Increased protein intake during recovery from zinc deficiency is accompanied by alterations in hypothalamic growth hormone releasing factor and somatostatin.

To determine if alterations in growth hormone releasing factor (GRF) and somatostatin (SRIF) occur during recovery from zinc deficiency and to examine the relationship between these peptides and the increased protein intake observed during zinc repletion, both the hypothalamic release and mRNA levels of GRF and SRIF were measured during zinc deficiency and zinc repletion. Groups of male rats (n = 4-8 each) were provided 3-choice macronutrient diets made either zinc adequate (Zn+) or deficient (Zn-; 30 vs. 1 mg Zn/kg diet). Pair fed, calorie restricted (PF) rats were also maintained. After 28 days, Zn- rats were repleted with Zn+ diets for 2 days and one PF group was allowed free access to the same Zn+ diets for 2 days. Additionally, groups of Zn- and PF rats were repleted 2 days with Zn+ carbohydrate and fat diets but no protein. Hypothalamic GRF and SRIF release was measured in vitro. Basal GRF secretion was not significantly different between Zn-, PF, or Zn+ groups although there was a significant increase (P<0.05) during zinc repletion. PF and Zn- rats repleted 2 days with diets devoid of protein had highest GRF secretion (P<0.01) compared to other groups. There were no differences in GRF mRNA levels among groups. Basal SRIF secretion was significantly lower in Zn- and PF groups compared to Zn+, and SRIF mRNA levels were significantly higher in zinc repleted groups compared to Zn-. These results demonstrate that during zinc repletion, GRF and SRIF secretion and SRIF gene expression are altered concomitantly with protein intake. The present data support involvement of GRF in protein intake changes during zinc repletion.

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.

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.

Zinc deficiency changes preferred macronutrient intake in subpopulations of Sprague-Dawley outbred rats and reduces hepatic pyruvate kinase gene expression.

Macronutrient selection patterns of male rats were analyzed using a 3-choice macronutrient selection system providing either adequate (+Zn) or deficient (-Zn) levels of zinc (30 or 1 mg Zn/kg). In study 1, rats were provided +Zn and -Zn diets for 28 d. All rats preferred carbohydrate (>50% carbohydrate intake) at the onset, consuming an average of 71% carbohydrate (cho), 17% protein (pro), and 12% fat. By the end of the study, 25% of the -Zn rats switched preference from cho to fat, whereas no +Zn rats changed. In study 2, -Zn rats preferring fat increased their total intake to normal levels, but only 50% reverted to carbohydrate preference after 35 d of zinc repletion. Hypothalamic concentrations of galanin were measured in groups of +Zn and -Zn cho- and fat-preferring rats. Galanin, which may be regulated with fat intake, was not different in -Zn rats preferring fat vs. -Zn rats preferring cho. Galanin concentrations were higher in +Zn than in -Zn rats (P < 0.05) and higher in +Zn rats preferring fat than in +Zn rats preferring cho (P < 0.05). Hepatic pyruvate kinase (PK) mRNA concentrations were related to cho preference, regardless of zinc status. When PK mRNA levels were measured in rats consuming a single AIN- 93-based diet, PK mRNA levels were significantly reduced by zinc deficiency (P < 0.05). Because PK is highly regulated by insulin, the effect of insulin may be reduced by zinc deficiency, making it more difficult for -Zn rats to catabolize dietary cho. This may explain why some -Zn rats switched from preferring cho to fat after developing zinc deficiency.

Food intake patterns are altered during long-term zinc deficiency in rats.

Rats experience anorexia and reduction or cessation in growth after being provided a zinc-deficient diet. While zinc deficient, intake levels may be reduced 50% or more compared to control rats. In the present report, diurnal food intake patterns of male Sprague-Dawley rats were measured during zinc deficiency. In Study 1, rats consuming a modified AIN-93 diet were tested during the dark phase using an automated food weighing system. In zinc-deficient animals (Zn-), the onset of the first meal of the dark phase was delayed compared to zinc-adequate rats (Zn+; 106+/-47 vs. 23+/-5 min; p<0.05) and the number of meals consumed during the dark phase was reduced in Zn- vs. Zn+ rats (3.9+/-0.5 vs 7.1+/-0.4; p<0.05). In Study 2, diurnal food intake patterns were tested using a three-choice macronutrient self-selection paradigm of carbohydrate-, protein-, and fat-containing diets made deficient or adequate in zinc (1 or 30 mg Zn/kg diet). Food intake was recorded in the early-, mid-, late-dark period (4 h each) and light period (12 h). Carbohydrate intake was 70% of total intake of both Zn+ and Zn- rats during the first 5 days, but decreased significantly to 50% in the Zn- group during the last 5 days. Fat intake increased significantly in the Zn- group during the last 5 days. This increase was the result of 4 of 15 Zn- rats increasing their intake of fat significantly. Results of this study indicate that zinc status alters dark phase and macronutrient selection patterns by delaying consumption of the initial meal of the dark phase, reducing the average meal number and by changing the dominant macronutrient preference of some Zn- rats.

Minimum thiamin requirement of weanling Sprague-Dawley outbred rats.

To determine the minimum thiamin requirement for maximal growth, two trials were conducted using male weanling Sprague-Dawley rats fed graded doses of thiamin from thiamin mononitrate as a component of a chemically defined diet. This diet included 16% amino acids, 72% sucrose and cornstarch and 5% soybean oil. Total weight gain and food intake were recorded over 2- (trial 1) or 3- (trial 2) wk periods. In trial 1, graded levels of thiamin were fed at 0, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg thiamin/kg diet, and growth rate reached a plateau in rats fed 0.50 mg thiamin/kg. In trial 2, lower doses of thiamin were fed (0, 0.25, 0.50, 0.75, 1.0, 4.0 and 5.0 mg/kg) to determine the minimum requirement for maximal growth. Using broken-line least-squares analysis, weight gain reached a plateau (6.8 g/d) at a thiamin concentration of 0.55 +/- 0.07 mg/kg. No differences (P > 0.05) in weight gain, food intake or gain:food ratio were observed at thiamin levels at or above 0.5 mg/kg, but food intake was substantially lower (P < 0.05) in rats fed 0 and 0.25 mg thiamin/kg (9.9 and 13.4 g/d, respectively) than in rats fed higher doses of thiamin (16.1 g/d). Hepatic transketolase, a measure of enzymatic thiamin status, increased with dietary thiamin in rats fed diets containing 0-5.0 mg/kg thiamin. However, an inflection point occurred at 0.53 mg thiamin/kg, with the slope being eight times greater below than above the inflection point. The data suggest that the thiamin requirement for maximal growth of weanling rats fed a chemically defined diet is approximately 0.55 mg thiamin/kg, which is substantially below the current National Research Council estimated requirement of 3.1 mg thiamin/kg diet.

Zinc status specifically changes preferences for carbohydrate and protein in rats selecting from separate carbohydrate-, protein-, and fat-containing diets.

This study examined how macronutrient intake preferences were specifically altered in the loss of appetite caused by experimentally produced zinc deficiency. Outbred female rats were allowed to freely select from simultaneously provided carbohydrate-, protein-, and fat-rich diets to provide themselves with an acceptable total diet. Rats were divided into two groups and provided the three diets containing either adequate (30 mg/kg; Zn+) or deficient (1 mg/kg; Zn-) levels of zinc (Zn). After 28 d, rats offered the Zn- diet were returned to a Zn+ diet (Zn repletion). Intakes from each of the three macronutrient diets were measured to determine macronutrient preferences of Zn-adequate, Zn-deficient, and Zn-repleted rats. In two 28-d studies involving a total of 66 rats, total metabolizable energy intake in Zn deficient rats was between 20 and 35% lower than in Zn+ rats, and carbohydrate intake accounted for essentially 100% of the lower energy intake. Fat and protein intakes were not affected by Zn deficiency. When Zn-deficient rats were repleted with Zn by providing diets containing adequate Zn, carbohydrate intake was restored to normal levels after 1 d of feeding. A transient difference in protein intake was noted during the repletion period, peaking during d 2-4 of repletion. Protein intake increased by more than 50% during this period. We hypothesize that specific changes in macronutrient intake patterns during development and recovery from Zn deficiency may be reflections, at least in part, of Zn-mediated changes in the central control of appetite.