Tooru M. Mizuno

Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting in ob/ob and db/db mice, but is stimulated by leptin

Reduction in the activity of the α-melanocyte-stimulating hormone (α-MSH) system causes obesity, and infusions of α-MSH can produce satiety, raising the possibility that α-MSH may mediate physiological satiety signals. Since α-MSH is coded for by the pro-opiomelanocortin (POMC) gene, we examined if POMC gene expression would be inhibited by fasting in normal mice or in models of obesity characterized by leptin insufficiency (ob/ob) or leptin insensitivity (db/db). In wild-type mice, hypothalamic POMC mRNA was decreased >60% after a 2-day fast and was positively correlated with leptin mRNA. Similarly, compared with controls, POMC mRNA was decreased by at least 60% in both db/db and ob/ob mice. POMC mRNA was negatively correlated with both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA. Finally, treatment of both male and female ob/ob mice with leptin stimulated hypothalamic POMC mRNA by about threefold. These results suggest that impairment in production, processing, or responsiveness to α-MSH may be a common feature of obesity and that hypothalamic POMC neurons, stimulated by leptin, may constitute a link between leptin and the melanocortin system.

Fasting regulates hypothalamic neuropeptide Y, agouti-related peptide, and proopiomelanocortin in diabetic mice independent of changes in leptin or insulin.

Fasting increases hypothalamic neuropeptide Y (NPY) and agouti-related peptide (AGRP) messenger RNA (mRNA) and reduces hypothalamic POMC mRNA, and is also characterized by a reduction in plasma leptin, insulin, and glucose, each of which has been implicated in the regulation of hypothalamic gene expression. To further evaluate the roles of leptin, insulin, and glucose in mediating effects of fasting, we examined hypothalamic gene expression in nondiabetic and streptozotocin (STZ)-induced diabetic mice both under ad lib fed and 48-h fasted conditions. In both diabetic and nondiabetic mice, fasting stimulated hypothalamic NPY and AGRP mRNA and inhibited hypothalamic POMC mRNA and adipose leptin mRNA. However, in diabetic mice fasting had no effect on plasma leptin and insulin while decreasing plasma glucose, whereas in nondiabetic mice fasting decreased plasma leptin, insulin, and glucose. Furthermore, in nondiabetic fasted mice, NPY and AGRP mRNA were higher, and POMC mRNA and plasma glucose were lower, th...

Targeted Deletion of the Vgf Gene Indicates that the Encoded Secretory Peptide Precursor Plays a Novel Role in the Regulation of Energy Balance

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Reducing hypothalamic AGRP by RNA interference increases metabolic rate and decreases body weight without influencing food intake

BackgroundSeveral lines of evidence strongly suggest that agouti-related peptide (AGRP) plays a key role in the regulation of metabolic function but ablation of the AGRP gene has no apparent effect on metabolic function. Since specific pharmacological antagonists of AGRP do not presently exist, we assessed if reduction of hypothalamic AGRP mRNA by RNA interference (RNAI) would influence metabolic function, an outcome suggesting that pharmacological antagonists might constitute useful reagents to treat obesity.ResultsThe RNAI protocol specifically reduced hypothalamic expression of AGRP mRNA by 50% and resulted in reduction of AGRP peptide immunoreactivity. Physiologically, the reduction in AGRP levels was associated with increased metabolic rate and reduced body weight without changes in food intake.ConclusionAGRP can function to increase body weight and reduce metabolic rate without influencing food intake. The present study demonstrates that RNAI protocols can be used to assess physiological function of neuronal genes in vivo.

Hyperphagia and weight gain after gold-thioglucose: relation to hypothalamic neuropeptide Y and proopiomelanocortin.

Genetic obesity is associated with increased neuropeptide Y (NPY) messenger RNA (mRNA) and decreased POMC mRNA in the hypothalamus of ob/ob and db/db mice, or impaired sensitivity to aMSH (derived from POMC) in the yellow agouti mouse. Acquired obesity can be produced by chemically lesioning the hypothalamus with either monosodium glutamate (MSG) in neonates or gold thioglucose (GTG) in adult mice. The present study examined whether elevated NPY mRNA and/or decreased POMC mRNA in the hypothalamus are associated with obesity due to hypothalamic lesions. GTG injection into adult mice produced a profound obese phenotype, including hyperphagia, increased body weight, and increased leptin mRNA and peptide, in association with reduced hypothalamic NPY mRNA and POMC mRNA. MSG treatment produced virtual elimination of NPY mRNA in the arcuate nucleus and a reduction of hypothalamic POMC mRNA, and led to elevated leptin. MSG pretreatment did not attenuate GTG-induced hyperphagia and obese phenotype. These results do not support a role for NPY-synthesizing neurons in the arcuate nucleus in mediating hypothalamic acquired obesity, but are consistent with the hypothesis that decreased activity of hypothalamic neurons synthesizing POMC play a role in mediating hypothalamic obesity. (Endocrinology 139: 4483–4488, 1998) E synthesis of neuropeptide Y (NPY) in the arcuate nucleus has been proposed to play an important role in mediating the obese phenotype in genetically obese animals (1, 2). This hypothesis is widely viewed as credible because 1) experimental elevation of hypothalamic NPY can induce obesity (1); 2) in some forms of genetic obesity, NPY messenger RNA (mRNA) in the arcuate nucleus is elevated (3–6); 3) leptin inhibits NPY mRNA in the arcuate nucleus (3, 7); and 4) obesity is attenuated in leptindeficient ob/ob mice that lack NPY (8). On the other hand, reduced POMC activity is also implicated in obesity, as 1) obesity in yellow agouti mice is associated with interference in response to aMSH, which is derived from POMC (9–13); 2) genetic deletion of an aMSH receptor produces obesity (14); 3) central administration of aMSH or a melanocortin-4 receptor agonist inhibits food intake, whereas a melanocortin-4 receptor antagonist stimulates food intake (13, 15); and 4) the expression of hypothalamic POMC mRNA is reduced in genetically obese ob/ob and db/db mice and is stimulated by leptin (16–18). Obesity can be acquired through several perturbations, including hypothalamic damage. Hypothalamic obesity has been reported in humans (19) as well as in other species, for example after neonatal administration of monosodium glutamate (MSG) (20) or treatment of adult mice with gold thioglucose (GTG) (21). An ip injection of GTG produces a lesion in the ventromedial hypothalamus whose localization is reproducible and which recapitulates the severe obese phenotype characteristic of lesions of the hypothalamic ventromedial nucleus produced by other means (e.g. an electrical current, an excitotoxin, or a tumor). Therefore, GTG has been used as a powerful tool to assess mechanisms of hypothalamic obesity (22–32). The hypothalamic lesion produced by GTG is dependent on insulin and the glucose moiety of the GTG molecule and is blocked by glucose uptake inhibitors, so GTG has been thought to primarily target the glucosesensitive neurons of the hypothalamus (21, 33). This hypothesis was supported by the observation that mice with GTG lesions are insensitive to the satiety effects of glucose and the induction of feeding by 2-deoxyglucose, but are sensitive to the satiety effects of cholecystokinin (34). The present study examined whether increased hypothalamic NPY mRNA and/or decreased hypothalamic POMC mRNA are associated with GTGor MSG-induced hypothalamic obesity. Materials and Methods

Acetylcholine release in the rat hippocampus as measured by the microdialysis method correlates with motor activity and exhibits a diurnal variation

Extracellular levels of acetylcholine were measured by the microdialysis method coupled to high performance liquid chromatography in the dorsal hippocampus of freely moving rats over a period of 24 h to examine whether the acetylcholine release in the hippocampus exhibited a diurnal variation. Spontaneous motor activity was simultaneously measured with an automatic animal activity monitor. The amount of acetylcholine collected per 20-min sample varied markedly, in a range from about 5 to 90 pmol. There appeared to be variations in the amount with a 2-4 h periodicity as well as an apparent diurnal periodicity. In all five rats studied, the overall mean value for the dark cycle (11.1-34.5, average 20.9 pmol/20 min) was significantly greater than that for the light cycle (5.1-21.3, average 12.3 pmol/20 min), showing a 70% average increase. Cross-correlation analysis performed between the amount of acetylcholine and the motor activity count for the animal during the sampling revealed a significant positive correlation coefficient in four rats studied. The present study demonstrates for the first time that the acetylcholine release shows a diurnal variation.

Resistance to diet-induced obesity is associated with increased proopiomelanocortin mRNA and decreased neuropeptide Y mRNA in the hypothalamus.

Mechanisms mediating genetic susceptibility to diet-induced obesity have not been completely elucidated. Elevated hypothalamic neuropeptide Y (NPY) and decreased hypothalamic proopiomelanocortin (POMC) are thought to promote the development and maintenance of obesity. To assess the potential role of hypothalamic neuropeptide gene expression in diet-induced obesity, the present study examined effects of a high-fat diet on hypothalamic NPY and POMC mRNA in three strains of mice that differ in susceptibility to develop diet-induced obesity. C57BL/6J, CBA, and A/J mice were fed either normal rodent chow or a high-fat diet for 14 weeks after which hypothalamic gene expression was measured. On the high-fat diet, C57BL/6J mice gained the most weight, whereas A/J mice gained the least weight. On the high-fat diet, NPY mRNA significantly decreased as body weight increased in CBA and A/J mice, but not in C57BL/6J mice. In addition, POMC mRNA significantly increased as body weight increased in A/J mice, but not in CBA and C57BL/6J mice. Since decreased NPY mRNA and increased POMC mRNA would presumably attenuate weight gain, these results suggest that a high-fat diet produces compensatory changes in hypothalamic gene expression in mice resistant to diet-induced obesity but not in mice susceptible to diet-induced obesity.

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

BackgroundFasting 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.ResultsFasting 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).ConclusionThese 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.

Age-related changes in diurnal acetylcholine release in the prefrontal cortex of male rats as measured by microdialysis

Extracellular levels of acetylcholine in the prefrontal cortex were measured using the micro-dialysis method in freely moving young (three to four months old) and old (23 to 24 months old) male rats over a period of 24 h to examine the effect of aging on prefrontal acetylcholine release. Prefrontal acetylcholine release during a 24 h period exhibited a diurnal variation with higher levels during the dark cycle than during the light cycle in young rats but not in old rats. In addition, prefrontal acetylcholine release was closely associated with spontaneous activity in young rats but not in old rats. The present study suggests that aging reduces diurnal changes in the prefrontal acetylcholine release and that there is a cross-correlation between the prefrontal acetylcholine release and spontaneous locomotor activity in male rats.

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.