Fasting regulates hypothalamic neuropeptide Y, agouti-related peptide, and proopiomelanocortin in diabetic mice independent of changes in leptin or insulin.

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.     

Inhibition of hypothalamic neuropeptide Y gene expression by insulin.

Insulin acts in the brain to suppress feeding, whereas neuropeptide Y (NPY) has the opposite effect. Since fasting lowers plasma insulin levels and increases hypothalamic synthesis of NPY, we proposed that insulin may inhibit hypothalamic NPY gene expression. To test this hypothesis, we used RIA and in situ hybridization histochemistry to determine if centrally administered insulin could reduce levels of both NPY and its messenger RNA (mRNA) in discreet hypothalamic regions during fasting. Three groups of Long-Evans rats were entered into a 72-h study protocol. One group was fed ad libitum during this period, while the others were fasted. Fed rats received intracerebroventricular (icv) injections of saline vehicle at 12-h intervals, whereas fasted groups received icv vehicle alone or with insulin (4 mU/12 h). In vehicle-only treated rats, fasting significantly increased expression of preproNPY mRNA in the arcuate nucleus to 179 +/- 20% of fed controls. Administration of icv insulin during fasting abolished this increase (99 +/- 14% of fed controls; P less than 0.05 vs. fasted, vehicle-treated rats). Central insulin administration during fasting also reduced immunoreactive NPY concentrations in samples punched from the paraventricular nucleus (PVN) (875 +/- 122 pg/punch) to levels below vehicle-only treated rats (1396 +/- 435 pg/punch; P less than 0.05), similar to free-feeding control values (814 +/- 170 pg/punch). By comparison, neither fasting nor central insulin administration altered NPY levels in four other hypothalamic regions (supraoptic, ventromedial, dorsomedial, and arcuate nuclei). Continuous icv insulin infusion at a lower dose (2 mU/day) produced a similar result during a shorter period (48 h) of food deprivation in Wistar rats. In this study, central insulin infusion also inhibited the fasting-related increase in arcuate preproNPY mRNA levels and did not affect plasma glucose or insulin levels. This suggests that insulin acts locally to inhibit hypothalamic NPY mRNA expression. We conclude that the increase of levels of NPY in the PVN and preproNPY mRNA in the arcuate nucleus during fasting are inhibited by icv insulin. Fasting, therefore, increases NPY biosynthesis along an arcuate nucleus-PVN pathway in the hypothalamus via a mechanism dependent on low insulin levels.     

Severity of the catabolic condition differentially modulates hypothalamic expression of growth hormone-releasing hormone in the fasted mouse: potential role of neuropeptide Y and corticotropin-releasing hormone.

To determine whether the severity of the catabolic condition differentially regulates the GH axis, male mice were either fed ad libitum or fasted for 12, 24, and 48 h. Hypothalami, pituitaries, and stomachs were collected for assessment of mRNA levels by quantitative real-time RT-PCR, and blood collected for measurement of plasma hormone and metabolite levels by commercial assay kits. Overnight (12 h) fasting resulted in a significant suppression of circulating glucose, insulin, IGF-I, and leptin levels and an increase in corticosterone, free fatty acids, and n-octanoyl ghrelin levels, and these directional changes were maintained at the 24- and 48-h time points. Fasting (24 h) also increased circulating GH levels, which was associated with an increase in pituitary mRNA levels for GHRH receptor and ghrelin receptor and a decrease in mRNA levels for somatostatin (SST) receptor (SSTR) subtypes, SSTR2, SSTR3, and SSTR5, where the changes in ghrelin receptor and SSTR expression persisted after 48 h fasting. Hypothalamic SST mRNA levels were not altered by fasting, whereas there was a transient rise in stomach SST mRNA levels 24 h after food withdrawal. In contrast, there was a biphasic effect of fasting on GHRH expression. GHRH mRNA levels were significantly elevated at 12 and 24 h but fell to approximately 50% of fed controls 48 h after food withdrawal. A sequential rise in hypothalamic neuropeptide Y (NPY) and CRH mRNA levels preceded the fall in GHRH expression, where fasting-induced changes in CRH and GHRH mRNA levels were not observed in 48-h-fasted NPY knockout mice. These observations, in light of previous reports showing both NPY and CRH can inhibit GHRH expression and GH release, suggest that these neuronal systems may work in concert to control the ultimate impact of fasting on GH axis function.     

Effects of leptin receptor mutation on Agrp gene expression in fed and fasted lean and obese (LA/N-faf) rats

Agouti-related protein provides an orexigenic signal, probably through interaction with central melanocortin receptors. Expression of Agrp is markedly increased in the hypothalamus of mice deficient in leptin (Lep(ob)/Lep(ob)) or its receptor (Lepr(db)/Lepr(db)), suggesting that leptin mediates signals suppressing Agouti-related protein production. The regulation of Agrp expression in the rat hypothalamus has not been reported. We, therefore, analyzed the expression of Agrp in the medial basal hypothalamus of lean (+/+, +/fa(f)) and obese leptin receptor-deficient (fa(f)/fa(f)) LA/N rats. Using a sensitive solution hybridization/S1 nuclease protection assay, we found no significant difference in Agrp messenger RNA (mRNA) levels (pg/microg total RNA +/- SEM) in obese rats (n = 5), compared with lean controls (n = 5): 0.46 +/- 0.06 vs. 0.47 +/- 0.06 (P = 0.9). Similarly, no difference in Agrp expression was found using in situ hybridization or semiquantitative RT-PCR. In contrast to Agrp, Pomc mRNA levels were significantly suppressed in the obese, compared with the lean, rats (P = 0.001). Thus, the ratio of Pomc to Agrp mRNA is decreased in the obese rats and may be an important modulator of food intake. To assess the physiological regulation of Agrp in rats, we examined the effect of food deprivation in lean Sprague Dawley (SD) rats. There was a 273% increase in medial basal hypothalamus Agrp mRNA in SD rats fasted for 48 h (n = 8), compared with rats fed ad libitum (n = 8): 0.82 +/- 0.23 vs. 0.30 +/- 0.08 (P = 0.0001). Lean LA/N rats (n = 7) fasted for 48 h also showed a 231% increase in Agrp expression, compared with fed lean controls (n = 8): 0.74 +/- 0.11 vs. 0.32 +/- 0.03 (P = 0.002), whereas Pomc expression was decreased by 32% in fasted animals from the same experiment (0.34 +/- 0.05 vs. 0.50 +/- 0.07; P = 0.03). There were no significant differences in Agrp or Pomc mRNA levels between fasted and fed obese LA/N-fa(f) rats. These results suggest that, in the rat, the Agrp response to fasting may involve leptin-mediated phenomena, but factors in addition to leptin must also be involved in the regulation of Agrp gene expression.     

Fasting induces a large, leptin-dependent increase in the intrinsic action potential frequency of orexigenic arcuate nucleus neuropeptide Y/Agouti-related protein neurons

The neuropeptide Y (NPY)/Agouti-related protein (AgRP) neurons of the hypothalamic arcuate nucleus are thought to promote feeding. Here, we demonstrate that feeding state in vivo, through a leptin-dependent process, induces large and persistent changes in the electrophysiological activity of these neurons as measured extracellularly in vitro. Consistent with an orexigenic role, fasting induced a 4-fold increase in the basal action potential frequency of NPY/AgRP neurons. Leptin, when injected into fasted wild-type mice, induced a dose- and time-dependent decrease in spike frequency, which approached fed levels 2-3 h post treatment. In leptin-deficient (lep(ob)/lep(ob)) and leptin receptor-deficient (lepr(db)/lepr(db)) mice, NPY/AgRP spike frequency was not significantly increased by fasting, and even in mutant mice fed ad libitum, spike frequency was at least as high as in fasted wild-type mice. All recordings included GABA(A) and ionotropic glutamate receptor antagonists, suggesting that expression of this modulation is potentially intrinsic and not synaptically dependent. Recorded neurons were unambiguously identified using NPY-Sapphire transgenic mice. This is a remarkably straightforward example of a very robust in vitro electrophysiogical effect produced by a simple behavioral manipulation, food restriction.     

Expression analysis of hypothalamic and pituitary components of the growth hormone axis in fasted and streptozotocin-treated neuropeptide Y (NPY)-intact (NPY+/+) and NPY-knockout (NPY-/-) mice

In the fasted and the streptozotocin (STZ)-induced diabetic male rat, hypothalamic growth hormone (GH)-releasing hormone (GHRH) mRNA levels, and pulsatile GH release are decreased. These changes are believed to be due to a rise in hypothalamic neuropeptide Y (NPY) that inhibits GHRH expression. To directly test if NPY is required for metabolic regulation of hypothalamic neuropeptides important in GH secretion, NPY, GHRH and somatostatin (SRIH) mRNA levels were determined in fasted (48 h) and STZ-treated wild-type (NPY(+/+)) and NPY-knockout (NPY(-/-)) mice by ribonuclease protection assay. In addition, pituitary receptor mRNA levels for GHRH (GHRH-R), ghrelin (GHS-R) and SRIH (sst2) were assessed by RT-PCR. Under fed conditions the GH axis of NPY(+/+) and NPY(-/-) did not differ. In the NPY(+/+) mouse, fasting resulted in a 23% weight loss and >250% increase in NPY mRNA accompanied by a significant reduction in both GHRH and SRIH mRNA. These changes were associated with increases in pituitary expression of GHRH-R and GHS-R and a concomitant suppression of sst2. In the NPY(-/-) mouse, fasting also resulted in a 23% weight loss and comparable changes in GHRH-R and sst2, but failed to alter GHRH, SRIH and GHS-R mRNA levels. Fasting resulted in an overall increase in circulating GH, which reached significance in the fasted NPY(-/-) mouse. Induction of diabetes in NPY(+/+) mice, using a single, high-dose, STZ injection (150 mg/kg), resulted in modest weight loss (5%), and a 158% increase NPY expression which was associated with reciprocal changes in pituitary GHS-R and sst2 expression, similar to that observed in the fasted state, but no change in hypothalamic GHRH or SRIF expression was observed. Induction of diabetes in NPY(+/+) and NPY(-/-) mice, using a multiple, low-dose, STZ paradigm (5 consecutive daily injections of 40 mg/kg), did not alter body weight, hypothalamic neuropeptide expression or pituitary receptor expression, with the exception that sst2 mRNA levels were suppressed and GH levels did rise in the NPY(-/-) mouse. These observations demonstrate that NPY is not required for basal regulation of the GH axis, but is required for fasting-induced suppression of GHRH and SRIH expression, as well as fasting-induced augmentation of pituitary GHS-R mRNA. In contrast to the rat, fasting clearly did not suppress circulating GH levels in mice, but resulted in an overall rise in mean GH levels, similar to that observed in other mammalian species. The fact that many of the fasting-induced changes in the GH axis were observed in the high-dose STZ-treated mice, but were not observed in the multiple, low-dose paradigm, suggests STZ-mediated modulation of GH axis function is dependent on the severity of the catabolic state and not hyperglycemia.     

Regulation of hypothalamic expression of KiSS-1 and GPR54 genes by metabolic factors: analyses using mouse models and a cell line

It is well established that reproductive function is metabolically gated. However, the mechanisms whereby energy stores and metabolic cues influence fertility are yet to be completely deciphered. Recently, the hypothalamic KiSS-1/GPR54 system has emerged as a fundamental regulator of the gonadotropic axis, which conveys the modulatory actions of sex steroids to GnRH neurons. Evidence is also mounting that KiSS-1 neurons may also represent the link between systemic metabolic signals and central control of reproduction. To further explore this possibility, we examined the impact of changes in energy status and key metabolic regulators on the hypothalamic expression of KiSS-1 and GPR54 genes, using different mouse models and the hypothalamic cell line N6. Time-course analysis of the effects of short-term fasting revealed a rapid (12- and 24-h) decline in KiSS-1 and GPR54 mRNA levels, which preceded that of GnRH (48 h). In contrast, diet-induced obesity or obesity associated with leptin deficiency (ob/ob vs. wild-type mice) failed to induce overt changes in hypothalamic expression of KiSS-1 and GPR54 genes. However, leptin infusion of ob/ob mice evoked a significant increase in KiSS-1 and GPR54 mRNA levels compared with pair-fed controls. Moreover, leptin, but not insulin or IGF-I, stimulated KiSS-1 mRNA expression in the mouse hypothalamic cell line N6. In addition, neuropeptide Y (NPY) null mice showed decreased KiSS-1 mRNA levels at the hypothalamus, whereas exposure to NPY increased expression of KiSS-1 in hypothalamic N6 cells. In sum, our present data further characterize the functional relevance and putative key mediators (such as leptin and NPY) of the metabolic regulation of the hypothalamic KiSS-1 system in the mouse.     

Galanin-like peptide mRNA alterations in arcuate nucleus and neural lobe of streptozotocin-diabetic and obese zucker rats. Further evidence for leptin-dependent and independent regulation

Galanin-like peptide (GALP) is a 60-amino-acid peptide with structural similarities to galanin and a high affinity for galanin receptors. GALP is expressed by a discrete population of neurons in the arcuate nucleus (ARC) and median eminence of the hypothalamus of several species, including the rat. GALP neurons express leptin receptors and GALP mRNA levels are decreased slightly in fasted rats and stimulated significantly by acute leptin treatment in combination with fasting. In studies to further explore the leptin dependence of GALP expression, we examined GALP mRNA levels in the hypothalamus of obese Zucker and streptozotocin-induced diabetic (STZ-DM) rats. In leptin receptor-deficient obese Zucker rats, with 75% higher body weight than lean littermates, GALP mRNA levels in the ARC were decreased by 75%, while neuropeptide Y (NPY) mRNA levels were increased 7-fold (n = 5, p < 0.001), consistent with earlier reports. In hypoleptinemic diabetic rats with 4.5-fold higher blood glucose and 15% lower body weight than controls, GALP mRNA levels in the ARC were decreased by 90%, while NPY mRNA levels were increased 9-fold (n = 5, p < 0.001). GALP is also expressed by pituicytes in the neural lobe of the rat pituitary gland and GALP expression is increased by osmotic stimulation such as dehydration and salt loading. Thus, in STZ-DM rats that are in a hyperosmotic state with elevated plasma vasopressin levels, GALP mRNA levels were increased by approximately 20-fold in the neural lobe relative to control (n = 4, p < 0.001). The current findings are consistent with a strong tonic influence of leptin receptor signalling on hypothalamic GALP expression under normal conditions, and possible abnormalities in GALP neuronal signalling and their putative targets, thyrotropin-releasing hormone and gonadotropin hormone-releasing hormone neurons, under pathophysiological conditions such as diabetes and obesity. Our data in STZ-DM rats also clearly demonstrate that GALP gene expression is differentially regulated in neurons and pituicytes.     

Fasting and leptin modulate adipose and muscle uncoupling protein: divergent effects between messenger ribonucleic acid and protein expression

Leptin is believed to act through hypothalamic centers to decrease appetite and increase energy utilization, in part through enhanced thermogenesis. In this study, we examined the effects of fasting for 2 days and exogenous s.c. leptin, 200 microg every 8 h for 2 days, on the regulation of uncoupling protein (UCP) subtypes in brown adipose tissue (BAT) and gastrocnemius muscle. Northern blot analysis (UCP-1) and ribonuclease protection (UCP-2 and 3) were used for quantitative messenger RNA (mRNA) analysis, and specific antibodies were used to measure UCP-1 and UCP-3 total protein expression. Leptin, compared with vehicle, did not alter BAT UCP-1 or UCP-3 mRNA or protein expression when administered to normal ad libitum fed rats. Fasting significantly decreased BAT UCP-1 and UCP-3 mRNA expression, to 31% and 30% of ad libitum fed controls, respectively, effects which were prevented by administration of leptin to fasted rats. Fasting also significantly decreased BAT UCP-1 protein expression, to 67% of control; however, that effect was not prevented by leptin treatment. Fasting also decreased BAT UCP-3 protein, to 85% of control, an effect that was not statistically significant. Fasting, with or without leptin administration, did not affect BAT UCP-2 mRNA; however, leptin administration to ad libitum fed rats significantly increased BAT UCP-2 mRNA, to 138% of control. Fasting significantly enhanced gastrocnemius muscle UCP-3 mRNA (411% of control) and protein expression (168% of control), whereas leptin administration to fasted rats did not alter either of these effects. In summary, UCP subtype mRNA and protein are regulated in tissue- and subtype-specific fashion by leptin and food restriction. Under certain conditions, the effects of these perturbations on UCP mRNA and protein are discordant.     

The regulation of stearoyl-CoA desaturase gene expression is tissue specific in chickens

Emerging evidence suggests a potential role of stearoyl-CoA desaturase (SCD)-1 in the control of body weight and energy homeostasis. The present study was conducted to investigate the effects of several energy balance-related factors (leptin, cerulenin, food deprivation, genotype, and gender) on SCD gene expression in chickens. In experiment 1, 6-week-old female and male broiler chickens were used. In experiment 2, two groups of 3-week-old broiler chickens were continuously infused with recombinant chicken leptin (8 micro g/kg/h) or vehicle for 6 h. In experiment 3, two groups of 2-week-old broiler chickens received i.v. injections of cerulenin (15 mg/kg) or vehicle. In experiment 4, two broiler chicken lines (fat and lean) were submitted to two nutritional states (food deprivation for 16 or 24 h and feeding ad libitum). At the end of each experiment, tissues were collected for analyzing SCD gene expression. Data from experiment 1 showed that SCD is ubiquitously expressed in chicken tissues with highest levels in the proventriculus followed by the ovary, hypothalamus, kidney, liver, and adipose tissue in female, and hypothalamus, leg muscle, pancreas, liver, and adipose tissue in male. Female chickens exhibited significantly higher SCD mRNA levels in kidney, breast muscle, proventriculus, and intestine than male chickens. However, hypothalamic SCD gene expression was higher in male than in female (P < 0.05). Leptin increased SCD gene expression in chicken liver (P < 0.05), whereas cerulenin decreased SCD mRNA levels in muscle. Both leptin and cerulenin significantly reduced food intake (P < 0.05). Food deprivation for either 16 or 24 h decreased the hepatic SCD gene expression in fat line and lean line chickens compared with their fed counterparts (P < 0.05). The hypothalamic SCD mRNA levels were decreased in both lines only after 24 h of food deprivation (P < 0.05). In conclusion, SCD is ubiquitously expressed in chickens and it is regulated by leptin, cerulenin, nutritional state, and gender in a tissue-specific manner.     

Inverse shift in circulating corticosterone and leptin levels elevates hypothalamic deiodinase type 2 in fasted rats

During food deprivation, plasma T(4) and T(3) levels are decreased. Under this metabolic condition, hypothalamic deiodinase type 2 (D2) activity and mRNA levels are elevated, whereas TRH mRNA levels are suppressed. Systemic T(4) administration does not reverse these hypothalamic changes. The mechanism(s) that underlies this paradoxical regulation of D2 during fasting is unknown. We hypothesize that leptin and/or glucocorticoids play a role in these mechanisms, and their interactions may be an important regulator of the hypothalamic-pituitary-thyroid axis. Thus, we assessed the effects of these hormones on D2 activity levels of food-deprived as well as fed animals using enzyme activity measurements. In food-deprived animals, corticosterone replacement reversed the inhibitory effect of adrenalectomy (ADX) on D2 induction, whereas ADX and ADX plus corticosterone replacement did not significantly affect D2 activity levels in rats fed ad libitum. Leptin administration to fed animals did not change D2 activity, whereas in fasted rats, leptin decreased D2 activity by reducing corticosterone plasma levels. When leptin was administered to fasted animals that were either ADX or ADX plus corticosterone treated at a high dose, D2 activity did not increase. Our results show that during fasting, diminishing leptin levels play a permissive role to enable glucocorticoid-induced up-regulation of D2. Thus, our observations suggest that appropriate induction of D2 activity during negative energy balance is dependent upon both leptin and glucocorticoid signaling.     

Cerebellin1 is a novel orexigenic peptide

Aim: Cerebellin1 (Cbln1) is highly expressed in the hypothalamus, a region of the brain involved in appetite regulation. However, the effects of Cbn1 on food intake are not known. The present study aimed to investigate the effect of Cbln1 on appetite regulation in rats. Methods: We determined the effect of (i) intracerebroventricular (ICV) injection of Cbln1 on food intake, behaviour and plasma pituitary hormone levels in male Wistar rats; (ii) Cbln1 on the release of hypothalamic neuropeptides known to modulate food intake from hypothalamic explants and (iii) fasting on hypothalamic Cbln1 mRNA expression. Results: (i) ICV administration of Cbln1 significantly increased food intake in rats and caused no adverse behaviours. ICV administration of Cbln1 significantly reduced plasma thyroid stimulating hormone (TSH) levels 10 min postinjection in rats. (ii) Cbln1 significantly increased the release of neuropeptide Y (NPY) from hypothalamic explants. (iii) Cbln1 mRNA expression levels were increased in the ventromedial nucleus of the hypothalamus in fasted rats. Conclusions: These data suggest that Cbln1 is a novel orexigenic peptide, which may mediate its effects via hypothalamic NPY.     

The human growth hormone-releasing hormone transgenic mouse as a model of modest obesity: differential changes in leptin receptor (OBR) gene expression in the anterior pituitary and hypothalamus after fasting and OBR localization in somatotrophs.

We reported previously an increase in leptin receptor (OBR) gene expression in the anterior pituitary of human GH-releasing hormone (hGHRH) transgenic mice. The primary goal of this study was to investigate the possible mechanisms regulating OBR expression in these mice. Compared with normal sibling controls, hGHRH transgenic mice had significantly greater amounts of abdominal fat, higher levels of leptin messenger RNA (mRNA), and a 2-fold increase in plasma leptin concentrations. Despite normal plasma glucose levels, hGHRH transgenic mice had 4.5-fold elevated levels of plasma insulin. Using a ribonuclease protection assay, we measured the mRNA levels of the OBR long form (OBR(L)) in the anterior pituitary and hypothalamus after 48 h of fasting. In the anterior pituitary, food deprivation induced dramatic increases in OBR(L) mRNA levels in both normal and transgenic mice. In contrast, in the hypothalamus, fasting resulted in a significant decrease in OBR(L) gene expression in normal mice, and no changes were detected in hGHRH transgenic mice. Using dual in situ hybridization, OBR(L) mRNA was detected in somatotrophs. Moreover, the number of OBR(L)-positive pituitary cells as well as the percentage of OBR(L)-positive cells that express GH mRNA were increased in transgenic mice. In conclusion, 1) the modest obesity in hGHRH transgenic mice is associated with increases in leptin synthesis and secretion as well as insulin secretion; 2) GH and/or GHRH as well as leptin and insulin may differentially contribute to the changes in OBR(L) gene expression in the anterior pituitary and the hypothalamus; 3) the response of OBR(L) gene expression in the hypothalamus to fasting is absent in the modestly obese hGHRH transgenic mice; and 4) somatotrophs are target cells for leptin, and the increase in OBR(L) gene expression in the pituitary of hGHRH transgenic mice is due at least in part to the increase in the number of cells expressing OBR(L).