Agouti-related Protein Gene Expression Research Articles

Abstract P249: ICV Infusion Of AgRP Modulates Feeding Behavior And Energy Expenditure In Mice

A subset of Agouti-related protein (AgRP) neurons within the arcuate nucleus express angiotensin type 1a receptors (AT1A), and genetic ablation of AT1A in these cells disinhibits AgRP gene expression and attenuates energy expenditure (EE) in mice. To further understand the role of AgRP in metabolic control, and to establish relevant dosing schedules in this species, here we tested the effect of intracerebroventricular infusion (icv) of recombinant AgRP on metabolic functions in C57BL/6J male mice. First, we examined the effects of AgRP(82-132) (21 days, 1 or 10 nmol/d, icv) or artificial cerebrospinal fluid (aCSF) using metabolic cages. High dose AgRP reduced body mass (aCSF n=12, +0.8±0.6 vs 1 nmol/d n=14, +0.6±0.6 vs 10 nmol/d n=11, -1.5±0.5 p<0.05 g/21d), without affecting food intake (15.1±1.2 vs 14.1±1.1 vs 16.3±1.1 kcal/d) or digestive efficiency (85.2±0.5 vs 84.5±0.7 vs 85.1±0.6 %), but a significant reduction in energy efficiency (+3.8±2.0 vs +2.7±2.8 vs -5.9±2.2 p<0.05 mg/kcal) indicated increased total EE. Next, we examined the effect of AgRP(82-132) (14 days, 1 nmol/d, icv) using a multiplexed system (Promethion, Sable). AgRP had no effect on body mass (25.1±1.2, n=8 vs 26.7±0.5, n=8), overall body composition (by NMR), heat production (Weir, 24h: 0.485±0.015 vs 0.490±0.022 kcal/h), or respiratory exchange ratio (0.88±0.01 vs 0.89±0.01). AgRP increased total food intake (10.1±0.6, n=8 vs 11.9±0.5, n=8, kcal/d, p=0.03) through a synergistic effect on number of meals and median meal mass. We conclude that AgRP (1-10 nmol/d, 2-3 wk, icv) infusion causes subtle changes in feeding behavior without effect on digestive efficiency. In contrast, EE is paradoxically increased by AgRP when infused at a high dose (10 nmol/d). We postulate that these differences may reflect differential accessibility of the peptide to relevant feeding vs autonomic control regions of the hypothalamus when infused into the cerebral ventricles, and/or compensatory increases in EE secondary to changes in feeding behavior. Future studies to deconvolute the role of AgRP in the control of EE in mice will require site-specific delivery of the peptide to relevant target regions or manipulation of its receptor in those regions. Funding: HL134850, HL084207

Plasma Agouti-Related Protein and Cortisol Levels in Cushing Disease: Evidence for the Regulation of Agouti-Related Protein by Glucocorticoids in Humans.

Glucocorticoids regulate energy balance, in part by stimulating the orexigenic neuropeptide agouti-related protein (AgRP). AgRP neurons express glucocorticoid receptors, and glucocorticoids have been shown to stimulate AgRP gene expression in rodents. We sought to determine whether there is a relationship between plasma AgRP and hypothalamic AgRP in rats and to evaluate the relationship between cortisol and plasma AgRP in humans. We retrospectively evaluated plasma AgRP levels prior to transsphenoidal surgery in 31 patients with Cushing disease (CD) vs 31 sex- and body mass index-matched controls from a separate study. We then prospectively measured plasma AgRP, before and 6 to 12 months after surgery, in a subgroup of 13 patients with CD. Plasma and hypothalamic AgRP were measured in adrenalectomized rats with and without corticosterone replacement. Plasma AgRP was stimulated by corticosterone in rats and correlated with hypothalamic AgRP expression. Plasma AgRP levels were higher in patients with CD than in controls (139 ± 12.3 vs 54.2 ± 3.1 pg/mL; P < 0.0001). Among patients with CD, mean 24-hour urine free cortisol (UFC) levels were 257 ± 39 μg/24 hours. Strong positive correlations were observed between plasma AgRP and UFC (r = 0.76; P < 0.0001). In 11 of 13 patients demonstrating surgical cure, AgRP decreased from 126 ± 20.6 to 62.5 ± 8.0 pg/mL (P < 0.05) postoperatively, in parallel with a decline in UFC. Plasma AgRP levels are elevated in CD, are tightly correlated with cortisol concentrations, and decline with surgical cure. These data support the regulation of AgRP by glucocorticoids in humans. AgRP's role as a potential biomarker and as a mediator of the adverse metabolic consequences of CD deserves further study.

Angiotensin‐(1‐7)‐Mediated Increases in Energy Expenditure Appear Independent of the Hypothalamic Melanocortin System

Obesity is a major public health problem that greatly increases risk for cardiovascular disease and type II diabetes. Obesity represents an energy imbalance where the amount of calories consumed exceeds the amount of calories expended. This energy balance is in part controlled by activation of proopiomelanocortin (POMC) neurons or inhibition of agouti‐related protein (AgRP) neurons within the arcuate nucleus (ARC) of the hypothalamus, which activates downstream melanocortin pathways to produce satiety and increase energy expenditure. Our preliminary data show that the beneficial renin‐angiotensin system hormone angiotensin (Ang)‐(1‐7) may be an important contributor to energy balance, as it attenuates high fat diet (HFD)‐induced obesity in mice by browning subcutaneous white fat to enhance energy expenditure. In this study, we tested the hypothesis that the beneficial effects of Ang‐(1‐7) on energy expenditure may be due to actions on POMC or AgRP neurons in the ARC. To test this hypothesis, we first examined the ability of an acute subcutaneous injection of Ang‐(1‐7) to induce neuronal activation in the ARC, as measured by c‐fos gene expression, in chow fed mice. We found that Ang‐(1‐7) significantly increased the number of c‐fos positive cells within the ARC compared with saline (43±5 Ang‐(1‐7) vs. 19±3 saline; p=0.022; n=3 per group). We then performed chronic experiments in which eight‐week old male C57BL/6J mice were implanted with subcutaneous osmotic mini‐pumps to deliver saline or Ang‐(1‐7) [400 ng/kg/min]. Immediately following mini‐pump implantation, mice were placed on a 60% high fat diet (HFD) or matched control chow diet for 12 weeks. The groups of mice included in this chronic study were: (1) chow diet with saline infusion (n=5); (2) HFD with saline infusion (n=6); and (3) HFD with Ang‐(1‐7) infusion [n=9]. At the end of treatment, the hypothalamus from each mouse was collected to assess for gene expression of mas receptor, POMC, and AgRP using quantitative real‐time polymerase chain reaction methods. We found that HFD reduced gene expression for POMC (1.1±0.3 chow vs. 0.4±0.1 HFD; p=0.005) in the hypothalamus, with no significant effect on mas receptor (1.2±0.3 chow vs. 0.7±0.1 HFD; p=0.134) or AgRP (1.3±0.5 chow vs. 1.1±0.2 HFD; p=0.856). There was no effect of Ang‐(1‐7) on POMC (0.4±0.1; p=0.998), mas receptor (0.7±0.1; p=0.995) or AgRP (1.0±0.1; p=0.996) gene expression in HFD mice. These findings provide evidence that Ang‐(1‐7) can acutely induce activation of neurons in the ARC. Chronic Ang‐(1‐7) treatment was not able to restore obesity‐induced reductions in POMC gene expression, however, despite enhancing energy expenditure. Further studies are needed to evaluate if beneficial effects of Ang‐(1‐7) on energy balance involve changes in POMC neuronal activity, or melanocortin‐independent pathways.Support or Funding InformationThis study was supported by NIH grant HL122507.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of Agouti-Related Protein and Pro-Opiomelanocortin Gene Expression in the Avian Arcuate Nucleus.

The arcuate nucleus is generally conserved across vertebrate taxa in its neuroanatomy and neuropeptide expression. Gene expression of agouti-related protein (AGRP), neuropeptide Y (NPY), pro-opiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART) has been established in the arcuate nucleus of several bird species and co-localization demonstrated for AGRP and NPY. The proteins encoded by these genes exert comparable effects on food intake in birds after central administration to those seen in other vertebrates, with AGRP and NPY being orexigenic and CART and α-melanocyte-stimulating hormone anorexigenic. We have focused on the measurement of arcuate nucleus AGRP and POMC expression in several avian models in relation to the regulation of energy balance, incubation, stress, and growth. AGRP mRNA and POMC mRNA are, respectively, up- and downregulated after energy deprivation and restriction. This suggests that coordinated changes in the activity of AGRP and POMC neurons help to drive the homeostatic response to replace depleted energy stores in birds as in other vertebrates. While AGRP and POMC expression are generally positively and negatively correlated with food intake, respectively, we review here situations in some avian models in which AGRP gene expression is dissociated from the level of food intake and may have an influence on growth independent of changes in appetite. This suggests the possibility that the central melanocortin system exerts more pleiotropic functions in birds. While the neuroanatomical arrangement of AGRP and POMC neurons and the sensitivity of their activity to nutritional state appear generally conserved with other vertebrates, detailed knowledge is lacking of the key nutritional feedback signals acting on the avian arcuate nucleus and there appear to be significant differences between birds and mammals. In particular, recently identified avian leptin genes show differences between bird species in their tissue expression patterns and appear less closely linked in their expression to nutritional state. It is presently uncertain how the regulation of the central melanocortin system in birds is brought about in the situation of the apparently reduced importance of leptin and ghrelin compared to mammals.

Evidence suggesting phosphodiesterase-3B regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons

Hypothalamic neurons expressing neuropeptide Y (NPY) and agouti related-protein (AgRP) are critical regulators of feeding behavior and body weight, and transduce the action of many peripheral signals including leptin and insulin. However, intracellular signaling molecules involved in regulating NPY/AgRP neuronal activity are incompletely understood. Since phosphodiesterase-3B (PDE3B) mediates the hypothalamic action of leptin and insulin on feeding, and is expressed in NPY/AgRP neurons, PDE3B could play a significant role in regulating NPY/AgRP neuronal activity. To investigate the direct regulation of NPY/AgRP neuronal activity by PDE3B, we examined the effects of gain-of-function or reduced function of PDE3B on NPY/AgRP gene expression in a clonal hypothalamic neuronal cell line, mHypoE-46, which endogenously express NPY, AgRP and PDE3B. Overexpression of PDE3B in mHypoE-46 cells with transfection of pcDNA-3.1-PDE3B expression plasmid significantly decreased NPY and AgRP mRNA levels and p-CREB levels as compared to the control plasmid. For the PDE3B knockdown study, mHypoE-46 cells transfected with lentiviral PDE3BshRNAmir plasmid or non-silencing lentiviral shRNAmir control plasmid were selected with puromycin, and stably transfected cells were grown in culture for 48h. Results showed that PDE3BshRNAmir mediated knockdown of PDE3B mRNA and protein levels (∼60–70%) caused an increase in both NPY and AgRP gene expression and in p-CREB levels. Together, these results demonstrate a reciprocal change in NPY and AgRP gene expression following overexpression and knockdown of PDE3B, and suggest a significant role for PDE3B in the regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons.

The effect of metformin on food intake and its potential role in hypothalamic regulation in obese diabetic rats

Metformin appears to be involved in altering energy expenditure and thermogenesis, and could affect hypothalamic feeding circuits. However, it is not clear whether metformin is able to cross the blood–brain barrier (BBB) to reach the hypothalamus and exert a direct effect on the central nervous system. Here we show the presence of metformin in cerebrospinal fluid (CSF) of diabetic rats administered orally with metformin which was confirmed by detecting the concentration of metformin with liquid chromatography-tandem mass spectrometry. Food intake of diabetic rats treated with metformin was reduced, and glucose homeostasis was gained. Expression of orexigenic peptides neuropeptide Y (NPY) and agouti-related protein (AgRP) decreased in the hypothalamus of metformin-treated diabetic rats, though anorexigenic peptides pro-opiomelanocortin (POMC) did not change significantly. The phosphorylation of signal transducer and activator of transcription 3 (STAT3) was increased but phosphorylated AMP-activated kinase (AMPK) was similar in the hypothalamus of metformin-treated diabetic rats. Our findings suggest that metformin may cross BBB and play a central mechanism on regulation of food intake in the hypothalamus. The anorexic effect of metformin may be mediated by inhibition of NPY and AgRP gene expression through the STAT3 signaling pathway.

Resveratrol, purified from the stem of Vitis coignetiae Pulliat, inhibits food intake in C57BL/6J Mice

Neuropeptide Y (NPY) and agouti-related protein (AgRP) have powerful stimulatory effects on food intake, which suggests that the downregulation of brain NPY or AgRP may help reduce obesity and diabetes by inhibiting food intake. To search for active compounds that inhibit NPY and AgRP expression, we made two luciferase reporter assay systems consisting of NPY and AgRP promoter-driven luciferase genes, together with the puromycin resistance gene, in a plasmid vector. Each plasmid was permanently transfected into N29-4 neuronal cells. Using the systems, resveratrol was purified from the stem of Vitis coignetiae Pulliat by activityguided fractionation. Resveratrol downregulated NPY and AgRP promoter-driven luciferase activity in a dose-dependent manner. The inhibitory concentrations (IC(50), 50% inhibition) of resveratrol against pNPY-luc and pAgRP-luc activities were 8.9 microM and 8.0 microM, respectively. Furthermore, one-time intraperitoneal injection of resveratrol (100 mg/kg) suppressed 20.0% and 17.2% of food intake during 24 and 48 h, respectively. These results indicated that resveratrol inhibited food intake, which may be related to the downregulation of NPY and AgRP gene expression.

Role of hypoleptinemia during cold adaptation in Brandt's voles (Lasiopodomys brandtii)

Brandt's voles Lasiopodomys brandtii exhibit large increases in nonshivering thermogenesis to cope with chronic cold exposure, resulting in compensatory hyperphagia and fat mobilization. These physiological events are accompanied by a remarkable reduction in serum leptin levels. However, the role of hypoleptinemia in cold adaptation in this species is still unknown. In the present study, we tested the hypothesis that hypoleptinemia contributes to increases in food intake and brown adipose tissue (BAT) thermogenesis by modifying hypothalamic neuropeptides in cold-exposed Brandt's voles. Adult male voles were transferred to 5 degrees C for 28 days. Accompanied by a decrease in serum leptin levels, hypothalamic agouti-related protein (AgRP) mRNA levels were significantly increased, but there were no changes in the long form of leptin receptor (Ob-Rb), suppressor of cytokine signaling 3 (SOCS3), neuropeptide Y (NPY) mRNA, proopiomelanocortin (POMC), and cocaine- and amphetamine-regulated peptide (CART) mRNA levels in the hypothalamus. When cold-exposed voles were returned to warm (23 degrees C) for 28 days, body mass, food intake, serum leptin, and AgRP mRNA were restored to control levels. Leptin administration in cold-exposed voles decreased food intake as well as hypothalamic AgRP mRNA levels. There were no significant effects of leptin administration on hypothalamic Ob-Rb, SOCS3, NPY, POMC, CART mRNA, and uncoupling protein 1 levels under cold conditions. These results suggest that hypoleptinemia partially contributes to cold-induced hyperphagia, which might involve the elevation of hypothalamic AgRP gene expression.

Enterostatin affects cyclic AMP and ERK signaling pathways to regulate Agouti-related Protein (AgRP) expression

Enterostatin, a gut–brain peptide, inhibits dietary fat intake in rats. The purpose of this study was to identify the intracellular signaling pathways that are responsive to enterostatin and that modulate the effects of enterostatin on the expression of Agouti-related protein (AgRP). We used the hypothalamic GT1-7 neuronal cell line to identify the effects of enterostatin on cyclic AMP and ERK signaling using conventional immunoassays or Western blots to assay the activity of these pathways. Enterostatin enhanced the level of cyclic AMP, PKA RIIβ and phospho-CREB and increased pERK levels in GT 1-7 cells. The effects on pERK were rapid (7.5 min) and dose-dependent. These signaling responses were blocked by an antibody to the enterostatin receptor (beta subunit of F1-ATPase), by the pERK inhibitor U0126 and by the P2Y receptor antagonist Suramin. Enterostatin showed a biphasic effect on AgRP mRNA, initially increasing but subsequently decreasing the levels. The cyclic AMP activator Sp-cAMP increased AgRP mRNA expression. Transfection of a wild type ERK construct reduced AgRP mRNA levels. Enterostatin inhibited expression of Krüppel-like factor 4 (KLF4), a transcriptional regulator of AgRP. KLF4 gene expression was increased by Sp-cAMP but decreased by wild-type ERK expression. U0126 blocked the effect of enterostatin on KLF4 expression. We conclude that enterostatin binding to its receptor activates the pERK pathway to inhibit AgRP gene expression but may enhance AgRP expression through activation of the cyclic AMP pathway. These pathways probably mediate the enterostatin inhibition of dietary fat intake.

Central role of the melanocortin-4 receptors in appetite regulation after endotoxin1

Melanocortin-4 receptors (MC4R) are key factors in the depression of appetite during disease. This study was designed to determine the role of agouti-related protein (AgRP) in the effect of endotoxin (lipopolysaccharide, LPS) on appetite. Sheep received an intracerebroventricular injection of either saline or AgRP (0.5 nmol/kg of BW) 1 h before intravenous injection of either saline or LPS (0.6 microg/kg of BW) at time 0 and again at 4 h. Agouti-related protein prevented the reduction in feed intake due to LPS (P < 0.05). In a second experiment, AgRP gene expression was unaffected at 3 h and increased (P < 0.01) at 6 h after LPS. Immunohistochemical evidence indicated that there was an increase in the percentage of AgRP neurons with c-Fos immunoreactive nuclei 6 h after sheep were injected with LPS (P < 0.04) and a corresponding decrease in a-melanocyte-stimulating hormone neurons coexpressing c-Fos (P < 0.001). In situ hybridization provided evidence for an increase in AgRP gene expression and a decrease in proopiomelanocortin gene expression 6 h after LPS (P < 0.05). In a final experiment, physiological elevation of orexigenic agents by short-term fasting kept feed intake at the same level as controls, in spite of the presence of LPS, similar to the effects of AgRP in Exp. 1. The AgRP inhibition of the MC4R prevents appetite inhibition in response to LPS and well after LPS inhibition of feed intake, both AgRP and a-melanocyte-stimulating hormone may change in a pattern that favors appetite increases. These studies support the notion of the MC4R as a critical component of the mechanism for appetite suppression due to endotoxin.

Differential Effects of Refeeding on Melanocortin-Responsive Neurons in the Hypothalamic Paraventricular Nucleus

To explore the effect of refeeding on recovery of TRH gene expression in the hypothalamic paraventricular nucleus (PVN) and its correlation with the feeding-related neuropeptides in the arcuate nucleus (ARC), c-fos immunoreactivity (IR) in the PVN and ARC 2 h after refeeding and hypothalamic TRH, neuropeptide Y (NPY) and agouti-related protein (AGRP) mRNA levels 4, 12, and 24 h after refeeding were studied in Sprague-Dawley rats subjected to prolonged fasting. Despite rapid reactivation of proopiomelanocortin neurons by refeeding as demonstrated by c-fos IR in ARC alpha-MSH-IR neurons and ventral parvocellular subdivision PVN neurons, c-fos IR was present in only 9.7 +/- 1.1% hypophysiotropic TRH neurons. Serum TSH levels remained suppressed 4 and 12 h after the start of refeeding, returning to fed levels after 24 h. Fasting reduced TRH mRNA compared with fed animals, and similar to TSH, remained suppressed at 4 and 12 h after refeeding, returning toward normal at 24 h. AGRP and NPY gene expression in the ARC were markedly elevated in fasting rats, AGRP mRNA returning to baseline levels 12 h after refeeding and NPY mRNA remaining persistently elevated even at 24 h. These data raise the possibility that refeeding-induced activation of melanocortin signaling exerts differential actions on its target neurons in the PVN, an early action directed at neurons that may be involved in satiety, and a later action on hypophysiotropic TRH neurons involved in energy expenditure, potentially mediated by sustained elevations in AGRP and NPY. This response may be an important homeostatic mechanism to allow replenishment of depleted energy stores associated with fasting.

Prenatal influences on susceptibility to diet-induced obesity are mediated by altered neuroendocrine gene expression

The escalating rates of obesity and type 2 diabetes have reached pandemic proportions. It has been proposed that the risk of developing metabolic disorders in adult life is influenced by environmental factors, which operate during the early periods of development. We have previously shown that an interaction between the prenatal and the postnatal dietary environment amplifies the propensity towards diet-induced obesity, although the mechanisms are unclear. In the present study, we investigated the interaction between prenatal undernutrition and postnatal high-fat nutrition on key genes of the hypothalamic appetite regulatory network. Pregnant Wistar rats were fed a standard chow diet either ad libitum (AD) or at 30% of AD intake throughout gestation (UN). From weaning, female AD and UN offspring were fed either a standard chow (ADC n = 8, UNC n = 8) or a high-fat diet (45% kcal as fat; ADHF n = 8, UNHF n = 8) ad libitum for the remainder of the study. At 24 weeks of age, body composition was assessed by dual energy X-ray absorptiometry analysis and total RNA was extracted from whole rat hypothalami. Real-time PCR was performed to characterise pro-opiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related protein (AgRP) and OBRb gene expression at the mRNA level. Our results demonstrate that the amplification of postnatal obesity develops as a consequence of an interaction between prenatal under-nutrition and postnatal high-fat nutrition. This phenotype also shows significant alterations in POMC, NPY, AgRP and OBRb gene expression together with elevations in circulating levels of both plasma leptin and insulin. These findings are consistent with the predictive adaptive response hypothesis that neuroendocrine development during fetal life may be based on predictions about postnatal environmental conditions. Increased susceptibility to diet-induced obesity develops if a mismatch between the anticipated and the actual conditions are encountered.

Effects of fasting, leptin, and insulin on AGRP and POMC peptide release in the hypothalamus

Agouti-related protein (AGRP) and proopiomelanocortin (POMC) have opposing effects on melanocortin receptor (MC-R) signaling and energy balance, and are important targets for leptin and insulin in the hypothalamus. While food intake and leptin have documented effects on POMC and AGRP gene expression, and insulin has effects on POMC gene expression, little is known about their effects on POMC or AGRP peptide release. Here we have examined the effects of fasting, leptin, and insulin on the release of AGRP and the POMC-derived peptide γ 3-MSH from the perifused rat hypothalamus in vitro. In the first experiment, fasting (48 h) resulted in a significant overall decrease in γ 3-MSH release measured every 20 min during a 3-h baseline perifusion period and after depolarization with 56 mM KCl ( p = 0.02); there was a trend towards an overall increase in the release of AGRP but this was not significant. When the ratio of γ 3-MSH/AGRP release was calculated at each time point, there was an overall decrease in γ 3-MSH/AGRP with fasting ( p < 0.01). Further examination of the ratio of γ 3-MSH/AGRP revealed a 34% reduction ( p < 0.05) in the basal area under the curve (AUC) and a 33% reduction ( p < 0.01) in the post-KCl stimulated AUC in fasted vs. fed animals. In the second experiment, perifusion of hypothalamic slices with 10 −8 or 10 −7 M leptin for 2 h resulted in a significant decrease in the release of AGRP noted primarily after depolarization with KCl ( p < 0.01); no effect was seen on γ 3-MSH release. Similarly, in a third experiment, perifusion with 10 −7 M insulin caused a significant decrease in AGRP release ( p < 0.001) without affecting γ 3-MSH release. Thus, there is a significant decrease in γ 3-MSH and the ratio of γ 3-MSH to AGRP released during fasting, consistent with a net inhibition of hypothalamic MC-R signaling. In contrast, short-term treatment with leptin and insulin may inhibit MC-R signaling primarily by decreasing the release of AGRP.

Hypothalamic gene expression in long-term fasted rats: relationship with body fat

Many hypothalamic neuropeptides are involved in the regulation of energy homeostasis and feeding behavior. We have investigated whether and to what extent neuropeptide Y (NPY), agouti-related protein (AGRP), melanin-concentrating hormone (MCH), and prepro-orexin (prepro-OX) as well as pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART) mRNA levels are affected in rat hypothalamus. An experimental model of long-term fasting rat characterized by three metabolic phases from changes in lipid and protein utilization was used. Except for prepro-OX and compared to fed group, starvation induced an increase in the orexigenic gene expressions that was much more marked in phase 3 (by 2.5-, 8.1-, and 13.5-fold for MCH, AGRP, and NPY, respectively) than in phase 2 (by about 1.5–2.2-fold as an average) of fasting. AGRP and NPY mRNA levels were inversely related to body fat content. Anorexigenic gene expression was only slightly affected at both fasting stages. We conclude that the regulation of NPY and AGRP gene expression is primarily involved during late fasting and could mediate the concomitant enhanced drive for refeeding.

Effects of streptozotocin-induced diabetes and insulin treatment on the hypothalamic melanocortin system and muscle uncoupling protein 3 expression in rats.

Hypothalamic melanocortins are among several neuropeptides strongly implicated in the control of food intake. Agonists for melanocortin 4 (MC-4) receptors such as alpha-melanocyte-stimulating hormone (alpha-MSH), a product of proopiomelanocortin (POMC), reduce food intake, whereas hypothalamic agouti-related protein (AgRP) is a MC-4 receptor antagonist that increases food intake. To investigate whether reduced melanocortin signaling contributes to hyperphagia induced by uncontrolled diabetes, male Sprague-Dawley rats were studied 7 days after administration of streptozotocin (STZ) or vehicle. In addition, we wished to determine the effect of diabetes on muscle uncoupling protein 3 (UCP-3), a potential regulator of muscle energy metabolism. STZ diabetic rats were markedly hyperglycemic (31.3 +/- 1.0 mmol/l; P < 0.005) compared with nondiabetic controls (9.3 +/- 0.2 mmol/l). Insulin treatment partially corrected the hyperglycemia (18.8 +/- 2.5 mol/l; P < 0.005). Plasma leptin was markedly reduced in STZ diabetic rats (0.4 +/- 0.1 ng/ml; P < 0.005) compared with controls (3.0 +/- 0.4 ng/ml), an effect that was also partially reversed by insulin treatment (1.8 +/- 0.3 ng/ml). Untreated diabetic rats were hyperphagic, consuming 40% more food (48 +/- 1 g/day; P < 0.005) than controls (34 +/- 1 g/day). Hyperphagia was prevented by insulin treatment (32 +/- 2 g/day). In untreated diabetic rats, hypothalamic POMC mRNA expression (measured by in situ hybridization) was reduced by 80% (P < 0.005), whereas AgRP mRNA levels were increased by 60% (P < 0.01), suggesting a marked decrease of hypothalamic melanocortin signaling. The change in POMC, but not in AgRP, mRNA levels was partially reversed by insulin treatment. By comparison, the effects of diabetes to increase hypothalamic neuropeptide Y (NPY) expression and to decrease corticotropin-releasing hormone (CRH) expression were normalized by insulin treatment, whereas the expression of mRNA encoding the long form of the leptin receptor in the arcuate nucleus was unaltered by diabetes or insulin treatment. UCP-3 mRNA expression in gastrocnemius muscle from diabetic rats was increased fourfold (P < 0.005), and the increase was prevented by insulin treatment. The effect of uncontrolled diabetes to decrease POMC, while increasing AgRP gene expression, suggests that reduced hypothalamic melanocortin signaling, along with increased NPY and decreased CRH signaling, could contribute to diabetic hyperphagia. These responses, in concert with increased muscle UCP-3 expression, may also contribute to the catabolic effects of uncontrolled diabetes on fuel metabolism in peripheral tissues.

Photoperiod regulates arcuate nucleus POMC, AGRP, and leptin receptor mRNA in Siberian hamster hypothalamus.

Siberian hamsters decreased body weight by 30% during 18 wk in short day (SD) vs. long day (LD) controls. Subsequent imposed food deprivation (FD; 24 h) caused a further 10% decrease. In the hypothalamic arcuate nucleus (ARC), SDs reduced proopiomelanocortin (POMC) gene expression and agouti-related protein (AGRP) mRNA was elevated, changes that summate to reduced catabolic drive through the melanocortin receptors. There was no effect of photoperiod on neuropeptide Y (NPY), melanin concentrating hormone, orexin, or corticotropin-releasing factor mRNAs. Superimposed FD increased AGRP gene expression and caused a localized elevation of NPY mRNA in the ARC. Both adipose tissue leptin and ARC leptin receptor (OB-Rb) mRNAs were downregulated in SDs, whereas FD increased OB-Rb gene expression. Thus OB-Rb mRNA is differentially regulated by acute and chronic changes in plasma leptin in this species. In a separate experiment in LDs, AGRP gene expression was increased by 24 or 48 h FD, whereas POMC mRNA was downregulated in the caudal ARC. AGRP and NPY mRNAs were extensively coexpressed in the ARC, and their differential regulation by photoperiod and FD is suggestive of transcript-specific regulation at the level of individual neurons.

Altered expression of agouti-related protein and its colocalization with neuropeptide Y in the arcuate nucleus of the hypothalamus during lactation.

During lactation, the levels of neuropeptide Y (NPY), which plays an important role in mediating food intake, are significantly elevated in a number of hypothalamic areas, including the arcuate nucleus (ARH). To identify additional hypothalamic systems that might be important in mediating the increase in food intake and alterations in energy homeostasis during lactation, the present studies examined the expression of agouti-related protein (AGRP), a recently described homologue of the skin agouti protein. AGRP is found in the hypothalamus and has been suggested to play an important role in the regulation of food intake. In the first experiment, animals were studied during diestrus of the estrous cycle, a stage of the cycle when estrogen levels are basal and similar to lactation, or during days 12-13 postpartum. Lactating animals had their litters adjusted to eight pups on day 2 postpartum. Brain tissue sections were used to measure AGRP messenger RNA (mRNA) levels by in situ hybridization. AGRP mRNA signal was found mostly in the ventromedial portion of the ARH, which has been shown to contain a high density of NPY neurons. A significant increase in AGRP mRNA content was observed in the mid- to caudal portion of the ARH of lactating animals compared with diestrous females. No difference was found in the rostral portion of the ARH. In the second experiment, double-label in situ hybridization for AGRP and NPY was performed in lactating animals to determine the extent of colocalization of the two peptides in the ARH, using 35S-labeled and digoxigenin-labeled antisense complementary RNA probes. It was found that almost all of the NPY-positive neurons throughout the ARH also expressed AGRP mRNA signal. Furthermore, AGRP expression was confined almost exclusively to NPY-positive neurons. Thus, the present study showed that during lactation, AGRP gene expression was significantly elevated in a subset of the AGRP neurons in the ARH. The high degree of colocalization of AGRP and NPY, coupled with previous reports from our laboratory demonstrating increased NPY expression in the ARH in response to suckling, suggests that AGRP and NPY are coordinately regulated and may be involved in the increase in food intake during lactation.