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.

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.

Effect of glucoprivation on glutamate decarboxylase activity in the ventromedial nucleus.

Activity of the GABA-synthesizing enzyme, glutamate decarboxylase (GAD), was measured in brain areas involved in glucoregulation 60 min after a glucoprivie challenge. The rate of GAD activity in the ventromedial nucleus of the hypothalamus (VMN) increased in a dose-dependent manner in response to intraperitoneal injection of 2-deoxy-D-glucose (2-DG). The increase in VMN GAD activity was significantly correlated with an increase in food intake (r2 = .77, p less than 0.01). The increase in VMN GAD activity was not due to the higher food intake since rats receiving 2-DG and denied access to food also had elevated rates of VMN GAD activity. VMN GAD activity was increased 28% and 32% after intracerebroventricular injection of 2-DG or 5-thioglucose, respectively. The rates of GAD activity in the lateral hypothalamus and area postrema were not affected by either peripherally or centrally administered 2-DG. The increase in VMN GAD activity after glucoprivation may be involved in the regulation of blood glucose by influencing food intake.

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.

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.

Timing and dose of amino acids injected into prepyriform cortex influence food intake.

The effects of time before feeding and dose of dietary-limiting amino acids (DLAA) injected into the prepyriform cortex (PPC) on intake of amino acid-imbalanced diets were evaluated. Intake of imbalanced diet was increased from approximately 50% to approximately 75% of baseline when an optimal amount of DLAA (1 nmol L-isoleucine or 2 nmol L-threonine) was injected immediately prior to feeding. Injections made several hours prior to feeding were more effective, increasing intake of imbalanced diets to approximately 85% of baseline. Delivering two half-optimal doses of DLAA, several hours apart, increased intake of imbalanced diet only to the same level as a single injection of the optimal dose immediately prior to feeding. The increase in intake of a threonine-imbalanced diet after injecting 2 nmol threonine 6 h prior to feeding was abolished if an additional 2 nmol threonine was injected immediately prior to feeding. It appears that it is the sum of the changes in tissue DLAA concentrations in the PPC that are recognized and influence food intake when amino acid imbalanced diets are fed.

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.

ICS 205-930 and feeding responses to amino acid imbalance: a peripheral effect?

Serotonin3 (5-HT3) receptor antagonists (ICS 205-930 and MDL 72222) have been shown to block or ameliorate the anorectic response of the rat to amino acid imbalanced (IMB) diets. Two experiments were conducted to determine whether the effects of these antagonists are mediated through central or peripheral 5-HT3 receptors. In Experiment One, ICS 205-930 (ICS) was injected centrally, into either the lateral ventricle (doses: 0.3 pmol to 10 nmol), or the cisterna magna (62 nmol). The intake of rats fed an isoleucine IMB diet was not affected by these injections. In Experiment Two, rats received an IP injection of either saline, ICS, or a quaternized derivative of ICS (Q-ICS) that should not cross the blood-brain barrier. Both ICS- and Q-ICS-injected rats ate significantly more (p less than 0.05) IMB diet than saline-injected rats. Intake of IMB diet was not different (p greater than 0.4) between ICS and Q-ICS groups. From these results, it appears that ICS restores intake of IMB through a peripheral component.

The early cancer anorexia paradigm: changes in plasma free tryptophan and feeding indexes.

Tumor growth is accompanied by an anorexia mediated by humoral factors that appear to influence appetitive mechanisms in the brain. Because tumor resection is followed by resumption of normal food intake, the circulating anorexigenic substance(s) are produced either by the neoplastic tissue or by the host in response to the tumor. Increased levels of plasma free tryptophan and plasma ammonia have been proposed to mediate cancer anorexia. With animal models, it is often difficult to ascertain whether changes in food intake depend upon metabolic changes or the progressively increasing tumor mass per se. The feeding patterns and biochemical changes that occur during tumor growth were evaluated in 96 male Fischer rats that were inoculated with 10(6) methylcholanthrene sarcoma cells or saline (controls). Rats were placed into metabolic cages equipped with an Automated Computerized Rat Eater Meter to continuously determine meal size and meal number. Plasma free tryptophan and ammonia were evaluated 6, 10, 16, 18, 22, and 26 days after tumor inoculation. Anorexia developed by day 17-18, when food intake started to decrease via a decrease in meal size but not meal number and reached 60% of control by day 26. However, long before anorexia developed, free tryptophan was significantly higher 6 days after tumor inoculation, and the greatest increase occurred after 18 days. Ammonia did not differ from control at any time. Data confirm tumor-associated increases in plasma free tryptophan that occurred before the manifestation of anorexia and support a possible role of brain serotonin in cancer anorexia.

Clonidine in the prepyriform cortex blocked anorectic response to amino acid imbalance.

Ingestion of imbalanced amino acid diets (IMB) has been associated with a decrease in norepinephrine (NE) concentration in the prepyriform cortex (PPC), an area essential for the anorectic response to IMB. Decreased NE could result from activity-induced release (and subsequent metabolism) of the transmitter. If activity of the NE system is important in the rat's anorectic response to IMB, reduced NE activity should result in increased IMB intake. Therefore, the feeding response to IMB was measured after injecting clonidine (Clon) into the PPC to inhibit NE release. At 3 and 6 h after Clon (1.0 and 1.5 micrograms/rat) injections, IMB intake was increased from 69 (the usual response to IMB in untreated animals) to > 100% of control intake. Effective injection sites did not include the gustatory neocortex, an area important for conditioned taste aversions. Thus activation of the NE system in the PPC may be associated with the initial reduced intake of IMB, suggesting that NE activity in the PPC has a role in the neural mechanisms that subserve recognition of amino acid deficiency.

Influence of fatty acid oxidation in lateral hypothalamus on food intake and body composition.

This study tested the concept that the level of fatty acid oxidation in the ventrolateral hypothalamus (VLH) reflects peripheral energy stores and elicits compensatory responses to changes in energy balance status. Fatty acid oxidation rates in the VLH were chronically altered over a 14-day period by infusing into the VLH either 0.1 mM 4-pentenoic acid (4-PA; 5 ng/h) or 1.0 mM L-carnitine (L-Carn; 98 ng/h). Fatty acid oxidation rates in the VLH were altered to a similar extent as by overfeeding (reduced 37% by 4-PA) and dietary restriction (increased 28% by L-Carn). Diffusion of infusates was limited, since there were normal rates of fatty acid oxidation in the ventromedial hypothalamus and cortex. There were no significant effects of altering fatty acid oxidation in the VLH on food intake, body weight, body composition, or serum levels of glucose, insulin, and free fatty acids. The results of this experiment indicate that the level of fatty acid oxidation in the VLH is unlikely to independently elicit changes in food intake or peripheral metabolism.

Increased GABA shunt activity in VMN of three hyperphagic rat models.

Metabolic signals have been proposed as controls of energy balance. Glucose utilization for gamma-aminobutyric acid (GABA) synthesis was evaluated in diet-restricted and streptozotocin-diabetic rats. Rates for glucose flux through the GABA shunt and glutamate decarboxylase (GAD), the rate-limiting enzyme in GABA synthesis, were measured in ventro-medial nucleus (VMN) and lateral hypothalamic (LHA) homogenates. Rates of GAD activity were elevated in the VMN of both restricted and diabetic rats but did not change in the LHA of either model. The rate of glucose flux through the GABA shunt in the VMN was increased in restricted rats but were decreased in the VMN of diabetic rats. Restricted rats allowed to feed ad libitum initially ate 160% of the amount the control rats ate. GAD activity rates in the VMN returned to near control levels after 2 days of refeeding. Obese Zucker rats (fa/fa) also had higher rates of GAD activity and glucose flux through the GABA shunt in the VMN but not in the LHA. The increased rate of GABA synthesis may represent increased GABAergic activity in the VMN and may possibly be involved in feeding behavior and energy balance.

Influence of serum glucose on glutamate decarboxylase activity in the ventromedial nucleus of rats.

Changes in the activity of glutamate decarboxylase (GAD), the rate-limiting enzyme in gamma-aminobutyric acid synthesis, in response to insulin-induced hypoglycemia were measured in several brain areas involved in glucoregulation. A 20% increase in GAD activity in the ventromedial nucleus (VMN) of rats was measured 30 min after injections of insulin. The increase was significantly correlated with the decrease in serum glucose (r = -0.74) and increased food intake (r = 0.44). The increase in VMN GAD activity was linear between 5 and 30 min after insulin injection and coincided with the decline in serum glucose. Pretreatment with a gastric preload of a 50% glucose solution attenuated the decline in serum glucose, the increase in food intake, and the increase in GAD activity. Inhibition of protein synthesis by puromycin did not block the decrease in glucose, but neither the increase in GAD activity in the VMN nor the increase in food intake in response to insulin was observed. The increase in VMN GAD activity also was present 6 h after insulin injection if rats were denied access to food. No consistent changes in GAD activity were measured in any brain area other than the VMN. The results of these trials suggest that the increase in VMN GAD activity is associated with the decrease in serum glucose and may be involved in the increased feeding associated with insulin administration.

Effect of dietary limiting amino acid in prepyriform cortex on food intake.

The mechanisms underlying the reduced intake of an amino acid-imbalanced diet (imbalanced diet) appears to involve a decrease in the content of the dietary limiting amino acid (DLAA) in the prepyriform cortex (PPC). Intake of imbalanced diet was increased from 45-50 to 70-75% of baseline after bilateral injection of the DLAA directly into the PPC, following an inverted U-shaped dose-response curve. Injections had no effect on intake of basal diets. Injection of the DLAA into the PPC reversed the aversion to imbalanced diet in choice studies, as rats selected an imbalanced diet over protein-free diet after such injections. Intake of imbalanced diet did not increase after a nonlimiting amino acid was injected into the PPC or after injections of the DLAA into other brain areas. Results were similar when either threonine or isoleucine was the DLAA. These results confirm that the decrease in the concentration of the DLAA in the PPC is involved in the reduction in intake of imbalanced diets.

Effect of dietary limiting amino acid in prepyriform cortex on meal patterns.

Microinjection of the dietary limiting essential amino acid (DLAA) into the prepyriform cortex (PPC) increased intake of a diet having an imbalance among the essential amino acids (imbalanced diet) from 50-55% of baseline, when artificial cerebrospinal fluid (aCSF) was injected, to 70-75% of baseline. The increase in intake of the imbalanced diet by DLAA injection became apparent after 3-6 h and was maintained throughout the dark period. Meal size, meal duration, and the number of meals returned to normal after bilateral injections of the DLAA into the PPC of rats fed the imbalanced diet. Injection of the DLAA 30 min before the onset of the dark phase increased intake of imbalanced diet to 70% of baseline intake. When injections of threonine or isoleucine were made 6 and 3 h, respectively, prior to onset of the dark phase, intake of imbalanced diet increased to 85% of baseline intake. Results suggest that some form of processing of the injected DLAA within the PPC is necessary to increase the intake of imbalanced diets.

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.

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.

Threonine concentration in the prepyriform cortex has separate effects on dietary selection and intake of a threonine-imbalanced diet by rats.

Dietary selection and intake of a threonine-imbalanced diet were evaluated after increasing the concentration of the dietary limiting amino acid in the prepyriform cortex. Selection against the threonine-imbalanced diet in favor of a protein-free diet was reversed when 2 or 4 nmol threonine was injected bilaterally into the prepyriform cortex. However, intake of the threonine-imbalanced diet was significantly increased only after injection of 2 nmol threonine. The reduced intake of the threonine-imbalanced diet, compared to the basal diet, after injection of 4 nmol threonine was not the result of an excess in the concentration of amino acid (or nitrogen) injected into the PPC because intake of the threonine-basal diet was not reduced when 4 nmol threonine was administered. Intake of the threonine-imbalanced diet was also increased after injection of 2 nmol threonine plus 2 nmol isoleucine but not after injections of 2 nmol isoleucine. The changes in food intake when an imbalanced diet is fed appear to be the result of at least two separable responses: recognition of a diet as having an amino acid imbalance, as indicated by dietary choice, and reduction in food intake. The results of this study indicate that changing the concentration of the dietary limiting amino acid in the prepyriform cortex influenced dietary selection and food intake separately.

Protein synthesis in the prepyriform cortex: effects on intake of an amino acid-imbalanced diet by Sprague-Dawley rats.

The effect of inhibiting protein synthesis within the prepyriform cortex (PPC) on intake of an amino acid imbalanced diet was evaluated in rats receiving bilateral injections of the dietary limiting amino acid (DLAA). Injection of the DLAA into the PPC increased intake of the imbalanced diet by 150% and incorporation of [3H]leucine into the trichloroacetic acid insoluble fraction of PPC homogenates by 248%. Coinjection of puromycin (100 mumol/L) or actinomycin D (10 mumol/L) blocked the increase in intake of the imbalanced diet but had no effect on intake of the basal diet. Puromycin blocked the increase in intake of the imbalanced diet whether injected with the DLAA or 6 h later. Selection of the imbalanced diet over a protein-free diet when the DLAA was administered was blocked by co-injecting puromycin or actinomycin D. The results indicated that the increased intake and the reversal of aversion to the imbalanced diet when the DLAA was injected into the PPC required de novo protein synthesis. The proteins necessary for the feeding response seemed to have a short half-life and seemed to require synthesis of new mRNA. We conclude that changes in concentration of the DLAA within the PPC influenced protein synthesis at the genomic level.