Orexin A in the rostrolateral hypothalamic area induces feeding by modulating GABAergic transmission.

The neuromodulatory peptides orexin A and B are important central nervous system regulators of appetite. We previously identified the rostral lateral portion of the hypothalamus as an area important to orexin A feeding regulation. As gamma-aminobutyric-acid (GABA) within the lateral hypothalamus also mediates feeding, we sought to determine the relationship between orexin and GABA signaling within this site. Adult male Sprague-Dawley rats were implanted with cannulae directed to the rostral lateral hypothalamus and saclofen (GABA-B receptor antagonist), biccuculine (GABA-A receptor antagonist) or muscimol (GABA-A receptor agonist) were injected prior to orexin A. Both GABA antagonists failed to significantly affect orexin A-induced feeding, but muscimol significantly and dose dependently inhibited orexin A-induced feeding. Using in vivo microdialysis GABA release within this region significantly dropped during the first hour following orexin A administration, coinciding with orexin A-induced feeding. Together, these data indicate that orexin A may influence food intake by decreasing GABAergic tone within the rostral lateral hypothalamus.

Zinc-deficient rats are insensitive to glucoprivation caused by 2-deoxy-D-glucose.

Three-choice macronutrient intake studies indicate that zinc-deficient (Zn-) rats selectively decrease intake of carbohydrate. Because glucoprivic stimuli increase food intake and selection for carbohydrate, the ability of Zn- rats to respond to glucoprivation induced by 2-deoxy-D-glucose (2-DG) was tested. Rats were fed a Zn-adequate (Zn+) or Zn- diet. In part 1, rats were challenged with 0, 250, or 400 mg 2-DG/kg BW (i.p.) after zinc deficiency was established. In part 2, rats received saline or 2-DG while zinc deficiency was being induced and then after deficiency was established. Food intake was increased after injection of 2-DG to Zn+ rats; however, food intake was not higher after 2-DG administration to Zn- rats. A dose-response test for 2-DG further confirmed these results. In part 2, it was found that Zn- rats lose the response to 2-DG administration when zinc deficiency-induced anorexia begins, after 3 days of consuming a zinc-deficient diet. It appears that the ability to sense blood glucose concentrations may be impaired during zinc deficiency, and this impairment could be a part of the anorexia that develops during zinc deficiency in the rat.

Noradrenergic and GABAergic systems in the medial hypothalamus are activated during hypoglycemia.

Noradrenergic and GABAergic systems in the medial hypothalamus influence plasma glucose and may be activated during glucoprivation. Microdialysis probes were placed into the ventromedial nucleus (VMH), lateral hypothalamus (LHA), and paraventricular nucleus (PVH) of male Sprague-Dawley rats to monitor extracellular concentrations of norepinephrine (NE) and GABA. During systemic hypoglycemia, induced by insulin (1.0 U/kg), NE concentrations increased in the VMH (P < 0.05) and PVH (P = 0.06) in a bimodal fashion during the first 10 min and 20-30 min after insulin administration. In the VMH, GABA concentrations increased (P < 0.05) in a similar manner as NE. Extracellular NE concentrations in the LHA were slightly lower (P = 0.13), and GABA levels remained at baseline. The increases in NE and GABA in the VMH were absent during euglycemic clamp; however, NE in the PVH still increased, reflecting a direct response to hyperinsulinemia. On the basis of these data, we propose that the activity of noradrenergic afferents to the medial hypothalamus is increased during hypoglycemia and influences the activity of local GABAergic systems to activate appropriate physiological compensatory mechanisms.

Growth hormone-releasing factor affects macronutrient intake during the anabolic phase of zinc repletion: total hypothalamic growth hormone-releasing factor content and growth hormone-releasing factor immunoneutralization during zinc repletion.

Growth hormone-releasing factor (GRF) is thought to perform two distinct functions within the brain. GRF synthesized in the median eminence (ME) stimulates the release of growth hormone (GH) from the pituitary, while GRF in the suprachiasmatic nucleus and median preoptic area (SCN/MPOA) may stimulate selection of dietary protein. These two functions may be coupled to regulate and enhance growth. During zinc repletion, a period characterized by increased protein intake and accelerated growth, we examined this coupling by measuring GRF peptide content in hypothalamic sites and neutralizing GRF function by infusing anti-GRF antibody into the hypothalamus during zinc repletion. Total GRF content and GRF content in the ME and SCN/MPOA were decreased in zinc-deficient (Zn-) rats compared to zinc-adequate (Zn+) rats (P < 0.05). There were no differences in GRF content during zinc repletion in either nuclei. Subsequently, we investigated the macronutrient feeding patterns of rats chronically infused with anti-GRF IgG into the lateral ventricle of the brain during zinc repletion. All Zn- and Zn+ rats administered anti-GRF IgG exhibited a reduction in protein intake during zinc repletion. The Zn- rats receiving anti-GRF-IgG consumed equal amounts of total diet compared to those receiving vehicle during the repletion period however they consumed less carbohydrate (P < 0.05) and considerably more fat (P < 0.02). There were no significant differences in carbohydrate or fat intake in Zn+ rats receiving anti-GRF antibody. These results suggest that GRF likely directs protein intake during normal growth, but may interact with additional appetite-controlling neuropeptides during zinc repletion.

Norepinephrine mediates glucoprivic-induced increase in GABA in the ventromedial hypothalamus of rats.

Noradrenergic mechanisms in the hypothalamus may be involved in counterregulatory responses to glucoprivic episodes. After 2-deoxy-D-glucose (2-DG; 1.2 mmol/kg iv), extracellular norepinephrine (NE) concentration in the ventromedial hypothalamus (VMN) increased in a bimodal fashion to 251 +/- 39% (P < 0.001) and 150 +/- 17% (P < 0.001) of baseline during the first 30 min. In the lateral hypothalamus (LHA), NE decreased by 30 min (61 +/- 4%, P < 0.001) and no consistent changes were measured in the paraventricular nucleus (PVN). Because the NE response in the VMN after 2-DG followed the same pattern as GABA, the interaction between NE and GABA was evaluated. In the VMN, GABA had little effect on extracellular NE concentrations but NE increased GABA concentrations 166 +/- 13%, (P < 0.01). In the presence of yohimbine (alpha(2)-adrenoceptor antagonist) the first GABA peak after 2-DG was absent, and the second GABA peak was absent in the presence of timolol (beta-adrenoceptor antagonist). These results support an interaction among noradrenergic and GABAergic systems in the VMN during glucoprivation and that increased NE mediates the increase in extracellular GABA after 2-DG.

Essential amino acids affect interstitial dopamine metabolites in the anterior piriform cortex of rats.

The anterior piriform cortex (APC) is essential for the anorectic reactions to an amino acid-imbalanced diet, and it also responds to repletion of the limiting amino acid. In the present study, we examine the dynamic changes of the interstitial dopamine metabolites in the APC following feeding of either an amino acid-corrected or -imbalanced diet. Microdialysates, collected from the APC, were analyzed using HPLC with electrochemical detection. The concentrations were 19.7 +/- 4.8 microg/L for 3, 4-dyhydroxyphenylacetic acid and 25.1 +/- 4.4 microg/L for homovanillic acid, respectively, in the baseline dialysates. After diet treatments, no significant changes occurred in 3, 4-dyhydroxyphenylacetic acid in the corrected (n = 7) or imbalanced (n = 9) groups vs. the basal group (n = 7). However, after feeding the threonine-corrected diet, the concentration of homovanillic acid was significantly less (P < 0.01) than after the basal and imbalanced diets. The homovanillic acid level in the corrected group was already significantly lower than in the basal group by 20 min (P < 0.05), and reached its lowest level at 70 min (P < 0.05). The concentrations of homovanillic acid in the corrected group remained at this low level until the end of the experiment. The present results introduce the idea that the dopaminergic system is involved in the feeding responses to essential amino acid repletion.

Leptin in the Anterior Piriform Cortex Affects Food Intake in Rats.

Leptin administered intracerebroventricularly (ICV) or intrahypothalamically inhibits food intake (FI), however, to our knowledge the effects of leptin administration have only been examined in one extrahypothalamic site (dorsal raphe nucleus). Our objectives were t. (1) determine the FI effects of leptin administration into the anterior piriform cortex (APC), an area linked to the control of FI in amino acid (AA) deficiency, (2) examine leptin action during short term anorexia that develops in response to AA deficiency. Bilateral injections of leptin (0.25 µ) into the APC suppressed FI of a balanced diet between 6 and 12 h by 36% (p < 0.01) and over the first 12 and 24 h by 15% (p < 0.05). Bilateral administration of leptin (0.1 µg) inhibited FI between 12 and 24 h by approximately 48% (p < 0.05) on a threonine-imbalanced diet without significantly affecting FI on a threonine-corrected diet. The increase of plasma leptin concentrations in response to feeding a threonine-basal diet was greater than that following an AA imbalanced diet, suggesting that suppression of FI by an AA imbalanced diet is not mediated by an increase of leptin. Our results suggest that (1) administration of leptin into a brain area outside the hypothalamus suppresses FI, and (2) leptin is unlikely to play a selective role in the short term anorectic response to AA deficiency. These data are consistent with the hypothesis that endogenous leptin can act within the APC to modulate FI.

Zinc deficiency increases hypothalamic neuropeptide Y and neuropeptide Y mRNA levels and does not block neuropeptide Y-induced feeding in rats.

Zinc deficiency reduces intake and produces an unusual approximately 3.5-d cycle of intake in rats. The mechanism underlying the anorexia and cycling has not yet been defined; current hypotheses suggest that alterations in amino acid metabolism and neurotransmitter concentrations may be a part of this anorexia. Recent reports indicate that appetite-stimulating neuropeptide Y (NPY) may be elevated during zinc deficiency. This suggests that a resistance to NPY may exist during zinc deficiency because NPY levels are high, yet appetite is low. The purpose of this study was to measure NPY peptide and mRNA concentrations during zinc deficiency in specific nuclei of the hypothalamus in which peptide and mRNA for NPY are known to be associated with appetite, and also to determine whether zinc-deficient rats are responsive to central infusions of NPY. Both NPY peptide levels in the paraventricular nucleus and NPY mRNA levels in the arcuate nucleus were higher (P < 0.05) in zinc-deficient rats than in zinc-adequate rats. When rats were administered exogenous NPY to the paraventricular nucleus, both zinc-deficient and zinc-adequate rats responded similarly by increasing food intake. These results suggest that NPY is elevated during zinc deficiency in an attempt to restore normal food intake levels, rather than being reduced and thereby contributing to the anorexia associated with zinc deficiency. During zinc deficiency, NPY receptors are able to bind NPY and initiate an orexigenic response.

Meal-induced changes in hepatic glycogen of fasted rats.

Hepatic metabolism of glucose and other nutrients influences feeding behavior. The present study was conducted to confirm prandial decreases in hepatic glycogen concentrations following a short-term fast. Male Sprague-Dawley rats were fasted 6-12 h during the light phase before having access to chow for one or two 20-min meals at the beginning of the dark phase. Plasma glucose and insulin concentrations in hepatic and portal venous blood and hepatic glycogen concentrations prior to and at the end of each meal were compared. Glucose concentration in the hepatic vein was greater than that in the portal vein prior to the meals but not at the end of the meal. Insulin levels were higher in the portal vein than the hepatic vein pre- and postprandially. Hepatic glycogen concentrations increased after each meal in younger (2-month-old) rats but not older (6-month-old) rats. Fasting levels of hepatic glycogen were lower in the younger rats than the older rats; however, the increase in hepatic glycogen was not due to differences in baseline glycogen concentrations at the start of the meal. The reported prandial decreases in hepatic glycogen of fasted rats were not apparent in this study. Because of the difference between 2- and 6-month-old rats in periprandial hepatic glycogen metabolism after a short-term fast, the age of the animal needs to be considered if the dynamics of liver glycogen metabolism are to be incorporated into a model of food intake regulation.

Basal and glucoprivic-induced changes in extracellular GABA in the ventral hypothalamus of Zucker rats.

The diminished sensitivity of genetically obese (fa/ fa) Zucker rats to the glucoprivic agent 2-deoxy-D-glucose (2-DG) may involve impaired release of the neurotransmitter gamma-aminobutyric acid (GABA) in discrete regions of the hypothalamus. Extracellular GABA concentrations in the medial (MH) and lateral (LH) hypothalamus of lean (Fa/Fa) and age-matched obese (fa/fa) male Zucker rats before and after 2-DG (1.2 mmol/kg i.v.). Basal GABA concentrations were higher (P < 0.05) in the MH of obese vs. lean rats. No differences were noted in LH GABA levels between lean and obese rats or in baseline extracellular GABA levels between brain regions in lean rats. In lean rats, a characteristic bimodal increase in GABA concentrations was apparent in the MH, whereas GABA concentrations decreased in the LH during the 60 min after 2-DG. No changes in GABA concentrations in dialysate from the MH or LH of obese rats were observed after 2-DG. The alterations in basal activity and responsiveness to glucoprivic stimuli by GABAergic system in the MH of obese rats may reflect a defect in central glucostatic control of food intake and, ultimately, in the hypothesized autonomic imbalance in fa/fa Zucker rat.

Carnitine supplementation accelerates normalization of food intake depressed during TPN.

When total parenteral nutrition (TPN; containing glucose, fat, and amino acids; caloric ratio 50:30:20) providing 100% of the rat's daily caloric intake is given for 3-4 days, food intake rapidly decreases by approximately 85%. After stopping TPN, there is a lag period of 3-4 days before food intake returns to previous level, which appears to be related to fatty acid oxidation and fat deposition. Carnitine plays a key role in the oxidation of fatty acids, and was demonstrated to reduce fat deposition in rats receiving TPN, by increasing beta oxidation. We therefore investigated whether rats receiving TPN supplemented with carnitine may prevent either the decrease or speed up the resumption or normalization of food intake, after TPN is stopped. Fourteen adult Fischer-344 rats had a central venous catheter inserted. After 10 recovery days, controls (n = 7) were infused with TPN providing 100% of rat's daily caloric intake for 3 consecutive days, followed by 4 more days of normal saline. The carnitine group (n = 7) received the same solution, but which provided 100 mg/kg/day carnitine. Daily food intake was measured and data were analyzed using ANOVA and Student's t-test. Both parenteral solutions depressed food intake maximally by almost 90% by day 3. Carnitine accelerated the normalization of food intake by decreasing the lag period by 1 day. We conclude that the addition of carnitine enhanced the normalization of post-TPN food intake and argue that this may be on the basis of enhanced fatty acid oxidation, a substrate known to play a significant role in the anorexia induced by TPN.

Mechanism of early tumor anorexia.

Whether in tumor-bearing rats a temporal relationship exists between an increase in plasma free tryptophan (FTRP), an increase in brain serotonin (5-HT), and onset of anorexia was studied. Rats were assigned to three groups: tumor-bearing (TB), pair fed (PF), and controls. Food intake was recorded daily. In TB rats anorexia developed on Day 18 and thereafter food intake decreased progressively until end of study. After tumor inoculation, tumor became palpable on Day 10 and continued to grow exponentially until end of study. Rats were killed on Days 6, 10, 16, 18, 22, and 26 to determine plasma FTRP, FTRP/LNAA, and brain 5HT and compared to PF and controls. On Day 6, before tumors became detectable, FTRP and FTRP/LNAA were increased (P < 0.05) in TB rats vs controls. Both continued to increase so that by Day 18 when food intake had started to decrease (P < 0.05), brain 5-HT increased and correlated with the onset of anorexia (R2 = 0.6, P < 0.05). Increases in plasma FTRP the precursor to brain 5-HT occurred in TB rats before physical appearance of tumor and increased until an increase in brain 5-HT occurred, leading to anorexia.

Glucose modulates gamma-aminobutyric acid release from the pancreatic beta TC6 cell line.

To determine if endogenous gamma-aminobutyric acid (GABA) is secreted by a pancreatic beta-cell-derived cell line and to determine the effects of glucose on GABA release, beta TC6 cultures were incubated in the presence of 1 or 10 mmol/l glucose for 12 h and then subjected to a 2-h secretion test in Krebs-Ringer buffer containing 1 or 10 mmol/l glucose. beta TC6-conditioned medium was collected at 15, 30, 60, and 120 min after glucose stimulation for GABA analysis by high pressure liquid chromatography-electrochemical detection. After 30 min, medium GABA concentrations were significantly higher (p < 0.05) in cultures that were exposed to high glucose during both the 12-h incubation period and the 2-h secretion test than in the remaining three glucose combinations. To address possible roles of beta-cell-derived GABA, the effect of GABA on glucagon secretion from pancreatic alpha TC6 cells was tested at concentrations released from beta TC6 cells. Inhibition of glucagon secretion by alpha TC6 cells was observed in the presence of GABA at concentrations equivalent to concentrations secreted by beta TC6 cells. The inhibitory effects of GABA on glucagon secretion by alpha TC6 cells were blocked by the GABAA receptor antagonist bicuculline and were dissociated from the inhibitory effects of glucose. Together, these results provide the first documentation that endogenous GABA is released from a highly differentiated beta-cell line and that glucose and GABA independently attenuate glucagon secretion by a pancreatic alpha-cell line.

Alterations in extracellular GABA in the ventral hypothalamus of rats in response to acute glucoprivation.

gamma-Aminobutyric acidergic (GABA) mechanisms in the ventral hypothalamus may be involved in counterregulatory responses to glucoprivic episodes. Microdialysis probes (1 mm) were placed into the ventromedial hypothalamus (VMH) or lateral hypothalamus (LHA) of male Sprague-Dawley rats 3.5 h before 2-deoxy-D-glucose (2-DG) administration (200 mg/kg i.v.). Probes were perfused (2 ml/min) with Ringer solution, and samples were collected every 10 min from 30 min before to 60 min after 2-DG. By 30 min after 2-DG, GABA concentration in VMH dialysate increased in a bimodal fashion to 204 +/- 36% (P < 0.01) of baseline, and GABA concentration in LHA dialysate decreased to 77 +/- 4% (P < 0.01) of baseline. The changes in dialysate GABA concentrations occurred concurrently with the animals eating and returned to baseline by 60 min. When animals were denied access to food after 2-DG, the decrease in LHA GABA was not apparent and VMH GABA remained approximately 15% above baseline at the end of the sample period. The results of the present study provide evidence that GABAergic systems in the ventral hypothalamus are responsive to alterations in glucose status.

Hepatic vagus does not mediate IL-1 alpha induced anorexia.

Peripherally infused interleukin-alpha (IL-1 alpha) reduces food intake. Since the innervated liver modulates eating activity via the vagus, we investigated the role of the hepatic vagus in the etiology of IL-1 alpha induced anorexia. Ten male Fischer 344 rats were randomly assigned to hepatic vagotomy (HX-IL-1 group) or sham operation (Sham-IL-1 group), and an internal jugular catheter was inserted in all rats. Another six sham operated rats receiving normal saline i.v. throughout the study period served as general controls. After a 10-day recovery period, HX-IL-1 and Sham-ILI-1 rats were infused with 3 micrograms day-1 of IL-1 alpha for 3 days, followed by a 4 day infusion of saline. During the IL-1 alpha infusion, food intake was reduced at a similar rate and by a similar amount in both vagotomized and sham-operated rats. When IL-1 alpha infusion was stopped, food intake normalized at a similar rate in both HX-ILI-1 and Sham-IL-1 groups. These data indicate that the hepatic vagus is not involved in the etiology of IL-1 alpha induced anorexia.

Surfeit calories during parenteral nutrition influences food intake and carcass adiposity in rats.

The relationship between surfeit caloric consumption during intravenous infusion of a parenteral nutrition solution providing 100% daily caloric needs (PN-100; glucose:fat:amino acid = 50:30:20), carcass adiposity, and postinfusion food intake was evaluated. Rats received saline (control) or PN-100 for 4 days via jugular vein. PN-100 rats were either allowed or denied access to chow during the infusion period. When food was available during the 4 days of PN-100, total cumulative caloric intake, eaten and infused, was 40% higher than controls (p < 0.01). Percent carcass adiposity was increased from 8.8% to 11.6% (p = 0.04), and there was a 3-day delay before food intake returned to baseline. When caloric intake was limited to that provided by PN-100, carcass adiposity was not increased and food intake was 86% of baseline during the first 24 h after stopping PN-100. Delayed return of normal food intake following PN-100 is the likely consequence of excess caloric ingestion during the PN-100 infusion period. The surfeit caloric intake resulted in increased carcass adiposity.

Factors influencing compensatory feeding during parenteral nutrition in rats.

During parenteral nutrition (PN) there is an incomplete compensatory decrease in oral caloric intake. The effects of two factors influencing nutrient utilization, diurnal timing of PN infusion and composition of available diet, on compensatory feeding were investigated in male rats receiving 100% of daily caloric need as PN (PN-100). Compensation for infused calories increased from 70 to 80% (P < 0.05) when the timing of infused kilocalories more closely matched diurnal pattern of oral kilocalories intake (e.g., 75% kcal daily during the dark phase, 25% during the light phase). There was a similar degree of compensation when the diet and PN-100 had the same caloric distribution (50% kcal from carbohydrate, 30% from fat, and 20% from protein). Adjusting the diurnal timing of infused calories and diet composition concurrently further increased compensation for infused calories from 70 to 90% (P < 0.05). In the rat PN-100 model, compensation for infused calories is improved by adjusting the temporal pattern of infusions and diet composition.

Metabolic influences on satiety in rats receiving parenteral nutrition.

Food intake is reduced during parenteral nutrition (PN) proportionally to the amount of calories or composition of the solution infused. The relative importance of infused glucose and lipid, 50 and 30% of PN kilocalories, respectively, in reducing food intake during PN was examined. Glycolysis, fatty acid oxidation, or both were acutely disrupted with 2-deoxy-D-glucose (2-DG) and mercaptoacetate (MA). Rats receiving intravenous infusions of saline or a PN solution providing 100% of total daily calories (PN-100) received a single intraperitoneal injection of saline, 2-DG, and/or MA during the early light phase. 2-DG (1.4 or 2.2 mmol/kg) did not initiate feeding in PN-100 rats, although hyperglycemia was evident in all rats 1 h after 2-DG. Food intake of PN-100 rats after MA (0.4 mmol/kg) was approximately 50% that of control rats. When 2-DG (1.4 mmol/kg) and MA (0.4 mmol/kg) were administered concomitantly, PN-100 and control rats consumed the same amount of food. During PN-100, rats appeared to be more sensitive to losing metabolic energy derived from lipid than from glucose.

Hepatic vagotomy effects on metabolic challenges during parenteral nutrition in rats.

During parenteral nutrition (PN) glucoprivic-induced feeding appeared to be neutralized by the oxidation of infused fatty acids. With the use of a latin-square design, the feeding response to 2-deoxy-D-glucose (2-DG) and/or 2-mercaptoacetate (MA) was evaluated in male Sprague-Dawley rats with hepatic branch vagotomy (HV) or sham operations (SO). Rats received continuous infusions of 0.9% saline or PN providing 100% of daily caloric needs (PN-100) for four consecutive days. During PN-100, food intake was stimulated by 2-DG in HV rats and when fatty acid oxidation was simultaneously inhibited by MA. 2-DG-induced hyperglycemia was apparent under all conditions. Lipoprivic-induced feeding and increased plasma free fatty acid concentrations were absent in HV rats, whether MA was administered alone or with 2-DG. The feeding response to glucoprivic challenges is influenced by the relative availability of alternate energy sources. The lack of feeding response to 2-DG during PN-100 is mediated by vagal input of hepatic fatty acid oxidation status.