Alexander Tups

Leptin Indirectly Regulates Gonadotropin-Releasing Hormone Neuronal Function

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 mug 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.

Leptin Rapidly Improves Glucose Homeostasis in Obese Mice by Increasing Hypothalamic Insulin Sensitivity

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/ob mice, 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.

High‐Fat Diet Induces Leptin Resistance in Leptin‐Deficient Mice

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, Lepob/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 Lepob/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 Lepob/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 Lepob/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 Lepob/ob mice on LFD (P ≤ 0.001) but was ineffective in mice on HFD. Furthermore, when leptin was administered centrally, the glucose tolerance of Lepob/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 Lepob/ob mice on LFD, HFD was associated with elevated pSTAT3 immunoreactivity in vehicle‐treated Lepob/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 Lepob/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.

Circulating Ghrelin Levels and Central Ghrelin Receptor Expression are Elevated in Response to Food Deprivation in a Seasonal Mammal (Phodopus sungorus)

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.

Loss of Hypothalamic Response to Leptin During Pregnancy Associated with Development of Melanocortin Resistance

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 Development of Diet-Induced Obesity and Glucose Intolerance in C57Bl/6 Mice on a High-Fat Diet Consists of Distinct Phases

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.

Central Inhibition of IKKβ/NF-κB Signaling Attenuates High-Fat Diet–Induced Obesity and Glucose Intolerance

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.

PC1/3 and PC2 Gene Expression and Post-Translational Endoproteolytic Pro-Opiomelanocortin Processing is Regulated by Photoperiod in the Seasonal Siberian Hamster (Phodopus sungorus)

A remarkable feature of the seasonal adaptation displayed by the Siberian hamster (Phodopus sungorus) is the ability to decrease food intake and body weight (by up to 40%) in response to shortening photoperiod. The regulating neuroendocrine systems involved in this adaptation and their neuroanatomical and molecular bases are poorly understood. We investigated the effect of photoperiod on the expression of prohormone convertases 1 (PC1/3) and 2 (PC2) and the endoproteolytic processing of the neuropeptide precursor pro‐opiomelanocortin (POMC) within key energy balance regulating centres of the hypothalamus. We compared mRNA levels and protein distribution of PC1/3, PC2, POMC, adrenocorticotrophic hormone (ACTH), α‐melanocyte‐stimulating hormone (MSH), β‐endorphin and orexin‐A in selected hypothalamic areas of long day (LD, 16 : 8 h light : dark), short day (SD, 8 : 16 h light : dark) and natural‐day (ND, photoperiod depending on time of the year) acclimated Siberian hamsters. The gene expression of PC2 was significantly higher within the arcuate nucleus (ARC, P < 0.01) in SD and in ND (versus LD), and is reflected in the day length profile between October and April in the latter. PC1/3 gene expression in the ARC and lateral hypothalamus was higher in ND but not in SD compared to the respective LD controls. The immunoreactivity of PC1/3 cleaved neuropeptide ACTH in the ARC and PC1/3‐colocalised orexin‐A in the latyeral hypothalamus were not affected by photoperiod changes. However, increased levels of PC2 mRNA and protein were associated with higher abundance of the mature neuropeptides α‐MSH and β‐endorphin (P < 0.01) in SD. This study provides a possible explanation for previous paradoxical findings showing lower food intake in SD associated with decreased POMC mRNA levels. Our results suggest that a major part of neuroendocrine body weight control in seasonal adaptation may be effected by post‐translational processing mediated by the prohormone convertases PC1/3 and PC2, in addition to regulation of gene expression of neuropeptide precursors.

Both p110α and p110β Isoforms of Phosphatidylinositol 3‐OH‐Kinase are Required for Insulin Signalling in the Hypothalamus

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 p110α and p110β. Total hypothalamic PI3K activity was inhibited 65% by a p110α inhibitor, and 35% by a p110β inhibitor, with a combination of inhibitors being equally effective as the broad‐spectrum PI3K inhibitor wortmannin. Individual i.c.v. administration of p110α and p110β 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 p110α and p110β 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 p110α and p110β inhibitors. The data suggest that p110α and p110β 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.

Hypothalamic WNT Signalling Is Impaired During Obesity and Reinstated by Leptin Treatment in Male Mice

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 bodys energy status encoded by leptin.