Regulation of the Fructose Transporter Gene Slc2a5 Expression by Glucose in Cultured Microglial Cells.

Microglia play a role in the regulation of metabolism and pathogenesis of obesity. Microglial activity is altered in response to changes in diet and the body's metabolic state. Solute carrier family 2 member 5 (Slc2a5) that encodes glucose transporter 5 (GLUT5) is a fructose transporter primarily expressed in microglia within the central nervous system. However, little is known about the nutritional regulation of Slc2a5 expression in microglia and its role in the regulation of metabolism. The present study aimed to address the hypothesis that nutrients affect microglial activity by altering the expression of glucose transporter genes. Murine microglial cell line SIM-A9 cells and primary microglia from mouse brain were exposed to different concentrations of glucose and levels of microglial activation markers and glucose transporter genes were measured. High concentration of glucose increased levels of the immediate-early gene product c-Fos, a marker of cell activation, Slc2a5 mRNA, and pro-inflammatory cytokine genes in microglial cells in a time-dependent manner, while fructose failed to cause these changes. Glucose-induced changes in pro-inflammatory gene expression were partially attenuated in SIM-A9 cells treated with the GLUT5 inhibitor. These findings suggest that an increase in local glucose availability leads to the activation of microglia by controlling their carbohydrate sensing mechanism through both GLUT5-dependent and -independent mechanisms.

Negative regulation of hepatic fat mass and obesity associated (Fto) gene expression by insulin.

AIMS: To investigate the role of glucose and insulin in the regulation of hepatic fat mass and obesity associated (Fto) gene expression and the role of hepatic Fto in the regulation of gluconeogenic gene expression. MAIN METHODS: To determine the effect of hyperglycemia on hepatic Fto expression, levels of Fto mRNA in liver were compared between normoglycemic/normoinsulinemic, hypereglycemic/hyperinsulinemic, and hyperglycemic/hypoinsulinemic mice. To determine the direct effect of insulin on Fto expression, levels of Fto, glucose-6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (Pepck) mRNA levels were compared between control and insulin-treated mouse liver tissues cultured ex vivo and immortalized mouse hepatocytes AML12. To determine the role of Fto in the regulation of gluconeogenic gene expression, we examined the effect of enhanced Fto expression on G6pase and Pepck mRNA levels in AML12 cells. KEY FINDINGS: Fto mRNA levels were significantly reduced in hyperglycemic/hyperinsulinemic mice compared to normoglycemic/normoinsulinemic mice, while they were indistinguishable between hyperglycemic/hypoinsulinemic mice and normoglycemic/normoinsulinemic mice. Insulin treatment reduced Fto, G6pase, and Pepck mRNA levels compared to control vehicle treatment in both ex vivo cultured mouse liver tissues and AML12 cells. Enhanced Fto expression significantly increased G6pase and Pepck mRNA level in AML12 cells. SIGNIFICANCE: Our findings support the hypothesis that hepatic Fto participates in the maintenance of glucose homeostasis possibly by mediating the inhibitory effect of glucose and insulin on gluconeogenic gene expression in liver. It is further suggested that impairments in nutritional and hormonal regulation of hepatic Fto expression may lead to impairments in glycemic control in diabetes.

Xenin delays gastric emptying rate and activates the brainstem in mice.

Xenin, a 25-amino acid gastrointestinal peptide, inhibits feeding by acting through the central nervous system. Gastrointestinal hormones reduce food intake partly by activating the brainstem and inhibiting gastric emptying. Therefore, we hypothesized that xenin delays gastric emptying through the activation of the brainstem cells. To address this hypothesis, we examined the effect of intraperitoneal (i.p.) injection of xenin on gastric emptying rate and brainstem Fos expression in mice. Gastric emptying rate was reduced by about 93% in xenin-treated mice compared to saline-treated control mice. The i.p. xenin injection significantly increased Fos-immunoreactive cells in the nucleus of the solitary tract (NTS) of the brainstem, but not area postrema (AP) and dorsal motor nucleus of the vagus (DMV). These findings support the hypothesis that xenin-induced anorexia is at least partly due to delayed gastric emptying and the activation of the NTS cells.

Involvement of RAGE, NADPH oxidase, and Ras/Raf-1 pathway in glycated LDL-induced expression of heat shock factor-1 and plasminogen activator inhibitor-1 in vascular endothelial cells.

Atherothrombotic cardiovascular diseases are the predominant causes of mortality of diabetic patients. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor for fibrinolysis, and it is also implicated in inflammation and tissue remodeling. Increased levels of PAI-1 and glycated low-density lipoprotein (glyLDL) were detected in patients with diabetes. Previous studies in our laboratory demonstrated that heat shock factor-1 (HSF1) is involved in glyLDL-induced PAI-1 overproduction in vascular endothelial cells (EC). The present study investigated transmembrane signaling mechanisms involved in glyLDL-induced HSF1 and PAI-1 up-regulation in cultured human vascular EC and streptozotocin-induced diabetic mice. Receptor for advanced glycation end products (RAGE) antibody prevented glyLDL-induced increase in the abundance of PAI-1 in EC. GlyLDL significantly increased the translocation of V-Ha-Ras Harvey rat sarcoma viral oncogene homologue (H-Ras) from cytoplasm to membrane compared with LDL. Farnesyltransferase inhibitor-277 or small interference RNA against H-Ras inhibited glyLDL-induced increases in HSF1 and PAI-1 in EC. Treatment with diphenyleneiodonium, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, blocked glyLDL-induced translocation of H-Ras, elevated abundances of HSF1 and PAI-1 in EC, and increased release of hydrogen peroxide from EC. Small interference RNA for p22(phox) prevented glyLDL-induced expression of NOX2, HSF1, and PAI-1 in EC. GlyLDL significantly increased V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) phosphorylation. Treatment with Raf-1 inhibitor blocked glyLDL-induced increase of PAI-1 mRNA in EC. The levels of RAGE, H-Ras, NOX4, HSF1, and PAI-1 were increased in hearts of streptozotocin-diabetic mice and positively correlated with plasma glucose. The results suggest that RAGE, NOX, and H-Ras/Raf-1 are implicated in the up-regulation of HSF1 or PAI-1 in vascular EC under diabetes-associated metabolic stress.

Xenin, a gastrointestinal peptide, regulates feeding independent of the melanocortin signaling pathway.

OBJECTIVE: Xenin, a 25-amino acid peptide, was initially isolated from human gastric mucosa. Plasma levels of xenin rise after a meal in humans, and administration of xenin inhibits feeding in rats and chicks. However, little is known about the mechanism by which xenin regulates food intake. Signaling pathways including leptin and melanocortins play a pivotal role in the regulation of energy balance. Therefore, we addressed the hypothesis that xenin functions as a satiety factor by acting through the melanocortin system or by interacting with leptin. RESEARCH DESIGN AND METHODS: The effect of intracerebroventricular and intraperitoneal administration of xenin on food intake was examined in wild-type, agouti, and ob/ob mice. The effect of intracerebroventricular injection of SHU9119, a melanocortin receptor antagonist, on xenin-induced anorexia was also examined in wild-type mice. To determine whether the hypothalamus mediates the anorectic effect of xenin, we examined the effect of intraperitoneal xenin on hypothalamic Fos expression. RESULTS: Both intracerebroventricular and intraperitoneal administration of xenin inhibited fasting-induced hyperphagia in wild-type mice in a dose-dependent manner. The intraperitoneal injection of xenin also reduced nocturnal intake in ad libitum-fed wild-type mice. The intraperitoneal injection of xenin increased Fos immunoreactivity in hypothalamic nuclei, including the paraventricular nucleus and the arcuate nucleus. Xenin reduced food intake in agouti and ob/ob mice. SHU9119 did not block xenin-induced anorexia. CONCLUSIONS: Our data suggest that xenin reduces food intake partly by acting through the hypothalamus but via signaling pathways that are independent of those used by leptin or melanocortins.

Tail suspension increases energy expenditure independently of the melanocortin system in mice.

Space travelers experience anorexia and body weight loss in a microgravity environment, and microgravity-like situations cause changes in hypothalamic activity. Hypothalamic melanocortins play a critical role in the regulation of metabolism. Therefore, we hypothesized that microgravity affects metabolism through alterations in specific hypothalamic signaling pathways, including melanocortin signaling. To address this hypothesis, the microgravity-like situation was produced by an antiorthostatic tail suspension in wild-type and agouti mice, and the effect of tail suspension on energy expenditure and hypothalamic gene expression was examined. Energy expenditure was measured using indirect calorimetry before and during the tail suspension protocol. Hypothalamic tissues were collected for gene expression analysis at the end of the 3 h tail suspension period. Tail suspension significantly increased oxygen consumption, carbon dioxide production, and heat production in wild-type mice. Tail suspension-induced increases in energy expenditure were not attenuated in agouti mice. Although tail suspension did not alter hypothalamic proopiomelanocortin (POMC) and agouti-related protein (AGRP) mRNA levels, it significantly increased hypothalamic interleukin 6 (Il-6) mRNA levels. These data are consistent with the hypothesis that microgravity increases energy expenditure and suggest that these effects are mediated through hypothalamic signaling pathways that are independent of melanocortins, but possibly used by Il-6.

Impaired anorectic effect of leptin in neurotensin receptor 1-deficient mice.

Neurotensin plays a role in regulating feeding behavior. Central injection of neurotensin reduces food intake and the anorectic effect of neurotensin is mediated through neurotensin receptor 1 (Ntsr1). Ntsr1-deficient mice are characterized by mild hyperphagia and overweight without hyperleptinemia. The mechanism by which Ntsr1-deficient mice develop these metabolic abnormalities is not well understood. Leptin, secreted by adipocytes, regulates food intake by acting on hypothalamic neurons including neurotensin-producing neurons. Since the anorectic effect of leptin is blocked by neurotensin receptor antagonist, we hypothesized that the anorectic effect of leptin is mediated through Ntsr1 in the central nervous system and that decreased sensitivity to the anorectic effect of leptin contributes to metabolic perturbations in Ntsr1-deficient mice. To address this hypothesis, we examined the effect of intracerebroventricular (i.c.v.) administration of leptin on food intake in Ntsr1-deficient mice. A single i.c.v. injection of leptin caused robust reductions in food intake in wild-type mice. These effects were markedly attenuated in Ntsr1-deficient mice. These data are consistent with our hypothesis that the anorectic effect of leptin is at least partly mediated through central Ntsr1 and that the leptin-Ntsr1 signaling pathway is involved in the regulation of food intake. Our data also suggest that the lack of Ntsr1 reduces sensitivity to the anorectic action of leptin, causing hyperphagia and abnormal weight gain.

VGF ablation blocks the development of hyperinsulinemia and hyperglycemia in several mouse models of obesity.

Targeted deletion of the gene encoding the neuronal and endocrine secreted peptide precursor called VGF (nonacronymic) produces a lean, hypermetabolic, hyperactive mouse. Because VGF mutant mice are resistant to specific forms of diet-, lesion-, and genetically induced obesity, we investigated the role that this polypeptide plays in glucose homeostasis. We report that VGF mutant mice have increased insulin sensitivity by hyperinsulinemic euglycemic clamp analysis, and by insulin and glucose tolerance testing. Blunted counterregulatory responses in VGF-deficient mice were likely influenced by their significantly lower liver glycogen levels. VGF deficiency lowered circulating glucose and insulin levels in several murine models of obesity that are also susceptible to adult onset diabetes mellitus, including A(y)/a agouti, ob/ob, and MC4R(-)/MC4R(-) mice. Interestingly, ablation of Vgf in ob/ob mice decreased circulating glucose and insulin levels but did not affect adiposity, whereas MC4R(-)/MC4R(-) mice that are additionally deficient in VGF have improved insulin responsiveness at 7-8 wk of age, when lean MC4R(-)/MC4R(-) mice already have impaired insulin tolerance but are not yet obese. VGF mutant mice also resisted developing obesity and hyperglycemia in response to a high-fat/high-carbohydrate diet, and after gold thioglucose treatment, which is toxic to hypothalamic glucose-sensitive neurons. Lastly, circulating adiponectin, an adipose-synthesized protein the levels of which are correlated with improved insulin sensitivity, increased in VGF mutant compared with wild-type mice. Modulation of VGF levels and/or VGF signaling may consequently represent an alternative means to regulate circulating glucose levels and insulin sensitivity.

Specific preservation of biosynthetic responses to insulin in adipose tissue may contribute to hyperleptinemia in insulin-resistant obese mice.

Obesity is characterized by whole-body insulin resistance, yet the expression of many insulin-stimulated genes, including leptin, is elevated in obesity. These observations suggest that insulin resistance may depend on tissue type and gene. To address this hypothesis, we examined the regulation of immediate-early gene expression in liver and adipose tissue after injection of insulin and glucose, in lean insulin-sensitive, and in A(y)/a obese insulin-sensitive and obese insulin-resistant mice. Expression of hepatic jun-B mRNA was robustly increased after insulin injection in lean insulin-sensitive a/a mice and insulin-sensitive A(y)/a mice. In contrast, induction of hepatic jun-B and c-fos gene expression by insulin was markedly attenuated in obese insulin-resistant mice. Surprisingly, induction of adipose jun-B and c-fos gene expression by insulin was markedly enhanced in obese insulin-resistant mice. Furthermore, the expressions of jun-B and leptin were also enhanced in insulin-resistant mice after injection of glucose. Leptin mRNA was positively correlated with blood glucose levels and jun-B mRNA in lean but not insulin-resistant mice. Multiple regression analysis indicated that the correlation between leptin mRNA and jun-B mRNA was significant even after removing the effect of blood glucose, but the correlation between leptin mRNA and glucose was no longer significant after removing the effect of jun-B mRNA. These data suggest that some impairments in biosynthetic responses to insulin are manifest primarily in the liver, leading to hyperinsulinemia and stimulating the expression of some adipose insulin-stimulated genes, including leptin. These studies demonstrate the utility of immediate-early gene expression in the analysis of biosynthetic mechanisms of insulin resistance.

Transgenic neuronal expression of proopiomelanocortin attenuates hyperphagic response to fasting and reverses metabolic impairments in leptin-deficient obese mice.

Hypothalamic proopiomelanocortin (POMC) gene expression is reduced in many forms of obesity and diabetes, particularly in those attributable to deficiencies in leptin or its receptor. To assess the functional significance of POMC in mediating metabolic phenotypes associated with leptin deficiency, leptin-deficient mice bearing a transgene expressing the POMC gene under control of the neuron-specific enolase promoter were produced. The POMC transgene attenuated fasting-induced hyperphagia in wild-type mice. Furthermore, the POMC transgene partially reversed obesity, hyperphagia, and hypothermia and effectively normalized hyperglycemia, glucosuria, glucose intolerance, and insulin resistance in leptin-deficient mice. Effects of the POMC transgene on glucose homeostasis were independent of the partial correction of hyperphagia and obesity. Furthermore, the POMC transgene normalized the profile of hepatic and adipose gene expression associated with gluconeogenesis, glucose output, and insulin sensitivity. These results indicate that central POMC is a key modulator of glucose homeostasis and that agonists of POMC products may provide effective therapy in treating impairments in glucose homeostasis when hypothalamic POMC expression is reduced, as occurs with leptin deficiency, hypothalamic damage, and aging.

The fatty acid synthase inhibitor cerulenin and feeding, like leptin, activate hypothalamic pro-opiomelanocortin (POMC) neurons.

Hypothalamic POMC neurons mediate catabolic responses such as decreased food intake and increased energy expenditure by, in part, monitoring levels of metabolic factors such as glucose, insulin and leptin. Recently, fatty acid synthase inhibitors were reported to reduce body weight, inhibit food intake, and increase metabolic rate, possibly by acting on hypothalamic neurons through a mechanism involving malonyl-CoA accumulation. Given the observation that leptin mediates similar catabolic effects by, in part, activating hypothalamic POMC neurons, it is possible that other catabolic signals such as feeding and fatty acid synthase inhibition may also activate POMC neurons. To test this hypothesis, hypothalamic sections from mice that were fed or injected with the fatty acid synthase inhibitor cerulenin were examined for Fos (a marker for neuronal activation) and POMC product immunoreactivity and compared with similarly processed sections from leptin-injected mice. Feeding increased Fos immunoreactivity in the lateral peri-arcuate area of the hypothalamus of both wild-type and leptin-deficient ob/ob mice (P<0.05), indicating that nutritional activation of the hypothalamus can be leptin-independent. Furthermore, feeding significantly induced Fos immunoreactivity in neurons expressing POMC (P<0.003), indicating that feeding, like leptin, activates POMC neurons. Injection with cerulenin, like feeding and leptin, also increased Fos immunoreactivity in the lateral peri-arcuate area (P<0.03) and, more specifically, in neurons expressing POMC. In contrast, injection with cerulenin had no grossly observable effects on cortical Fos immunoreactivity and appeared to suppress fasting-induced Fos immunoreactivity by about 35% (although the decrease did not reach statistical significance) in the medial arcuate nucleus, an area associated with anabolic responses such as increased food intake. Injection with cerulenin also decreased Fos immunoreactivity in the granular layer of the dentate gyrus of the hippocampus by about 30% (P<0.05), further suggesting that cerulenin does not non-specifically activate wide varieties of neurons. These results suggest that activation of hypothalamic POMC neurons may help to mediate some of the catabolic effects associated with feeding, cerulenin and leptin.

Adiponectin is stimulated by adrenalectomy in ob/ob mice and is highly correlated with resistin mRNA.

Plasma levels of the adipocyte product adiponectin, a putative insulin-sensitizing agent, are reduced in obesity, whereas plasma levels of resistin, an agent that some believe to confer insulin resistance, are thought to increase with obesity. Because adrenalectomy can increase insulin sensitivity, we hypothesized that adrenalectomy would increase expression of adiponectin and decrease expression of resistin. Therefore, we measured adiponectin mRNA, adiponectin peptide, and resistin mRNA in adrenalectomized ob/ob mice. Adrenalectomy restored adiponectin expression in ob/ob mice to wild-type levels and stimulated adiponectin peptide to above wild-type levels. Surprisingly, expression of adiponectin and resistin was highly positively correlated even after statistical removal of effects of insulin, glucose, and adiposity. In addition, adiponectin and resistin expression were also highly correlated in diet-induced obese mice. The data support a role for adiponectin in mediating some effects of adrenalectomy on insulin sensitivity.

VGF is required for obesity induced by diet, gold thioglucose treatment, and agouti and is differentially regulated in pro-opiomelanocortin- and neuropeptide Y-containing arcuate neurons in response to fasting.

Targeted deletion of the gene encoding the neuronal and neuroendocrine secreted polypeptide VGF (nonacronymic) produces a lean, hypermetabolic mouse. Consistent with this phenotype, VGF mRNA levels are regulated in the hypothalamic arcuate nucleus in response to fasting. To gain insight into the site(s) and mechanism(s) of action of VGF, we further characterized VGF expression in the hypothalamus. Double-label studies indicated that VGF and pro-opiomelanocortin were coexpressed in lateral arcuate neurons in the fed state, and that VGF expression was induced after fasting in medial arcuate neurons that synthesize neuropeptide Y (NPY). Like NPY, VGF mRNA induction in this region of the hypothalamus in fasted mice was inhibited by exogenous leptin. In leptin-deficient ob/ob and receptor-mutant db/db mice, VGF mRNA levels in the medial arcuate were elevated. To identify neural pathways that are functionally compromised by Vgf ablation, VGF mutant mice were crossed with obese A(y)/a (agouti) and ob/ob mice. VGF deficiency completely blocked the development of obesity in A(y)/a mice, whereas deletion of Vgf in ob/ob mice attenuated weight gain but had no impact on adiposity. Hypothalamic levels of NPY and agouti-related polypeptide mRNAs in both double-mutant lines were dramatically elevated 10- to 15-fold above those of wild-type mice. VGF-deficient mice were also found to resist diet- and gold thioglucose-induced obesity. These data and the susceptibility of VGF mutant mice to monosodium glutamate-induced obesity are consistent with a role for VGF in outflow pathways, downstream of hypothalamic and/or brainstem melanocortin 4 receptors, that project via the autonomic nervous system to peripheral metabolic tissues and regulate energy homeostasis.