IRS2 signaling in LepR-b neurons suppresses FoxO1 to control energy balance independently of leptin action

Irs2-mediated insulin/IGF1 signaling in the CNS modulates energy balance and glucose homeostasis; however, the site for Irs2 function is unknown. The hormone leptin mediates energy balance by acting on leptin receptor (LepR-b)-expressing neurons. To determine whether LepR-b neurons mediate the metabolic actions of Irs2 in the brain, we utilized Lepr(cre) together with Irs2(L/L) to ablate Irs2 expression in LepR-b neurons (Lepr(ΔIrs2)). Lepr(ΔIrs2) mice developed obesity, glucose intolerance, and insulin resistance. Leptin action was not altered in young Lepr(ΔIrs2) mice, although insulin-stimulated FoxO1 nuclear exclusion was reduced in Lepr(ΔIrs2) mice. Indeed, deletion of Foxo1 from LepR-b neurons in Lepr(ΔIrs2) mice normalized energy balance, glucose homeostasis, and arcuate nucleus gene expression. Thus, Irs2 signaling in LepR-b neurons plays a crucial role in metabolic sensing and regulation. While not required for leptin action, Irs2 suppresses FoxO1 signaling in LepR-b neurons to promote energy balance and metabolism.     

Insulin action in AgRP-expressing neurons is required for suppression of hepatic glucose production

Insulin action in the central nervous system regulates energy homeostasis and glucose metabolism. To define the insulin-responsive neurons that mediate these effects, we generated mice with selective inactivation of the insulin receptor (IR) in either pro-opiomelanocortin (POMC)- or agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus. While neither POMC- nor AgRP-restricted IR knockout mice exhibited altered energy homeostasis, insulin failed to normally suppress hepatic glucose production during euglycemic-hyperinsulinemic clamps in AgRP-IR knockout (IR^ΔAgRP) mice. These mice also exhibited reduced insulin-stimulated hepatic interleukin-6 expression and increased hepatic expression of glucose-6-phosphatase. These results directly demonstrate that insulin action in POMC and AgRP cells is not required for steady-state regulation of food intake and body weight. However, insulin action specifically in AgRP-expressing neurons does play a critical role in controlling hepatic glucose production and may provide a target for the treatment of insulin resistance in type 2 diabetes.     

Enhanced leptin sensitivity and improved glucose homeostasis in mice lacking suppressor of cytokine signaling-3 in POMC-expressing cells

Suppressor of cytokine signaling-3 (Socs-3) negatively regulates the action of various cytokines, as well as the metabolic hormones leptin and insulin. Mice with haploinsufficiency of Socs-3, or those with neuronal deletion of Socs-3, are lean and more leptin and insulin sensitive. To examine the role of Socs-3 within specific neurons critical to energy balance, we created mice with selective deletion of Socs-3 within pro-opiomelanocortin (POMC)-expressing cells. These mice had enhanced leptin sensitivity, measured by weight loss and food intake after leptin infusion. On chow diet, glucose homeostasis was improved despite normal weight gain. On a high-fat diet, the rate of weight gain was reduced, due to increased energy expenditure rather than decreased food intake; glucose homeostasis and insulin sensitivity were substantially improved. These studies demonstrate that Socs-3 within POMC neurons regulates leptin sensitivity and glucose homeostasis, and plays a key role in linking high-fat diet to disordered metabolism.     

PDK1-Foxo1 in agouti-related peptide neurons regulates energy homeostasis by modulating food intake and energy expenditure

Insulin and leptin intracellular signaling pathways converge and act synergistically on the hypothalamic phosphatidylinositol-3-OH kinase/3-phosphoinositide-dependent protein kinase 1 (PDK1). However, little is known about whether PDK1 in agouti-related peptide (AGRP) neurons contributes to energy homeostasis. We generated AGRP neuron-specific PDK1 knockout (AGRPPdk1(-/-)) mice and mice with selective expression of transactivation-defective Foxo1 (Δ256Foxo1(AGRP)Pdk1(-/-)). The AGRPPdk1(-/-) mice showed reductions in food intake, body length, and body weight. The Δ256Foxo1(AGRP)Pdk1(-/-) mice showed increased body weight, food intake, and reduced locomotor activity. After four weeks of calorie-restricted feeding, oxygen consumption and locomotor activity were elevated in AGRPPdk1(-/-) mice and reduced in Δ256Foxo1(AGRP)Pdk1(-/-) mice. In vitro, ghrelin-induced changes in [Ca(2+)](i) and inhibition of ghrelin by leptin were significantly attenuated in AGRPPdk1(-/-) neurons compared to control neurons. However, ghrelin-induced [Ca(2+)](i) changes and leptin inhibition were restored in Δ256Foxo1(AGRP)Pdk1(-/-) mice. These results suggested that PDK1 and Foxo1 signaling pathways play important roles in the control of energy homeostasis through AGRP-independent mechanisms.     

Enhanced leptin-stimulated Pi3k activation in the CNS promotes white adipose tissue transdifferentiation

The contribution of different leptin-induced signaling pathways in control of energy homeostasis is only partly understood. Here we show that selective Pten ablation in leptin-sensitive neurons (Pten^ΔObRb) results in enhanced Pi3k activation in these cells and reduces adiposity by increasing energy expenditure. White adipose tissue (WAT) of Pten^ΔObRb mice shows characteristics of brown adipose tissue (BAT), reflected by increased mitochondrial content and Ucp1 expression resulting from enhanced leptin-stimulated sympathetic nerve activity (SNA) in WAT. In contrast, leptin-deficient ob/ob-Pten^ΔObRb mice exhibit unaltered body weight and WAT morphology compared to ob/ob mice, pointing to a pivotal role of endogenous leptin in control of WAT transdifferentiation. Leanness of Pten^ΔObRb mice is accompanied by enhanced sensitivity to insulin in skeletal muscle. These data provide direct genetic evidence that leptin-stimulated Pi3k signaling in the CNS regulates energy expenditure via activation of SNA to perigonadal WAT leading to BAT-like differentiation of WAT.     

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

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

Expression of a constitutively active insulin receptor in Drosulfakinin (Dsk) neurons regulates metabolism and sleep in Drosophila

The ability of organisms to sense their nutritional environment and adjust their behavior accordingly is critical for survival. Insulin-like peptides (ilps) play major roles in controlling behavior and metabolism; however, the tissues and cells that insulin acts on to regulate these processes are not fully understood. In the fruit fly, Drosophila melanogaster, insulin signaling has been shown to function in the fat body to regulate lipid storage, but whether ilps act on the fly brain to regulate nutrient storage is not known. In this study, we manipulate insulin signaling in defined populations of neurons in Drosophila and measure glycogen and triglyceride storage. Expressing a constitutively active form of the insulin receptor (dInR) in the insulin-producing cells had no effect on glycogen or triglyceride levels. However, activating insulin signaling in the Drosulfakinin (Dsk)-producing neurons led to triglyceride accumulation and increased food consumption. The expression of ilp2, ilp3 and ilp5 was increased in flies with activated insulin signaling in the Dsk neurons, which along with the feeding phenotype, may cause the triglyceride storage phenotypes observed in these flies. In addition, expressing a constitutively active dInR in Dsk neurons resulted in decreased sleep in the fed state and less starvation-induced sleep suppression suggesting a role for insulin signaling in regulating nutrient-responsive behaviors. Together, these data support a role for insulin signaling in the Dsk-producing neurons for regulating behavior and maintaining metabolic homeostasis.     

Intracerebroventricular leptin infusion improves glucose homeostasis in lean type 2 diabetic MKR mice via hepatic vagal and non-vagal mechanisms

MKR mice, lacking insulin-like growth factor 1 receptor (IGF-1R) signaling in skeletal muscle, are lean yet hyperlipidemic, hyperinsulinemic, and hyperglycemic, with severe insulin resistance and elevated hepatic and skeletal muscle levels of triglycerides. We have previously shown that chronic peripheral administration of the adipokine leptin improves hepatic insulin sensitivity in these mice independently of its effects on food intake. As central leptin signaling has been implicated in the control of peripheral glucose homeostasis, here we examined the ability of central intracerebroventricular leptin administration to affect energy balance and peripheral glucose homeostasis in non-obese diabetic male MKR mice. Central leptin significantly reduced food intake, body weight gain and adiposity, as well as serum glucose, insulin, leptin, free fatty acid and triglyceride levels relative to ACSF treated controls. These reductions were accompanied by increased fat oxidation as measured by indirect calorimetry, as well as increased oxygen consumption. Central leptin also improved glucose tolerance and hepatic insulin sensitivity determined using the euglycemic-hyperinsulinemic clamps relative to pair fed vehicle treated controls, as well as increasing the rate of glucose disappearance. Hepatic vagotomy only partially reversed the ability of central leptin to improve glucose tolerance. These results demonstrate that central leptin dramatically improves insulin sensitivity independently of its effects on food intake, in a lean mouse model of type 2 diabetes. The findings also suggest that: 1) both hepatic vagal and non-vagal pathways contribute to this improvement, and 2) central leptin alters glucose disposal in skeletal muscle in this model.     

Adipocyte-specific protein tyrosine phosphatase 1B deletion increases lipogenesis, adipocyte cell size and is a minor regulator of glucose homeostasis

Protein tyrosine phosphatase 1B (PTP1B), a key negative regulator of leptin and insulin signaling, is positively correlated with adiposity and contributes to insulin resistance. Global PTP1B deletion improves diet-induced obesity and glucose homeostasis via enhanced leptin signaling in the brain and increased insulin signaling in liver and muscle. However, the role of PTP1B in adipocytes is unclear, with studies demonstrating beneficial, detrimental or no effect(s) of adipose-PTP1B-deficiency on body mass and insulin resistance. To definitively establish the role of adipocyte-PTP1B in body mass regulation and glucose homeostasis, adipocyte-specific-PTP1B knockout mice (adip-crePTP1B(-/-)) were generated using the adiponectin-promoter to drive Cre-recombinase expression. Chow-fed adip-crePTP1B(-/-) mice display enlarged adipocytes, despite having similar body weight/adiposity and glucose homeostasis compared to controls. High-fat diet (HFD)-fed adip-crePTP1B(-/-) mice display no differences in body weight/adiposity but exhibit larger adipocytes, increased circulating glucose and leptin levels, reduced leptin sensitivity and increased basal lipogenesis compared to controls. This is associated with decreased insulin receptor (IR) and Akt/PKB phosphorylation, increased lipogenic gene expression and increased hypoxia-induced factor-1-alpha (Hif-1α) expression. Adipocyte-specific PTP1B deletion does not beneficially manipulate signaling pathways regulating glucose homeostasis, lipid metabolism or adipokine secretion in adipocytes. Moreover, PTP1B does not appear to be the major negative regulator of the IR in adipocytes.     

The ATM cofactor ATMIN protects against oxidative stress and accumulation of DNA damage in the aging brain

Progressive accumulation of DNA damage is causally involved in cellular senescence and organismal aging. The DNA damage kinase ATM plays a central role in maintaining genomic stability. ATM mutations cause the genetic disorder ataxia telangiectasia, which is primarily characterized by progressive neurodegeneration and cancer susceptibility. Although the importance of ATM function to protect against oxidative DNA damage and during aging is well described, the mechanism of ATM activation by these stimuli is not known. Here we identify ATM interactor (ATMIN) as an essential component of the ATM signaling pathway in response to oxidative stress and aging. Embryos lacking ATMIN (atmin(Δ/Δ)) died in utero and showed increased numbers of cells positive for phosphorylated histone H2aX, indicative of increased DNA damage. atmin(Δ/Δ) mouse embryonic fibroblasts accumulated DNA damage and prematurely entered senescence when cultured at atmospheric oxygen levels (20%), but this defect was rescued by addition of an antioxidant and also by culturing cells at physiological oxygen levels (3%). In response to acute oxidative stress, atmin(Δ/Δ) mouse embryonic fibroblasts showed slightly lower levels of ATM phosphorylation and reduced ATM substrate phosphorylation. Conditional deletion of ATMIN in the murine nervous system (atmin(ΔN)) resulted in reduced numbers of dopaminergic neurons, as does ATM deficiency. ATM activity was observed in old, but not in young, control mice, but aging-induced ATM signaling was impaired by ATMIN deficiency. Consequently, old atmin(ΔN) mice showed accumulation of DNA damage in the cortex accompanied by gliosis, resulting in increased mortality of aging mutant mice. These results suggest that ATMIN mediates ATM activation by oxidative stress, and thereby ATMIN protects the aging brain by preventing accumulation of DNA damage.     

Altered glucose homeostasis in mice lacking the receptor protein tyrosine phosphatase sigma

Several protein tyrosine phosphatases (PTPs) expressed in insulin sensitive-tissues are proposed to attenuate insulin action and could act as key regulators of the insulin receptor (IR) signaling pathway. Among these PTPs, RPTPsigma is expressed in relatively high levels in insulin-target tissues. We show that RPTPsigma-/- knockout mice have reduced plasma glucose and insulin concentrations in the fasted state compared with their wild-type siblings. The knockout animals were also more sensitive to exogenous insulin as assayed by insulin-tolerance tests. Despite increased whole-body insulin sensitivity, tyrosine phosphorylation of the IR was not increased in muscle of RPTPsigma-/- animals, as would be expected in insulin-sensitive animals. Instead, the levels of IR tyrosine phosphorylation and PI3-kinase activity were reduced in the muscle of knockout animals stimulated with insulin in vivo. However, insulin-stimulated Akt serine phosphorylation was essentially identical between both groups of mice. Accordingly, muscles isolated from RPTPsigma-/- mice did not have a significant increase in glucose uptake in response to insulin, suggesting that RPTPsigma did not play a direct role in this process. Taken together, our results suggest an indirect modulation of the IR signaling pathways by RPTPsigma. Since low dose injection of growth hormone (GH) normalized the response to exogenous insulin in RPTPsigma-/- mice, we propose that the insulin hypersensitivity observed in RPTPsigma-/- mice is secondary to their neuroendocrine dysplasia and GH/IGF-1 deficiency.     

Methionine restriction restores a younger metabolic phenotype in adult mice with alterations in fibroblast growth factor 21

Methionine restriction (MR) decreases body weight and adiposity and improves glucose homeostasis in rodents. Similar to caloric restriction, MR extends lifespan, but is accompanied by increased food intake and energy expenditure. Most studies have examined MR in young animals; therefore, the aim of this study was to investigate the ability of MR to reverse age‐induced obesity and insulin resistance in adult animals. Male C57BL/6J mice aged 2 and 12 months old were fed MR (0.172% methionine) or control diet (0.86% methionine) for 8 weeks or 48 h. Food intake and whole‐body physiology were assessed and serum/tissues analyzed biochemically. Methionine restriction in 12‐month‐old mice completely reversed age‐induced alterations in body weight, adiposity, physical activity, and glucose tolerance to the levels measured in healthy 2‐month‐old control‐fed mice. This was despite a significant increase in food intake in 12‐month‐old MR‐fed mice. Methionine restriction decreased hepatic lipogenic gene expression and caused a remodeling of lipid metabolism in white adipose tissue, alongside increased insulin‐induced phosphorylation of the insulin receptor (IR) and Akt in peripheral tissues. Mice restricted of methionine exhibited increased circulating and hepatic gene expression levels of FGF21, phosphorylation of eIF2a, and expression of ATF4, with a concomitant decrease in IRE1α phosphorylation. Short‐term 48‐h MR treatment increased hepatic FGF21 expression/secretion and insulin signaling and improved whole‐body glucose homeostasis without affecting body weight. Our findings suggest that MR feeding can reverse the negative effects of aging on body mass, adiposity, and insulin resistance through an FGF21 mechanism. These findings implicate MR dietary intervention as a viable therapy for age‐induced metabolic syndrome in adult humans.     

Altered insulin signaling in retinal tissue in diabetic states

Both type 1 and type 2 diabetes can lead to altered retinal microvascular function and diabetic retinopathy. Insulin signaling may also play a role in this process, and mice lacking insulin receptors in endothelial cells are protected from retinal neovascularization. To define the role of diabetes in retinal function, we compared insulin signaling in the retinal vasculature of mouse models of type 1 (streptozotocin) and type 2 diabetes (ob/ob). In streptozotocin mice, in both retina and liver, insulin receptor (IR) and insulin receptor substrate (IRS)-2 protein and tyrosine phosphorylation were increased by insulin, while IRS-1 protein and its phosphorylation were maintained. By contrast, in ob/ob mice, there was marked down-regulation of IR, IRS-1, and IRS-2 protein and phosphorylation in liver; these were maintained or increased in retina. In both mice, Phosphatidylinositol 3,4,5-trisphosphate generation by acute insulin stimulation was enhanced in retinal endothelial cells. On the other hand, protein levels and phosphorylation of PDK1 and Akt were decreased in retina of both mice. Interestingly, phosphorylation of p38 mitogen-activated protein kinase and ERK1 were responsive to insulin in retina of both mice but were unresponsive in liver. HIF-1alpha and vascular endothelial growth factor were increased and endothelial nitric-oxide synthase was decreased in retina. These observations indicate that, in both insulin-resistant and insulin-deficient diabetic states, there are alterations in insulin signaling, such as impaired PDK/Akt responses and enhanced mitogen-activated protein kinases responses that could contribute to the retinopathy. Furthermore, insulin signaling in retinal endothelial cells is differentially altered in diabetes and is also differentially regulated from insulin signaling in classical target tissues such as liver.     

GLUT2 in pancreatic and extra-pancreatic gluco-detection (review).

Detection of variations in blood glucose concentrations by pancreatic beta-cells and a subsequent appropriate secretion of insulin are key events in the control of glucose homeostasis. Because a decreased capability to sense glycemic changes is a hallmark of type 2 diabetes, the glucose signalling pathway leading to insulin secretion in pancreatic beta-cells has been extensively studied. This signalling mechanism depends on glucose metabolism and requires the presence of specific molecules such as GLUT2, glucokinase and the K(ATP) channel subunits Kir6.2 and SUR1. Other cells are also able to sense variations in glycemia or in local glucose concentrations and to modulate different physiological functions participating in the general control of glucose and energy homeostasis. These include cells forming the hepatoportal vein glucose sensor, which controls glucose storage in the liver, counterregulation, food intake and glucose utilization by peripheral tissues and neurons in the hypothalamus and brainstem whose firing rates are modulated by local variations in glucose concentrations or, when not protected by a blood-brain barrier, directly by changes in blood glucose levels. These glucose-sensing neurons are involved in the control of insulin and glucagon secretion, food intake and energy expenditure. Here, recent physiological studies performed with GLUT2-/- mice will be described, which indicate that this transporter is essential for glucose sensing by pancreatic beta-cells, by the hepatoportal sensor and by sensors, probably located centrally, which control activity of the autonomic nervous system and stimulate glucagon secretion. These studies may pave the way to a fine dissection of the molecular and cellular components of extra-pancreatic glucose sensors involved in the control of glucose and energy homeostasis.     

An Obligate Role of Oxytocin Neurons in Diet Induced Energy Expenditure

Oxytocin neurons represent one of the major subsets of neurons in the paraventricular hypothalamus (PVH), a critical brain region for energy homeostasis. Despite substantial evidence supporting a role of oxytocin in body weight regulation, it remains controversial whether oxytocin neurons directly regulate body weight homeostasis, feeding or energy expenditure. Pharmacologic doses of oxytocin suppress feeding through a proposed melanocortin responsive projection from the PVH to the hindbrain. In contrast, deficiency in oxytocin or its receptor leads to reduced energy expenditure without feeding abnormalities. To test the physiological function of oxytocin neurons, we specifically ablated oxytocin neurons in adult mice. Our results show that oxytocin neuron ablation in adult animals has no effect on body weight, food intake or energy expenditure on a regular diet. Interestingly, male mice lacking oxytocin neurons are more sensitive to high fat diet-induced obesity due solely to reduced energy expenditure. In addition, despite a normal food intake, these mice exhibit a blunted food intake response to leptin administration. Thus, our study suggests that oxytocin neurons are required to resist the obesity associated with a high fat diet; but their role in feeding is permissive and can be compensated for by redundant pathways.     

Role of Insulin Signaling in Maintaining Energy Homeostasis

ObjectiveTo examine the role that insulin signaling plays in modulating metabolic functions involving both peripheral and hypothalamic systems.MethodsWe review the literature regarding insulin signaling as it relates to energy homeostasis.ResultsInsulin signaling in the periphery is known to affect hepatic glucose production and glucose uptake in muscle and adipose tissue. In the brain, insulin is involved in a variety of signaling pathways that control positive and negative aspects of food intake and energy metabolism. Disruption of insulin signaling can affect key cellular pathways that serve to maintain energy balance and glucose homeostasis, which can then lead to insulin resistance and progression toward various metabolic disorders, including cardiovascular disease, obesity, and type 2 diabetes. The use of exogenous insulin as therapy for patients with type 2 diabetes is traditionally associated with increases in weight.ConclusionAn enhanced understanding of how these insulin signaling pathways function may provide answers about how to control weight gain associated with exogenous insulin use. Pharmacologic agents, such as the long-acting insulin analogues and particularly insulin detemir, that may reduce these weight effects hold considerable advantage. (Endocr Pract. 2008;14:373-380)     

PDK1 deficiency in POMC-expressing cells reveals FOXO1-dependent and -independent pathways in control of energy homeostasis and stress response

Insulin- and leptin-stimulated phosphatidylinositol-3 kinase (PI3K) activation has been demonstrated to play a critical role in central control of energy homeostasis. To delineate the importance of pathways downstream of PI3K specifically in pro-opiomelanocortin (POMC) cell regulation, we have generated mice with selective inactivation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in POMC-expressing cells (PDK1(DeltaPOMC) mice). PDK1(DeltaPOMC) mice initially display hyperphagia, increased body weight, and impaired glucose metabolism caused by reduced hypothalamic POMC expression. On the other hand, PDK1(DeltaPOMC) mice exhibit progressive, severe hypocortisolism caused by loss of POMC-expressing corticotrophs in the pituitary. Expression of a dominant-negative mutant of FOXO1 specifically in POMC cells is sufficient to ameliorate positive energy balance in PDK1(DeltaPOMC) mice but cannot restore regular pituitary function. These results reveal important but differential roles for PDK1 signaling in hypothalamic and pituitary POMC cells in the control of energy homeostasis and stress response.