the profile of elevated NPY and AGRP mRNA, and reduced POMC mRNA is likely to predispose to obesity (37). Because appropriate regulation of hypothalamic gene expression appears to require independent actions of glucose, insulin, and leptin, hypothalamic resistance to effects of any of these three agents could independently produce obesity.

	Acknowledgments

 
We thank Joseph Beasley for his assistance with the hormone assays.

	Footnotes

 
¹ This work was supported by a grant from the National Institutes of Health DK-50110–01.

Received January 19, 1999.

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