Estrogen receptor ␣ (ER␣) plays a pivotal role in the regulation of food intake and energy expenditure by estrogens. Although it is well documented that a disruption of ER␣ signaling in ER␣ knockout (ERKO) mice leads to an obese phenotype, the sites of estrogen action and mechanisms underlying this phenomenon are still largely unknown. In the present study, we exploited RNA interference mediated by adeno-associated viral vectors to achieve focused silencing of ER␣ in the ventromedial nucleus of the hypothalamus, a key center of energy homeostasis. After suppression of ER␣ expression in this nucleus, female mice and rats developed a phenotype characteristic for metabolic syndrome and marked by obesity, hyperphagia, impaired tolerance to glucose, and reduced energy expenditure. This phenotype persisted despite normal ER␣ levels elsewhere in the brain. Although an increase in food intake preceded weight gain, our data suggest that a leading factor of obesity in this model is likely a decline in energy expenditure with all three major constituents being affected, including voluntary activity, basal metabolic rate, and diet-induced thermogenesis. Together, these findings indicate that ER␣ in the ventromedial nucleus of the hypothalamus neurons plays an essential role in the control of energy balance and the maintenance of normal body weight.adeno-associated virus ͉ body weight ͉ energy metabolism ͉ obesity ͉ RNA interference E strogen receptor ␣ (ER␣) is the main mediator of estrogen effects on energy homeostasis. ER␣ knockout (ERKO) mice with targeted deletion of this receptor develop several hallmark features often associated with obesity including increased visceral adiposity, elevated insulin levels, and impaired glucose tolerance (1). Although the nature of events leading to this phenotype is unclear, hyperphagia does not seem to be the cause because food consumption was not altered in ERKO mice. Instead, several observations suggest that the weight gain in this model is due to a decrease in energy expenditure, which given the normal food intake would result in a state of positive energy balance (1, 2). In this respect, this condition partially resembles an obesity syndrome following lesions of the ventromedial nucleus of hypothalamus (VMN), which is also marked by a significant weight gain due to an accumulation of visceral fat, impaired glucose homeostasis, and reduced energy expenditure (3, 4). Although VMN-lesioned animals initially become hyperphagic as well, when tube-pair-fed with control animals to ensure equal food consumption, lesioned rats still gained more weight and accumulated more fat, albeit to a lesser extent than rats fed ad libitum (3, 5). There are additional lines of evidence that are consistent with a functional role of VMN ER␣ in the regulation of body weight. VMN has a high density of estrogenbinding sites (6), and the neurons in this nucleus express ER␣ at high levels (7). Although VMN lesioning or ovariectomy both lead to increased food intake and body weight, the effect does not appear...

Glucose-responsive neurons in the ventromedial hypothalamus (VMH) are stimulated when glucose increases from 5 to 20 mmol/l and are thought to play an essential role in regulating metabolism. The present studies examined the role of glucose metabolism in the mechanism by which glucose-responsive neurons sense glucose. The pancreatic, but not hepatic, form of glucokinase was expressed in the VMH, but not cerebral cortex, of adult rats. In brain slice preparations, the transition from 5 to 20 mmol/l glucose stimulated approximately 17% of the neurons (as determined by single-cell extracellular recording) from VMH but none in cortex. In contrast, most cells in both VMH and cortex were silent below 1 mmol/l and active at 5 mmol/l glucose. Glucosamine, 2-deoxyglucose, phloridzin, and iodoacetic acid blocked the activation of glucose-responsive neurons by the transition from 5 to 20 mmol/l glucose. Adding 15 mmol/l mannose, galactose, glyceraldehyde, glycerol, and lactate to 5 mmol/l glucose stimulated glucose-responsive neurons. In contrast, adding 15 mmol/l pyruvate to 5 mmol/l glucose failed to activate glucose-responsive neurons, although pyruvate added to 0 mmol/l glucose permitted neurons to maintain activity. Tolbutamide activated glucose-responsive neurons; however, diazoxide only blocked the effect of glucose in a minority of neurons. These data suggest that glucose-responsive neurons sense glucose through glycolysis using a mechanism similar to the mechanism of pancreatic beta-cells, except that glucose-responsive neurons are stimulated by glycerol and lactate, and diazoxide does not generally block the effect of glucose.

Interest in brain glucose-sensing mechanisms is motivated by two distinct neuronal responses to changes in glucose concentrations. One mechanism is global and ubiquitous in response to profound hypoglycemia, whereas the other mechanism is largely confined to specific hypothalamic neurons that respond to changes in glucose concentrations in the physiological range. Although both mechanisms use intracellular metabolism as an indicator of extracellular glucose concentration, the two mechanisms differ in key respects. Global hyperpolarization (inhibition) in response to 0 mM glucose can be reversed by pyruvate, implying that the reduction in ATP levels acting through ATP-dependent potassium (K-ATP) channels is the key metabolic signal for the global silencing in response to 0 mM glucose. In contrast, neuroendocrine hypothalamic responses in glucoresponsive and glucose-sensitive neurons (either excitation or inhibition, respectively) to physiological changes in glucose concentration appear to depend on glucokinase; neuroendocrine responses also depend on K-ATP channels, although the role of ATP itself is less clear. Lactate can substitute for glucose to produce these neuroendocrine effects, but pyruvate cannot, implying that NADH (possibly leading to anaplerotic production of malonyl-CoA) is a key metabolic signal for effects of glucose on glucoresponsive and glucose-sensitive hypothalamic neurons.

Neurons in the ventromedial hypothalamus mediate some counterregulatory responses to hypoglycemia and 2-deoxyglucose, but the mechanisms that mediate these responses to glucose are unclear. In the present study, ventromedial hypothalamus neurons were identified on the basis of their inhibition by the transition from 5 to 20 mmol/l glucose. Tolbutamide, which activates glucose-stimulated neurons, failed to inhibit or activate glucose-inhibited neurons. Inhibitors of glucose transport and glycolysis, in particular by the glucokinase inhibitor glucosamine, blocked the effect of glucose on glucose-inhibited neurons. Furthermore, the glucoseinhibited neurons were activated by 2-deoxyglucose, which also activates counterregulatory responses. Conversely, glucose-inhibited neurons were inhibited by glycolytic metabolites, including lactate, but not by pyruvate. These data indicate that hypoglycemia induces electrical activity in glucose-inhibited neurons by attenuating glycolysis in those neurons. Thus, counterregulatory failure could be due to relatively enhanced glycolysis in glucose-stimulated neurons during hypoglycemia and attenuation of glycolysis in glucose-inhibited neurons might reverse counterregulatory failure. Diabetes 53: [67][68][69][70][71][72][73] 2004 F ailure in the counterregulatory responses to hypoglycemia is considered to be a limiting factor in the optimal treatment of type 1 diabetes (1). The ventromedial hypothalamus (VMH) seems to mediate some counterregulatory responses to hypoglycemia because local attenuation of glucose metabolism by 2-deoxyglucose (2-DG) in the VMH stimulates peripheral counterregulatory responses (2). Furthermore, counterregulatory responses to systemic glucopenia are blocked by lesions in the VMH (3) and by infusion of glucose into the VMH (4). Although some neurons in the VMH are activated by increasing glucose concentration, other VMH neurons are directly inhibited as glucose increases (5,6). Neurons that are sensitive to glucose at plasma concentrations (ϳ3 to ϳ10 -20 mmol/l glucose) are referred to as glucose stimulated and glucose inhibited, respectively (7). Glucose-inhibited neurons are presumably activated by and may mediate some responses to hypoglycemia. Although glucokinase may mediate effects of glucose to inhibit VMH neurons (6), as stated by Schuit et al. (7), "relatively little is known about the mechanism underlying this type of glucose inhibition." Extensive studies of pancreatic ␤-cells provided key guidance to studying mechanisms of glucose stimulation of VMH neurons (7,8). However, much less is known about how glucose inhibits, for example, glucagon-producing ␣-cells, and inhibition of glucagon secretion by glucose seems to be complex and may involve indirect mechanisms (9,10). Of particular interest, in intact islets, metabolic pathways that regulate glucagon secretion seem to be distinct from those that regulate insulin secretion (11). Therefore, in the present studies, we used methods previously used to map metabolic pathways in glucose-stimulate...

Nutrient-sensitive hypothalamic neurons regulate energy balance and glucose homeostasis, but the molecular mechanisms mediating hypothalamic responses to nutritional state remain incompletely characterized. To address these mechanisms, the present studies used quantitative PCR to characterize the expression of a panel of genes the hypothalamic expression by nutritional status of which had been suggested by DNA microarray studies. Although these genes regulate a variety of function, the most prominent set regulate intermediary metabolism, and the overall pattern clearly indicated that a 48-h fast produced a metabolic reprogramming away from glucose metabolism and toward the utilization of alternative fuels, particularly lipid metabolism. This general reprogramming of intermediary metabolism by fasting was observed both in cortex and hypothalamus but most prominently in hypothalamus. The effect of fasting on the expression of these genes may be mediated by reduction in plasma glucose or glucose metabolism, rather than leptin, because they were generally recapitulated by hypoglycemia even in the presence of elevated insulin and in vitro by low glucose but were not recapitulated in ob/ob mice. These studies suggest that fasting reduces glucose metabolism and thus minimizes the production of hypothalamic malonyl-coenzyme A. However, because the reprogramming of glucose metabolism by fasting was also observed in cortex, this apparent substrate competition may mediate more general responses to nutritional deprivation, including those responsible for the protective effects of dietary restriction. The present studies also provide a large panel of novel glucose-regulated genes that can be used as markers of glucose action to address mechanisms mediating hypothalamic responses to nutritional state.