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Endocrinology Vol. 148, No. 1 310-316
Copyright © 2007 by The Endocrine Society

Altered Hypothalamic Leptin, Insulin, and Melanocortin Binding Associated with Moderate-Fat Diet and Predisposition to Obesity

Boman G. Irani, Ambrose A. Dunn-Meynell and Barry E. Levin

Departments of Neurology and Neurosciences (B.G.I., A.A.D.-M., B.E.L.), New Jersey Medical School, Newark, New Jersey 07103; and Neurology Service (A.A.D.-M., B.E.L.), Veterans Administration Medical Center, East Orange, New Jersey 07018

Address all correspondence and requests for reprints to: Barry E. Levin, M.D., Neurology Service (127C), Veterans Administration Medical Center, 385 Tremont Avenue, East Orange, New Jersey 07018-1095. E-mail: levin{at}umdnj.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rats with a genetic predisposition to develop diet-induced obesity (DIO) have a preexisting reduction in central leptin and insulin sensitivity. High-fat diets also reduce sensitivity to leptin, insulin, and melanocortin agonists. We postulated that such reduced sensitivities would be associated with decreased binding to the hypothalamic leptin, insulin, and melanocortin receptors in selectively bred DIO rats and in rats fed a high-energy (HE; 31% fat) diet for 7 wk. On HE diet, DIO rats gained 15% more weight and had 121% heavier fat pads and 70% higher leptin levels than low fat chow-fed DIO rats. Diet-resistant (DR) rats gained no more weight on HE diet but had 48% heavier fat pads and 70% higher leptin levels than chow-fed DR rats. Compared with DR rats, DIO 125I-leptin binding was 41, 36, and 40% lower in the hypothalamic dorsomedial, arcuate, and dorsomedial portion of the ventromedial nuclei, respectively, and arcuate 125I-insulin binding was 31% lower independent of diet. In contrast, hypothalamic melanocortin binding did not differ between DIO and DR rats. However, HE diet intake lowered lateral hypothalamic melanocortin-3 and melanocortin-4 receptor and hippocampal insulin binding of both DIO and DR rats and hypothalamic paraventricular nucleus melanocortin-4 receptor binding only in DR rats. Neither genotype nor diet affected substantia nigra or ventral tegmental area binding. These results corroborate our previous findings demonstrating a preexisting decrease in DIO hypothalamic leptin and insulin signaling and demonstrate that HE diet intake reduces hypothalamic melanocortin and hippocampal insulin binding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DIET-INDUCED OBESITY (DIO) in rats shares several features of human obesity which include leptin and insulin resistance (1, 2, 3). Approximately half of the outbred rats fed a moderate-fat (31%), high-energy (HE) diet increase their caloric intake and become obese, whereas the remaining rats are diet resistant (DR), gaining no more weight than low-fat chow-fed controls (4, 5, 6). As in many human beings, the DIO and DR phenotypes are inherited as polygenic traits that can be selectively bred from an outbred population (7, 8). Therefore, these rat models serve as an excellent surrogate to study the underlying causes of human obesity and the central pathways that regulate energy homeostasis.

Adiposity signals such as leptin and insulin are secreted into the blood in proportion to body fat as signals to the brain (3, 9, 10). They cross the blood-brain barrier via a saturable transport mechanism (11, 12) to interact with central pathways that regulate energy homeostasis (13). Chronic leptin (14, 15) and insulin (16) administration reduces food intake and body weight, and reduction of central leptin (17) and insulin (18, 19) signaling results in hyperphagia and obesity. However, chronic obesity often raises leptin and insulin levels, leading to a reduced sensitivity to their central catabolic effects (1, 2, 3, 5, 20, 21).

DIO rats have reduced central leptin and insulin sensitivity before they are exposed to HE diet and develop obesity, and this may predispose them to become obese when the caloric density and fat content of their diet is increased (5, 20, 22, 23, 24). Similarly, defective melanocortin-3 (MC3) and/or melanocortin-4 receptor (MC4R) signaling predisposes rodents to develop DIO (25, 26, 27, 28, 29). At least for leptin, the reduced sensitivity of DIO rats may be due to reduced central leptin signaling. DIO rats have reduced mRNA expression of the long (signaling) form of leptin receptor (Lepr-b) and leptin-induced phosphorylation of STAT3 in the hypothalamus before the onset of obesity (5, 23). However, it is unknown whether the decreased Lepr-b mRNA expression is associated with reduced receptor binding or whether DIO rats also have defects in insulin or melanocortin binding. Also, although intake of a high-fat diet is associated with reduced sensitivity to the anorectic effects of leptin, insulin, and melanocortin agonists (5, 20, 23, 30, 31, 32), it is unknown whether this might be mediated by altered function of the respective receptors that initiate signaling of these hormones and peptides. Given these prior findings, we postulated that DIO rats would have reduced binding to hypothalamic insulin and leptin receptors compared with DR rats and that intake of a diet relatively high in fat would reduce binding to leptin, insulin, and melanocortin receptors. We found that DIO rats did indeed have reduced 125I-leptin and 125I-insulin but not melanocortin binding in the hypothalamus, whereas 7-wk intake of HE diet primarily led to reduced MC3 and/or MC4R binding in the hypothalamus and 125I-insulin binding in the hippocampus that was largely independent of genotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and diets
Animal usage was in compliance with Animal Care Committee of the East Orange Veterans Affairs Medical Center. All rats were housed at 23–24 C on a 12-h light, 12-h dark cycle (lights off at 1800 h). Male selectively bred DIO and DR rats raised in our own vivarium were used (7), and all litters were culled to 10 pups per dam at birth. DIO and DR rats (n = 12 per genotype) were weaned at 21 d of age and fed Purina rat chow ad libitum, which contains 3.30 kcal/g with 23.4% as protein, 4.5% as fat, and 72.1% as carbohydrate, which is primarily in the form of complex polysaccharide (33).

DIO
At 5 wk of age, half of each genotype was kept on chow, and the other half of each genotype was switched to HE diet (no. C11024F, Research Diets, New Brunswick, NJ), which contains 4.47 kcal/g with 21% of the metabolizable energy content as protein, 31% as fat, and 48% as carbohydrate, 50% of which is sucrose (33). After 7 wk on their respective diets, rats were decapitated in the nonfasting state during the 2 h after lights on. Brains were quickly removed, frozen on dry ice, and stored at –70 C until processed for quantitative receptor autoradiography. Trunk blood was collected, and the bilateral retroperitoneal, perirenal, epididymal, mesenteric, and inguinal adipose depots were removed and weighed.

Plasma glucose, leptin, and insulin levels
Baseline blood samples (0.5 ml) at the start of the study were obtained by tail nip into heparinized tubes for glucose, leptin, and insulin assays between 1000 and 1100 h, making sure all rats were provided only two food pellets at dark onset on the previous day. From both tail nip and trunk blood, glucose was assayed by automated glucose oxidase method (Beckman Coulter, Fullerton, CA), and insulin and leptin levels were analyzed by RIAs (Linco, St. Charles, MO) using antibodies to authentic rat insulin and leptin, respectively.

Tissue sections
Rat brains were cut in a cryostat in seven sets of 14-µm coronal sections through the hypothalamic paraventricular (PVN), arcuate (ARC), ventromedial (VMN), and dorsomedial (DMN) nuclei and lateral hypothalamic area (LHA) and the midbrain region containing the ventral tegmental area (VTA) and substantia nigra, pars compacta (SNc). The sections were freeze-thawed onto gel-coated slides and then dried.

Receptor binding autoradiography
The procedure for 125I-leptin binding was adapted from the original method of Baskin et al. (34, 35). The slides were brought to room temperature, dipped in cold 0.5% paraformaldehyde, and then rinsed several times in cold Tris buffer. Binding was carried out by incubating the slides for 1 h at room temperature in a solution containing 0.25 nM murine 125I-leptin (PerkinElmer Life Sciences, Boston, MA), Tris HCl (pH 7.2), 1% BSA (Sigma, St. Louis, MO), 0.05% leupeptin (Sigma), and 0.001% pepstatin (Sigma). Nonspecific binding was defined as that seen in the presence of a 1000-fold excess of unlabeled murine leptin (National Hormone and Peptide Program, Torrance, CA), and this accounted for less than 30% of total binding. After 1-h incubation in the respective radioactive solutions, total and nonspecific labeled slides were rinsed in cold Tris HCl and dipped in cold 4% paraformaldehyde, followed by a dip in Tris HCl. The slides were dipped in cold distilled water, dried overnight, and exposed to BioMax MR film (Kodak, Rochester, NY) for 3–5 d.

Binding of 125I-insulin was carried out by modifications of the methods of Hill et al. (36). Slides were incubated overnight at 4 C in a solution containing 0.05 nM murine 125I-insulin (PerkinElmer Life Sciences), HEPES buffer (Sigma), and 100 KIU/ml aprotinin (Sigma). Nonspecific binding was defined as that seen in presence of 1000-fold excess regular insulin (Lilly, Indianapolis, IN), and this accounted for less than 5% of total binding. The next day, slides were rinsed several times in cold HEPES buffer, followed by a dip in cold 4% paraformaldehyde. The slides were dipped in cold distilled water, dried under a stream of cold air and exposed to BioMax MR film (Kodak) for 2–3 d.

Binding to MC3 receptors (MC3Rs) and MC4Rs was carried out by modifications of the methods of Lindblom et al. (37) by incubating the sections in buffer (50 mM Tris HCl, 2 mM MgCl2, 1 mM CaCl2, 5 mM KCl, and 0.5% BSA) containing 20 pmol 125I-[Nle4, D-Phe7]-MSH (PerkinElmer Life Sciences) in the presence of 10 nM HS014 (Phoenix Pharmaceuticals, Belmont, CA), an MC4R-selective peptide antagonist, or with 1 µM {gamma} 1-MSH (Phoenix Pharmaceuticals), an MC3R-selective peptide, respectively. Nonspecific binding was defined as that seen in the presence of 1 µM MTII (Phoenix Pharmaceuticals), a nonselective MC3/4R agonist, and this accounted for less than 10% of total binding. After 2 h incubation at room temperature, sections were then washed three times in cold buffer, dipped briefly in cold distilled water, dried under a stream of cold air, and exposed to BioMax MR film (Kodak) for 7–10 d.

After the films were developed, an observer blinded to the experimental groups read the developed films. The anatomical areas of interest first were defined on the same cresyl violet-stained section that had been used to generate that specific autoradiogram. This image was superimposed digitally on the autoradiographic image to assess binding density in the selected brain regions using computer-assisted densitometry (Drexel University, Philadelphia, PA). Film density was equated to the density of known 125I standards placed on each film, and binding was calculated directly from density and the specific activities of 125I-insulin, leptin, and [Nle4, D-Phe7]-MSH, respectively.

Statistical analysis
Comparisons of body weight, fat pad, plasma hormones, glucose, and receptor binding were carried out by two-way ANOVA (genotype, diet). When significant differences were found by two-way ANOVA, further comparisons were made by one-way ANOVA with post hoc Bonferroni corrections (P ≤ 0.05). All data are expressed as means ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight gain, adiposity, plasma hormone, and glucose levels (Table 1Go)
Initially, there was a significant genotype effect; DIO rats were heavier compared with DR rats [F(1,17) = 38.4; P = 0.001] but had comparable plasma leptin and insulin levels in keeping with our past data showing that DIO rats are heavier but not fatter when fed chow from weaning (38). After 7 wk on HE diet, body weight gain in DR rats was comparable with chow-fed DR rats. However, they did have 48% heavier fat pads and 70% higher leptin levels, suggesting that they had become obese on HE diet compared with chow-fed DR rats. This suggests that they might have actually lost some lean body mass on the HE diet. In contrast, DIO rats fed HE diet gained 15% more body weight and had 121% heavier fat pads and 70% higher leptin levels than chow-fed DIO rats. In addition, intake of HE diet had no effect on plasma glucose or insulin levels overall, although there was a genotype-specific effect whereby DIO rats had higher insulin levels compared with DR rats regardless of diet [F(1,18) = 8.98; P = 0.008]. Thus, HE diet intake produced greater obesity in DIO than DR rats over a 7-wk period.


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  		TABLE 1. Body weight gain, adipose depot weights, glucose, plasma leptin, and insulin levels in DIO and DR rats fed either chow or HE diet for 7 wk

 
Leptin, insulin, and melanocortin receptor binding
Figures 1AGo and 2AGo demonstrate representative 125I-leptin binding autoradiograms for the hypothalamus and midbrain, respectively. Overall, there was a genotype-specific effect whereby DIO rats had reduced 125I-leptin binding compared with DR rats in the ARC, dorsomedial VMN (dVMN), and DMN [F(1,85) = 62.4; P = 0.001] independent of dietary exposure (Fig. 3AGo). When individual nuclei were assessed, 125I-leptin binding was 36% lower in the ARC [F(1,16) = 56.4; P = 0.001], 40% lower in the dVMN [F(1,16) = 53.3; P = 0.001], and 41% lower in the DMN [F(1,15) = 115; P = 0.001] than DR rats regardless of diet. A comparison of leptin binding in the subnuclei of the VMN revealed significantly higher leptin binding in the dVMN compared with ventral VMN [F(1,34) = 78.4; P = 0.001]. Neither genotype nor diet altered leptin binding in the thalamus, VTA, or SNc (Fig. 3BGo).


Figure 1
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  		FIG. 1. 125I-leptin (A) and 125I-insulin binding (B) in the hypothalamus of chow-fed DR rats. Dotted areas, ARC, VMN, and DMN identified on the cresyl violet-stained slides used to generate the autoradiogram. Leptin binding was assessed in both the vVMN and dVMN, whereas insulin binding was measured in the entire VMN.

 

Figure 2
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  		FIG. 2. 125I-leptin (A) and 125I-insulin binding (B) in the midbrain region of chow-fed DR rats. Dotted areas, Midbrain nuclei including VTA, SNc, and substantia nigra, pars reticulata (SNr) identified on the cresyl violet-stained slides used to generate the autoradiogram.

 

Figure 3
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  		FIG. 3. 125I-leptin binding in the hypothalamus, midbrain region, and thalamus. A, Leptin binding in the ARC, dVMN, vVMN, and DMN. B, Leptin binding in the midbrain VTA and SNc and the thalamus; n = 6 per group. Bars for a given brain area with differing superscripts differ significantly from each other at P ≤ 0.05 by post hoc analysis after intergroup differences were found by ANOVA.

 
Figures 1BGo and 2BGo demonstrate representative 125I-insulin binding autoradiograms for the hypothalamus and midbrain, respectively. Among all the hypothalamic nuclei analyzed, 125I-insulin binding was significantly lower (31%) only in the ARC (Fig. 4AGo) of DIO rats compared with DR rats regardless of diet [F(1,16) = 12; P = 0.003]. On the other hand, there was a diet-specific effect on 125I-insulin binding in the CA1 region of the hippocampus (Fig. 4BGo), whereby binding was reduced in both DIO and DR rats fed HE vs. chow diet [F(1,17) = 5.47; P = 0.032]. There was no effect of diet or genotype on 125I-insulin binding in the VTA or SNc (Fig. 4BGo).


Figure 4
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  		FIG. 4. 125I-insulin binding in the hypothalamus, midbrain region, and hippocampus. A, Insulin binding in ARC, VMN, and DMN. B, Insulin binding in the midbrain VTA and SNc and the hippocampus; n = 6 per group. Bars for a given brain area with differing superscripts differ significantly from each other at P ≤ 0.05 by post hoc analysis after intergroup differences were found by ANOVA.

 
Intake of HE diet was associated with approximately 39% lower MC4R binding in the PVN of only DR rats [F(1,19) = 7.27; P = 0.01] and of both DIO (27%) and DR (26%) rats in the LHA [F(1,20) = 5.58; P = 0.02] (Table 2Go). In addition, intake of HE diet was associated with lower MC3R binding in the LHA for both DIO (25%) and DR (39%) compared with chow-fed rats [F(1,14) = 8.38; P = 0.01] (Table 3Go). Also, two-way ANOVA indicated that MC3 and MC4R binding was lower in the medial habenular nucleus (MHb) of DIO rats compared with DR rats regardless of diet [F(1,14) = 7.31; P = 0.01] (Tables 2Go and 3Go). There was no significant difference in MC3R vs. MC4R binding either in the ventrolateral VMN (vVMN) or dVMN. However, both MC3R [F(1,28) = 10.0; P = 0.004] and MC4R [F(1,28) = 9.48; P = 0.005] binding was higher in the dVMN compared with the vVMN across all four groups.


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  		TABLE 2. Hypothalamic and medial habenular nucleus MC4R binding (femtomoles per milligram) of selectively bred DR and DIO rats on chow and HE diet

 

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  		TABLE 3. Hypothalamic and medial habenular nucleus MC3R binding (femtomoles per milligram) of selectively bred DR and DIO rats on chow and HE diet

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously reported that DIO rats have attenuated central leptin and insulin signaling before the development of obesity on HE diet. Compared with DR rats, preobese DIO rats have reduced expression of Lepr-b mRNA in the ARC, VMN, and DMN (5, 23), reduced leptin-induced anorexia and phosphorylation of STAT3 (21, 23), and a reduced anorectic effect of centrally administered insulin (20). Here, we provide important confirmatory data for the previously demonstrated reduction in Lepr-b mRNA by showing that 125I-leptin binding was similarly reduced in the ARC, dVMN, and DMN. In addition, we show that 125I-insulin binding was reduced selectively in the ARC of DIO compared with DR rats regardless of diet or the development of obesity. We also demonstrate the presence of leptin and insulin binding in the SNc and VTA in keeping with the presence of mRNA for both receptors in those areas (39). However, binding to either receptor was not affected by genotype or dietary intake. Finally, unlike leptin and insulin binding, there were no differences between DIO and DR rats in hypothalamic MC3 or MC4R binding, although DIO rats did have lower binding of both in the MHb, a nucleus without a known function in the regulation of energy homeostasis. These results suggest that reduced medial hypothalamic binding of leptin and insulin is an important trait of DIO rats that is present before they become obese and may be a major reason for their decreased responding to the anorectic effects of central leptin and insulin. This reduced binding is unlikely to be due to desensitization because of raised plasma levels because neither plasma leptin nor insulin levels were higher in DIO than that in DR rats under baseline conditions.

The preexisting reduction of hypothalamic leptin and insulin binding and downstream signaling in DIO rats is likely to be an important factor that predisposes them to become obese when the caloric density and fat content of their diet is increased. Such reduced sensitivity would make them less responsive to the inhibitory feedback of rising leptin and insulin levels associated with excess intake of such diets (5). In addition to impaired leptin and insulin signaling, DIO rats also have multiple defects in their ability to sense and respond to glucose (40). Importantly, glucose (41), leptin (40, 42), and insulin (41, 43) converge on select groups of VMN hypothalamic metabolic-sensing neurons to regulate their activity (40, 41, 44, 45, 46). The effect of leptin and insulin on such neurons is highly dependent upon their transmitter/peptide phenotype (47, 48), anatomical location (41, 44, 45, 46), and ambient glucose levels (41). DIO rats have fewer hypothalamic neurons that specifically sense glucose (46), and many of these same neurons show reduced leptin-induced STAT3 phosphorylation than those in DR rats (23). Furthermore, DIO rats have reduced binding to the low-affinity sulfonylurea receptor subunits of the ATP-sensitive potassium channel (22). This channel mediates glucosensing and is the final common pathway upon which leptin and insulin signaling converge to alter hypothalamic neuronal activity at high glucose levels (42, 43). Thus, reduced responsiveness of metabolic-sensing neurons to a variety of hormonal and metabolic signals that inform the brain of the metabolic and energy storage status of the body may allow DIO rats to eat beyond their metabolic needs when food is abundant.

We also assessed the effects of chronic HE diet intake on central leptin, insulin, and melanocortin binding with the expectation that all would be reduced. In fact, only PVN DR MC4R, hippocampal CA1 insulin, and LHA MC3R and MC4R DIO and DR binding were reduced by intake of HE diet. Intake of a high-fat diet reduces the anorectic, signaling effects and/or blood-brain barrier transport of leptin (5, 23, 31, 32, 49), insulin (20), and melanocortin agonists (30). High fat intake also reduces reproductive function (50) and insulin-induced facilitation of memory (51). Such attenuated responses might be a direct effect of either the high fat content of the diet or the metabolic consequences of obesity. Transport of both leptin and insulin is reduced as their plasma levels rise during development of obesity. This probably occurs because transport is a saturable process (52, 53) and because the elevated levels of plasma triglycerides that occur with both intake of high-fat diet and the development of obesity can also reduce transport (54). Chronic intake of HE diet can, in addition to altering hippocampal insulin and hypothalamic melanocortin binding, also reduce binding to the hypothalamic {alpha}2-adrenoceptor (24) and sulfonylurea receptors (22) in DIO and DR rats. Such altered binding may occur as a result of changes in the fatty acid composition of neuronal plasma membranes (24), which could affect membrane fluidity or the formation of lipid rafts (55). However, it should be pointed out that the chow and HE diets also differ in protein and carbohydrate composition, and these differences might also contribute to altered binding. The site- and genotype-specific alterations in leptin, insulin, and melanocortin receptor binding are also likely to reflect the diversity of cell types that populate different brain regions of DIO vs. DR rats.

Although our results generally support a role for reduced receptor binding as a critical cause of reduced leptin and insulin signaling in DIO rats, there are some caveats to the interpretation of the data presented here. First, 125I-leptin binds to all splice variants of the leptin receptor and is not selective for the long (signaling) form (56, 57). Similarly, 125I-insulin can potentially bind to both insulin and insulin-like growth factor receptors (58). In addition, there are no currently available ligands that bind to the MC3R and MC4R with sufficient selectivity to ensure that our data fully separate binding to these two related receptors (37). For this reason, we used two commercially available, relatively selective compounds (59). Finally, because we used only a single concentration of ligand, genotype- and diet-induced differences in binding might represent differences in affinity and/or number of receptors.

In conclusion, although there are some caveats, the current results strongly support our previous data showing that DIO rats have a preexisting reduction in both central leptin and insulin signaling that is associated with reduced binding to both receptors before the development of obesity on HE diet. The reduced hypothalamic leptin and insulin binding demonstrated here reinforces our previous demonstration of reduced Lepr-b mRNA (21, 23) and leptin-induced anorexia (5, 21, 23) and STAT3 phosphorylation (23) and insulin-induced anorexia (20) in DIO rats. On the other hand, intake of HE diet was associated with reduced MC3R and MC4R binding in the LHA and insulin hippocampal binding of both DIO and DR rats and PVN MC4R binding in DR rats. Such changes were highly selective because binding in areas such as the SNc and VTA differed as a function of neither genotype nor diet. Although there may be many other differences in downstream signaling pathways of these receptors, altered binding suggests that there is a primary effect of genotype and/or dietary content on the first step of signaling for these hormones and peptides. Such data suggest that drugs that target binding to these receptors might be an effective way to prevent and treat the development of obesity.


   Acknowledgments
 
We thank Odeal Gordon, Laura Petrie, Antoinette Moralishvilli, and Charlie Salter for technical assistance and Dr. Xiaoming Guan (Merck Research Laboratories) for expert advice on the melanocortin binding experiment.


   Footnotes
 
This work was supported by the Research Service of the Department of Veterans Affairs (to B.E.L., A.A.D.-M.), by the National Institute of Diabetes, Digestive, and Kidney Disorders (Grant DK-30066 to B.E.L.), and by an award from the American Heart Association (to B.G.I.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 5, 2006

Abbreviations: ARC, Hypothalamic arcuate nucleus; DIO, diet-induced obesity; DMN, hypothalamic dorsomedial nucleus; DR, diet resistant; dVMN, dorsomedial VMN; HE, high energy; LHA, lateral hypothalamic area; MC3, melanocortin-3; MC3R, MC3 receptor; MC4R, melanocortin-4 receptor; MHb, medial habenular nucleus; PVN, hypothalamic paraventricular nucleus; SNc, substantia nigra, pars compacta; VMN, hypothalamic ventromedial nucleus; VTA, ventral tegmental area; vVMN, ventrolateral VMN.

Received August 16, 2006.

Accepted for publication September 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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