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Insights on Zinc Regulation of Food Intake and Macronutrient Selection MING-YAN JING,1 JIAN-YI SUN,*,1 AND XIAO-YAN WENG2 1

The Key Laboratory of Molecular Animal Nutrition, Ministry of Education, College of Animal Science, and 2College of Life Science, Zhejiang University, Hangzhou 310029, People’s Republic of China

Received May 2, 2006; Revised June 1, 2006; Accepted June 16, 2006

ABSTRACT Zinc (Zn) is an essential trace element required for human beings and animals. This divalent cation is involved in many physiological functions, including immune and antioxidant function, growth, and reproduction. Deficiency of Zn produces several pathological disorders and abnormalities in its metabolism, such as anorexia, weight loss, poor efficiency, and growth retardation. Although it has been known for more than 50 yr that Zn deficiency regularly and consistently causes anorexia in many animal species, the mechanism that causes this phenomenon still remains an enigma. The present review describes recent research investigating the relationship between Zn deficiency and the regulation of food intake, as well as macronutrient selection. Index Entries: Zinc; anorexia; food intake; macronutrient selection.

INTRODUCTION Zinc (Zn) is regarded as an essential nutrient for human beings and animals. This divalent cation is vital for the normal growth, development, and function. Numerous proteins, crucial enzymes, and transcription factors bind Zn and are thought to be dependent on Zn for their functions. Zn is a cofactor for a wide range of enzymes, including those associated with cell division, protein synthesis, and carbohydrate metabolism in animals and humans (1–3). It is vital to the activity of a variety of hormones, including glucagon, insulin, growth hormone, thyroid hormone, and the sex hormone. It also plays a key role in the immune system; deficiency *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research

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results in reduced immunocompetence and decreased resistance to infections (4). The human body has 1.5–2.5 g of Zn, making it nearly as abundant as iron. Zn requirements in total diets of beef cattle, dairy cattle, swine, horses, sheep, goats, and chickens are 30, 40, 50, 40, 20–33, 40–75, and 40–50 mg/kg, respectively (5,6). Zn has a wide spectrum of biological activities and its deficiency has been related to various dysfunctions and alterations of normal cell metabolism that include anorexia, weight loss, poor food efficiency, growth retardation, and disorders of the skin (7–9). The anorexia resulting from Zn deficiency consistently results in decreased food intake with subsequent poor weight. However, the cause of this anorexia is not well understood. Feeding is a complex behavior that is controlled by the neuroendocrine system consisting of peripheral signals like leptin and central signals—in particular, neuropeptides. Zn deficiency results in a reduction in food intake, suggesting that Zn status might affect the complex mechanisms underlying normal food intake. This review describes recent research on the relationship between Zn deficiency and the regulation of food intake, as well as macronutrient preference.

ZINC DEFICIENCY AND REGULATION OF FOOD INTAKE Zinc deficiency was initially discovered in humans and reported by Prasad et al. (10). Symptoms reported to accompany Zn deficiency included dwarfism, hypogonadism, and poor appetite. In these studies, similarities were noted between Zn-deficient human subjects and known characteristics of Zn-deficient animals. Although Zn deficiency has been studied in many different species, including rats, pigs, chicks, lambs, monkeys, rabbits, and guinea pigs, studies in the rat have provided most of the experimental data regarding Zn deficiency and changes in food intake. The young growing rat is very responsive to the consumption of a Zn-deficient diet. One of the first observable signs of Zn deficiency in the rat is a reduction in food intake within 3–5 d (3,7–9). Sustained Zn deficiency often results in food intake that is less than 50% of normal, and force-feeding the deficient rats rapidly induces illness. The biochemical mechanism for food intake depression in the Zn-deficient rat is still unknown, but some substances, such as neuropeptides and hormones, have been associated with feeding behavior. Neuropeptide Y (NPY) is one of the most potent appetite-regulating neuropeptides and stimulates food intake behavior when injected into the paraventricular nucleus (PVN) (11). This peptide has received much attention with regard to food intake regulation in Zn deficiency. Studies were therefore designed to test the hypothesis that Zn deficiency induces anorexia by inhibiting NPY gene expression and/or peptide synthesis. The initial report showed that NPY mRNA was elevated in the hypothalamus of Zn-deficient rats (12). Despite the elevations in NPY mRNA, NPY pepBiological Trace Element Research

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tide levels in the hypothalamus were not increased by Zn deficiency (13), suggesting that Zn deficiency impairs NPY translation. However, later work clarified the regulation of NPY translation by examining the effect of Zn deficiency. Investigators found that not only did Zn deficiency increase NPY mRNA abundance in the arcuate nucleus (ARC) but also NPY peptide levels in the PVN were higher in Zn-deficient rats (12). Succeeding research showed that NPY levels are equally high in Zndeficient and pair-fed rats, suggesting that the increase in NPY and NPY mRNA levels appears to be the effect of reduced food intake rather than Zn deficiency and is a compensatory mechanism to restore normal food intake in response to Zn-induced anorexia. However, when pair-fed rats were provided with food, they immediately engaged in feeding behaviors; Zn-deficient rats were anorexia despite high levels of NPY (14). This suggests that Zn-deficient rats are insensitive to increase in NPY (viz., a resistance to NPY might occur during Zn deficiency). Interestingly, when exogenous NPY was administered to Zn-deficient rats, it elicited increased food intake (13), suggesting that the hypothesis of NPY resistance in Zn deficiency is unreasonable and untenable. Subsequent findings give new insights on the Zn regulation of NPY. A recent report showed that although NPY was released from terminals in the PVN of the hypothalamus of food-restricted animals, this release was significantly impaired in Zn-deficient animals. Zn deficiency might therefore cause anorexia by inhibiting the release of NPY that is required for receptor activation (15,16). In conclusion, rather than disrupting NPY synthesis, processing, or receptor function as previous hypothesized, Zn deficiency appears to impair NPY release from the PVN. This is s significant step forward in our understanding of the Zn-dependent mechanisms that are responsible for feeding behavior and macronutrient selection. However, the mechanism of Zn regulation of food intake and its effect on NPY is complex and still needs further study. Obviously, the next step might start with exploring the role of Zn in the mechanisms that are responsible for the release of NPY from the PNV and possibly other terminals in the central nervous system. Cholecystokinin (CCK), another neuropeptide, is secreted into the blood from endocrine cells in the duodenum upon stimulation by partially digested food, especially certain proteins. CCK then acts through specific receptors to regulate gallbladder contractions and pancreatic secretion, to delay gastric emptying, and, through both peripheral and central mechanisms, to suppress food intake (17,18). CCK is metabolized by a Zndependent enzyme, aminopeptidase A (19), the activity of which might be reduced in Zn-deprived rats. In addition, Blanchard and Cousins (20) found that the relative concentration of intestinal CCK mRNA was increased in the Zn-deprived rat, giving a greater potential for CCK production. A sustained elevation of plasma CCK might lead to anorexia in the Zn-deficient rat. However, anorexia would reduce CCK release, and once it was cleared from the plasma, feeding would begin again. Biological Trace Element Research

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The fact that CCK is a satiety factor controlling food intake involves the question of whether it is a major factor in causing the anorexia associated with Zn deficiency. Some other peptides might be responsible for the loss of appetite in Zn-deficient rats. Galanin is a 29-amino-acid peptide that, like NPY, is widespread in the brain but with particularly high concentrations in the PVN. Injections of galanin into the PVN acutely stimulate food intake in rats. Reduced galanin peptide levels have been measured during Zn deficiency, which might have a role in decreased appetite (21). Little is known regarding other appetite-regulating factors such as calcitonin gene-related peptide (CGRP) during Zn deficiency. CGRP is generally considered a satiety factor, and its inhibitory feeding effect is stronger than that of CCK. Our previous studies showed that pituitary CGRP mRNA levels are upregulated during Zn deficiency by means of cDNA microarrays (3). It is now clear that monoaminergic neurotransmitters act in conjunction with neuropeptides and peripheral hormones to control physiologic states such as hunger, satiation, and satiety. Serotonin (5-HT), for example, has a suppressive effect on food intake and body weight (22). Injection of serotoninergic agents into the PVN, ventromedial nucleus (VMH), anterior hypothalamic area, and dorsomedial nucleus (DMN) decreases food intake. In addition to decreasing food intake, serotoninergic stimulation of the PVN enhances energy metabolism (23). It has been reported that CGRP might be coupled to the serotoninergic system to modulate food intake (24). Our previous study showed that serotonin mRNA levels were upregulated in rats fed the Zn-deficient diet (3). The relationships of hormones to food intake regulation in the Zndeficient rats have been studied. Leptin is a cytokine that is secreted by adipose tissue and is involved in appetite regulation and body weight maintenance via the regulation of hypothalamic factors (25). Numerous investigators have found an association among Zn, food intake, and leptin (26,27). Leptin is found to change NPY levels in the hypothalamus. High levels of leptin, presumably reflecting high or adequate levels of body fat, were found to downregulate the production of hypothalamic NPY through the receptor in the brain, which, in turn, suggested a decrease in appetite in response to the signal of adequate body energy reserves (28). During fasting, circulating leptin levels are reduced, whereas secretion, cellular peptide content, and mRNA levels for hypothalamic NPY are all increased, suggesting that circulating leptin levels and hypothalamic NPY are inversely related (29). Thus, the discovery of leptin has defined a “leptin–NPY–appetite” axia, which is one component of a complex appetite regulation system. Recent studies have investigated the regulation of leptin levels during Zn deficiency. Circulating leptin concentrations were reduced during Zn deficiency in the rat (30)and in humans (31). This reduction appears to be the result of a decrease in the amount of body fat present during Zn deficiency. Plasma Zn concentration was inversely related to plasma leptin in Biological Trace Element Research

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humans (32). However, work by Gaetke et al. (33) suggested that the low plasma leptin concentration in Zn-deprived rats was not caused by the deficiency per se but by anorexia that normally accompanies the deficiency. Mangian et al. (30) concluded that leptin was not a dominant factor in the development of Zn deficiency-induced anorexia. Although low plasma leptin concentrations are usually inversely correlated with hypothalamic NPY in the Zn-deprived rat, there is no clear association with food intake regulation in this model. Because the secretion of leptin from adipose tissue is reduced by Zn deficiency and insulin action is a major factor stimulating the synthesis and secretion of leptin (34), we hypothesize that a reduction in the effect of insulin resulting from Zn deficiency might be partially responsible for the reductions in leptin. Zn is required for normal insulin metabolism. It seems reasonable, therefore, that changes in body Zn status could affect the production, storage, and secretion of insulin (35–37). Some investigators showed that insulin action was reduced during Zn deficiency. The link between Zn deficiency and insulin resistance was found in many studies (21). Apparently, the relationship between Zn deficiency and insulin signal transduction pathways, or signal transduction in general, might ultimately help explain many of the physiological pathologies associated with Zn deficiency.

ZINC DEFICIENCY AND MACRONUTRIENT SELECTION Nutrient selection is altered during Zn deficiency in rats. When Zndeficient rats were given a choice among carbohydrate, fat, and protein, the intake of fat and protein remained constant, whereas carbohydrate intake fell significantly. In fact, 100% of the reduction in food intake could be explained by a reduction in carbohydrate intake. Refeeding with a Znadequate diet resulted in a reversal of carbohydrate intake within 1 d. During the first several days of refeeding, protein intakes appeared to be transiently increased (38). Hence, Zn not only regulates food intake but appears to regulate nutrient selection as well. There exist some inconsistent findings regarding Zn deficiency and macronutrient preference. Some investigators found that rats fed Zn-deficient diets selectively reduced carbohydrate consumption and increased fat and/or protein expenditure compared to the Zn-adequate rats (21,38–40). Another work suggested that Zn-deficient rats might reduce food intake in an attempt to prevent protein or amino acid accumulation. Reeves and O’Dell (41) found that rats given ad libitum access to Zn-deficient diets containing 5% or 20% protein consumed a greater amount of the low-protein diet. Subsequently, Reeves (42) reported that Zn-deficient rats reduced their selection of proteins from 12% to 8% of the diet. At the same time, they increased their intake of carbohydrates from 69% to 73% of the diet, but the intake of fat was not significantly affected. It is not immediately apparent why there are Biological Trace Element Research

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so many discrepant results; however, differences in the composition of the diets might explain the lack of consistent responses. Changes in food intake patterns might be explained by the fact that key enzymes required for nutrients metabolism might be lacking. Reductions of hydrolase activity are associated with reductions in the digestibility of dietary nutrients. It is possible, according to our previous research, that impaired carbohydrate digestion (reflected in amylase activity) and defective protein absorption (reflected in protease activity) might contribute to Zn deficiency, which consequently leads to shift in nutrient selection and reduces food intake. Food intake patterns correlate to both hypothalamic and metabolic factors. NPY not only increases total food intake but also seems to be associated with increased carbohydrate selection when rats are offered a choice among three diets with different concentrations of the macronutrients (43).

BRIEF SUMMARY Zinc plays a crucial role in the regulation of food intake, the NIH workshop has achieved a broad consensus on characterizing the role of Zn in the food intake that is considered as priority research areas regarding Zn in the future. Because so many peptides such as NPY, orexins, melanin-concentrating hormone (MCH), galanin, ghrelin, CCK, CGRP, cocaine- and amphetamine-regulated transcript (CART) and serotonin, and hormones like leptin and insulin are involved in appetite regulation, the biochemical mechanism for food intake depression during Zn deficiency is complicated and still unknown. Consequently, more comprehensive studies should be conducted to clarify this enigma. Possible avenues of exploration include examination of cellular secretion, signal transduction, peptide processing and releasing, and regulation of a large number of appetite-regulating neuropeptides during Zn deficiency. With recent reports describing changes in concentrations of neural and peripheral factors during Zn deficiency, it is anticipated that mechanisms causing the anorexia resulting from Zn deficiency might soon be understood at the cellular level, which has a potential impact on our fundamental understanding of the role of Zn in neurophysiology. Moreover, elucidating the mechanism for Zn regulation of food intake will facilitate obtaining an efficient target for treatment of anorexia nervosa and other disorders associated with Zn deficiency.

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