Role of Hypothalamic Creb-Binding Protein in Obesity and Molecular Reprogramming of Metabolic Substrates.

We have reported a correlation between hypothalamic expression of Creb-binding protein (Cbp) and lifespan, and that inhibition of Cbp prevents protective effects of dietary restriction during aging, suggesting that hypothalamic Cbp plays a role in responses to nutritional status and energy balance. Recent GWAS and network analyses have also implicated Cbp as the most connected gene in protein-protein interactions in human Type 2 diabetes. The present studies address mechanisms mediating the role of Cbp in diabetes by inhibiting hypothalamic Cbp using a Cre-lox strategy. Inhibition of hypothalamic Cbp results in profound obesity and impaired glucose homeostasis, increased food intake, and decreased body temperature. In addition, these changes are accompanied by molecular evidence in the hypothalamus for impaired leptin and insulin signaling, a shift from glucose to lipid metabolism, and decreased Pomc mRNA, with no effect on locomotion. Further assessment of the significance of the metabolic switch demonstrated that enhanced expression of hypothalamic Cpt1a, which promotes lipid metabolism, similarly resulted in increased body weight and reduced Pomc mRNA.

Regulation of peripheral metabolism by substrate partitioning in the brain.

All organisms must adapt to changing nutrient availability, with nutrient surplus promoting glucose metabolism and nutrient deficit promoting alternative fuels (in mammals, mainly free fatty acids). A major function of glucose-sensing neurons in the hypothalamus is to regulate blood glucose. When these neurons sense glucose levels are too low, they activate robust counterregulatory responses to enhance glucose production, primarily from liver, and reduce peripheral metabolism. Some hypothalamic neurons can metabolize free fatty acids via ß-oxidation, and ß-oxidation generally opposes effects of glucose on hypothalamic neurons. Thus hypothalamic ß-oxidation promotes obese phenotypes, including enhanced hepatic glucose output.

Hypothalamic Fkbp51 is induced by fasting, and elevated hypothalamic expression promotes obese phenotypes.

To discover hypothalamic genes that might play a role in regulating energy balance, we carried out a microarray screen for genes induced by a 48-h fast in male C57Bl/6J mouse hypothalamus. One such gene was Fkbp51 (FK506 binding protein 5; Locus NP_034350). The product of this gene is of interest because it blocks glucocorticoid action, suggesting that fasting-induced elevation of this gene in the hypothalamus may reduce glucocorticoid negative feedback, leading to elevated glucocorticoid levels, thus promoting obese phenotypes. Subsequent analysis demonstrated that a 48-h fast induces Fkbp51 in ventromedial, paraventricular, and arcuate hypothalamic nuclei of mice and rats. To assess if hypothalamic Fkbp51 promotes obesity, the gene was transferred to the hypothalamus via an adeno-associated virus vector. Within 2 wk following Fkbp51 overexpression, mice on a high-fat diet exhibited elevated body weight, without hyperphagia, relative to mice receiving the control mCherry vector. Body weight remained elevated for more than 8 wk and was associated with elevated corticosterone and impaired glucose tolerance. These studies suggest that elevated hypothalamic Fkbp51 promotes obese phenotypes.

Estradiol impairs hypothalamic molecular responses to hypoglycemia.

In rats and humans estradiol attenuates neuroendocrine responses to hypoglycemia. Since neuroendocrine responses to hypoglycemia are mediated by hypothalamic neurons, we assessed if estradiol attenuates hypoglycemia-induced gene expression in the hypothalamus in female ovariectomized mice. As expected, estradiol-implanted ovariectomized mice exhibited increased plasma estradiol, increased uterine weight, decreased body weight, decreased visceral adiposity, and enhanced glucose tolerance with decreased plasma insulin. Estradiol-implanted mice exhibited attenuated hypoglycemia-induced gene expression of both glucose transporter 1 (Glut1) and inhibitor of kappa beta signaling (IkappaB) in the hypothalamus but not in the liver. Estradiol also attenuated hypoglycemia-induced plasma glucagon, pituitary proopiomelanocortin (POMC), and adrenal c-fos, consistent with impaired counterregulatory responses to hypoglycemia. In addition, estradiol inhibited hypothalamic expression of carnitine palmitoyltransferase (CPT1a and CPT1c) and pyruvate dehydrogenase kinase 4 (PDK4), effects that would be expected to enhance the accumulation of long-chain fatty acids and glycolysis. Taken together, these findings suggest hypothalamic mechanisms mediating attenuation of hypoglycemia-induced neuroendocrine responses.

Inhibition of agouti-related peptide expression by glucose in a clonal hypothalamic neuronal cell line is mediated by glycolysis, not oxidative phosphorylation.

The regulation of neuroendocrine electrical activity and gene expression by glucose is mediated through several distinct metabolic pathways. Many studies have implicated AMP and ATP as key metabolites mediating neuroendocrine responses to glucose, especially through their effects on AMP-activated protein kinase (AMPK), but other studies have suggested that glycolysis, and in particular the cytoplasmic conversion of nicotinamide adenine dinucleotide (NAD+) to reduced NAD (NADH), may play a more important role than oxidative phosphorylation for some effects of glucose. To address these molecular mechanisms further, we have examined the regulation of agouti-related peptide (AgRP) in a clonal hypothalamic cell line, N-38. AgRP expression was induced monotonically as glucose concentrations decreased from 10 to 0.5 mm glucose and with increasing concentrations of glycolytic inhibitors. However, neither pyruvate nor 3-beta-hydroxybutyrate mimicked the effect of glucose to reduce AgRP mRNA, but on the contrary, produced the opposite effect of glucose and actually increased AgRP mRNA. Nevertheless, 3beta-hydroxybutyrate mimicked the effect of glucose to increase ATP and to decrease AMPK phosphorylation. Similarly, inhibition of AMPK by RNA interference increased, and activation of AMPK decreased, AgRP mRNA. Additional studies demonstrated that neither the hexosamine nor the pentose/carbohydrate response element-binding protein pathways mediate the effects of glucose on AgRP expression. These studies do not support that either ATP or AMPK mediate effects of glucose on AgRP in this hypothalamic cell line but support a role for glycolysis and, in particular, NADH. These studies support that cytoplasmic or nuclear NADH, uniquely produced by glucose metabolism, mediates effects of glucose on AgRP expression.

Impaired glucose signaling as a cause of obesity and the metabolic syndrome: the glucoadipostatic hypothesis.

Since nutrition-sensitive feedback signals normally act to maintain relatively stable levels of both available and stored nutritional resources, failure in one or more of these feedback signals could plausibly lead to obese phenotypes. The glucostatic hypothesis in its original form posited that glucose serves as a physiological satiety factor (in the sense that post-prandial increases in plasma glucose cause meal termination), but in this form the hypothesis has been difficult to prove, and, especially since the discovery of leptin, the glucostatic hypothesis has largely been abandoned. Nevertheless, reduction of plasma glucose levels or glucose signaling produces a profile of neuroendocrine responses similar to those produced by leptin deficiency. Since leptin is not a physiological satiety factor (because it does not increase before meal termination), yet leptin deficiency causes obesity, we suggest that the glucostatic hypothesis be re-formulated without reference to satiety (i.e., short-term effects on food intake). Instead we argue that like leptin signaling, glucose signaling regulates long-term energy balance, in part by regulating metabolic rate but also by chronically regulating food intake. We further speculate that high-fat diets produce obesity in part because carbohydrates are, per calorie, more effective than lipids to reduce food intake and increase metabolic rate. In support of this glucoadipostatic hypothesis, the 5 present review examines evidence that obesity and the metabolic syndrome may be due to reduction in neuroendocrine sensitivity to glucose leading to increased metabolic efficiency.

D-chiro-Inositol enhances effects of hypothalamic toxin gold-thioglucose.

D-chiro-Inositol (DCI) enhances reproductive function in insulin-resistant women with polycystic ovarian disease and enhances the effects of insulin in the periphery, suggesting that this compound may act in part by sensitizing the hypothalamus to effects of insulin. Effects of gold-thioglucose (GTG) to produce hypothalamic lesions and subsequent obesity are insulin-dependent, suggesting that responses to GTG may be a marker of hypothalamic sensitivity to insulin. To assess these hypotheses, the present study assessed if DCI would enhance the ability of a subthreshhold dose of GTG to produce hypothalamic lesions and subsequent obesity. At the subthreshhold dose used (0.4 mg/kg i.p.), injection of GTG produced no subsequent effect on body weight compared to saline; similarly, at the dose of DCI used (10 mg/kg/day in drinking water), DCI produced no effect on body weight. In contrast, when given to mice exposed to DCI, this dose of GTG produced significant increase in body weight and evidence of an enhanced medial arcuate hypothalamic lesion.

Role of glucocorticoids in mediating effects of fasting and diabetes on hypothalamic gene expression.

BACKGROUND: Fasting and diabetes are characterized by elevated glucocorticoids and reduced insulin, leptin, elevated hypothalamic AGRP and NPY mRNA, and reduced hypothalamic POMC mRNA. Although leptin replacement can reverse changes in hypothalamic gene expression associated with fasting and diabetes, leptin also normalizes corticosterone; therefore the extent to which the elevated corticosterone contributes to the regulation of hypothalamic gene expression in fasting and diabetes remains unclear. To address if elevated corticosterone is necessary for hypothalamic responses to fasting and diabetes, we assessed the effects of adrenalectomy on hypothalamic gene expression in 48-hour-fasted or diabetic mice. To assess if elevated corticosterone is sufficient for the hypothalamic responses to fasting and diabetes, we assessed the effect of corticosterone pellets implanted for 48 hours on hypothalamic gene expression. RESULTS: Fasting and streptozotocin-induced diabetes elevated plasma glucocorticoid levels and reduced serum insulin and leptin levels. Adrenalectomy prevented the rise in plasma glucocorticoids associated with fasting and diabetes, but not the associated reductions in insulin or leptin. Adrenalectomy blocked the effects of fasting and diabetes on hypothalamic AGRP, NPY, and POMC expression. Conversely, corticosterone implants induced both AGRP and POMC mRNA (with a non-significant trend toward induction of NPY mRNA), accompanied by elevated insulin and leptin (with no change in food intake or body weight). CONCLUSION: These data suggest that elevated plasma corticosterone mediate some effects of fasting and diabetes on hypothalamic gene expression. Specifically, elevated plasma corticosterone is necessary for the induction of NPY mRNA with fasting and diabetes; since corticosterone implants only produced a non-significant trend in NPY mRNA, it remains uncertain if a rise in corticosterone may be sufficient to induce NPY mRNA. A rise in corticosterone is necessary to reduce hypothalamic POMC mRNA with fasting and diabetes, but not sufficient for the reduction of hypothalamic POMC mRNA. Finally, elevated plasma corticosterone is both necessary and sufficient for the induction of hypothalamic AGRP mRNA with fasting and diabetes.