ATP synthesis is impaired in isolated mitochondria from myotubes established from type 2 diabetic subjects

Leptin resistance associated with hyperleptinemia in high-fat-diet-induced obese rats and aged obese rats is well established, but it is not clear whether hyperphagia-induced obese rats also develop leptin resistance. We investigated whether Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which are a strain of hyperphagia-induced obese rats, develop leptin resistance and whether caloric restriction reversed this leptin resistance-induced leptin receptor (ObRb) deficit. Twenty male OLETF rats, 20 male Long-Evans Tokushima Otsuka (LETO) rats, and 10 male Sprague Dawley (SD) rats were used. All rats were initially studied at 10 weeks of age and were freely fed with standard rat chow and water until they were 38 weeks of age. Daily food intake, body weight, and plasma leptin levels of OLETF rats were remarkably increased compared to LETO or SD rats from 10 to 38 weeks of age. When they were 38 weeks of age, all OLETF rats were randomly divided into two groups. One group was freely fed with standard rat chow (FD, or free diet group), and the other group (RD, or restricted diet group) was fed with only 70% of the amount consumed by the FD group. The LETO and SD rats were dismissed from further study. After 4 weeks of caloric restriction, the average body weight (636 ± 33 g vs. 752 ± 24 g, P < 0.05) and abdominal adipose tissue weight (10.6 ± 3.2 g vs. 15.8 ± 1.5 g, P < 0.05) of the RD group were decreased compared with those of the FD group. Plasma leptin levels of the RD group were significantly decreased compared with those of the FD group (3.47 ± 1.40 ng/mL vs. 11.55 ± 1.16 ng/mL, P < 0.05). The mRNA expression of ObRb and leptin-related suppressor of cytokine signaling 3 (SOCS3) in the hypothalamus, liver, and skeletal muscles of the RD group were significantly decreased compared with those of the FD group. Caloric restriction did not improve leptin receptor (ObRb) deficit or the downstream signaling of leptin in the liver, skeletal muscles, and hypothalamus. Thus, we demonstrated that OLETF rats, which are a strain of hyperphagia-induced obese rats, did not develop central or peripheral leptin resistance. We suggest that hyperleptinemia in OLETF rats is a compensatory mechanism to overcome obesity induced by hyperphagia.     

SOCS-3 expression in leptin-sensitive neurons of the hypothalamus of fed and fasted rats

Treatment of rodents with exogenous leptin increases SOCS-3 mRNA levels in the arcuate nucleus (ARC) and dorsomedial nucleus (DMN) of the hypothalamus. To determine if SOCS-3 gene activity in the hypothalamus could be influenced by changes in physiological levels of circulating leptin, we performed in situ hybridization (ISH) and immunostaining for SOCS-3 expression in fed vs. fasted (48 h) rats. The ARC and DMN were the only regions of the diencephalon that showed SOCS-3 ISH and the autoradiographic ISH signal for SOCS-3 mRNA was visibly less in the ARC and DMN of fasted rats. The ISH signal for SOCS-3 mRNA was decreased 70% in the ARC and 90% in the DMN (to background levels) when animals were fasted (P<0.01), consistent with decreased immunostaining for SOCS-3 protein observed in the fasted rats. Double fluorescence ISH (FISH) analyses showed colocalization of SOCS-3 mRNA with mRNAs for NPY and POMC in the ARC. These findings are consistent with increased leptin signaling to the NPY and POMC neurons in the ARC by physiological levels of circulating leptin during normal feeding. Therefore, changes in SOCS-3 mRNA levels in the ARC and DMN can be viewed as an indicator of relative physiological leptin signaling to the hypothalamus and also identify cells responding directly to leptin signaling through its cognate receptor.     

Leptin treatment increases suppressors of cytokine signaling in central and peripheral tissues.

Leptin concentrations are elevated in the majority of obese individuals raising the possibility that leptin resistance contributes to their obesity. Peripheral leptin administration for 48 h caused a several-fold increase in mRNA encoding the suppressors of cytokine signaling SOCS-3 and CIS in hypothalamus and peripheral tissues. Paradoxically, CIS and SOCS-3 mRNAs are also elevated in the leptin-deficient ob/ob mouse. Forced expression of CIS in insulinoma cells prevented transactivation mediated by leptin. Thus tissues continuously exposed to leptin and/or other factors associated with obesity accumulate excessive amounts of SOCS-3 and CIS which could provide a potential mechanism for leptin resistance.     

Diet-induced obesity leads to the development of leptin resistance in vagal afferent neurons

Ingestion of high-fat, high-calorie diets is associated with hyperphagia, increased body fat, and obesity. The mechanisms responsible are currently unclear; however, altered leptin signaling may be an important factor. Vagal afferent neurons (VAN) integrate signals from the gut in response to ingestion of nutrients and express leptin receptors. Therefore, we tested the hypothesis that leptin resistance occurs in VAN in response to a high-fat diet. Sprague-Dawley rats, which exhibit a bimodal distribution of body weight gain, were used after ingestion of a high-fat diet for 8 wk. Body weight, food intake, and plasma leptin levels were measured. Leptin signaling was determined by immunohistochemical localization of phosphorylated STAT3 (pSTAT3) in cultured VAN and by quantifaction of pSTAT3 protein levels by Western blot analysis in nodose ganglia and arcuate nucleus in vivo. To determine the mechanism of leptin resistance in nodose ganglia, cultured VAN were stimulated with leptin alone or with lipopolysaccharide (LPS) and SOCS-3 expression measured. SOCS-3 protein levels in VAN were measured by Western blot following leptin administration in vivo. Leptin resulted in appearance of pSTAT3 in VAN of low-fat-fed rats and rats resistant to diet-induced obesity but not diet-induced obese (DIO) rats. However, leptin signaling was normal in arcuate neurons. SOCS-3 expression was increased in VAN of DIO rats. In cultured VAN, LPS increased SOCS-3 expression and inhibited leptin-induced pSTAT3 in vivo. We conclude that VAN of diet-induced obese rats become leptin resistant; LPS and SOCS-3 may play a role in the development of leptin resistance.     

The role of SOCS-3 in leptin signaling and leptin resistance.

We earlier demonstrated that leptin induces expression of SOCS-3 mRNA in the hypothalamus. Furthermore, transfection data suggest that SOCS-3 is an inhibitor of leptin signaling. However, little is known about the regulation of SOCS-3 expression by leptin and the mechanism by which SOCS-3 inhibits leptin action. We here show that in CHO cells stably expressing the long form of the leptin receptor (CHO-OBRl), leptin induces transient expression of endogenous SOCS-3 mRNA but not of CIS, SOCS-1, or SOCS-2 mRNA. SOCS-3 protein levels were maximal after 2-3 h of leptin treatment and remained elevated at 20 h. Furthermore, in leptin-pretreated CHO-OBRl cells, proximal leptin signaling was blocked for more than 20 h after pretreatment, thus correlating with increased SOCS-3 expression. Leptin pretreatment did not affect cell surface expression of leptin receptors as measured by (125)I-leptin binding assays. In transfected COS cells, forced expression of SOCS-3 results in inhibition of leptin-induced tyrosine phosphorylation of JAK2. Finally, JAK2 co-immunoprecipitates with SOCS-3 in lysates from leptin-treated COS cells. These results suggest that SOCS-3 is a leptin-regulated inhibitor of proximal leptin signaling in vivo. Excessive SOCS-3 activity in leptin-responsive cells is therefore a potential mechanism for leptin resistance, a characteristic feature in human obesity.     

Hypothalamic response to leptin changes during a hormonally induced estrous cycle in rats

The leptin system regulates body fat mass through a feedback loop between adipose tissue and the hypothalamus. To test if leptin responsiveness may be regulated we assayed hypothalamic response to leptin during the estrous cycle; when changes in food intake are known to occur. Immature rats were treated with pregnant mare’s serum gonadotropin (PMSG) to induce synchronized follicular development, ovulation and corpus luteum formation. Leptin response was estimated by measuring the in vitro induction of tis11, a primary response gene activated by STAT3-dependent cytokines in hypothalamic explants after leptin stimulation. In addition, mRNA levels of the suppressor cytokine signaling-3 (SOCS-3), a possible mediator of leptin resistance, were analyzed. Serum leptin levels did not change between days 2 and day 3 (corresponding to proestrus and estrus, respectively) but the response to leptin was higher on day 2 than on day 3 (p=0.05). Food intake displayed a tendency towards downregulation between day 1 and day 2 (p=0.057), and a tendency towards upregulation between day 2 and day 3 (p=0.072), although the body weight increased on day of the study (p<0.0001). There was no significant difference in hypothalamic expression of SOCS-3 between day 2 and day 3. In conclusion, we have shown that leptin responsiveness changes during a hormonally induced estrous cycle in rats. Our data suggest that a change in the hypothalamic response to leptin may cause the cyclic feeding behavior seen in rats.     

The role of leptin in striped hamsters subjected to food restriction and refeeding

Food restriction(FR) and refeeding(Re) have been suggested to impair body mass regulation and thereby making it easier to regain the lost weight and develop over-weight when FR ends. However, it is unclear if this is the case in small mammals showing seasonal forging behaviors. In the present study, energy budget, body fat and serum leptin level were measured in striped hamsters that were exposed to FR-Re. The effects of leptin on food intake, body fat and genes expressions of several hypothalamus neuropeptides were determined. Body mass, fat content and serum leptin level decreased during FR and then increased during Re. Leptin supplement significantly attenuated the increase in food intake during Re, decreased genes expressions of neuropepetide Y(NPY) and agouti-related protein(AgRP) of hypothalamus and leptin of white adipose tissue(WAT). Hormone-sensitive lipase(HSL) gene expression of WAT increased in leptin-treated hamsters that were fed ad libitum, but decreased in FR-Re hamsters. This indicates that the adaptive regulation of WAT HSL gene expression may be involved in the mobilization of fat storage during Re, which partly contributes to the resistance to FR-Re-induced overweight. Leptin may be involved in the down regulations of hypothalamus orexigenic peptides gene expression and consequently plays a crucial role in controlling food intake when FR ends.     

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.     

Induction of leptin resistance by activation of cAMP-Epac signaling

Leptin regulates energy balance and glucose homeostasis. Shortly after leptin was identified, it was established that obesity is commonly associated with leptin resistance, though the molecular mechanisms remain to be identified. To explore potential mechanisms of leptin resistance, we employed organotypic brain slices to identify candidate signaling pathways that negatively regulate leptin sensitivity. We found that elevation of adenosine 3', 5'-monophosphate (cAMP) levels impairs multiple signaling cascades activated by leptin within the hypothalamus. Notably, this effect is independent of protein kinase A activation. In contrast, activation of Epac, a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap1, was sufficient to impair leptin signaling with concomitant induction of SOCS-3 expression. Epac activation also blunted leptin-induced depolarization of hypothalamic POMC neurons. Finally, central infusion of an Epac activator blunted the anorexigenic actions of leptin. Thus, activation of hypothalamic cAMP-Epac pathway is sufficient to induce multiple indices of leptin resistance.     

SOCS-1 deficiency does not prevent diet-induced insulin resistance

Obesity is associated with inflammation and increased expression of suppressor of cytokine signaling (SOCS) proteins, which inhibit cytokine and insulin signaling. Thus, reducing SOCS expression could prevent the development of obesity-induced insulin resistance. Using SOCS-1 knockout mice, we investigated the contribution of SOCS-1 in the development of insulin resistance induced by a high-fat diet (HFD). SOCS-1 knockout mice on HFD gained 70% more weight, displayed a 2.3-fold increase in epididymal fat pads mass and increased hepatic lipid content. This was accompanied by increased mRNA expression of leptin and the macrophage marker CD68 in white adipose tissue and of SREBP1c and FAS in liver. HFD also induced hyperglycemia in SOCS-1 deficient mice with impairment of glucose and insulin tolerance tests. Thus, despite the role of SOCS proteins in obesity-related insulin resistance, SOCS-1 deficiency alone is not able to prevent insulin resistance induced by a diet rich in fat.     

Downregulation of melanocortin-4 receptor during refeeding and its modulation by adrenalectomy in rats

Melanocortin system and corticotropin releasing hormone (CRH) are implicated in the control of feeding behavior. Besides its anorexigenic effect on food intake, CRH is one of the most important regulators of hypothalamic-pituitary-adrenal (HPA) axis activity. Therefore, there could be an interplay between HPA axis activity and melanocortin system. We investigated the expression of melanocortin-4 receptor (MC4-R) mRNA in the hypothalamus of rats after 14 days of food restriction or after a fasting-refeeding regimen, in sham or adrenalectomized rats. Male Wistar rats were subjected to free access to food or food ingestion restricted for 2 h a day (8-10 AM) during 14 d, when plasma corticosterone, ACTH, insulin, leptin concentrations, and MC4-R mRNA expression were determined before and after refeeding. Another set of rats was fasted for 48 h, followed by refeeding during 2 or 4 h on the seventh day after adrenalectomy (ADX) or sham surgery. On the day of the experiment, rats were anesthetized and perfused and the brain processed for MC4-R mRNA by in situ hybridization. Long-term reduction of food intake, either secondary to food restriction or adrenalectomy, reduced body weight gain and also leptin and insulin plasma concentrations. Food ingestion reduced MC4-R expression in the paraventricular nucleus in naive rats subjected to food restriction and also in sham rats fasted for 48 h. However, after ADX, MC4-R expression was not changed by refeeding. In conclusion, the present data indicate that MC4-R expression is downregulated by food ingestion and this response could be modulated by glucocorticoid withdrawal.     

Expression of hypothalamic neuropeptides and the desensitization of pituitary-adrenal axis and hypophagia in the endotoxin tolerance

Repeated exposure to lipopolysaccharide (LPS) induces desensitization of hypothalamus-pituitary-adrenal axis (HPA) responses and hypophagia. We investigated the interplay between the neural circuitries involved in the control of food intake and HPA axis activity following single or repeated LPS injections. Male Wistar rats received a single or repeated i.p. injection of LPS (100 microg/kg) for 6 days and were subdivided into four groups: 6 saline, 5 saline+1 LPS, 5 LPS+1 saline and 6 LPS. Animals with a single exposure to LPS showed increased plasma levels of ACTH, CORT, PRL, TNF-alpha and also CRF mRNA in the paraventricular nucleus of the hypothalamus. These animals exhibited a reduced food intake and body weight associated with an increase of CART expression in the arcuate nucleus (ARC). Leptin plasma levels were not altered. On the other hand, repeated LPS administration did not alter ACTH, CORT, PRL and TNF-alpha, but it reduced leptin level, compared to single LPS or saline treatment. Furthermore, repeated LPS administration did not increase CRF or CART mRNA expression. Food intake and weight gain after repeated LPS injections were not different from saline-treated animals. There was no change in NPY and POMC mRNA expression in the ARC after single or repeated injections of LPS. In conclusion, desensitization induced by repeated exposure to LPS involves the blockade of HPA axis activation and anorexigenic response, which are both associated with an unresponsiveness of TNF-alpha production and CRF and CART expression in the hypothalamus.     

A Dietary Fat Excess Alters Metabolic and Neuroendocrine Responses Before the Onset of Metabolic Diseases

Early changes in neuroendocrine pathways are essential in the development of metabolic pathologies. Thus, it is important to have a better understanding of the signals involved in their initiation. Long-term consumption of high-fat diets induces insulin resistance, obesity, diabetes. Here, we have investigated early neural and endocrine events in the hypothalamus and hippocampus induced by a short-term high fat, low carbohydrate diet in adult male Wistar rats. The release of serotonin, which is closely associated with the actions of insulin and leptin, was measured, by electrochemical detection following reverse-phase liquid chromatography (HPLC), in the extracellular space of the medial hypothalamus and the dorsal hippocampus in samples obtained from non-anesthetized animals, by microdialysis. The high-fat diet had a specific effect on the hypothalamus. Serotonin release induced by food intake was reduced after 1 week, and effectively ceased after 6 weeks of the diet. After 1 week, there was an increased gene expression of the insulin receptor and the insulin receptor substrates IRS1 and IRS2, as measured by real-time PCR. After 6 weeks of diet, insulin gene expression increased. Leptinemia increased in all cases. This new data support the concept that high-fat diets, in addition to have peripheral effects, cause a rapid alteration in specific central mechanisms involved in energy and glucose homeostasis. The changes in the gene expression of insulin and signaling elements represent possible adaptations aimed at counterbalancing the reduced responsiveness of the serotonergic system to nutritional signals and maintaining homeostasis. Sophie M. Banas and Claude Rouch have contributed equally to this work.     

Sex-associated differences in the leptin and ghrelin systems related with the induction of hyperphagia under high-fat diet exposure in rats

Leptin and ghrelin are known to be main hormones involved in the control of food intake, with opposing effects. Here we have explored whether changes in the leptin and ghrelin system are involved in the long-term effects of high-fat (HF) diet feeding in rats and whether sex-associated differences exist. Male and female Wistar rats were fed until the age of 6 months with a normal-fat (NF) or an HF-diet. Food intake and body weight were followed. Gastric and serum levels of leptin and ghrelin, and mRNA levels of leptin (in stomach and adipose tissue), ghrelin (in stomach), and NPY, POMC, and leptin and ghrelin receptors (OB-Rb and GHS-R) (in the hypothalamus) were measured. In both males and females, total caloric intake and body weight were greater under the HF-diet feeding. In females, circulating ghrelin levels and leptin mRNA expression in the stomach were higher under HF-diet. HF-diet feeding also resulted in higher hypothalamic NPY/POMC mRNA levels, more marked in females, and in lower OB-Rb mRNA levels, more marked in males. In addition, in females, serum ghrelin levels correlated positively with hypothalamic NPY mRNA levels, and these with caloric intake. In males, hypothalamic OB-Rb mRNA levels correlated positively with POMC mRNA levels and these correlated negatively with caloric intake and with body weight. These data reflect differences between sexes in the effects of HF-diet feeding on food intake control systems, suggesting an impairment of the anorexigenic leptin-POMC system in males and an over-stimulation of the orexigenic ghrelin-NPY system in females.     

Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area.

Recent studies have reinforced the view that the lateral hypothalamic area (LHA) regulates food intake and body weight. We identified leptin-sensitive neurons in the arcuate nucleus of the hypothalamus (Arc) that innervate the LHA using retrograde tracing with leptin administration. We found that retrogradely labeled cells in the Arc contained neuropeptide Y (NPY) mRNA or proopiomelanocortin (POMC) mRNA. Following leptin administration, NPY cells in the Arc did not express Fos but expressed suppressor of cytokine signaling-3 (SOCS-3) mRNA. In contrast, leptin induced both Fos and SOCS-3 expression in POMC neurons, many of which also innervated the LHA. These findings suggest that leptin directly and differentially engages NPY and POMC neurons that project to the LHA, linking circulating leptin and neurons that regulate feeding behavior and body weight homeostasis.