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Over the last years, hypothalamic inflammation has been linked to the development and progression of obesity and its sequelae. There is accumulating evidence that this inflammation not only impairs energy balance but also contributes to obesity-associated insulin resistance. Elevated activation of key inflammatory mediators such as JNK and IκB kinase (IKK) occurs rapidly upon consumption of a high-fat diet, even prior to significant weight gain. This activation of hypothalamic inflammatory pathways results in the uncoupling of caloric intake and energy expenditure, fostering overeating and further weight gain. In addition, these inflammatory processes contribute to obesity-associated insulin resistance and deterioration of glucose metabolism via altered neurocircuit functions. An understanding of the contributions of different neuronal and non-neuronal cell types to hypothalamic inflammatory processes, and delineation of the differences and similarities between acute and chronic activation of these inflammatory pathways, will be critical for the development of novel therapeutic strategies for the treatment of obesity and metabolic syndrome., Introduction For many years obesity has been known to induce a state of systemic low-grade inflammation that contributes to the development of numerous comorbidities, such as type 2 diabetes, dyslipidemia, [...]

The transcription factor FOXO1 is identified as a crucial checkpoint of vascular growth, coupling the metabolic and proliferative activities of endothelial cells. FOXO1 is a checkpoint of vascular growth The mechanisms that balance the metabolism of endothelial cells and their growth state are not known. Here Michael Potente and colleagues identify the transcription factor FOXO1 as a crucial checkpoint of vascular growth, coupling the metabolic and proliferative activities of endothelial cells. They find that FOXO1 expression in endothelial cells is required to keep the cells quiescent, through suppressing c-MYC signalling, thereby reducing glycolysis and mitochondrial respiration. Endothelial-specific deletion of FOXO1 in mice induces vessel hyperplasia and enlargement. Endothelial cells (ECs) are plastic cells that can switch between growth states with different bioenergetic and biosynthetic requirements.sup.1. Although quiescent in most healthy tissues, ECs divide and migrate rapidly upon proangiogenic stimulation.sup.2,3. Adjusting endothelial metabolism to the growth state is central to normal vessel growth and function.sup.1,4, yet it is poorly understood at the molecular level. Here we report that the forkhead box O (FOXO) transcription factor FOXO1 is an essential regulator of vascular growth that couples metabolic and proliferative activities in ECs. Endothelial-restricted deletion of FOXO1 in mice induces a profound increase in EC proliferation that interferes with coordinated sprouting, thereby causing hyperplasia and vessel enlargement. Conversely, forced expression of FOXO1 restricts vascular expansion and leads to vessel thinning and hypobranching. We find that FOXO1 acts as a gatekeeper of endothelial quiescence, which decelerates metabolic activity by reducing glycolysis and mitochondrial respiration. Mechanistically, FOXO1 suppresses signalling by MYC (also known as c-MYC), a powerful driver of anabolic metabolism and growth.sup.5,6. MYC ablation impairs glycolysis, mitochondrial function and proliferation of ECs while its EC-specific overexpression fuels these processes. Moreover, restoration of MYC signalling in FOXO1-overexpressing endothelium normalizes metabolic activity and branching behaviour. Our findings identify FOXO1 as a critical rheostat of vascular expansion and define the FOXO1-MYC transcriptional network as a novel metabolic checkpoint during endothelial growth and proliferation., Author(s): Kerstin Wilhelm [sup.1] , Katharina Happel [sup.1] , Guy Eelen [sup.2] [sup.3] , Sandra Schoors [sup.2] [sup.3] , Mark F. Oellerich [sup.1] , Radiance Lim [sup.1] , Barbara Zimmermann [...]

Polymorphisms in the fat mass and obesity-associated gene (FTO) are associated with human obesity and obesity-prone behaviors, including increased food intake and a preference for energy-dense foods. FTO demethylates [N.sup.6]-methyladenosine, [...]

Insulin resistance represents a hallmark during the development of type 2 diabetes mellitus and in the pathogenesis of obesity-associated disturbances of glucose and lipid metabolism (1-3). MicroRNA (miRNA)-dependent post-transcriptional gene [...]

Pubertal onset, initiated by pulsatile gonadotropin-releasing hormone (GnRH), only occurs in a favorable, anabolic hormonal milieu. Anabolic factors that may signal nutritional status to the hypothalamus include the growth factors insulin and IGF-1. It is unclear which hypothalamic neuronal subpopulation these factors affect to ultimately regulate GnRH neuron function in puberty and reproduction. We examined the direct role of the GnRH neuron in growth factor regulation of reproduction using the Cre/lox system. Mice with the IR or IGF-1R deleted specifically in GnRH neurons were generated. Male and female mice with the IR deleted in GnRH neurons displayed normal pubertal timing and fertility, but male and female mice with the IGF-1R deleted in GnRH neurons experienced delayed pubertal development with normal fertility. With IGF-1 administration, puberty was advanced in control females, but not in females with the IGF-1R deleted in GnRH neurons, in control males, or in knockout males. These mice exhibited developmental differences in GnRH neuronal morphology but normal number and distribution of neurons. These studies define the role of IGF-1R signaling in the coordination of somatic development with reproductive maturation and provide insight into the mechanisms regulating pubertal timing in anabolic states., Introduction At pubertal onset, the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator becomes activated to trigger the hormonal cascade necessary for sexual maturation. It has been estimated that 50%-80% of the [...]

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses (1,2). The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components (3). Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase (4)) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to [p22.sub.phox], the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and [p22.sub.phox] is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase., TNFR1 was recently shown to activate NADPH oxidase 1 (Nox1), resulting in ROS production and necrotic cell deaths. TNF-induced Nox1 activation was suggested to occur through TRADD (5), a crucial [...]

Several independent, genome-wide association studies have identified a strong correlation between body mass index and polymorphisms in the human FTO gene (1-4). Common variants in the first intron define a risk allele predisposing to obesity, with homozygotes for the risk allele weighing approximately 3 kilograms more than homozygotes for the low risk allele (1). Nevertheless, the functional role of FTO in energy homeostasis remains elusive. Here we show that the loss of Fto in mice leads to postnatal growth retardation and a significant reduction in adipose tissue and lean body mass. The leanness of Fto-deficient mice develops as a consequence of increased energy expenditure and systemic sympathetic activation, despite decreased spontaneous locomotor activity and relative hyperphagia. Taken together, these experiments provide, to our knowledge, the first direct demonstration that Fto is functionally involved in energy homeostasis by the control of energy expenditure., Previously, we have identified the murine homologue Fto as part of a 1.6-megabase (Mb) deletion on chromosome 8 in the Fused toes mouse mutant, a mutant with severe developmental defects [...]

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive [K.sup.+] ([K.sub.ATP]) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic [beta] cells. These [beta]-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from [beta]-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of [K.sub.ATP] channels in pancreatic [beta] cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of [K.sub.ATP] channels, closed [K.sub.ATP] channels, elevated intracellular calcium levels, and stimulated insulin release in [beta]-V59M [beta] cells, indicating that events downstream of [K.sub.ATP] channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic [beta] cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. [beta]-V59M islets also displayed a reduced percentage of [beta] cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of [beta]-V59M mice. Their cause requires further investigation., Introduction Neonatal diabetes (ND) is a rare disease that affects about 1 in 200,000 live births (1). It is defined as diabetes that manifests within the first 6 months of [...]

Insulin resistance is a hallmark of type 2 diabetes, and many insights into the functions of insulin have been gained through the study of mice lacking the IR. To gain a better understanding of the role of insulin action in the brain versus peripheral tissues, we created 2 mouse models with inducible IR inactivation, 1 in all tissues including brain ([IR.sup.[DELTA]wb]), and 1 restricted to peripheral tissues ([IR.sup.[DELTA]per]). While downregulation of IR expression resulted in severe hyperinsulinemia in both models, hyperglycemia was more pronounced in [IR.sup.[DELTA]wb] mice. Both strains displayed a dramatic upregulation of hepatic leptin receptor expression, while only [IR.sup.[DELTA]per] mice displayed increased hepatic Stat3 phosphorylation and Il6 expression. Despite a similar reduction in IR expression in white adipose tissue (WAT) mass in both models, [IR.sup.[DELTA]wb] mice had a more pronounced reduction in WAT mass and severe hypoleptinemia. Leptin replacement restored hepatic Stat3 phosphorylation and normalized glucose metabolism in these mice, indicating that alterations in glucose metabolism occur largely as a consequence of lipoathrophy upon body-wide IR deletion. Moreover, chronic intracerebroventricular insulin treatment of control mice increased fat mass, fat cell size, and adipose tissue lipoprotein lipase expression, indicating that CNS insulin action promotes lipogenesis. These studies demonstrate that central insulin action plays an important role in regulating WAT mass and glucose metabolism via hepatic Stat3 activation., Introduction Resistance to the biological effects of the peptide hormone insulin represents one of the hallmarks during the development of type 2 diabetes (1), (2). Apart from the direct effect [...]

As obesity, diabetes, and associated comorbidities are on a constant rise, large efforts have been put into better understanding the cellular and molecular mechanisms by which nutrients and metabolic signals influence central and peripheral energy regulation. For decades, peripheral organs as a source and a target of such cues have been the focus of study. Their ability to integrate metabolic signals is essential for balanced energy and glucose metabolism. Only recently has the pivotal role of the central nervous system in the control of fuel partitioning been recognized. The rapidly expanding knowledge on the elucidation of molecular mechanisms and neuronal circuits involved is the locus of this review. central nervous system; insulin; glucose metabolism

Development of obesity-associated insulin resistance and diabetes mellitus type 2 has been linked to activation of proinflammatory pathways in the liver, leading to impaired insulin signal transduction. To further define the role of hepatic NF-[kappa]B activation in this process, we have analyzed glucose metabolism in mice with liver-specific inactivation of the NF-[kappa]B essential modulator gene ([NEMO.sup.L-KO] mice) exposed to a high-fat diet (HFD). These animals are protected from the development of obesity-associated insulin resistance, highlighting the importance of hepatic NF-[kappa]B activation in this context. However, hepatic NEMO deficiency synergizes with HFD in the development of liver steatosis as a consequence of decreased peroxisome proliferator-activated receptor (PPAR-[alpha]) and increased PPAR-[gamma], expression. Steatosis interacts with increased inflammation, causing elevated apoptosis in the livers of these mice under HFD. These changes result in liver tumorigenesis of [NEMO.sup.L-KO] mice under normal diet, a process that is largely aggravated when these mice are exposed to HFD. These data directly demonstrate the interaction of hepatic inflammation, dietary composition, and metabolism in the development of liver tumorigenesis. insulin sensitivity | spontaneous hepatic carcinoma | steatosis | hepatic NEMO deficiency

Ciliary neurotrophic factor (CNTF) exerts anorectic effects by overcoming leptin resistance via activation of hypothalamic neurons. However, the exact site of CNTF action in the hypothalamus has not yet been identified. Using Cre-loxP-mediated recombination in vivo, we have selectively ablated the common cytokine signaling chain gp130, which is required for functional CNTF signaling, in proopiomelanocortin (POMC)-expressing neurons. POMC-specific gp130 knockout mice exhibit unaltered numbers of POMC cells and normal energy homeostasis under standard and high fat diet. Endotoxin (LPS) and stress-induced anorexia and adrenocorticotropin regulation were unaffected in these animals, Strikingly, the anorectic effect of centrally administered CNTF was abolished in POMC-specific gp130 knockout mice. Correspondingly, in these animals, CNTF failed to activate STAT3 phosphorylation in POMC neurons and to induce c-Fos expression in the paraventricular nucleus. These data reveal POMC neurons as a critical site of CNTF action in mediating its anorectic effect. CNS | obesity

Leptin and insulin have been identified as fuel sensors acting in part through their hypothalamic receptors to inhibit food intake and stimulate energy expenditure. As their intracellular signaling converges at [...]

Insulin has pleiotropic biological effects in virtually all tissues. However, the relevance of insulin signaling in peripheral tissues has been studied far more extensively than its role in the brain. [...]

G protein-coupled receptors (GPCRs) are essential for normal central CNS function and represent the proximal site(s) of action for most neurotransmitters and many therapeutic drugs, including typical and atypical antipsychotic drugs. Similarly, protein kinases mediate many of the downstream actions for both ionotropic and metabotropic receptors. We report here that genetic deletion of p90 ribosomal S6 kinase 2 (RSK2) potentiates GPCR signaling. Initial studies of 5-hydroxytryptamine [(5-HT).sub.2A] receptor signaling in fibroblasts obtained from RSK2 wild-type (+/+) and knockout (-/-) mice showed that [5-HT.sub.2A] receptor-mediated phosphoinositide hydrolysis and both basal and 5-HT-stimulated extracellular signal-regulated kinase 1/2 phosphorylation are augmented in RSK2 knockout fibroblasts. Endogenous signaling by other GPCRs, including P2Y-purinergic, PAR-1-thrombinergic, [beta]1-adrenergic, and bradykinin-B receptors, was also potentiated in RSK2-deficient fibroblasts. Importantly, reintroduction of RSK2 into RSK2-/fibroblasts normalized signaling, thus demonstrating that RSK2 apparently modulates GPCR signaling by exerting a 'tonic brake' on GPCR signal transduction. Our results imply the existence of a novel pathway regulating GPCR signaling, modulated by downstream members of the extracellular signal-related kinase/ mitogen-activated protein kinase cascade. The loss of RSK2 activity in humans leads to Coffin-Lowry syndrome, which is manifested by mental retardation, growth deficits, skeletal deformations, and psychosis. Because RSK2-inactivating mutations in humans lead to Coffin-Lowry syndrome, our results imply that alterations in GPCR signaling may account for some of its clinical manifestations. serotonin | signal transduction

Impairment of insulin signaling in the brain has been linked to neurodegenerative diseases. To test the hypothesis that neuronal insulin resistance contributes to defects in neuronal function, we have performed a detailed analysis of brain/neuron-specific insulin receptor knockout (NIRKO) mice. We find that NIRKO mice exhibit a complete loss of insulin-mediated activation of phosphatidylinositol 3-kinase and inhibition of neuronal apoptosis. In intact animals, this loss results in markedly reduced phosphorylation of Akt and GSK3 [beta], leading to substantially increased phosphorylation of the microtubule-associated protein Tau, a hallmark of neurodegenerative diseases. Nevertheless, these animals exhibit no alteration in neuronal proliferation/survival, memory, or basal brain glucose metabolism. Thus, lack of insulin signaling in the brain may lead to changes in Akt and GSK3/] activity and Tau hyperphosphorylation but must interact with other mechanisms for development of Alzheimer's disease.

Research was conducted to characterize the phosphorylation of recombinant human Grb2-associated binder-1 (Gab-1) protein by epidermal growth factor receptor (EGFR) in vitro. The kinetic data for phosphorylation of the proteins and the recombinant EGFR receptor kinase domain were determined by using hte pGEX system. Results indicate that human Gab-1 is a high-affinity substrate for the EGFR. Findings also reveal that the major tyrosine phosphorylation site in the C terminus is a specific binding site for the tyrosine phosphatase Syp.

Mice which are double heterozygous for null alleles in the insulin receptor and insulin receptor substrate-1 genes which rendered them highly insulin resistant, were used as experimental disease model for non-insulin-dependent diabetes mellitus (NIDDM). The mice were observed to develop NIDDM in an age-dependent manner. The results of immunoprecipitation assay and Western blot analysis revealed that the genetic and molecular mechanisms behind NIDDM involve the combination of two mild effects in the insulin signaling cascade, leading to insulin resistance and subsequent progression to a diabetic phenotype.

Cutting-edge experiments show that the hormone leptin, which is secreted by fat cells, promotes fat loss by activating the release of catecholamine signalling molecules from neurons wrapped around the fat [...]

Insulin receptors (IRs) and insulin signaling proteins are widely distributed throughout the central nervous system (CNS). To study the physiological role of insulin signaling in the brain, we created mice with a neuron-specific disruption of the IR gene (NIRKO mice). Inactivation of the IR had no impact on brain development or neuronal survival However, female NIRKO mice showed increased food intake, and both male and female mice developed diet-sensitive obesity with increases in body fat and plasma leptin levels, mild insulin resistance, elevated plasma insulin levels, and hypertriglyceridemia. NIRKO mice also exhibited impaired spermatogenesis and ovarian follicle maturation because of hypothalamic dysregulation of luteinizing hormone. Thus, IR signaling in the CNS plays an important role in regulation of energy disposal fuel metabolism, and reproduction., Insulin receptors (IR) are expressed in most tissues of the body, including classic insulin-sensitive tissues (liver, muscle, and fat), as well as 'insulin-insensitive' tissue, such as red blood cells and [...]

Ribosomal subunit kinases (Rsk) have been implicated in the regulation of transcription by phosphorylating and thereby activating numerous transcription factors, such as c-Fos, cAMP responsive element binding protein (CREB), and nuclear receptors. Here we describe the generation and characterization of immortalized embryonic fibroblast cell lines from mice in which the Rsk-2 gene was disrupted by homologous recombinant gene targeting. Rsk2-deficient (knockout or KO) cell lines have no detectable Rsk-2 protein, whereas Rsk-1 expression is unaltered as compared with cell lines derived from wild-type control mice. KO cells exhibit a major reduction in platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1-stimulated expression of the immediate-early gene c-Fos. This results primarily from a reduced transcriptional activation of the ternary complex factor Elk-1 and reduced activation of the serum response factor. The reduced Elk-1 activation in KO cells occurs despite normal activation of the mitogen-activated protein kinase pathway and normal PDGF- and IGF-l-stimulated Elk-1 phosphorylation. By contrast, PDGF- and IGF-l-stimulated phosphorylation and transcriptional activation of CREB is unaltered in KO cells. Thus Rsk-2 is required for growth factor-stimulated expression of c-Fos and transcriptional activation of Elk-1 and the serum response factor, but not for activation of CREB or the mitogen-activated protein kinase pathway in response to PDGF and IGF-1 stimulation.

Author(s): Tamas L Horvath [1]; Jens C Bruning [2] Metabolic disorders, such as obesity and type 2 diabetes, have become major causes of morbidity and mortality in Western societies. Although [...]

The role of insulin signaling in mice pancreatic beta cells was investigated using the Cre-loxP system to inactivate the insulin receptor gene in the beta cells. Mice lacking in the insulin receptor gene were found to display a selective loss of insulin secretion in response to glucose and an impairment in glucose tolerance, indicating that the insulin receptor plays an important role in glucose sensing by the beta cells. Results also suggested that impairments in insulin signaling at the beta cell level influence the changes in insulin secretion in type 2 diabetes.