Introduction

Leptin, a cytokine-like hormone produced primarily in adipose tissue, plays a very important role in the regulation of feeding and energy homeostasis (1, 2). Leptin modulates feeding primarily by down-regulating the orexigenic neuropeptides, neuropeptide Y (NPY)¹/agouti-related protein (AGRP), and by up-regulating the anorexigenic neuropeptides, proopiomelanocortin (POMC)/cocaine- and amphetamine-regulated transcript (CART), in the arcuate nucleus (ARC) of the hypothalamus and hindbrain regions (3, 4, 5). Leptin initiates a series of complicated signaling pathways, primarily the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway (6). The suppressor of cytokine signaling 3 (SOCS3) is likely to play a critical role in the negative feedback regulation of the hypothalamic leptin signaling system (7). Leptin is also involved in mobilization of stored lipid and mediation of adipocyte apoptosis, and all of these functions are directly or indirectly mediated by the hypothalamus (8, 9).

The ob/ob mice, with a point mutation of the leptin gene, are genetically leptin deficient. It is hypothesized that the lack of functional circulating leptin in ob/ob mice might up-regulate hypothalamic leptin receptors (10). Moreover, ob/ob mice are more responsive to leptin than lean mice in reducing feeding, body weight, serum insulin, and glucose levels (11). Furthermore, prolonged leptin treatment reduces leptin uptake in the central nervous system and induces the development of leptin resistance, in which SOCS3 might be involved (7, 11). Thus, we hypothesized that, given widespread distribution of leptin receptors in the hypothalamus, lack of leptin signaling in ob/ob mice might be associated with an alteration of hypothalamic gene profile pattern, and ob/ob mice might respond differently to leptin than lean mice. In this study, we treated both lean and ob/ob mice with 14 days of subcutaneous (sc) leptin injection. We dissected the mouse hypothalami and extracted the total RNA. Then we used real-time reverse transcription polymerase chain reaction (RT-PCR) with microfluidic cards to investigate the effects of sc administration of leptin in lean or ob/ob mice on a series of potential biomarkers, whose mRNA expressions in the hypothalamus are critical in regulation of feeding, cellular signaling, inflammation, and several other biological functions.

Research Methods and Procedures

Animals and Leptin Administration

Fifteen-week old female obese ob/ob (strain B6.V-Lep^ob, n = 16) and wild-type lean mice (n = 16) were purchased from Jackson Laboratories, Inc. Mice were initially housed individually in suspended cages in a room with a 12-hour/12-hour light/dark cycle, 22 1 °C ambient temperature, and 50% humidity. They were provided with food and water ad libitum throughout the study. All mice were surgically implanted with osmotic mini-pumps (Model 1002, 0.25 L/h; Alzet Corp., Cupertino, CA) for subcutaneous (sc) infusion of treatment solutions. Mice were anesthetized briefly with 0.5% oxygen/isoflurane and the prefilled and primed pumps were inserted into a subcutaneous pocket as described previously (12). Then the 16 mice within each genotype were randomly divided into two groups treated with either phosphate-buffered saline solution or recombinant mouse leptin (R&D Systems, Minneapolis, MN) at a dose of 10 g/d by sc infusion for 14 continuous days. Thus, there were 8 mice in each group (control/lean: lean mice sc treated with phosphate-buffered saline; control/obob: ob/ob mice sc treated with phosphate-buffered saline; leptin/lean: lean mice sc treated with leptin; leptin/obob: ob/ob mice sc treated with leptin). Mice were weighed at the beginning and end of the study. Mice were killed by decapitation after sedation in a CO₂ chamber at the end of the 14th day of injection (12). All of the animals and surgical procedures in this study were approved by the Animal Care and Use Committee of the University of Georgia.

RNA Extraction

The brains were removed rapidly after decapitation and immediately frozen by placing them on plastic cassettes on top of powdered dry ice. Once completely frozen, they were stored at - 80 °C. The brains were thawed to - 5 °C before the hypothalamic dissection. Tissue homogenization and total RNA extraction were performed according to the protocol from Invitrogen (Carlsbad, CA). Briefly, each dissected hypothalamic block was homogenized in 1 mL of Trizol reagent using the power homogenizer. Then the homogenized samples were incubated for 5 minutes at room temperature and 0.2 mL of chloroform was added. The samples were centrifuged at 12,000g for 15 minutes at 4 °C and the aqueous phase was removed and mixed with 0.5 mL of isopropyl alcohol. After centrifugation at 12,000g for 10 minutes at 4 °C, the pellet was washed with 1 mL of 75% ethanol and then dissolved in RNase-free water. The integrity of the RNA produced from all samples was verified and quantified using a RNA 6000 Nano Assay and the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA).

Reverse Transcription and Real-time PCR

A total of 100 ng of total RNA in a 20 L reaction was reverse transcribed using the cDNA Archive Kit (part #4322171; Applied Biosystems, Inc., Foster City, CA) according to the manufacturer's protocols. Reactions were incubated initially at 25 °C for 10 minutes and subsequently at 37 °C for 120 minutes. Quantitative PCR (Taqman) assays were performed using 384-well MicroFluidic cards on the ABI PRISM 7900 Sequence Detection System. All of the oligonucleotide primer and fluorogenic probe sets for Taqman real-time PCR were made by ABI (Table 1). The cycle conditions were: 94.5 °C for 15 minutes, followed by 40 cycles at 97 °C for 30 seconds, 59.7 °C for 1 minute. mRNA expressions were normalized by using -actin (-ACT) as an endogenous control to correct the differences in the amount of total RNA added to each reaction. The relative quantification values from each gene were used to compare the hypothalamic gene expression of all of the groups.

Table 1 - Probes used in real-time PCR.

Full table

Statistical Analysis

Data were expressed as mean standard error (SE) of relative quantification values from each scientific dataset file for all of the genes. Statistical significance was assessed by a general linear model two-way ANOVA to determine the possible effects of genotype and leptin treatment. The one-way ANOVA with Duncan's multiple range tests also was performed even when the two-way ANOVA did not show an interaction between genotype and leptin treatment, as the large biological variation observed in some genes distorted significant treatment effects. Multivariate analysis between genes was also performed with Pearson correlation.

Results

Our results showed that ob/ob mice were more sensitive than lean mice to leptin-induced body weight loss (Table 2). The genes we detected fell into different categories according to their reported physiological and cellular functions in the hypothalamus (Table 3). These candidate genes have been reported as being involved in feeding and body weight regulation, cellular signal transduction, and various other activities, such as temperature and fluid balance. Overall, our results indicated that the major hypothalamic mRNA expression difference between ob/ob and lean control mice was the divergent gene profile for those biomarkers involved in feeding regulation. While leptin dramatically modified hypothalamic mRNAs of these feeding-related genes in both genotypes, leptin altered hypothalamic expression of only some of the other genes assayed (Table 3).

Table 2 - Body weight change (g; mean SE) in ob/ob and lean mice after 14 days of sc leptin treatment (10 g/d).

Full table

Table 3 - Hypothalamic mRNA expressions of lean and ob/ob mice treated with sc leptin.

Full table

mRNA Expression of Hypothalamic Signaling Molecules Involved in Leptin Pathways in Lean and ob/ob Mice

The results showed similar gene expression patterns of the selected signaling molecules between the ob/ob and lean control mice except for cyclic adenosine monophosphate response element binding protein (CREB)-1 (Figure 1). Leptin injections increased only the hypothalamic mRNA level of FBJ murine osteosarcoma oncogene homolog (FOS) by 26.8% in lean mice (p < 0.01) and mRNA level of SOCS3 by 32.1% in ob/ob mice (p < 0.05). In ob/ob control mice, the hypothalamic mRNA level of CREB1 was lower by 23.7% than that of lean control mice (p < 0.01). With leptin treatment, CREB1 mRNA levels were higher in ob/ob mice, resulting in no significant difference between ob/ob leptin-treated mice and lean leptin-treated mice. There was no effect of leptin treatment on mRNA levels of JAK2, tyrosine hydroxylase, STAT3, and mitogen-activated protein kinase-1.

Figure 1.

Hypothalamic mRNA expression of ob/ob and lean mice after 14 days of sc leptin treatment (10 g/d) as determined by real-time RT-PCR. Data are mean SE for each group normalized to the -ACT values and then expressed as relative quantification. Within each gene, bars denoted with a common letter are not different. a, b: p < 0.05; w, x: p < 0.01.

Full figure and legend (177K)

Strong positive correlations were observed between CREB1, JAK2, and STAT3 in all of the treatment combinations. The correlation coefficient was 0.92 between JAK2 and CREB1 mRNA levels, 0.75 between STAT3 and CREB1 mRNA levels, and 0.84 between JAK2 and STAT3 mRNA levels.

mRNA Expression of Hypothalamic Molecules Regulating Food Intake and Body Weight in Lean and ob/ob Mice

The data indicated that, for ob/ob control mice, the hypothalamic mRNA levels of anorectic neuropeptides were lower and mRNA levels of orexigenic neuropeptides were higher when compared with those of lean controls (Figure 2). Leptin treatment increased the hypothalamic mRNA levels of anorectic neuropeptides and reduced mRNA levels of orexigenic neuropeptides in ob/ob mice, but it modified only some of these markers in lean mice. For hypothalamic POMC1, there were effects of treatment (p < 0.01) and genotype (p < 0.05), and an interaction between these two factors (p < 0.05). The hypothalamic POMC1 mRNA level was lower in ob/ob control mice by 53.7% compared with lean control mice (p < 0.01). It was dramatically up-regulated by leptin treatment in both ob/ob and lean mice (8- and 135-fold increase in lean and ob/ob mice, respectively, both p < 0.01). The hypothalamic CART mRNA level in ob/ob control mice was lower by 15.6% than in lean control mice (p < 0.05); sc leptin treatment increased hypothalamic CART mRNA in ob/ob (p < 0.05) but not in lean mice. For hypothalamic NPY, there were effects of treatment (p < 0.01) and genotype (p < 0.01), as well as an interaction between the two factors (p < 0.01). The hypothalamic NPY mRNA level in ob/ob control mice was higher by 64.6% than in lean control mice. While NPY was not changed in lean mice with sc leptin administration, its mRNA level was down-regulated by 25.8% in leptin-treated ob/ob mice (p < 0.01). For hypothalamic AGRP, there were effects of treatment (p < 0.01) and genotype (p < 0.01) as well as an interaction (p < 0.01). The hypothalamic AGRP mRNA level in ob/ob control mice was higher (by 386.9% ) than in lean control mice. The hypothalamic AGRP mRNA level was down-regulated by 45.0% in leptin-treated ob/ob mice (p < 0.01) but was up-regulated by 28.3% in leptin-treated lean mice (p < 0.05). No effects of treatment and genotype were observed for hypothalamic mRNA level of gamma-aminobutyric acid A receptor delta.

Figure 2.

Hypothalamic mRNA expression of ob/ob and lean mice after 14 days of sc leptin treatment (10 g/d) as determined by real-time RT-PCR. Data are mean SE for each group normalized to the -ACT values and then expressed as relative quantification. Within each gene, bars denoted with a common letter are not different. a, b: p < 0.05; w, x, y, z: p < 0.01.

Full figure and legend (121K)

mRNA Expression of Hypothalamic Molecules Regulating Inflammation and Cachexia in Lean and ob/ob Mice

Leptin had no effect on the hypothalamic mRNA levels of two cachectic factors, tumor necrosis factor and prostaglandin E synthase (Figure 3). For both tumor necrosis factor and prostaglandin E synthase, there were no differences in the hypothalamic mRNA levels between lean and ob/ob control mice, and leptin did not alter the hypothalamic mRNA levels of these two cytokines in either mouse genotype.

Figure 3.

Hypothalamic mRNA expression of ob/ob and lean mice after 14 days of sc leptin treatment (10 g/d) as determined by real-time RT-PCR. Data are mean SE for each group normalized to the -ACT values and then expressed as relative quantification.

Full figure and legend (123K)

mRNA Expression of Hypothalamic Molecules Regulating Other Biological Functions in Lean and ob/ob Mice

The results showed that ob/ob control mice have a higher hypothalamic mRNA level of arginine vasopressin (AVP) and lower level of urocortin (UCN)-3 when compared with their lean counterparts (Figure 4). Of the five genes in this category, leptin-induced hypothalamic mRNA change was only observed with AVP in ob/ob mice. The data showed that there were genotype effects on the hypothalamic mRNA level of AVP (p < 0.01) and a trend for a treatment effect (p = 0.07), as well as a genotype treatment interaction (p < 0.01). While the hypothalamic AVP mRNA level was higher in ob/ob control mice than in lean control mice by 95.8% (p < 0.01), sc leptin treatment dramatically reduced its mRNA level by 32% in ob/ob mice (p < 0.01) but not in lean mice. Strong positive correlation (r = 0.83) was observed between NPY and AVP mRNA levels (Figure 5d). Although the hypothalamic mRNA level of UCN3 was lower in ob/ob control mice than in lean control mice by 22.9% (p < 0.01), no leptin treatment effect was observed. There were no changes in expression of oxytocin, stem cell transplant, and vasoactive intestinal peptide between the two genotypes, and sc leptin treatment did not change the mRNA expression of these genes.

Figure 4.

Hypothalamic mRNA expression of ob/ob and lean mice after 14 days of sc leptin treatment (10 g/d) as determined by real-time RT-PCR. Data are mean SE for each group normalized to the -ACT values and then expressed as relative quantification. Within each gene, bars denoted with a common letter are not different. w, x: p < 0.01.

Full figure and legend (155K)

Figure 5.

Relationship between hypothalamic mRNA levels of (A) CREB1 and STAT3, (B) CREB1 and JAK2, (C) STAT3 and JAK2, and (D) NPY and AVP in lean and ob/ob mice with 14 days of sc leptin treatment.

Full figure and legend (81K)

In addition, we also investigated the relationship between these mRNA changes and the phenotypic changes (such as body weight), and we observed a strong positive correlation (r = 0.68, p < 0.01) between body weight loss and mRNA level of POMC.

Discussion

The widespread presence of leptin receptor in the hypothalamus, a center for the regulation of energy metabolism and various other biological activities, suggests the hypothalamic involvement in the leptin-induced physiological functions (13). Therefore, ob/ob mice, with a mutated ob gene and the resulting lack of leptin signaling, might have an altered hypothalamic gene profile, and respond differently than lean mice when subjected to exogenous leptin stimulation. Our previous studies have shown that ob/ob mice had higher overall sensitivity to leptin than lean mice on feeding and leptin-induced adipose tissue apoptosis (12). In this study, we speculate that similar phenomena might also occur in the hypothalamic gene expression. Our data not only confirm the findings of previous reports showing a clear difference between ob/ob and lean mice in mRNA levels of hypothalamic neuropeptides, such as NPY and CART, but show some novel findings as well. We found that there were differences between two genotypes in mRNA levels of hypothalamic AVP, CREB1, and UCN3. We found that not only do ob/ob mice have hypothalamic gene patterns different from lean mice, but also that specific genes (e.g., CART, POMC1, NPY, and AVP) were more sensitive to the effects of leptin stimulation in ob/ob mice (Table 3). Also, with leptin treatment, the up-regulated mRNA level of AGRP in lean mice and the extraordinary increase of mRNA level of POMC1 in ob/ob mice may demonstrate the importance of these peptides in energy balance regulation.

Leptin-induced hypothalamic JAK-STAT signaling has been intensively studied. STAT3 and leptin receptor long form, Ob-Rb, have been shown to be co-localized in many hypothalamic ARC neurons, and leptin increased hypothalamic STAT3 phosphorylation and up-regulated SOCS3 expression (6, 14). In our study, hypothalamic mRNA levels of SOCS3 were significantly up-regulated with sc leptin treatment in ob/ob mice, but not in their lean counterparts. STAT3 mRNA levels in the ARC in ob/ob mice have been shown to be lower than in lean mice (15). This difference, however, was not observed in our study, although our use of whole hypothalamus may have prevented us from detecting site-specific changes in STAT3 expression. The mRNAs of tyrosine hydroxylase and mitogen-activated protein kinase-1 were unchanged with leptin treatment in both lean and ob/ob mice. This is not unexpected because these signaling molecules might be involved in leptin signaling by phosphorylation of the protein rather than up-regulation of the gene. We cannot eliminate the possibility, however, that their expression changes could be blunted by leptin-induced negative feedback signals such as SOCS3 during the prolonged sc leptin treatment.

CREB is a transcription factor and usually considered a marker for neuron activation (16). In our study, hypothalamic mRNA level of CREB1 in ob/ob control mice was lower than that in lean control mice, but its mRNA level was unaltered with sc leptin treatment in either genotype. The up-regulated hypothalamic mRNA level of another neuronal activation marker, c-FOS, in lean but not ob/ob mice indicated that neuronal activity was different between the two genotypes in response to leptin treatment. Regional differences in c-FOS expression in the hypothalamus have been demonstrated in response to leptin treatment (17). Thus, the c-FOS difference observed in the whole hypothalamus in our study reflects only an average value that prevents us from drawing specific conclusions about the role of c-FOS expression in the response of ob/ob mice to leptin. Even though our results showed that leptin failed to alter the hypothalamic mRNA levels of JAK2, STAT3, and CREB1 in either genotype, the strong positive correlation between these molecules in all of the groups suggests that their expression might be closely inter-related under our experimental conditions. It must be stated that a high correlation may not necessarily mean definite causation because the development of leptin resistance in ob/ob mice could distort any normal relationship between one biomarker and the other.

Leptin has been shown to induce febrile and inflammatory responses, and there are strong relationships between leptin and the immune system, although the mechanisms of leptin's involvement in inflammation and cachexia are still poorly understood (18). In our study, sc leptin treatment had no effect on hypothalamic mRNA levels of two inflammatory factors, tumor necrosis factor and prostaglandin E synthase. Considering the redundant inflammatory pathways, it is quite possible that tumor necrosis factor and prostaglandin E synthase are not the only cytokines induced by leptin, and the lack of leptin signaling could induce alterations in other inflammatory molecules that might, in turn, modulate the expression of both cachectic molecules.

Both AVP and oxytocin in the hypothalamic ARC have been shown to be involved in the communication between brain and white adipose tissue (19). In our study, the mRNA level of AVP in ob/ob control mice was significantly higher than that in lean control mice, and leptin dramatically decreased its mRNA level in ob/ob but not in lean mice. It has been shown that food restriction in rats alters the circadian rhythms and the hypothalamic-pituitary-adrenal axis, thus reducing AVP level in the blood or in the suprachiasmatic nucleus (20, 21). Although decreased plasma leptin level and increased NPY level in the hypothalamus have been reported in both food-restricted rats and ob/ob mice, there is an apparent disparity on AVP expression. We speculate that in ob/ob mice, the intrinsic mutation of functional leptin might cause early disruption of a series of metabolic and endocrine factors, e.g., the increased hypothalamic AVP and NPY. In contrast, during food restriction, the reduction in leptin level and the resulting changes in hypothalamic neuropeptides regulated by leptin might be temporary and dynamic. In addition, it has been found that the hypothalamic AVP is related to stress, and chronic stress results in weight loss through corticotropin releasing hormone-induced anorexia (22, 23, 24). This suggests the possibility that the higher mRNA levels of hypothalamic AVP in ob/ob mice may reflect increased stress in this genotype. Although we did not measure the mRNA levels of corticotropin releasing hormone in this study, previous reports have shown that leptin inhibits corticotropin releasing hormone synthesis and release in the hypothalamus (25, 26). Other studies have demonstrated the anatomical relationship between nerve terminals containing NPY and AVP in the hypothalamus (27). The strong correlation between hypothalamic NPY and AVP in this study supports these observations.

UCN has been found to regulate gastric emptying and reduce feeding as well as body weight in ob/ob mice (28). In this study, although the hypothalamic mRNA level of UCN3 was higher in ob/ob control mice than lean control mice, its mRNA level was unchanged with leptin treatment in either genotype. Thus, UCN3, a weak anorectic peptide, may not be as important as POMC in the compensatory mechanism after prolonged leptin stimulation. There were no significant treatment or genotype effects on expression of stem cell transplant and vasoactive intestinal peptide. Whether the use of whole hypothalamic tissue in our study resulted in dilution of site-specific differential expression changes of these molecules is unknown.

It is accepted that leptin regulates feeding by modulating the expression of orexigenic and anorectic peptides in the hypothalamus (2). POMC is the precursor molecule of -melanocyte stimulating hormone, and the latter is a primary hypothalamic anorectic neuropeptide (29). In our study, leptin treatment drastically increased its level in both genotypes, although the increase in ob/ob mice was more pronounced. In contrast, the mRNA level of another hypothalamic anorectic neuropeptide, CART, was increased only in ob/ob, but not in lean mice, with sc leptin treatment. Thus, although results from anatomical studies suggested that -melanocyte stimulating hormone and CART-producing neurons are morphologically co-localized (30), they may differ in sensitivity to leptin signaling. Previous studies have shown that CART is involved in leptin's regulation of bone resorption (31). We have shown that ob/ob mice have lower whole body mineral content and bone mineral density, and that leptin treatment increased body mineral content and bone mineral density in ob/ob mice but had no effect in lean mice (32). In this study, the decreased hypothalamic CART mRNA in ob/ob mice and the increased CART level with leptin treatment in ob/ob mice but not in lean mice supports this. It has been suggested that the reduction of gamma-aminobutyric acid might be involved indirectly in leptin's effect on POMC expression (33). In this study, we measured the mRNA level of hypothalamic gamma-aminobutyric acid receptor and found it was unchanged in either genotype with leptin treatment. Whether the dramatic up-regulation of POMC1 might, in turn, blunt the hypothalamic gamma-aminobutyric acid receptor mRNA expression is unknown.

NPY and AGRP are the primary orexigenic neuropeptides in the hypothalamic arcuate nucleus, and fasting has been shown to increase the mRNA levels of both peptides in the hypothalamus (34). In our study, the mRNA levels of hypothalamic NPY and AGRP were higher in ob/ob control mice than lean control mice. The mRNA level of NPY was significantly reduced in ob/ob mice with leptin treatment. This suggests that NPY neurons of ob/ob mice have increased sensitivity to leptin, and is in agreement with the previous literature, which suggested an enhanced interaction between leptin and NPY signaling in ob/ob mice (35).

It has been hypothesized that, in lean mice, prolonged leptin treatment might suppress endogenous leptin secretion and induce leptin resistance (36). Also in lean mice, reduction in feeding and body weight that occurs with continuous leptin injection might stimulate hypothalamic orexigenic mechanisms to compensate for the energy loss. Intraperitoneal injection of leptin in mice for 48 hours has been found to reduce body weight and hypothalamic mRNA levels of AGRP (37). Our finding of increased hypothalamic mRNA level of orexigenic AGRP induced by 12 days of leptin injections suggests that the changes of gene expression are very dynamic processes and the relatively long-term leptin treatment induces compensatory mechanism. It also provides an explanation for the lack of change in body weight of lean mice with leptin treatment (Table 2). In ob/ob mice, the lack of leptin signaling results in increased expression of orexigenic and decreased expression of anorectic neuropeptides, and leptin treatment appears to correct this abnormality, resulting in reversal of obesity (38). Because POMC is the most powerful catabolic factor, it would be the first candidate neuropeptide to be used in the hypothalamus that helps normalize the food intake and body weight of ob/ob mice undergoing leptin treatment. In this study, the strong positive correlation between body weight loss and mRNA level of POMC1 but not other feeding-related neuropeptides supports this.

Previous studies have demonstrated that, in ob/ob mice, the lack of leptin up-regulates expression of the leptin receptor and increases its binding affinity for leptin (10, 39). It is possible that in lean mice, leptin receptors in the hypothalamus are down-regulated in response to continuous leptin treatment, while in ob/ob mice the inherent absence of leptin and the corresponding up-regulation of leptin receptors may cause them to be very sensitive to exogenous leptin stimulation (10, 39). Up-regulation of the hypothalamic leptin receptor has also been found during fasting (40). Considering the widespread localization of leptin receptors in the hypothalamus and various extra-hypothalamic regions, as well as the possible diversified regulation of gene expression in these different areas, further investigation, such as injections of leptin receptor inhibitors in specific areas such as hypothalamic ARC, may help decipher the role of leptin receptors in depth.

In summary, our data demonstrate that ob/ob and lean mice have different hypothalamic mRNA expression patterns (particularly for those feeding-related genes), and some of the selected genes (e.g., CART, POMC1, NPY, and AVP) in ob/ob mice are more sensitive to exogenous leptin stimulation compared with lean mice. Our results both confirm previous reports and establish a number of candidate hypothalamic biomarkers (e.g., AVP) whose changes in expression may warrant further study. The hypothalamic gene expression profiles we obtained may be useful in deciphering the pathogenesis of obesity and thus be of potential clinical importance for prevention or treatment of obesity.

Notes

¹ Nonstandard abbreviations: NPY, neuropeptide Y; AGRP, agouti-related protein; POMC, proopiomelanocortin; CART, cocaine- and amphetamine-regulated transcript; ARC, arcuate nucleus; JAK, Janus kinase; STAT, signal transducers and activators of transcription; SOCS3, suppressor of cytokine signaling 3; RT-PCR, reverse transcription polymerase chain reaction; sc, subcutaneous; ACT, actin; SE, standard error; CREB, cyclic adenosine monophosphate response element binding protein; FOS, FBJ murine osteosarcoma oncogene homolog; AVP, arginine vasopressin; UCN, urocortin.

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Acknowledgments

This study was supported in part by Georgia Research Alliance Eminent Scholar endowment (held by C.A.B.).