Hypothalamic integration of body fluid regulation.

The progression of animal life from the paleozoic ocean to rivers and diverse econiches on the planet's surface, as well as the subsequent reinvasion of the ocean, involved many different stresses on ionic pattern, osmotic pressure, and volume of the extracellular fluid bathing body cells. The relatively constant ionic pattern of vertebrates reflects a genetic "set" of many regulatory mechanisms--particularly renal regulation. Renal regulation of ionic pattern when loss of fluid from the body is disproportionate relative to the extracellular fluid composition (e.g., gastric juice with vomiting and pancreatic secretion with diarrhea) makes manifest that a mechanism to produce a biologically relatively inactive extracellular anion HCO3- exists, whereas no comparable mechanism to produce a biologically inactive cation has evolved. Life in the ocean, which has three times the sodium concentration of extracellular fluid, involves quite different osmoregulatory stress to that in freshwater. Terrestrial life involves risk of desiccation and, in large areas of the planet, salt deficiency. Mechanisms integrated in the hypothalamus (the evolutionary ancient midbrain) control water retention and facilitate excretion of sodium, and also control the secretion of renin by the kidney. Over and above the multifactorial processes of excretion, hypothalamic sensors reacting to sodium concentration, as well as circumventricular organs sensors reacting to osmotic pressure and angiotensin II, subserve genesis of sodium hunger and thirst. These behaviors spectacularly augment the adaptive capacities of animals. Instinct (genotypic memory) and learning (phenotypic memory) are melded to give specific behavior apt to the metabolic status of the animal. The sensations, compelling emotions, and intentions generated by these vegetative systems focus the issue of the phylogenetic emergence of consciousness and whether primal awareness initially came from the interoreceptors and vegetative systems rather than the distance receptors.

Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice

Leptin is a circulating protein involved in the long-term regulation of food intake and body weight. Cholecystokinin (CCK) is released postprandially and elicits satiety signals. We investigated the interaction between leptin and CCK-8 in the short-term regulation of food intake induced by 24-hr fasting in lean mice. Leptin, injected intraperitoneally (i.p.) at low doses (4–120 μg/kg), which did not influence feeding behavior for the first 3 hr postinjection, decreased food intake dose dependently by 47–83% during the first hour when coinjected with a subthreshold dose of CCK. Such an interaction was not observed between leptin and bombesin. The food-reducing effect of leptin injected with CCK was not associated with alterations in gastric emptying or locomotor behavior. Leptin–CCK action was blocked by systemic capsaicin at a dose inducing functional ablation of sensory afferent fibers and by devazepide, a CCK-A receptor antagonist but not by the CCK-B receptor antagonist, L-365,260. The decrease in food intake which occurs 5 hr after i.p. injection of leptin alone was also blunted by devazepide. Coinjection of leptin and CCK enhanced the number of Fos-positive cells in the hypothalamic paraventricular nucleus by 60%, whereas leptin or CCK alone did not modify Fos expression. These results indicate the existence of a functional synergistic interaction between leptin and CCK leading to early suppression of food intake which involves CCK-A receptors and capsaicin-sensitive afferent fibers.

Corticotropin-releasing factor-binding protein ligand inhibitor blunts excessive weight gain in genetically obese Zucker rats and rats during nicotine withdrawal

Elevation of the neuropeptide corticotropin-releasing factor (CRF) in the brain is associated with a reduction of food intake and body weight gain in normal and obese animals. A protein that binds CRF and the related peptide, urocortin, with high affinity, CRF-binding protein (CRF-BP), may play a role in energy homeostasis by inactivating members of this peptide family in ingestive and metabolic regulatory brain regions. Intracerebroventricular administration in rats of the high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which dissociates CRF or urocortin from CRF-BP and increases endogenous brain levels of “free” CRF or urocortin significantly blunted exaggerated weight gain in Zucker obese subjects and in animals withdrawn from chronic nicotine. Chronic administration of CRF suppressed weight gain nonselectively by 60% in both Zucker obese and lean control rats, whereas CRF-BP ligand inhibitor treatment significantly reduced weight gain in obese subjects, without altering weight gain in lean control subjects. Nicotine abstinent subjects, but not nicotine-naive controls, experienced a 35% appetite suppression and a 25% weight gain reduction following acute and chronic administration, respectively, of CRF-BP ligand inhibitor. In marked contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand inhibitor did not stimulate adrenocorticotropic hormone secretion or elevate heart rate and blood pressure. These results provide support for the hypothesis that the CRF-BP may function within the brain to limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP may represent a novel and functionally selective target for the symptomatic treatment of excessive weight gain associated with obesity of multiple etiology.

Obesity and mild hyperinsulinemia found in neuropeptide Y-Y1 receptor-deficient mice

To elucidate the role of neuropeptide Y (NPY)-Y1 receptor (Y1-R) in food intake, energy expenditure, and other possible functions, we have generated Y1-R-deficient mice (Y1-R−/−) by gene targeting. Contrary to our hypothesis that the lack of NPY signaling via Y1-R would result in impaired feeding and weight loss, Y1-R−/− mice showed a moderate obesity and mild hyperinsulinemia without hyperphagia. Although there was some variation between males and females, typical characteristics of Y1-R−/− mice include: greater body weight (females more than males), an increase in the weight of white adipose tissue (WAT) (approximately 4-fold in females), an elevated basal level of plasma insulin (approximately 2-fold), impaired insulin secretion in response to glucose administration, and a significant changes in mitochondrial uncoupling protein (UCP) gene expression (up-regulation of UCP1 in brown adipose tissue and down-regulation of UCP2 in WAT). These results suggest either that the Y1-R in the hypothalamus is not a key molecule in the leptin/NPY pathway, which controls feeding behavior, or that its deficiency is compensated by other receptors, such as NPY-Y5 receptor. We believe that the mild obesity found in Y1-R−/− mice (especially females) was caused by the impaired control of insulin secretion and/or low energy expenditure, including the lowered expression of UCP2 in WAT. This model will be useful for studying the mechanism of mild obesity and abnormal insulin metabolism in noninsulin-dependent diabetes mellitus.

Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R

Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive Gαq coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2–16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.

Defective STAT signaling by the leptin receptor in diabetic mice.

Leptin and its receptor, obese receptor (OB-R), comprise an important signaling system for the regulation of body weight. Splice variants of OB-R mRNA encode proteins that differ in the length of their cytoplasmic domains. We cloned a long isoform of the wild-type leptin receptor that is preferentially expressed in the hypothalamus and show that it can activate signal transducers and activators of transcription (STAT)-3, STAT-5, and STAT-6. A point mutation within the OB-R gene of diabetic (db) mice generates a new splice donor site that dramatically reduces expression of this long isoform in homozygous db/db mice. In contrast, an OB-R protein with a shorter cytoplasmic domain is present in both db/db and wild-type mice. We show that this short isoform is unable to activate the STAT pathway. These data provide further evidence that the mutation in OB-R causes the db/db phenotype and identify three STAT proteins as potential mediators of the anti-obesity effects of leptin.

Obese gene expression: reduction by fasting and stimulation by insulin and glucose in lean mice, and persistent elevation in acquired (diet-induced) and genetic (yellow agouti) obesity.

Mutations in the obese (ob) gene lead to obesity. This gene has been recently cloned, but the factors regulating its expression have not been elucidated. To address the regulation of the ob gene with regard to body weight and nutritional factors, Northern blot analysis was used to assess ob mRNA in adipose tissue from mice [lean, obese due to diet, or genetically (yellow agouti) obese] under different nutritional conditions. ob mRNA was elevated in both forms of obesity, compared to lean controls, correlated with elevations in plasma insulin and body weight, but not plasma glucose. In lean C57BL/6J mice, but not in mice with diet-induced obesity, ob mRNA decreased after a 48-hr fast. Similarly, in lean C57BL/6J controls, but not in obese yellow mice, i.p. glucose injection significantly increased ob mRNA. For up to 30 min after glucose injection, ob mRNA in lean mice significantly correlated with plasma glucose, but not with plasma insulin. In a separate study with only lean mice, ob mRNA was inhibited >90% by fasting, and elevated approximately 2-fold 30 min after i.p. injection of either glucose or insulin. These results suggest that in lean animals glucose and insulin enhance ob gene expression. In contrast to our results in lean mice, in obese animals ob mRNA is elevated and relatively insensitive to nutritional state, possibly due to chronic exposure to elevated plasma insulin and/or glucose.

Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.

Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzodiazepine (BZ) site of gamma-aminobutyric acid type A (GABAA) receptors. In vivo, BZ sites are potential targets for endogenous ligands regulating the corresponding central nervous system states. To assess the physiological relevance of BZ sites, mice were generated containing GABAA receptors devoid of BZ sites. Following targeted disruption of the gamma 2 subunit gene, 94% of the BZ sites were absent in brain of neonatal mice, while the number of GABA sites was only slightly reduced. Except for the gamma 2 subunit, the level of expression and the regional and cellular distribution of the major GABAA receptor subunits were unaltered. The single channel main conductance level and the Hill coefficient were reduced to values consistent with recombinant GABAA receptors composed of alpha and beta subunits. The GABA response was potentiated by pentobarbital but not by flunitrazepam. Diazepam was inactive behaviorally. Thus, the gamma 2 subunit is dispensable for the assembly of functional GABAA receptors but is required for normal channel conductance and the formation of BZ sites in vivo. BZ sites are not essential for embryonic development, as suggested by the normal body weight and histology of newborn mice. Postnatally, however, the reduced GABAA receptor function is associated with retarded growth, sensorimotor dysfunction, and drastically reduced life-span. The lack of postnatal GABAA receptor regulation by endogenous ligands of BZ sites might contribute to this phenotype.

Appearance of insulin-like growth factor mRNA in the liver and pyloric ceca of a teleost in response to exogenous growth hormone.

Augmentation of vertebrate growth by growth hormone (GH) is primarily due to its regulation of insulin-like growth factor I (IGF I) and IGF II levels. To characterize the effect of GH on the levels of IGF I and IGF II mRNA in a teleost, 10 micrograms of bovine GH (bGH) per g of body weight was administered to juvenile rainbow trout (Oncorhynchus mykiss) through i.p. injection. The levels of IGF I and IGF II mRNA were determined simultaneously, by using RNase protection assays, in the liver, pyloric ceca, kidney, and gill at 0, 1, 3, 6, 12, 24, 48, and 72 hr after injection. In the liver, IGF I mRNA levels were significantly elevated at 6 and 12 hr (approximately 2- to 3-fold, P < or = 0.01), while IGF II mRNA levels were significantly elevated at 3 and 6 hr (approximately 3-fold, P < or = 0.01). In the pyloric ceca, IGF II mRNA levels were significantly elevated at 12, 24, and 48 hr (approximately 3-fold, P < or = 0.01), while IGF I mRNA was below the limits of assay accuracy. GH-dependent IGF mRNA appearance was not detected in the gill and kidney. Serum bGH levels, determined by using a radioimmunoassay, were significantly elevated at 3 and 6 hr (P < 0.005). In primary hepatocyte culture, IGF I and IGF II mRNA levels increased in a bGH dose-dependent fashion, with ED50 values of approximately 45 and approximately 6 ng of bGH per ml, respectively. The GH-dependent appearance of IGF II mRNA in the liver and pyloric ceca suggests important roles for this peptide hormone exclusive of IGF I.