Peptidergic systems in the medial hypothalamus (MH) are involved in feeding and energy homeostasis. High protein diets have been suggested to aid in weight loss by increasing satiety and decreasing food intake in human studies as well as animal models. The objective of this study was to evaluate the pattern of changes in orexigenic and anorexigenic neuropeptides (NPs) in the MH of rats fed a high protein diet (HP; 30% protein: 38% CHO: 32% fat) versus a standard protein diet (SP; 15:59:26). Our study focused on the pattern of NP change in the MH when the high protein diet was fed. After 3 weeks rats were sacrificed after an overnight fast, the animals were placed into 3 groups: immediately sacrificed, 1hr or 3hr following a 3g meal. At sacrifice the MH was removed and homogenized in acetic acid. Following centrifugation, proteins were extracted and analyzed for Neuropeptide Y (NPY), Orexin A (OxA; also known as Hypocretin-1), Cocaine-Amphetamine Related Transcript (CART), and α-Melanocortin Stimulating Hormone (α-MSH) by radioimmunoassay. Total food intake levels during the last week were ~10% (p=0.01) lower in the HP group. NPY was 20% lower and α-MSH was 12% higher after 3 weeks of HP. This relationship also remained at 1 hour and to a lesser extent 3 hours post-meal. Our results indicate that the expression of the orexigenic NPs (NPY and OxA) was decreased in rats chronically fed the HP diet, while the expression of anorexigenic NPs (CART and α-MSH) was elevated in these same animals. The comprehensive pattern of changes in the appetite-related NPs might contribute to the reduced food intake in animals fed a HP diet. NIH DK59755

Adaptations in brain signaling pathways may underlie the altered detection and response to hypoglycemia seen in hypoglycemic unawareness. Glycogen synthase kinase-3beta (GSK3ß), a key regulator of numerous metabolic processes, responds to changes in insulin and glucose. The objective of this study was to examine the phosphorylation of GSK3ß by Akt (protein kinase B) in the rat medial basal hypothalamus (MBH) following acute and recurrent episodes of insulin-induced hypoglycemia. MBH brain regions were obtained from male Sprague-Dawley rats 30 minutes after a first-time (acute) or fourth-time (recurrent) 2 U/kg IV insulin injection. Expression of Ser-9 phosphorylated and total GSK3ß and Thr-308 phosphorylated and total Akt were measured from homogenates by Western blot analysis. Following acute hypoglycemia, phosphorylation of GSK3ß was decreased ~40% (p=0.05) as compared to saline-treated controls. The phosphorylation of Akt was decreased ~50% (p=0.03) after first-time hypoglycemia. Levels of phosphorylated GSK3ß were similar (p=0.34) to saline controls after recurrent hypoglycemia. There was no change (p=.98) in Akt phosphorylation to a 4th hypoglycemic episode. Total GSK3ß and Akt levels were not changed by acute or recurrent hypoglycemia. The decrease in phosphorylation and inactivation of hypothalamic GSK3ß to insulin-induced hypoglycemia was absent after 3 previous episodes of hypoglycemia. These results are consistent with a possible role of GSK3ß in hypoglycemic unawareness. NIH-DK59755

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-aminobutyricacid (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.

During insulin-induced hypoglycemia, there is an increase in extracellular norepinephrine (NE) in the ventromedial hypothalamus (VMH). This brain area is known to play an important role in integrated hormonal and behavioral responses to systemic hypoglycemia. Selective glucoprivation restricted to the VMH is both necessary and sufficient to initiate secretion of counterregulatory hormones. The present study was designed to investigate whether increased release of NE in the VMH depends on detection of glucoprivation localized in this area. In awake, chronically catheterized male Sprague-Dawley rats, extracellular NE in the VMH was monitored using 1-mm microdialysis probes perfused with Krebs Ringer buffer (KRB) or KRB + 100 mM d-glucose (d-Glc). During insulin-induced hypoglycemia (glycemic nadir approximately 2.4 mM) extracellular NE was increased to >160% of baseline (P < 0.01) only in the KRB + insulin group. There was no increase in NE from baseline when glucose was added to the perfusate to maintain euglycemia at the periprobe environment. The sympathoadrenal response to hypoglycemia, present in the KRB + insulin group, was attenuated in the d-Glc + insulin group. The present results confirm that noradrenergic activation in the VMH during systemic hypoglycemia depends on detection of glucoprivation locally in this area. These data provide additional support for the importance of increased noradrenergic activity in the VMH in the counterregulatory hormonal responses to hypoglycemia.

Bartonella quintana, an emerging gram-negative pathogen, may cause trench fever, endocarditis, cerebral abscess and bacillary angiomatosis usually with the absence of septic shock in humans. B. quintana lipopolysaccharide (LPS), a deep rough endotoxin with strong reactivity in the limulus amebocyte lysate (LAL)-assay, was studied in human whole blood and in a rat model. A significant (P<0.05) increase of interleukin-8 (IL-8) concentration, comparable to the level induced by enterobacterial LPS, was stimulated in the human whole blood by B. quintana LPS. Isolated human neutrophils delayed their apoptotic behavior in the presence of B. quintana LPS. In the rat, B. quintana LPS induced a significant (P<0.001) increase in white blood cell count, both 30 and 60 min after intravenous injection. Such leukocytosis was inhibited by pretreatment with prazosin, an α-adrenergic antagonist. B. quintana LPS did not significantly change heart rate (HR), hematocrit (HCT) and platelet count in the above reported in vivo model, and regarding mean blood pressure (MAP) only a very early (5 min after LPS) and mild (yet significant) hypotension was observed. In contrast, a long-lasting decrease of MAP was found in Salmonella minnesota R595 LPS-treated animals. Blood TNFα levels did not change significantly from the baseline in rats injected with either saline or with B. quintana LPS, on the contrary S. minnesota R595 LPS-injected animals showed substantial increase of TNFα levels up to 2924 pg/ml at 60 min after LPS injection. B. quintana LPS as well as Salmonella LPS-injected rats exhibited an increase of the blood levels of GRO/CINC-1, particularly at 240 min after LPS administration. Apical part of rat gut villi showed several TUNEL-positive cells in tissue sections from B. quintana LPS-treated animals. Taken together, our data demonstrates that B. quintana LPS is able to selectively stimulate some inflammatory mediators. B. quintana LPS-induced leukocytosis appears mediated by an α-adrenergic receptor. The delayed apoptotic process of leukocytes and the chemokine increase may explain the apoptotic cells found in the rat gut and the inflammatory reactions in some human Bartonella diseases. This peculiar inflammatory pattern induced by B. quintana LPS, may partially account for the lack of severe septic shock, observed in human B. quintana infections.

This study evaluated whether attenuation of sympathoadrenal responses to recurrent hypoglycemia is mediated by diminished noradrenergic activity in the hypothalamus. Male Sprague-Dawley rats received either once daily insulin (1.0 units/kg) injections or an equal administration of saline for 3 days. Both groups received an administration of insulin on the fourth day, during which blood glucose and plasma catecholamines were determined, and extracellular norepinephrine (NE) in the ventromedial hypothalamus (VMH) or paraventricular hypothalamic nucleus (PVN) was monitored with microdialysis. The peak response of plasma epinephrine to insulin-induced hypoglycemia (nadir approximately 3.2 mmol/l) was significantly reduced during the fourth hypoglycemic episode (774 +/- 134 pg/ml) compared with the first episode (2,561 +/- 410 pg/ml, P < 0.001). Baseline levels of extracellular NE were elevated approximately 25% (P = 0.07) in the VMH and approximately 46% (P = 0.03) in the PVN after multiple hypoglycemic episodes. There was no difference in noradrenergic activity during the first or fourth hypoglycemic episode in either brain area. The reduced sympathoadrenal output after recurrent hypoglycemia is likely postsynaptic from hypothalamic NE release or is mediated via a collateral pathway.

The activity of neurons in the ventromedial hypothalamus (VMH) important for initiating compensatory responses to hypoglycemia is influenced by ambient glucose concentration. In the present study, we used in vivo microdialysis to evaluate interstitial glucose concentrations in rat VMH under various glycemic conditions. Using the zero-net-flux method, steady-state glucose concentration in the VMH was approximately 20% of blood glucose (approximately 1.4 mmol/l) in fed rats but approximately 14% of blood glucose (approximately 0.7 mmol/l) in overnight-fasted rats. During moderate hypoglycemia VMH glucose declined in parallel with blood glucose; however, VMH glucose decreased to a greater degree than blood glucose during a more severe hypoglycemic episode, falling to 10 +/- 1.2% of blood levels (P < 0.01). To determine whether VMH glucose concentrations were influenced by recurrent episodes of hypoglycemia a second zero-net-flux study was conducted. Steady-state glucose concentrations in the VMH were approximately 20% lower after three episodes of recurrent hypoglycemia, a value 17.8 +/- 0.8% of blood glucose, although the relative change in VMH glucose levels during the first and fourth hypoglycemic episodes were similar. From these results, we conclude that interstitial glucose concentrations in the VMH are not maintained at a constant level and are more dynamic than previously proposed.

Alterations in neurochemical activity in the paraventricular nucleus (PVN) of the hypothalamus may account for decreased intake of zinc-deficient diets. Male Sprague-Dawley rats were fed zinc-deficient (ZD) or zinc-adequate (ZA) diet for 14 d before samples of extracellular fluid in the PVN were collected by microdialysis or push-pull perfusion. A third set of rats was pair-fed (PF) an amount of ZA diet equal to the intake of ZD rats. Samples were collected over a 2-h period spanning the transition from light to dark. All rats then consumed the zinc adequate diet ad libitum for 3 d before a second set of samples was collected. The increase in extracellular norepineprhrine (NE) during h 1 of the dark period to 147 +/- 13% of baseline (P < 0.05) was apparent only in ZA rats at d 14. After the 3-d repletion period, the increase in NE at dark onset occurred in all three groups. An increase in extracellular neuropeptide Y (NPY) at dark onset to 174 +/- 32% of baseline in rats fed ZA (P < 0.01) was measured in all three groups at both d 14 and 17. Basal NPY concentrations were significantly elevated in PF rats on d 14 (7.45 +/- 2.01 vs. 0.58 +/- 0.23 pmol/L, P = 0.01) and returned to ZA levels by d 17. The activities of the NE and NPY systems in the PVN were altered in rats fed a zinc-deficient diet; however, it is unclear whether the disruption in the NE and NPY neural systems in the PVN results in the altered feeding behavior accompanying zinc deficiency.

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 Znadequate (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.

Forty-eight growing-finishing pigs were used in a split-plot design with a 2×2 factorial arrangement of treatments to investigate the effect of sire line and sex (castrates and gilts) on feeding patterns. Sire line A was of Pietrain ancestry and sire line B was a synthetic line that included Large White, Landrace, Duroc, and Pietrain. Sires from line A (n=8) and line B (n=9) were mated with PIC Camborough 22 females. Growth performance and feeding patterns of the progeny were measured from 40.2±2.04 to 120.0±2.85 kg body weight (BW). Pigs were housed in groups of eight, with two pigs from each line×sex subclass in each pen, at a floor space allowance of 0.9 m2 per pig. Feeding patterns were monitored by a computerized feed intake recording equipment (FIRE) system. Line B progeny had higher growth rates (1036 versus 956 g per day, S.E.=15, P<0.01), higher feed intakes (2657 versus 2470 g per day, S.E.=61.2, P<0.05), but similar gain:feed ratios, as compared to the progeny of line A. Line B pigs tended to have smaller loin-eye depths (P<0.10), and had greater lean growth rates (P<0.01) than line A pigs. Line B pigs, compared to line A pigs, had a greater feed intake per visit (207 versus 172 g, S.E.=9.9, P<0.05), and a higher feed consumption rate (36.7 versus 28.7 g/min, S.E.=2.41, P<0.05). Line B pigs tended to have lower feeder occupation times per day than line A pigs (P<0.10). There were no differences between the two sire lines for number of feeder visits or feeder occupation time per visit (P>0.10). Castrates had higher growth rates (P<0.01) and feed intakes (P<0.001), lower gain:feed ratios (P<0.01), but similar (P>0.05) feeding patterns compared to gilts. Feeding patterns showed significant changes with increasing BW: number of feeder visits and feeder occupation time per visit, and per day decreased linearly, while feed intake per visit and feed consumption rate increased linearly with increasing pig BW. The rates of change in a number of feeding pattern traits differed (P<0.05) between the two genetic lines. The results of this study highlight the influence of genetic ancestry and, particularly, BW on growth performance and feeding patterns in growing-finishing pigs.

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.

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.