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The prevalence of obesity is rising, creating an urgent need for efficacious therapies. Recent clinical trials show that tirzepatide, a dual agonist of receptors for the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), yields more weight loss than selective GLP-1 receptor (GLP-1R) agonists. Incretin receptors in the central nervous system (CNS) may contribute to these effects. Yet exactly how each receptor regulates body weight from within the CNS is not clearly understood. It remains especially unclear how GIP receptor (GIPR) signalling contributes to the effects of tirzepatide because both stimulation and inhibition of CNS GIPRs yield weight loss in preclinical models. We summarise current knowledge on CNS incretin receptor pharmacology to provide insight into the potential mechanisms of action of dual GIPR/GLP-1R agonists, with tirzepatide as the exemplar. In addition, we discuss recent developments in incretin-based dual- and tri-agonism for inducing weight loss in obese individuals.

The importance of fatty acids (FAs) for healthy brain development and function has become more evident in the past decades. However, most studies focus on the hypothalamus as an important FA-sensing brain region involved in energy homeostasis. Less work has been done to evaluate the effects of FAs on brain regions such as the hippocampus or cortex, two important centres of learning, memory formation, and cognition. Furthermore, the mechanisms of how FAs modulate the neuronal development and function are incompletely understood. Therefore, this study examined the effects of the saturated FA palmitic acid (PA) and the polyunsaturated FA docosahexaenoic acid (DHA) on primary hippocampal and cortical cultures isolated from P0/P1 Sprague Dawley rat pups. Exposure to PA, but not DHA, resulted in severe morphological changes in primary neurons such as cell body swelling, axonal and dendritic blebbing, and a reduction in synaptic innervation, compromising healthy cell function and excitability. Pharmacological assessment revealed that the PA-mediated alterations were caused by overactivation of neuronal insulin signaling, demonstrated by insulin stimulation and phosphoinositide 3-kinase inhibition. Remarkably, co-exposure to DHA prevented all PA-induced morphological changes. This work provides new insights into how FAs can affect the cytoskeletal rearrangements and neuronal function via modulation of insulin signaling.

Leptin is best known for its role in adipostasis, but it also regulates blood glucose levels. The molecular mechanism by which leptin controls glucose homeostasis remains largely unknown. Here, we use a zebrafish model to show that Wnt signaling mediates the glucoregulatory effects of leptin. Under normal feeding conditions, leptin regulates glucose homeostasis but not adipostasis in zebrafish. In times of nutrient excess, however, we found that leptin also regulates body weight and size. Using a Wnt signaling reporter fish, we show that leptin activates the canonical Wnt pathway in vivo. Utilizing two paradigms for hyperglycemia, it is revealed that leptin regulates glucose homeostasis via the Wnt pathway, as pharmacological inhibition of this pathway impairs the glucoregulatory actions of leptin. Our results may shed new light on the evolution of the physiological function of leptin.

Synchronization between biologic clocks and metabolism is crucial for most species. Here, we examined the ability of leptin, important in the control of energy metabolism, to induce leptin signaling at the molecular as well as the behavioral level throughout the 24-h day in mice fed either a control or a high-fat diet (HFD). Furthermore, we investigated the effects of time-restricted feeding (TRF; a limitation of HFD access to 6 h each day) on energy metabolism during different periods throughout the 24-h day. In control mice, molecular leptin sensitivity was highest at zeitgeber time (ZT)0 (lights on), declining during the light phase, and increasing during the dark phase. Surprisingly, leptin resistance in HFD-fed mice was only present from the middle of the dark to the middle of the light period. Specifically, when TRF occurred from ZT21 to ZT3 (when leptin resistance in HFD-fed mice was most profound), it resulted in a disruption of the daily rhythms of locomotor activity and energy expenditure and in increased plasma insulin levels compared with other TRF periods. These data provide evidence that leptin sensitivity is controlled by the circadian rhythm and that TRF periods may be most efficient when aligned with the leptin-sensitive period.—Boucsein, A., Rizwan, M. Z., Tups, A. Hypothalamic leptin sensitivity and health benefits of time-restricted feeding are dependent on the time of day in male mice.

Recent evidence suggests that the circadian timing system plays a role in energy and glucose homeostasis, and disruptions to this system are a risk factor for the development of metabolic disorders. We exposed animals to a constantly shifting lighting environment comprised of a 6-hour advance, occurring every 6 days, to chronically disrupt their circadian timing system. This treatment caused a gradual increase in body weight of 12 ± 2% after 12 phase shifts, compared with a 6 ± 1% increase in mice under control lighting conditions. Additionally, after the fifth phase shift, light cycle–disrupted (CD) animals showed a reversal in their diurnal pattern of energy homeostasis and locomotor activity, followed by a subsequent loss of this rhythm. To investigate potential molecular mechanisms mediating these metabolic alterations, we assessed central leptin and insulin sensitivity. We discovered that CD mice had a decrease in central leptin signaling, as indicated by a reduction in the number of phosphorylated signal transducer and activator of transcription 3 immunoreactive cells in the arcuate nucleus of the hypothalamus. Furthermore, CD animals exhibited a marked increase in fasting blood glucose (269.4 ± 21.1 mg/dL) compared with controls (108.8 ± 21.3 mg/dL). This dramatic increase in fasting glucose levels was not associated with an increase in insulin levels, suggesting impairments in pancreatic insulin release. Peripheral hyperglycemia was accompanied by central alterations in insulin signaling at the level of phospho Akt and insulin receptor substrate 1, suggesting that light cycle disruption alters central insulin signaling. These results provide mechanistic insights into the association between light cycle disruption and metabolic disease.

Metabolic syndrome and Alzheimer's disease (AD) are two major health issues in modern society causing an extraordinary financial burden for the global healthcare systems. A tight link between the pathologies of obesity and type 2 diabetes (T2D), and more recently between T2D and AD, has been discovered. Furthermore, in recent years it has become apparent that the circadian clock has an important function in controlling metabolism. This review integrates the role of the circadian clock in the development of these metabolic derangements and vice versa. Common features such as central insulin resistance, altered glycogen synthase kinase 3β (GSK3β) signalling, and central inflammation are discussed, and therapeutic interventions targeting those mechanisms are mentioned briefly.

Energy homeostasis is controlled by an intricate regulatory system centred in the brain. The peripheral adiposity signals insulin and leptin play a crucial role in this system by informing the brain of the energy status of the body and mediating their catabolic effects through signal transduction in hypothalamic areas that control food intake, energy expenditure and glucose metabolism. Disruptions of insulin and leptin signalling can result in diabetes and obesity. The central signalling cross-talk between insulin and leptin is essential for maintenance of normal healthy energy homeostasis. An important role of leptin in glucoregulation has been revealed. Typically regarded as being controlled by insulin, the control of glucose homeostasis critically depends on functional leptin action. Leptin, on the other hand, is able to lower glucose levels in the absence of insulin, although insulin is necessary for long-term stabilisation of euglycaemia. Evidence from rodent models and human patients suggests that leptin improves insulin sensitivity in type 1 diabetes. The signalling cross-talk between insulin and leptin is likely conveyed by the WNT/β-catenin pathway. Leptin activates WNT/β-catenin signalling, leading to inhibition of glycogen synthase kinase-3β, a key inhibitor of insulin action, thereby facilitating improved insulin signal transduction and sensitisation of insulin action. Interestingly, insights into the roles of insulin and leptin in insects and fish indicate that leptin may have initially evolved as a glucoregulatory hormone and that its anorexigenic and body weight regulatory function was acquired throughout evolution. Furthermore, the regulation of both central and peripheral control of energy homeostasis is tightly controlled by the circadian clock, allowing adaptation of homeostatic processes to environmental cues.

SummaryLeptin is a body weight regulatory hormone, but it is arguably even more potent at regulating blood glucose levels. To further our understanding of the molecular mechanisms by which leptin controls glucose homeostasis, we have used transgenic zebrafish models and conditional deletion of beta catenin in the mediobasal hypothalamus of adult mice to show that Wnt signalling in the brain mediates glucoregulatory effects of leptin. In zebrafish, under normal feeding conditions, leptin regulates glucose homeostasis but not adipostasis. In times of nutrient excess, we found that leptin also regulates body weight and size in this species. Using a Wnt signalling reporter fish, we show that leptin directly activates the canonical Wnt pathway in vivo. Pharmacological inhibition of this pathway prevented the leptin-induced improvement in glucose tolerance. In adult mice, conditional deletion of the key Wnt effector molecule, β-catenin, in the mediobasal hypothalamus of male mice confirmed the essential role of the Wnt pathway in mediating leptin action and the neuroendocrine regulation of glucose homeostasis. Adult-onset β-catenin deletion in the mediobasal hypothalamus led to glucose intolerance, exacerbation of caloric intake and body weight gain under high fat diet, as well as resistance to exogenous leptin.

Obesity has emerged as a major risk factor for insulin resistance leading to the development of type 2 diabetes (T2D). The condition is characterized by high circulating levels of the adipose-derived hormone leptin and a state of chronic low-grade inflammation. Pro-inflammatory signaling in the hypothalamus is associated with a decrease of central leptin- and insulin action leading to impaired systemic glucose tolerance. Intriguingly, leptin not only regulates body weight and glucose homeostasis but also acts as a pro-inflammatory cytokine. Here we demonstrate that increasing leptin levels (62,5 µg/kg/d, PEGylated leptin) in mice fed a high-fat diet (HFD) exacerbated body weight gain and aggravated hypothalamic micro- as well as astrogliosis. In contrast, administration of a predetermined dose of a long-acting leptin antagonist (100 µg/kg/d, PESLAN) chosen to block excessive leptin signaling during diet-induced obesity (DIO) showed the opposite effect and significantly improved glucose tolerance as well as decreased the total number of microglia and astrocytes in the hypothalamus of mice fed HFD. These results suggest that high levels of leptin, such as in obesity, worsen HFD-induced micro-and astrogliosis, whereas the partial reduction of hyperleptinemia in DIO mice may have beneficial metabolic effects and improves hypothalamic gliosis.

To induce diet-induced obesity (DIO) in rodents, diets high in saturated fat and/or carbohydrates are commonly used. In the laboratory, standardised diets evolved over time without paying particular attention to the effect of fat composition on metabolic alterations. In the present study, customised high-fat diets (HFD) enriched with a combination of lard and different concentrations of New Zealand green-lipped mussel (Perna canaliculus) oil or MSC Hoki (Macruronus novaezelandiae, blue grenadier) liver oil, important sources of n-3 PUFA, in comparison with a solely lard-based diet, were fed to lean and DIO male C57BL/6 mice and their effects on metabolic parameters were monitored. Intriguingly, an isoenergetic HFD containing 63 % of total fat in the form of mussel oil and only 28 % in the form of lard attenuated HFD-induced body weight gain after 1 and 4 weeks, respectively. Consistently, changing a lard-enriched HFD to the mussel oil diet reduced body weight markedly even after mice had been exposed to the former diet for 10 months. The weight-reducing effect of the diet was not caused by altered energy intake or expenditure, but was associated with reduced visceral fat mass. Collectively, these data suggest a novel weight-reducing potential of green-lipped mussel oil.

The ability of the brain to directly control glucose levels in the blood independently of its effects on food intake and body weight has been known ever since 1854 when Claude Bernard, a French physiologist, discovered that lesioning the floor of the fourth ventricle in rabbits led to a rise of sugar in the blood. Despite this outstanding discovery at that time, it took more than 140 years before progress started to be made in identifying the underlying mechanisms of brain-mediated control of glucose homeostasis. Technological advances including the generation of brain insulin receptor null mice revealed that insulin action specifically in the central nervous system is required for the regulation of glucose metabolism, particularly in the modulation of hepatic glucose production. Furthermore, it was established that the hormone leptin, known for its role in regulating food intake and body weight, actually exerts its most potent effects on glucose metabolism, and that this function of leptin is mediated centrally. Under certain circumstances, high levels of leptin can replicate the actions of insulin, thus challenging the idea that life without insulin is impossible. Disruptions of central insulin signaling and glucose metabolism not only lead to impairments in whole body glucose homeostasis, they also have other serious consequences, including the development of Alzheimer's disease which is sometimes referred to as type 3 diabetes reflecting its common etiology with type 2 diabetes. © 2017 American Physiological Society. Compr Physiol 7:471-764, 2017.

Saturated fatty acids are implicated in the development of metabolic diseases, including obesity and type 2 diabetes. There is evidence, however, that polyunsaturated fatty acids can counteract the pathogenic effects of saturated fatty acids. To gain insight into the early molecular mechanisms by which fatty acids influence hypothalamic inflammation and insulin signalling, we performed time-course experiments in a hypothalamic cell line, using different durations of treatment with the saturated fatty acid palmitate, and the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA). Western blot analysis revealed that palmitate elevated the protein levels of phospho(p)AKT in a time-dependent manner. This effect is involved in the pathogenicity of palmitate, as temporary inhibition of the PI3K/AKT pathway by selective PI3K inhibitors prevented the palmitate-induced attenuation of insulin signalling. Similar to palmitate, DHA also increased levels of pAKT, but to a weaker extent. Co-administration of DHA with palmitate decreased pAKT close to the basal level after 8 h, and prevented the palmitate-induced reduction of insulin signalling after 12 h. The monounsaturated fatty acid oleate had a similar effect on the palmitate-induced attenuation of insulin signalling, the polyunsaturated fatty acid linoleate had no effect. Measurement of the inflammatory markers pJNK and pNFκB-p65 revealed tonic elevation of both markers in the presence of palmitate alone. DHA alone transiently induced elevation of pJNK, returning to basal levels by 12 h treatment. Co-administration of DHA with palmitate prevented palmitate-induced inflammation after 12 h, but not at earlier timepoints.

Saturated fatty acids are implicated in the development of metabolic diseases, including obesity and type 2 diabetes. There is evidence, however, that polyunsaturated fatty acids can counteract the pathogenic effects of saturated fatty acids. To gain insight into the early molecular mechanisms by which fatty acids influence hypothalamic inflammation and insulin resistance, we performed time-course experiments in a hypothalamic cell line, using different durations of treatment with the saturated fatty acid palmitate, and the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA). Western blot analysis revealed that palmitate elevated the protein levels of phospho(p)AKT in a time-dependent manner. This effect seems involved in the pathogenicity of palmitate, as temporary inhibition of the PI3K/AKT pathway by selective PI3K inhibitors prevented palmitate-induced insulin resistance. Similarly to palmitate, DHA also increased levels of pAKT, but to a weaker extent. Co-administration of DHA with palmitate decreased pAKT close to the basal level after 8 h, and prevented palmitate-induced insulin resistance after 12 h. Measurement of the inflammatory markers pJNK and pNFκB-p65 revealed tonic elevation of both markers in the presence of palmitate alone. DHA alone transiently induced elevation of pJNK, returning to basal levels by 12 h treatment. Co-administration of DHA with palmitate prevented palmitate-induced inflammation after 12 h, but not at earlier time points.

β-catenin is a multifunctional protein that can act in the canonical Wnt/β-catenin pathway to regulate gene expression but can also bind to cadherin proteins in adherens junctions where it plays a key role in regulating cytoskeleton linked with these junctions. Recently, evidence has been presented indicating an essential role for β-catenin in regulating trafficking of insulin vesicles in β-cells and showing that changes in nutrient levels rapidly alter levels of β-catenin in these cells. Given the importance of neuroendocrine hormone secretion in the regulation of whole body glucose homeostasis, the objective of this study was to investigate whether β-catenin signalling is regulated in the hypothalamus during the normal physiological response to food intake. Rats were subjected to a fasting/re-feeding paradigm, and then samples collected at specific timepoints for analysis of β-catenin expression by immunohistochemistry and Western blotting. Changes in gene expression were assessed by RT-qPCR. Using immunohistochemistry, feeding acutely increased detectable cytoplasmic levels of β-catenin ('stabilized β-catenin') in neurons in specific regions of the hypothalamus involved in metabolic regulation, including the arcuate, dorsomedial and paraventricular nuclei of the hypothalamus. Feeding-induced elevations in β-catenin in these nuclei were associated with increased transcription of several genes that are known to be responsive to Wnt/β-catenin signalling. The effect of feeding was mimicked by administration of the GLP-1 agonist exendin-4, and was characterized by cAMP-dependent phosphorylation of β-catenin at serine residues 552 and 675. The data suggest that β-catenin/TCF signalling is involved in metabolic sensing in the hypothalamus. This article is protected by copyright. All rights reserved.

The WNT pathway was shown to play an important role in the adult central nervous system. We previously identified the WNT pathway as a novel integration site of the adipokine leptin in mediating its neuroendocrine control of metabolism in obese mice. Here we investigated the implication of WNT signaling in seasonal body weight regulation exhibited by the Djungarian hamster (Phodopus sungorus), a seasonal mammal that exhibits profound annual changes in leptin sensitivity. We furthermore investigated whether crucial components of the WNT pathway are regulated in a diurnal manner. Gene expression of key components of the WNT pathway in the hypothalamus of hamsters acclimated to either long day (LD) or short day (SD) photoperiod was analyzed by in situ hybridization. We detected elevated expression of the genes WNT-4, Axin-2, Cyclin-D1, and SFRP-2, in the hypothalamic arcuate nucleus, a key energy balance integration site, during LD compared with SD as well as a diurnal regulation of Axin-2, Cyclin-D1, and DKK-3. Investigating the effect of photoperiod as well as leptin on the activation (phosphorylation) of the WNT coreceptor LRP-6-(Ser1490) by immunohistochemistry, we found elevated activity in the arcuate nucleus during LD relative to SD as well as after leptin treatment (2 mg/kg body weight). These findings indicate that differential WNT signaling may be associated with seasonal body weight regulation and is partially regulated in a diurnal manner in the adult brain. Furthermore, they suggest that this pathway plays a key role in the neuroendocrine regulation of body weight and integration of the leptin signal.

Background Energy homeostasis is regulated by the hypothalamus but fails when animals are fed a high-fat diet (HFD), and leptin insensitivity and obesity develops. To elucidate the possible mechanisms underlying these effects, a microarray-based transcriptomics approach was used to identify novel genes regulated by HFD and leptin in the mouse hypothalamus. Results Mouse global array data identified serpinA3N as a novel gene highly upregulated by both a HFD and leptin challenge. In situ hybridisation showed serpinA3N expression upregulation by HFD and leptin in all major hypothalamic nuclei in agreement with transcriptomic gene expression data. Immunohistochemistry and studies in the hypothalamic clonal neuronal cell line, mHypoE-N42 (N42), confirmed that alpha 1-antichymotrypsin (α1AC), the protein encoded by serpinA3, is localised to neurons and revealed that it is secreted into the media. SerpinA3N expression in N42 neurons is upregulated by palmitic acid and by leptin, together with IL-6 and TNFα, and all three genes are downregulated by the anti-inflammatory monounsaturated fat, oleic acid. Additionally, palmitate upregulation of serpinA3 in N42 neurons is blocked by the NFκB inhibitor, BAY11, and the upregulation of serpinA3N expression in the hypothalamus by HFD is blunted in IL-1 receptor 1 knockout (IL-1R1−/−) mice. Conclusions These data demonstrate that serpinA3 expression is implicated in nutritionally mediated hypothalamic inflammation LMW, CG, ACM, EB, AM, BdR, MVB, MM and C-DM were supported by a project grant from NuGO; LMW, AM and FHM were supported by a project support grant from the British Society for Neuroendocrinology; DS was supported by a SULSA studentship; FHM was supported by a EASTBIO DTP BBSRC studentship; LMW, CG, ACM, EB, BdR, C-DM, FMC, PN, JED and NH were funded by the Scottish Government’s Rural and Environment Science and Analytical Services Division (RESAS). AT and KC were funded by the German Ministry of Research and Education (Ref. No: 0315087). HMR was supported by the Irish Department of Agriculture, Food and Marine ImmunoMet Programme (14/F/828). FCM and HMR were supported by Science Foundation Ireland Principal Investigator Programme (11/PI/1119). RN and CD were supported by grants from Ministerio de Economia y Competitividad (CD: BFU2011-29102; RN: BFU2012-35255), Xunta de Galicia (RN: EM 2012/039 and 2012-CP069). Centro de Investigación Biomédica en Red (CIBER) de Fisiopatología de la Obesidad y Nutrición (CIBERobn). CIBERobn is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain which is supported by FEDER funds. The research leading to these results has also received funding from the European Community’s Seventh Framework Programme under the following grants: no 245009: NeuroFAST to CD and ERC StG-2011-OBESITY53-281408 to RN SI

β-catenin is a multifunctional protein that not only acts in the canonical Wnt/β-catenin pathway to regulate gene expression but also binds to cadherin proteins in adherens junctions, where it plays a key role in regulating cytoskeleton linked with these junctions. Recently, evidence has been presented indicating an essential role for β-catenin in regulating the trafficking of insulin vesicles in β-cells and showing that changes in nutrient levels rapidly alter levels of β-catenin in these cells. Given the importance of neuroendocrine hormone secretion in the regulation of whole body glucose homeostasis, the present study aimed to investigate whether β-catenin signalling is regulated in the hypothalamus during the normal physiological response to food intake. Rats were subjected to a fasting/re-feeding paradigm, and then samples were collected at specific timepoints for analysis of β-catenin expression by immunohistochemistry and western blotting. Changes in gene expression were assessed by a quantitative reverse transcriptase-polymerase chain reaction. Using immunohistochemistry, feeding acutely increased detectable cytoplasmic levels of β-catenin (‘stabilised β-catenin’) in neurones in specific regions of the hypothalamus involved in metabolic regulation, including the arcuate, dorsomedial and paraventricular nuclei of the hypothalamus. Feeding-induced elevations in β-catenin in these nuclei were associated with an increased transcription of several genes known to be responsive to Wnt/β-catenin signalling. The effect of feeding was mimicked by administration of the GLP-1 agonist exendin-4 and was also characterised by cAMP-dependent phosphorylation of β-catenin at serine residues 552 and 675. These data suggest that β-catenin/T cell factor signalling is involved in metabolic sensing in the hypothalamus. Refereed/Peer-reviewed

Metabolic inflammation in the central nervous system might be causative for the development of overnutrition-induced metabolic syndrome and related disorders, such as obesity, leptin and insulin resistance, and type 2 diabetes. Here we investigated whether nutritive and genetic inhibition of the central IκB kinase β (IKKβ)/nuclear factor-κB (NF-κB) pathway in diet-induced obese (DIO) and leptin-deficient mice improves these metabolic impairments. A known prominent inhibitor of IKKβ/NF-κB signaling is the dietary flavonoid butein. We initially determined that oral, intraperitoneal, and intracerebroventricular administration of this flavonoid improved glucose tolerance and hypothalamic insulin signaling. The dose-dependent glucose-lowering capacity was profound regardless of whether obesity was caused by leptin deficiency or high-fat diet (HFD). To confirm the apparent central role of IKKβ/NF-κB signaling in the control of glucose and energy homeostasis, we genetically inhibited this pathway in neurons of the arcuate nucleus, one key center for control of energy homeostasis, via specific adeno-associated virus serotype 2–mediated overexpression of IκBα, which inhibits NF-κB nuclear translocation. This treatment attenuated HFD-induced body weight gain, body fat mass accumulation, increased energy expenditure, and reduced arcuate suppressor of cytokine signaling 3 expression, indicative for enhanced leptin signaling. These results reinforce a specific role of central proinflammatory IKKβ/NF-κB signaling in the development and potential treatment of DIO-induced comorbidities.

The occurrence of type II diabetes is highly correlated with obesity, although the mechanisms linking the two conditions are incompletely understood. Leptin is a potent insulin sensitiser and, in leptin-deficient, insulin insensitive, Lep(ob/ob) mice, leptin improves glucose tolerance, indicating that leptin resistance may link obesity to insulin insensitivity. Leptin resistance occurs in response to a high-fat diet (HFD) and both hyperleptinaemia and inflammation have been proposed as causative mechanisms. Scrutinising the role of hyperleptinaemia in this process, central hyperleptinaemia in Lep(ob/ob) mice was induced by chronic i.c.v. infusion of leptin (4.2 μg/day) over 10 days. This treatment led to a dramatic decline in body weight and food intake, as well as an improvement in glucose tolerance. Transfer to HFD for 4 days markedly arrested the beneficial effects of leptin on these parameters. Because Lep(ob/ob) mice are exquisitely sensitive to leptin, the possibility that leptin could reverse HFD-induced glucose intolerance in these animals was investigated. HFD led to increased body weight and glucose intolerance compared to a low-fat diet (LFD). Older and heavier Lep(ob/ob) mice were used as body weight-matched controls. Mice in each group received either i.p. leptin (1.25 mg/kg) or vehicle, and glucose tolerance, food intake and the number of phosphorylated signal transducer and activator of transcription (pSTAT)3 immunoreactive cells in the arcuate nucleus (ARC) and ventromedial hypothalamus (VMH) were analysed. Leptin improved glucose tolerance (P = 0. 019) and reduced food intake in Lep(ob/ob) mice on LFD (P ≤ 0.001) but was ineffective in mice on HFD. Furthermore, when leptin was administered centrally, the glucose tolerance of Lep(ob/ob) mice on HFD was significantly impaired (P = 0.007). Although leptin induced the number of pSTAT3 immunoreactive cells in the ARC and VMH of Lep(ob/ob) mice on LFD, HFD was associated with elevated pSTAT3 immunoreactivity in vehicle-treated Lep(ob/ob) mice that was unaffected by leptin treatment, suggesting central leptin resistance. Negating central inflammation by co-administering a c-Jun n-terminal kinase (JNK) inhibitor reinstated the glucose-lowering effects of leptin. These findings demonstrate that Lep(ob/ob) mice develop leptin resistance on a HFD independent of hyperleptinaemia and also indicate that the JNK inflammatory pathway plays a key role in the induction of diet-induced glucose intolerance.

In common forms of obesity, leptin fails to convey its regulatory effect. This so called "leptin resistance" is not well understood, and solving this puzzle is a key to understanding how obesity develops. In the present study, we investigated the temporal and regional onset of leptin resistance in response to a diet enriched with long-chain saturated fatty acids (high-fat diet; HFD) in mice. Mice were exposed to either a low-fat diet (LFD) or a HFD for 4 hours, 24 hours, 10 days and 28 days. Mice in each group received an i.p. injection of either phosphate-buffered saline or leptin and the number of phosphorylated signal transducer and activator of transcription-3 (pSTAT3) immunoreactive (-IR) cells in the arcuate nucleus (ARC), ventromedial nucleus of the hypothalamus (VMH) and dorsomedial nucleus of the hypothalamus (DMH) was analysed 30 or 120 minutes after treatment. In the ARC, as soon as 24 hours of HFD, the molecular leptin response was reduced by 40% (P≤.01). Compared to at 24 hours, after 10 days, the number of leptin-induced pSTAT3-IR cells was elevated after 120 minutes, suggesting a sustained response and a partial return of leptin sensitivity. After 28 days, leptin failed to induce the number of pSTAT3-IR over control levels, suggesting a markedly reduced sensitivity to leptin. In the VMH after 24 hours, we observed a 50% reduction in leptin-induced pSTAT-3-IR cells, followed by a further decline after 10 days. However, after 28 days, there was a significant increase in pSTAT-3-IR cells (P≤.05), indicating partial recovery of leptin sensitivity. By contrast to these two regions, in the DMH, no loss of leptin sensitivity was observed at any time-point. These findings demonstrate that a loss of sensitivity to leptin occurs rapidly after exposure to HFD in the ARC and VMH but not the DMH. However, there appears to be a biphasic pattern of leptin responsiveness, with a partial return of leptin sensitivity occurring after 10 days in the arcuate nucleus, and after 28 days in the VMH. By 28 days, the response to leptin in the arcuate nucleus was completely lost. These findings suggest that the molecular responses to leptin are altered after high-fat feeding in a time- and region-specific manner.

Wnt signalling and its downstream effectors are well known for their roles in embryogenesis and tumourigenesis, including the regulation of cell proliferation, survival and differentiation. In the nervous system, Wnt signalling has been described mainly during embryonic development, although accumulating evidence suggests that it also plays a major role in adult brain morphogenesis and function. Studies have predominantly concentrated on memory formation in the hippocampus, although recent data indicate that Wnt signalling is also critical for neuroendocrine control of the developed hypothalamus, a brain centre that is key in energy balance regulation and whose dysfunction is implicated in metabolic disorders such as type 2 diabetes and obesity. Based on scattered findings that report the presence of Wnt molecules in the tanycytes and ependymal cells lining the third ventricle and arcuate nucleus neurones of the hypothalamus, their potential importance in key regions of food intake and body weight regulation has been investigated in recent studies. The present review brings together current knowledge on Wnt signalling in the hypothalamus of adult animals and discusses the evidence suggesting a key role for members of the Wnt signalling family in glucose and energy balance regulation in the hypothalamus in diet-induced and genetically obese (leptin deficient) mice. Aspects of Wnt signalling in seasonal (photoperiod sensitive) rodents are also highlighted, given the recent evidence indicating that the Wnt pathway in the hypothalamus is not only regulated by diet and leptin, but also by photoperiod in seasonal animals, which is connected to natural adaptive changes in food intake and body weight. Thus, Wnt signalling appears to be critical as a modulator for normal functioning of the physiological state in the healthy adult brain, and is also crucial for normal glucose and energy homeostasis where its dysregulation can lead to a range of metabolic disorders.

GSK3β (glycogen synthase kinase 3β) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3β activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3β in leptin-deficient Lepob/ob mice and show that intracerebroventricular injection of a GSK3β inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3β inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3β in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3β signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.

Secondary metabolites of herbs and spices are widely used as an alternative strategy in the therapy of various diseases. The polyphenols naringenin, quercetin and curcumin have been characterised as anti-diabetic agents. Conversely,in vitro,naringenin and quercetin are described to inhibit phosphoinositide-3-kinase (PI3K), an enzyme that is essential for the neuronal control of whole body glucose homoeostasis. Using bothin vitroandin vivoexperiments, we tested whether the inhibitory effect on PI3K occurs in neurons and if it might affect whole body glucose homoeostasis. Quercetin was found to inhibit basal and insulin-induced phosphorylation of Akt (Ser473), a downstream target of PI3K, in HT-22 cells, whereas naringenin and curcumin had no effect. In Djungarian hamsters (Phodopus sungorus) naringenin and quercetin (10 mg/kg administered orally) diminished insulin-induced phosphorylation of Akt (Ser473) in the arcuate nucleus, indicating a reduction in hypothalamic PI3K activity. In agreement with this finding, glucose tolerance in naringenin-treated hamsters (oral) and mice (oral and intracerebroventricular) was reduced compared with controls. Dietary quercetin also impaired glucose tolerance, whereas curcumin was ineffective. Circulating levels of insulin and insulin-like growth factor-binding protein were not affected by the polyphenols. Oral quercetin reduced the respiratory quotient, suggesting that glucose utilisation was impaired after treatment. These data demonstrate that low doses of naringenin and quercetin acutely and potently impair glucose homoeostasis. This effect may be mediated by inhibition of hypothalamic PI3K signalling. Whether chronic impairments in glucose homoeostasis occur after long-term application remains to be identified.

Both insulin and leptin action in the brain are considered to involve activation of phosphoinositide 3-kinase (PI3K), although the roles of different PI3K isoforms in insulin signalling in the hypothalamus are unknown. In the present study, we characterised the roles of these isoforms in hypothalamic insulin and leptin signalling and investigated the cross-talk of both hormones. To evaluate PI3K levels in the hypothalamus, PI3K was immunoprecipitated using an antibody directed against the p85 subunit, and then total PI3K activity was measured in the presence of novel isoform-selective pharmacological inhibitors of each isoform of PI3K. Subsequently, these inhibitors were administered into the lateral ventricle of male Sprague-Dawley rats, followed by vehicle, insulin, leptin or both hormones 45 min later. PI3K activity was determined by immunohistochemical detection of phosphorylated AKT (S473). In a separate study, the effects of the inhibitors on the anorexigenic action of insulin and leptin were determined. Hypothalamic insulin signalling was specifically mediated by the combined actions of the class Ia isoforms p110alpha and p110beta. Total hypothalamic PI3K activity was inhibited 65% by a p110alpha inhibitor, and 35% by a p110beta inhibitor, with a combination of inhibitors being equally effective as the broad-spectrum PI3K inhibitor wortmannin. Individual i.c.v. administration of p110alpha and p110beta inhibitors partly prevented insulin-induced phosphorylated AKT (S473) in the arcuate nucleus, whereas simultaneous application completely blocked insulin action. Unlike insulin, leptin did not induce phosphorylated AKT in the hypothalamus, as detected by immunohistochemistry, and the anorectic effects of leptin were not affected by pre-treatment with a combination of p110alpha and p110beta inhibitors. The enhanced anorectic effect of a combined i.c.v. application of both insulin and leptin could be prevented by pre-treatment with the combination of p110alpha and p110beta inhibitors. The data suggest that p110alpha and p110beta isoforms of PI3K are necessary to mediate insulin action in the hypothalamus. The role of PI3K in leptin action is less clear, but it may be involved by means of an insulin-dependent sensitisation of leptin action.

In common forms of obesity, animals and humans become leptin resistant associated with impaired regulation of energy homeostasis. Over the last decade, significant advances in delineating the underlying mechanisms have been achieved. As well as the obvious scientific progress obtained by novel transgenic animals, natural and physiological models of leptin resistance such as the Siberian hamster (Phodoups sungorus), the field vole (Microtus agrestis) or the rat during pregnancy have also provided invaluable insight into the dynamic long-term control of energy homeostasis. Seasonal (in the hamster) and pregnancy-induced leptin resistance are characterised by a modulation of the leptin signalling cascade downstream of its receptor in the hypothalamus. In this state, leptin-induced phosphorylation of the important transcription factor, signal transducer and activator of transcription 3 (STAT3), is impaired in the arcuate nucleus and the ventromedial hypothalamus (only during pregnancy). This is accompanied by elevated levels of leptin signalling inhibitors such as the suppressor of cytokine signalling (SOCS3) and the protein tyrosine phosphatase 1B (PTP1B). The janus kinase 2 (JAK2)-STAT3 signalling pathway might be modulated by a dual function of the tyrosine residue Tyr(985) in the intracellular domain of the leptin receptor. In seasonal animals, SOCS3, most importantly seems to act as a 'molecular switch' enabling a photoperiod-induced alteration in leptin signalling and subsequent adjustments in energy homeostasis to allow attainment of a new body weight set-point. These physiological models show that animals can exhibit leptin resistance as an adaptive response to meet new physiological or environmental challenges, promoting the survival of the species during times of increased metabolic demand. The molecular mechanisms mediating physiological and/or pathological leptin resistance, like during diet induced obesity, might be very similar involving hypothalamic SOCS3. Investigation of these models might further provide new insight into the dynamic complexity of energy homeostasis.

During the last 5 years it has been well established that photoperiod-induced changes in body weight in the seasonal hamster, Phodopus sungorus, are accompanied by a marked seasonal cycle in leptin sensitivity. In the present study, we investigated the possible involvement of insulin signaling in seasonal body weight regulation. We analyzed the expression pattern and relative intensity of insulin receptor (IR), phosphatidylinositol 3-kinase (PI3-kinase), and protein tyrosine phosphatase 1B (PTP1B) mRNAs by in situ hybridization in the brains of juvenile female hamsters acclimated to either long- (LD) or short-day length (SD) for 8 wk, with or without superimposed food deprivation for 48 h. Furthermore, the hypothalamic concentration and distribution of phospho-AKT, a marker of PI3-kinase activity was determined by immunoblotting and immunohistochemistry. Eight weeks of acclimation to SD led to a substantial downregulation of IR, PTP1B gene expression, and phospho-AKT concentration in this brain region, whereas PI3-kinase mRNA was unchanged. Food deprivation induced a decrease in PTP1B and a trend toward lowered IR gene expression in LD but not in SD. Additionally, a striking increase in PTP1B gene expression in the thalamus was observed after food deprivation in both photoperiods. The direction of change in neuronal insulin signaling contrasts to the central catabolic nature of this pathway described in other species. SD-induced reduction in insulin signaling may be due to decline in body fat stores mediated by enhanced central leptin sensitivity. Increased anorexigenic tone of leptin may overwrite central insulin signaling to prevent catabolic overdrive.

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHSR). However, the functional interaction of ligand and receptor is not very well understood. We demonstrate that GHSR mRNA is up-regulated after food deprivation (48 h) in the hypothalamic arcuate nucleus and ventromedial nucleus of the seasonal Siberian hamster, Phodopus sungorus. This increase is accompanied by a two-fold elevation of circulating ghrelin concentration. Chronic changes in feeding state imposed by food restriction over a period of 12 weeks during long day-length induced increased GHSR gene expression, whereas food restriction for 6 weeks had no effect. Phodopus sungorus reveals remarkable seasonal changes in body weight, fat mass and circulating leptin levels. Ghrelin is generally regarded as having opposing effects on appetite and body weight with respect to those exhibited by leptin. However, our study revealed that seasonal adaptations were not accompanied by changes in either GHSR gene expression or circulating ghrelin concentration. Therefore, we suggest that ghrelin only plays a minor role in modulating long-term seasonal body weight cycles. Our findings imply that ghrelin predominantly acts as a short-term regulator of feeding.

We present the first evidence that suppressor of cytokine signaling-3 (SOCS3), a protein inhibiting Janus kinase/signal transducer and activator of transcription (STAT) signaling distal of the leptin receptor, conveys seasonal changes in leptin sensitivity in the Siberian hamster. Food deprivation (48 h) reduced SOCS3 gene expression in hamsters acclimated to either long (LD) or short (SD) photoperiods, suggesting that leptin signals acute starvation regardless of photoperiod. However, SOCS3 mRNA levels were substantially lower in the hypothalamic arcuate nucleus of hamsters acclimated to SD than in those raised in LD. In juveniles raised in LD, a rapid increase in SOCS3 mRNA was observed within 4 d of weaning, which was completely prevented by transfer to SD on the day of weaning. The early increase in SOCS3 gene expression in juvenile hamsters in LD clearly preceded the establishment of different body weight trajectories in LD and SD. In adult LD hamsters, SOCS3 mRNA was maintained at an elevated level despite the chronic food restriction imposed to lower body weight and serum leptin to or even below SD levels. A single injection of leptin in SD hamsters elevated SOCS3 mRNA to LD levels, whereas leptin treatment had no effect on SOCS3 gene expression in LD hamsters. Our results suggest that the development of leptin resistance in LD-acclimated hamsters involves SOCS3-mediated suppression of leptin signaling in the arcuate nucleus. Increased SOCS3 expression in LD hamsters is independent of body fat and serum leptin levels, suggesting that the photoperiod is able to trigger the biannual reversible switch in leptin sensitivity.

Recent evidence suggests that peripheral ghrelin regulates glucose metabolism. Here, we designed experiments to examine how central acyl ghrelin infusion affects peripheral glucose metabolism under pair-fed or ad libitum feeding conditions. Mice received intracerebroventricular (icv) infusion of artificial cerebrospinal fluid (aCSF), ghrelin, and allowed to eat ad libitum (icv ghrelin ad lib) or ghrelin and pair-fed to the aCSF group (icv ghrelin pf). Minipumps delivered acyl ghrelin at a dose of 0.25 μg/h at 0.5 μL/h for 7 days. There was no difference in daily blood glucose, insulin, glucagon, triglycerides, or nonesterified fatty acids. Body weight gain and food intake was significantly higher in icv ghrelin ad lib mice. However, both icv ghrelin ad lib and icv ghrelin pf groups exhibited heavier white adipose mass. Icv ghrelin pf mice exhibited better glucose tolerance than aCSF or icv ghrelin ad lib mice during a glucose tolerance test, although both icv ghrelin ad lib and icv ghrelin pf increased insulin release during the glucose tolerance test. Central acyl ghrelin infusion and pair feeding also increased breakdown of liver glycogen and triglyceride, and regulated genes involved in hepatic lipid and glucose metabolism. Icv ghrelin pf mice had an increase in plasma blood glucose during a pyruvate tolerance test relative to icv ghrelin ad lib or aCSF mice. Our results suggest that under conditions of negative energy (icv ghrelin pf), central acyl ghrelin engages a neural circuit that influences hepatic glucose function. Metabolic status affects the ability of central acyl ghrelin to regulate peripheral glucose homeostasis.

High-fat diet (HFD) feeding causes ghrelin resistance in arcuate neuropeptide Y (NPY)/Agouti-related peptide neurons. In the current study, we investigated the time course over which this occurs and the mechanisms responsible for ghrelin resistance. After 3 weeks of HFD feeding, neither peripheral nor central ghrelin increased food intake and or activated NPY neurons as demonstrated by a lack of Fos immunoreactivity or whole-cell patch-clamp electrophysiology. Pair-feeding studies that matched HFD calorie intake with chow calorie intake show that HFD exposure does not cause ghrelin resistance independent of body weight gain. We observed increased plasma leptin in mice fed a HFD for 3 weeks and show that leptin-deficient obese ob/ob mice are still ghrelin sensitive but become ghrelin resistant when central leptin is coadministered. Moreover, ob/ob mice fed a HFD for 3 weeks remain ghrelin sensitive, and the ability of ghrelin to induce action potential firing in NPY neurons was blocked by leptin. We also examined hypothalamic gliosis in mice fed a chow diet or HFD, as well as in ob/ob mice fed a chow diet or HFD and lean controls. HFD-fed mice exhibited increased glial fibrillary acidic protein–positive cells compared with chow-fed mice, suggesting that hypothalamic gliosis may underlie ghrelin resistance. However, we also observed an increase in hypothalamic gliosis in ob/ob mice fed a HFD compared with chow-fed ob/ob and lean control mice. Because ob/ob mice fed a HFD remain ghrelin sensitive, our results suggest that hypothalamic gliosis does not underlie ghrelin resistance. Further, pair-feeding a HFD to match the calorie intake of chow-fed controls did not increase body weight gain or cause central ghrelin resistance; thus, our evidence suggests that diet-induced hyperleptinemia, rather than diet-induced hypothalamic gliosis or HFD exposure, causes ghrelin resistance.

Adiponectin, an adipocyte-derived hormone, regulates glucose and lipid metabolism. It is also antiinflammatory. During obesity, adiponectin levels and sensitivity are reduced. Whereas the action of adiponectin in the periphery is well established the neuroendocrine role of adiponectin is largely unknown. To address this we analyzed the expression of adiponectin and the 2 adiponectin receptors (AdipoR1 and AdipoR2) in response to fasting and to diet-induced and genetic obesity. We also investigated the acute impact of adiponectin on central regulation of glucose homeostasis. Adiponectin (1 μg) was injected intracerebroventricularly (ICV), and glucose tolerance tests were performed in dietary and genetic obese mice. Finally, the influence of ICV adiponectin administration on central signaling cascades regulating glucose homeostasis and on markers of hypothalamic inflammation was assessed. Gene expression of adiponectin was down-regulated whereas AdipoR1 was up-regulated in the arcuate nucleus of fasted mice. High-fat (HF) feeding increased AdipoR1 and AdipoR2 gene expression in this region. In mice on a HF diet and in leptin-deficient mice acute ICV adiponectin improved glucose tolerance 60 minutes after injection, whereas normoglycemia in control mice was unaffected. ICV adiponectin increased pAKT, decreased phospho-AMP-activated protein kinase, and did not change phospho-signal transducer and activator of transcription 3 immunoreactivity. In HF-fed mice, ICV adiponectin reversed parameters of hypothalamic inflammation and insulin resistance as determined by the number of phospho-glycogen synthase kinase 3 β(Ser9) and phospho-c-Jun N-terminal kinase (Thr183/Tyr185) immunoreactive cells in the arcuate nucleus and ventromedial hypothalamus. This study demonstrates that the insulin-sensitizing properties of adiponectin are at least partially based on a neuroendocrine mechanism that involves centrally synthesized adiponectin.

High–fat (HF) diet-induced obesity and insulin insensitivity are associated with inflammation, particularly in white adipose tissue (WAT). However, insulin insensitivity is apparent within days of HF feeding when gains in adiposity and changes in markers of inflammation are relatively minor. To investigate further the effects of HF diet, C57Bl/6J mice were fed either a low (LF) or HF diet for 3 days to 16 weeks, or fed the HF-diet matched to the caloric intake of the LF diet (PF) for 3 days or 1 week, with the time course of glucose tolerance and inflammatory gene expression measured in liver, muscle and WAT. HF fed mice gained adiposity and liver lipid steadily over 16 weeks, but developed glucose intolerance, assessed by intraperitoneal glucose tolerance tests (IPGTT), in two phases. The first phase, after 3 days, resulted in a 50% increase in area under the curve (AUC) for HF and PF mice, which improved to 30% after 1 week and remained stable until 12 weeks. Between 12 and 16 weeks the difference in AUC increased to 60%, when gene markers of inflammation appeared in WAT and muscle but not in liver. Plasma proteomics were used to reveal an acute phase response at day 3. Data from PF mice reveals that glucose intolerance and the acute phase response are the result of the HF composition of the diet and increased caloric intake respectively. Thus, the initial increase in glucose intolerance due to a HF diet occurs concurrently with an acute phase response but these effects are caused by different properties of the diet. The second increase in glucose intolerance occurs between 12 - 16 weeks of HF diet and is correlated with WAT and muscle inflammation. Between these times glucose tolerance remains stable and markers of inflammation are undetectable.

The WNT pathway has been well characterized in embryogenesis and tumorigenesis. In humans, specific polymorphisms in the T cell-specific transcription factor 7 and the WNT coreceptor, low-density lipoprotein receptor-related protein-6 (LRP-6), both prominent components of this pathway, correlate with a higher incidence of type 2 diabetes, suggesting that the WNT pathway might be involved in the control of adult glucose homeostasis. We previously demonstrated that glycogen-synthase-kinase-3β (GSK-3β), the key enzyme of the WNT pathway, is increased in the hypothalamus during obesity and exacerbates high-fat diet-induced weight gain as well as glucose intolerance. These data suggest that WNT action in the hypothalamus might be required for normal glucose homeostasis. Here we characterized whether WNT signaling in general is altered in the hypothalamus of adult obese mice relative to controls. First we identified expression of multiple components of this pathway in the murine arcuate nucleus by in situ hybridization. In this region mRNA of ligands and target genes of the WNT pathway were down-regulated in obese and glucose-intolerant leptin-deficient mice. Similarly, the number of cells immunoreactive for the phosphorylated (active) form of the WNT-coreceptor LRP-6 was also decreased in leptin-deficient mice. Leptin treatment normalized expression of the WNT-target genes Axin-2 and Cylin-D1 and increased the number of phospho-LRP-6-immunoreactive cells reaching levels of lean controls. Leptin also increased the levels of phosphorylated (inactive) GSK-3β in the arcuate nucleus, and this effect was colocalized to neuropeptide Y neurons, suggesting that inactivation of GSK-3β may contribute to the neuroendocrine control of energy homeostasis. Taken together our findings identify hypothalamic WNT signaling as an important novel pathway that integrates peripheral information of the body's energy status encoded by leptin.

The profound seasonal cycle in body weight exhibited by the Djungarian hamster (Phodopus sungorus) is associated with the development of hypothalamic leptin resistance during long day photoperiod (LD, 16:8 h light dark cycle), when body weight is elevated relative to short day photoperiod (SD, 8:16 h light dark cycle). We previously have shown that this seasonal change in physiology is associated with higher levels of mRNA for the potent inhibitor of leptin signaling, suppressor of cytokine signaling-3 (SOCS3), in the arcuate nucleus (ARC) of LD hamsters relative to hamsters in SD. The alteration in SOCS3 gene expression preceded the body weight change suggesting that SOCS3 might be the molecular switch of seasonal body weight changes. To functionally characterize the role of SOCS3 in seasonal body weight regulation, we injected SOCS3 expressing recombinant adeno-associated virus type-2 (rAAV2-SOCS3) constructs into the ARC of leptin sensitive SD hamsters immediately after weaning. Hamsters that received rAAV2 expressing enhanced green fluorescent protein (rAAV2-EGFP) served as controls. ARC-directed SOCS3 overexpression led to a significant increase in body weight over a period of 12 weeks without fully restoring the LD phenotype. This increase was partially due to elevated brown and white adipose tissue mass. Gene expression of pro-opiomelanocortin was increased while thyroid hormone converting enzyme DIO3 mRNA levels were reduced in SD hamsters with SOCS3 overexpression. In conclusion, our data suggest that ARC-directed SOCS3 overexpression partially overcomes the profound seasonal body weight cycle exhibited by the hamster which is associated with altered pro-opiomelanocortin and DIO3 gene expression.

The hypothalamus has been identified as a main insulin target tissue for regulating normal body weight and glucose metabolism. Recent observations suggest that c-Jun-N-terminal kinase (JNK)-signalling plays a crucial role in the development of obesity and insulin resistance because neuronal JNK-1 ablation in the mouse prevented high-fat diet-induced obesity (DIO) and increased energy expenditure, as well as insulin sensitivity. In the present study, we investigated whether central JNK inhibition is associated with sensitisation of hypothalamic insulin signalling in mice fed a high-fat diet for 3 weeks and in leptin-deficient mice. We determined whether i.c.v. injection of a pharmacological JNK-inhibitor (SP600125) improved impaired glucose homeostasis. By immunohistochemistry, we first observed that JNK activity was increased in the arcuate nucleus (ARC) and the ventromedial hypothalamus (VMH) in both mouse models, relative to normoglycaemic controls. This suggests that up-regulation of JNK in these regions is associated with glucose intolerance and obesity, independent of leptin levels. Acute i.c.v. injection of SP600125 ameliorated glucose tolerance within 30 min in both leptin-deficient and DIO mice. Given the acute nature of i.c.v. injections, these effects cannot be attributed to changes in food intake or energy balance. In a hypothalamic cell line, and in the ARC and VMH of leptin-deficient mice, JNK inhibition by SP600125 consistently improved impaired insulin signalling. This was determined by a reduction of phospho-insulin receptor substrate-1 [IRS-1(Ser612)] protein in a hypothalamic cell line and a decline in the number of pIRS-1(Ser612) immunoreactive cells in the ARC and VMH. Serine 612 phosphorylation of IRS-1 is assumed to negatively regulate insulin signalling. In leptin-deficient mice, in both nuclei, central inhibition of JNK increased the number of cells immunoreactive for phospho-Akt (Ser473) and phospho-GSK-3β (Ser9), which are important markers of insulin signalling. Collectively, our data suggest that the acute inhibition of central JNK improves impaired glucose homeostasis and is associated with sensitisation of hypothalamic insulin signalling.

Branched-chain amino acids, particularly leucine, are thought to activate nutrient sensing pathways in the hypothalamus that regulate food intake and energy homeostasis. In the light of recent controversial findings of leucine’s effect on energy homeostasis further clarification of the metabolic impact of dietary leucine supplementation is required. We examined the pharmacological and dietary effects of leucine on energy metabolism in the Djungarian hamster (Phodopus sungorus), a well-established model for studies of alterations in leptin sensitivity and energy metabolism. We acutely administered leucine into the lateral ventricle (1.1 μg) of hamsters to characterize whether leucine exhibits anorexigenic properties in this species as has been described in other rodents. Next the catabolic effect of dietary administered leucine via supplemented rodent diet (15 % leucine), drinking water (17 g/L leucine) and oral gavages (10 mg/day); as well as the effect of subcutaneously (0.1 and 3 mg/day) and intraperitoneally (0.1, 3 and 6 mg/day) injected leucine which avoids the gastrointestinal-track was analyzed. Centrally administered leucine reduced 24 h food intake (by 32 %) and body weight. Both parameters were also reduced in hamsters with leucine supplemented diet, but this catabolic response was based on a pronounced taste aversion to the leucine-diet. In all other experiments, dietary leucine and peripheral injections of leucine had no effect on food intake, body weight and basal blood glucose levels. Our data suggest that in the Djungarian hamster dietary leucine fails to exhibit catabolic effects that would override the evolutionary conserved adaptations of the species which is critical for its survival.

The Siberian hamster, Phodopus sungorus, undergoes a striking seasonal cycle of leptin sensitivity and body weight regulation, but the molecular mechanism and relevance to human leptin insensitivity are unknown. Here we show that nuclear translocation of phospho-STAT3 in the hypothalamus is rapidly stimulated by leptin to a greater extent in hamsters held in short-day length (SD) as compared to long-day length (LD). Intriguingly, effects of leptin on STAT3 appeared to be in part limited to nuclear translocation of phospho-STAT3 associated with the cell surface rather than phosphorylation of STAT3. The number of phospho-ERK cells within the hypothalamus was unaffected by either photoperiod or leptin. However, proximal to ERK phosphorylation, hypothalamic SH2-containing tyrosine phosphatase (SHP2) and the small growth factor receptor-binding protein (GRB2), which act as competitive negative modulators on binding of SOCS3 to leptin receptor (LRb)-associated Tyr985, were increased in SD compared to LD. Our findings suggest that activation of STAT3 by leptin may be dependent on interaction of stimulatory SHP2/GRB2 as well as inhibitory SOCS3 on the level of competitive binding to LRb-associated Tyr985. This hypothetical mechanism may represent the molecular identity of seasonally induced adjustments in leptin sensitivity and may be applied to investigating leptin sensitivity in other rodent models.

We have shown that high-fat diet-induced insulin insensitivity is seen within 3 days in mice. To further investigate the mechanismsunderlying this induction we fed 12 week old male C57Bl/6J mice a low- or high-fat diet: 10% or 60% (Kcal) from fat (D12450B andD12492 respectively, Research Diets, USA) for 3 days. Insulin sensitivity was assessed by intraperitioneal glucose tolerance tests (IPGTT)and proteomics was used to determine changes in hypothalamic proteins. Body composition was measured using MRI, liver lipid contentmeasured by oil red O staining. Mice on the high-fat diet showed increased adiposity, liver lipid content and insulin insensitivity with a48% increase in area under the curve (AUC) compared to control animals. Hypothalamic protein samples analysed by 2D eletrophoresisrevealed profound changes after 3 days on the high-fat diet, which when identiÞed by LC/MS/MS were found to be proteins associatedwith neuronal damage particularly insammation, ischemia and apoptosis including collapsin response mediated protein 2 (CRMP-2),cyclophilin A, alpha-enolase and apolipoprotein E

Obesity is associated with resistance to the actions of both leptin and insulin via mechanisms that remain incompletely understood. To investigate whether leptin resistance per se contributes to insulin resistance and impaired glucose homeostasis, we investigated the effect of acute leptin administration on glucose homeostasis in normal as well as leptin- or leptin receptor-deficient mice. In hyperglycemic, leptin-deficient Lepob/obmice, leptin acutely and potently improved glucose metabolism, before any change of body fat mass, via a mechanism involving the p110α and β isoforms of phosphatidylinositol-3-kinase (PI3K). Unlike insulin, however, the anti-diabetic effect of leptin occurred independently of phospho-AKT, a major downstream target of PI3K, and instead involved enhanced sensitivity of the hypothalamus to insulin action upstream of PI3K, through modulation of IRS1 (insulin receptor substrate 1) phosphorylation. These data suggest that leptin resistance, as occurs in obesity, reduces the hypothalamic response to insulin and thereby impairs peripheral glucose homeostasis, contributing to the development of type 2 diabetes.

Hypothalamic leptin resistance during pregnancy is an important adaptation that facilitates the state of positive energy balance required for fat deposition in preparation for lactation. Within the arcuate nucleus, pro-opiomelanocortin (POMC) neurones and neuropeptide Y (NPY)/agouti-related gene protein (AgRP) neurones are first-order leptin responsive neurones involved in the regulation of energy balance. The present study aimed to investigate whether the regulation of these neuropeptides is disrupted during pregnancy in association with the development of leptin resistance. As measured by quantitative in situ hybridisation, POMC and AgRP mRNA levels were not significantly different during pregnancy, whereas NPY mRNA levels increased such that, by day 21 of pregnancy, levels were significantly higher than in nonpregnant, animals. These data suggest that these neurones were not responding normally to the elevated leptin found during pregnancy. To further characterise the melanocortin system during pregnancy, double-label immunohistochemistry was used to quantify leptin-induced phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) in POMC neurones, using α-melanocyte-stimulating hormone (MSH) as a marker. The percentage of α-MSH neurones containing leptin-induced pSTAT3 did not significantly differ from nonpregnant animals, indicating that there was no change in the number of POMC neurones that respond to leptin during pregnancy. Treatment with α-MSH significantly reduced food intake in nonpregnant rats, but not in pregnant rats, indicating resistance to the satiety actions of α-MSH during pregnancy. The data suggest that multiple mechanisms contribute to leptin resistance during pregnancy. As well as a loss of responses in first-order leptin-responsive neurones in the arcuate nucleus, there is also a downstream disruption in the melanocortin system.

The adipose-derived hormone leptin communicates information about metabolic status to the hypothalamic GnRH neuronal system. It is unclear whether leptin can act directly on GnRH neurons. To examine this, we used three approaches. First, the presence of leptin-induced signal transducer and activator of transcription-3 activation was examined in GnRH neurons in male and female rats. Intracerebroventricular treatment with 4 μg leptin-induced robust signal transducer and activator of transcription-3 expression within the anteroventral periventricular nucleus but not in GnRH neurons. Second, fertility was assessed in male and female CRE-loxP transgenic mice with conditional leptin receptor (Lepr) deletion from either all forebrain neurons or GnRH neurons only. Forebrain neuron LEPR deletion prevented the onset of puberty resulting in infertility in males and females and blocked estradiol-induced LH surge. However, mice with GnRH neuron-selective Lepr deletion exhibited normal fertility apart from a slight puberty delay in males. Lastly, the highly sensitive technique of single-cell nested PCR was used to test for Lepr transcript presence in individual GnRH neurons, identified in situ using GnRH-green fluorescent protein transgenics. Whereas 75% of positive control (proopiomelanocortin) neurons contained Lepr mRNA, no (none of 18) GnRH neurons were Lepr mRNA positive. Collectively, these results show that leptin does not act directly on GnRH neurons in rats and mice. Leptin appears to regulate GnRH function via forebrain neurons that are afferent to GnRH because forebrain neuronal LEPR deletion caused infertility. The location and phenotype of these leptin-responsive neurons remains to be elucidated.

Central regulation of energy balance in seasonal mammals such as the Siberian hamster is dependent on the precise integration of short-term satiety information arising from the gastrointestinal tract with long-term signals on the status of available energy reserves (e.g. leptin) and prevailing photoperiod. Within the central nervous system, the brainstem nucleus of the solitary tract (NTS) and the parabrachial nucleus (PBN) are major relay nuclei that transmit information from the gastrointestinal tract to higher forebrain centres. We extended studies on the seasonal programming of the hypothalamus to examine the effect of the photoperiod on neuropeptidergic circuitries of this gut-brain axis. In the NTS and PBN we performed gene expression and immunoreactivity (-ir) studies on selected satiety-related neuropeptides and receptors: alpha-melanocyte stimulating hormone, melanocortin-3 receptor, melanocortin-4 receptor (MC4-R), growth hormone secretagogue-receptor, cocaine- and amphetamine-regulated transcript, preproglucagon (PPG), glucagon-like peptide 1 (GLP-1), cholecystokinin (CCK), peptide YY, galanin, neurotensin, and corticotrophin releasing hormone (CRH). Gene expression of PPG and MC4-R, and -ir of CCK and GLP-1, in the NTS were up-regulated after 14 weeks in long-day photoperiod (16 h light:8 h dark) compared to short-days (8 h light:16 h dark), whereas CRH-ir and NT-ir were increased in short-days within the PBN. We suggest that brainstem neuroendocrine mechanisms contribute to the long-term regulation of body mass in the Siberian hamster by a photoperiod-related modulation of satiety signalling.

We have previously shown that cold-acclimated (8 degrees C) male field voles (Microtus agrestis) transferred from short day (SD, 8 h light) to long day (LD, 16 h light) photoperiod exhibit an increase in body mass lasting 4 weeks, after which they stabilise at a new plateau approximately 7.5 g (24.8%) higher than animals maintained in SD. By infusing voles with exogenous leptin, we have also demonstrated that SD voles respond to the hormone by reducing body mass and food intake, whereas LD animals increasing body mass are resistant to leptin treatment. In the present study, we investigated whether seasonal changes in body mass could be linked to modulation of the leptin signal by suppressor of cytokine signalling-3 (SOCS3). We used in situ hybridisation to examine hypothalamic arcuate nucleus (ARC) expression of SOCS3, neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) genes in 90 voles exposed to either SD or LD for up to 11 weeks. LD voles increasing body mass had significantly higher levels of SOCS3 mRNA than SD or LD voles with a stable body mass. There were no associated changes in expression of NPY, AgRP, POMC and CART genes. These results suggest that voles that regulate body mass at either the lower (SD) or upper (LD) plateau remain sensitive to leptin action, whereas SOCS3-mediated leptin resistance is a short-term mechanism that enables animals to move between the stable body mass plateaus. Our data provide evidence that expression of SOCS3 in the ARC is involved in the modulation of the strength of the leptin signal to facilitate seasonal cycles in body mass and adiposity.