Rapid inhibition of leptin signaling by glucocorticoids in vitro and in vivo

Elevated secretion of glucocorticoids (GCs) or hypersensitivity to GCs has a permissive effect on the development of obesity and leads to abnormalities of body fat distribution. Recent studies demonstrated GCs act as antagonists of leptin in rodents. However, little is known about the interaction between GCs and leptin signaling. In the present study, we investigated the effects of GCs on leptin action in vitro and in vivo. GCs rapidly inhibited the leptin-induced STAT3 phosphorylation in a dose- and time-dependent manner, as assayed by Western blotting using anti-phosphospecific-STAT3 in human hepatoma cell lines (Huh7) transiently expressing long form leptin receptor. GCs also inhibited the leptin-induced JAK2 tyrosine phosphorylation but unaltered the specific binding of (125)I-leptin to the cells. Parallel experiments, however, demonstrated that the inhibitory effects of GCs were not observed in either IL-6- or LIF-induced STAT3 phosphorylation. Furthermore, we examined the feeding behavior and hypothalamic leptin signaling following intracerebroventricular (icv) infusion of GCs prior to icv leptin infusion in Sprague-Dawley rats. The food intake after 24 h of icv leptin injection increased 3-fold in GCs-treated animals. In addition, central infusion of GCs resulted in a marked reduction of hypothalamic STAT3 phosphorylation in response to icv infusion of leptin. To clarify the molecular mechanism by which GCs rapidly reduce leptin-induced JAK/STAT signaling, we examined the intracellular signal transduction pathway potentially mediated by GCs. PD98059, a specific MEK inhibitor, blocked the inhibitory effects of GCs on leptin-induced JAK/STAT activation in Huh7 cells. These results suggest GCs antagonize leptin action by a rapid inhibition of the leptin-induced JAK/STAT pathway partly via MAPK cascade.     

Interaction between leptin and insulin signaling pathways differentially affects JAK-STAT and PI 3-kinase-mediated signaling in rat liver

Chronic leptin treatment markedly enhances the effect of insulin on hepatic glucose production unproportionally with respect to body weight loss and increased insulin sensitivity. In the present study the cross-talk between insulin and leptin was evaluated in rat liver. Upon stimulation of JAK2 tyrosine phosphorylation, leptin induced JAK2 co-immunoprecipitation with STAT3, STAT5b, IRS-1 and IRS-2. This phenomenon parallels the leptin-induced tyrosine phosphorylation of STAT3, STAT5b, IRS-1 and IRS-2. Acutely injected insulin stimulated a mild increase in tyrosine phosphorylation of JAK2, STAT3 and STAT5b. Leptin was less effective than insulin in stimulating IRS phosphorylation and their association with PI 3-kinase. Simultaneous treatment with both hormones yielded no change in maximal phosphorylation of STAT3, IRS-1, IRS-2 and Akt, but led to a marked increase in tyrosine phosphorylation of JAK2 and STAT5b when compared with isolated administration of insulin or leptin. This indicates that there is a positive cross-talk between insulin and leptin signaling pathways at the level of JAK2 and STAT5b in rat liver.     

Critical role of STAT3 in leptin's metabolic actions

Leptin has pleiotropic effects on glucose homeostasis and feeding behavior. Here, we validate the use of a cell-permeable phosphopeptide that blocks STAT3 activation in vivo. The combination of this biochemical approach with stereotaxic surgical techniques allowed us to pinpoint the contribution of hypothalamic STAT3 to the acute effects of leptin on food intake and glucose homeostasis. Leptin's ability to acutely reduce food intake critically depends on intact STAT3 signaling. Likewise, hypothalamic signaling of leptin through STAT3 is required for the acute effects of leptin on liver glucose fluxes. Lifelong obliteration of STAT3 signaling via the leptin receptor in mice (s/s mice) results in severe hepatic insulin resistance that is comparable to that observed in db/db mice, devoid of leptin receptor signaling. Our results demonstrate that the activation of the hypothalamic STAT3 pathway is an absolute requirement for the effects of leptin on food intake and hepatic glucose metabolism.     

Diet-induced obesity alters AMP kinase activity in hypothalamus and skeletal muscle

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance and of the effects of leptin on food intake and fatty acid oxidation. Obesity is usually associated with resistance to the effects of leptin on food intake and body weight. To determine whether diet-induced obesity (DIO) impairs the AMPK response to leptin in muscle and/or hypothalamus, we fed FVB mice a high fat (55%) diet for 10-12 weeks. Leptin acutely decreased food intake by approximately 30% in chow-fed mice. DIO mice tended to eat less, and leptin had no effect on food intake. Leptin decreased respiratory exchange ratio in chow-fed mice indicating increased fatty acid oxidation. Respiratory exchange ratio was low basally in high fat-fed mice, and leptin had no further effect. Leptin (3 mg/kg intraperitoneally) increased alpha2-AMPK activity 2-fold in muscle in chow-fed mice but not in DIO mice. Leptin decreased acetyl-CoA carboxylase activity 40% in muscle from chow-fed mice. In muscle from DIO mice, acetyl-CoA carboxylase activity was basally low, and leptin had no further effect. In paraventricular, arcuate, and medial hypothalamus of chow-fed mice, leptin inhibited alpha2-AMPK activity but not in DIO mice. In addition, leptin increased STAT3 phosphorylation 2-fold in arcuate of chow-fed mice, but this effect was attenuated because of elevated basal STAT3 phosphorylation in DIO mice. Thus, DIO in FVB mice alters alpha2-AMPK in muscle and hypothalamus and STAT3 in hypothalamus and impairs further effects of leptin on these signaling pathways. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.     

Neotomodon alstoni mice present sex differences between lean and obese in daily hypothalamic leptin signaling

This article compared the effects of spontaneous obesity on the daily profile in the relative amount of the leptin receptor (LepRb), and its output. That is the precursor Pro-opiomelanocortin (POMC) over a 24-hour period and compared with differences in locomotion and food intake in periods of artificial light. Differences between lean and obese mice were examined, as were sex differences. Body weight, food intake and locomotor activity were monitored in freely moving lean and obese mice. Hypothalamic tissue was collected at 5 h, 10 h, 15 h, 19 h and 24 h. Samples were analyzed by western blotting to determine the relative presence of protein for LepRb, STAT3 phosphorylation (by pSTAT3/STAT3 ratio) and POMC. Obese mice were 60% less active in locomotion than lean mice during the night. While both locomotor activity and food intake were noticeably greater during the day in obese mice than in lean mice, the hypothalamus in obese mice showed a lower relative abundance of POMC and reduced pSTAT3/STAT3 ratio and leptin receptors. Behavioral and biochemical differences were more evident in obese females than in obese males. These results indicate that obesity in N. alstoni affects hypothalamic leptin signaling according to sex.     

Molecular and functional resistance to insulin in hypothalamus of rats exposed to cold

Insulin and leptin act in the hypothalamus, providing robust anorexigenic signals. The exposure of homeothermic animals to a cold environment leads to increased feeding, accompanied by sustained low levels of insulin and leptin. In the present study, the initial and intermediate steps of the insulin-signaling cascade were evaluated in the hypothalamus of cold-exposed Wistar rats. By immunohistochemistry, most insulin receptor (IR) and insulin receptor substrate-2 (IRS-2) immunoreactivity localized to the arcuate nucleus. Basal levels of tyrosine phosphorylation of IR and IRS-2 were increased in cold-exposed rats compared with rats maintained at room temperature. However, after an acute, peripheral infusion of exogenous insulin, significantly lower increases of IR and IRS-2 tyrosine phosphorylation were detected in the hypothalamus of cold-exposed rats. Insulin-induced association of p85/phosphatidylinositol 3-kinase with IRS-2, Ser473 phosphorylation of Akt, and tyrosine phosphorylation of ERK was significantly reduced in the hypothalamus of cold-exposed rats. To test the hypothesis of functional impairment of insulin signaling in the hypothalamus, intracerebroventricularly cannulated rats were acutely treated with insulin, and food ingestion was measured over a period of 12 h. Cold-exposed animals presented a significantly lower insulin-induced reduction in food consumption compared with animals maintained at room temperature. Hence, the present studies reveal that animals exposed to cold are resistant, both at the molecular and the functional level, to the actions of insulin in the hypothalamus.     

Leptin-induced changes in body composition in high fat-fed mice

Female C57BL/6J mice were adapted to 10% or 45% kcal fat diets for 8 weeks. Continuous intraperitoneal infusion of 10 micro g of leptin/day from a miniosmotic pump transiently inhibited food intake in low fat-fed but not high fat-fed mice. In contrast, both low and high fat-fed leptin-infused mice were less fat than their phosphate-buffered saline (PBS) controls after 13 days. Leptin infusion inhibited insulin release but did not change glucose clearance in low fat-fed mice during a glucose tolerance test. A single intraperitoneal injection of 30 micro g of leptin inhibited 24-hr energy intake and inhibited weight gain in both low and high fat-fed mice. Insulin responsiveness was improved in high fat-fed mice during an insulin sensitivity test due to an exaggerated elevation of circulating insulin concentrations. Thus, leptin infusion reduced adiposity independently of energy intake in high fat-fed mice and improved insulin sensitivity in low fat-fed mice, whereas leptin injections, which produced much greater, but transient, increases in serum leptin concentration, inhibited energy intake in both low and high fat-fed mice.     

AdipoR1 mediates the anorexigenic and insulin/leptin-like actions of adiponectin in the hypothalamus

Adiponectin exerts an insulin-sensitizing effect, improving insulin action in peripheral tissues and restraining insulin resistance. Here, we explore the hypothesis that adiponectin can reproduce some of the actions of insulin/leptin in the hypothalamus. The presence of AdipoR1 and AdipoR2 was mapped to the arcuate and lateral hypothalamic nuclei. Icv adiponectin reduced food intake, which was accompanied by activation/engagement of IRS1/2, ERK, Akt, FOXO1, JAK2 and STAT3. All these actions were dependent on AdipoR1, since inhibition of this receptor, and not of AdipoR2, completely reversed the effects described above. Thus, adiponectin acts in the hypothalamus, activating elements of the canonical insulin and leptin signaling pathways and promoting reduction of food intake.     

Effects of intracerebroventricular leptin administration on feeding and sexual behaviors in lean and obese female Zucker rats

Obese Zucker rats (fa/fa) are characterized by inadequate leptin signaling caused by a mutation in the leptin receptor gene. Obese Zucker females are infertile and hyporesponsive to the inductive effects of ovarian hormones on sexual behaviors. Leptin treatment reverses aspects of reproductive dysfunction due to perturbations in energy balance in other animal models. Our first experiment tested the hypothesis that intracerebroventricular (icv) leptin administration would enhance the display of sexual behaviors in obese Zucker females. A second experiment compared lean and obese Zucker females' responses to leptin, during fed and fasted conditions. Ovariectomized (OVX) Zucker rats were implanted with lateral ventricular cannulae. In Experiment 1, fasted, obese females received estradiol benzoate, progesterone, and icv injections of 3, 18, or 36 microg murine leptin or vehicle. Leptin administration reduced food intake, but did not enhance sexual behaviors. In Experiment 2, steroid-replaced, OVX lean and obese females (from a different source than those in Experiment 1) received icv injections of vehicle or 3 or 36 microg leptin under fed and fasted conditions. Leptin treatment reduced food intake and weight gain in the fed, but not the fasted, condition in both genotypes. Sexual receptivity and locomotion were not affected, but icv leptin injections reduced proceptive behaviors in ad libitum-fed rats. These data confirm previous reports that centrally administered leptin decreases food intake and weight gain in obese Zucker rats; results from Experiment 2 suggest that lean and obese females are similarly responsive to these actions of leptin. Contrary to our hypothesis, leptin treatment did not stimulate sexual behaviors; rather, the hormone appears to inhibit the display of sexual proceptivity in ad libitum-fed lean and obese Zucker female rats.     

Central Exercise Action Increases the AMPK and mTOR Response to Leptin

AMP-activated protein kinase (AMPK) and mammalian Target of Rapamycin (mTOR) are key regulators of cellular energy balance and of the effects of leptin on food intake. Acute exercise is associated with increased sensitivity to the effects of leptin on food intake in an IL-6-dependent manner. To determine whether exercise ameliorates the AMPK and mTOR response to leptin in the hypothalamus in an IL-6-dependent manner, rats performed two 3-h exercise bouts, separated by one 45-min rest period. Intracerebroventricular IL-6 infusion reduced food intake and pretreatment with AMPK activators and mTOR inhibitor prevented IL-6-induced anorexia. Activators of AMPK and fasting increased food intake in control rats to a greater extent than that observed in exercised ones, whereas inhibitor of AMPK had the opposite effect. Furthermore, the reduction of AMPK and ACC phosphorylation and increase in phosphorylation of proteins involved in mTOR signal transduction, observed in the hypothalamus after leptin infusion, were more pronounced in both lean and diet-induced obesity rats after acute exercise. Treatment with leptin reduced food intake in exercised rats that were pretreated with vehicle, although no increase in responsiveness to leptin-induced anorexia after pretreatment with anti-IL6 antibody, AICAR or Rapamycin was detected. Thus, the effects of leptin on the AMPK/mTOR pathway, potentiated by acute exercise, may contribute to appetite suppressive actions in the hypothalamus.     

Brain insulin system dysfunction in streptozotocin intracerebroventricularly treated rats generates hyperphosphorylated tau protein

The intracerebroventricular (icv) application of streptozotocin (STZ) in low dosage was used in 3-month-old rats to explore brain insulin system dysfunction. Three months following STZ icv treatment, the expression of insulin-1 and -2 mRNA was significantly reduced to 11% in hippocampus and to 28% in frontoparietal cerebral cortex, respectively. Insulin receptor (IR) mRNA expression decreased significantly in frontoparietal cerebral cortex and hippocampus (16% and 33% of control). At the protein/activity level, different abnormalities of protein tyrosine kinase activity (increase in hippocampus), total IR beta-subunit (decrease in hypothalamus) and phosphorylated IR tyrosine residues (increase) became apparent. The STZ-induced disturbance in learning and memory capacities was not abolished by icv application of glucose transport inhibitors known to prevent STZ-induced diabetes mellitus. The discrepancy between reduced IR gene expression and increase in both phosphorylated IR tyrosine residues/protein tyrosine kinase activity may indicate imbalance between phosphorylation/dephosphorylation of the IR beta-subunit causing its dysfunction. These abnormalities may point to a complex brain insulin system dysfunction after STZ icv application, which may lead to an increase in hyperphosphorylated tau-protein concentration. Brain insulin system dysfunction is discussed as possible pathological core in the generation of hyperphosphorylated tau protein as a morphological marker of sporadic Alzheimer's disease.     

The pancreatic beta cell is a key site for mediating the effects of leptin on glucose homeostasis

The hormone leptin plays a crucial role in maintenance of body weight and glucose homeostasis. This occurs through central and peripheral pathways, including regulation of insulin secretion by pancreatic beta cells. To study this further in mice, we disrupted the signaling domain of the leptin receptor gene in beta cells and hypothalamus. These mice develop obesity, fasting hyperinsulinemia, impaired glucose-stimulated insulin release, and glucose intolerance, similar to leptin receptor null mice. However, whereas complete loss of leptin function causes increased food intake, this tissue-specific attenuation of leptin signaling does not alter food intake or satiety responses to leptin. Moreover, unlike other obese models, these mice have reduced fasting blood glucose. These results indicate that leptin regulation of glucose homeostasis extends beyond insulin sensitivity to influence beta cell function, independent of pathways controlling food intake. These data suggest that defects in this adipoinsular axis could contribute to diabetes associated with obesity.     

Hypothalamic clamp on insulin release by leptin-transgene expression

The effects of sustained leptin action locally in the hypothalamus on the functional link between fat accrual and insulin secretion after chronic high fat diet (HFD) consumption in leptin-deficient ob/ob mice, and on the post-prandial insulin response in rats consuming regular chow diet (RCD), was examined in this study. A single intracerebroventricular (icv) injection of recombinant adeno-associated virus vector encoding leptin gene (rAAV-lep) enhanced hypothalamic leptin-transgene expression in ob/ob mice consuming RCD and suppressed the time-related weight gain and fat accumulation concomitant with abrogation of hyperinsulinemia and enhanced glucose tolerance. This increased hypothalamic leptin-transgene expression continued to impose insulinopenia and increased glucose tolerance but was ineffective in suppressing weight gain and fat accumulation after these mice were switched to chronic HFD consumption. A similar icv rAAV-lep pretreatment in rats consuming RCD markedly attenuated the post-prandial rise in insulin release concomitant with suppressed weight and fat depots. These results show for the first time that a sustained hypothalamic leptin action can stably clamp pancreatic insulin secretion independent of the status of fat accrual engendered by diets of varying caloric enrichment. Thus, the efficacy of increased leptin afferent signaling in the hypothalamus to persistently restrain pancreatic insulin release and insulin resistance can be explored as an adjunct therapeutic modality to alleviate pathophysiological derrangements that confer type 2 diabetes.     

Intermittent fasting,adipokines, insulin sensitivity,and hypothalamic neuropeptides in a dietary overload with high-fat or high-fructose diet in mice

The intermittent fasting (IF) might have benefits on metabolism and food intake. Twelve-week old C57BL/6 J mice were fed a control diet (C, 10% kcal fat), a high-fat diet (HF, 50% kcal fat) or a high-fructose diet (HFru, 50% kcal fructose) for 8 weeks, then half of the animals in each group underwent IF (24 h fed, 24 h fasting) for an additional 4 weeks. Although food intake on the fed day remained the same for all groups, all fasting groups showed a reduction in body mass compared to their counterparts. IF reduced total cholesterol, triacylglycerol, fasting glucose, fasting insulin resistance index, and plasma leptin, but increased plasma adiponectin. IF reduced Leptin gene expression in the HF-IF group, but increased proinflammatory markers in the hypothalamus, also in the C-IF group. Both groups HFru-IF and C-IF, showed alterations in the leptin signaling pathway (Leptin, OBRb, and SOCS3), mainly in the HFru-IF group, suggesting leptin resistance. NPY and POMC neuropeptides labeled the neurons of the hypothalamus by immunofluorescence, corroborating qualitatively other quantitative findings of the study. In conclusion, current results are convincing in demonstrating the IF effect on central regulation of food intake control, as shown by NPY and POMC neuropeptide expressions, resulting in a lower weight gain. Besides, IF improves glycemia, lipid metabolism, and consequently insulin and leptin resistance. However, there is increased expression of inflammatory markers in mouse hypothalamus challenged by the HF and HFru diets, which in the long term may induce adverse effects.     

Leptin regulation of prepro-orexin and orexin receptor mRNA levels in the hypothalamus

The aim of this study was to determine the effects of leptin treatment on prepro-orexin and orexin receptor expression in the rat hypothalamus. Adult male rats, food-deprived for 48 and 72 h, were treated one time with vehicle or leptin (10 microg, icv). Prepro-orexin mRNA content was measured by semiquantitative RT-PCR, Northern blot, and in situ hybridization; orexin receptor 1 and 2 mRNA content was quantified by Northern blot and/or semiquantitative RT-PCR. Our results indicate that leptin inhibits a fasting-induced increase in prepro-orexin mRNA and orexin receptor 1 mRNA levels in the rat hypothalamus, while orexin receptor 2 mRNA levels were unchanged in all situations evaluated. These data provide direct evidence for an additional mechanism of adaptation of the hypothalamus to food deprivation and for a new effect of leptin in the regulation of food intake.     

Glucose, insulin, and leptin signaling pathways modulate nitric oxide synthesis in glucose-inhibited neurons in the ventromedial hypothalamus

Glucose-sensing neurons in the ventromedial hypothalamus (VMH) are involved in the regulation of glucose homeostasis. Glucose-sensing neurons alter their action potential frequency in response to physiological changes in extracellular glucose, insulin, and leptin. Glucose-excited neurons decrease, whereas glucose-inhibited (GI) neurons increase, their action potential frequency when extracellular glucose is reduced. Central nitric oxide (NO) synthesis is regulated by changes in local fuel availability, as well as insulin and leptin. NO is involved in the regulation of food intake and is altered in obesity and diabetes. Thus this study tests the hypothesis that NO synthesis is a site of convergence for glucose, leptin, and insulin signaling in VMH glucose-sensing neurons. With the use of the NO-sensitive dye 4-amino-5-methylamino-2',7'-difluorofluorescein in conjunction with the membrane potential-sensitive dye fluorometric imaging plate reader, we found that glucose and leptin suppress, whereas insulin stimulates neuronal nitric oxide synthase (nNOS)-dependent NO production in cultured VMH GI neurons. The effects of glucose and leptin were mediated by suppression of AMP-activated protein kinase (AMPK). The AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) increased both NO production and neuronal activity in GI neurons. In contrast, the effects of insulin on NO production were blocked by the phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Furthermore, decreased glucose, insulin, and AICAR increase the phosphorylation of VMH nNOS, whereas leptin decreases it. Finally, VMH neurons express soluble guanylyl cyclase, a downstream mediator of NO signaling. Thus NO may mediate, in part, glucose, leptin, and insulin signaling in VMH glucose-sensing neurons.