The Journal of Nutrition Vol. 128 No. 1 January 1998, pp. 136-142

Reduced Food Intake in Zinc Deficient Rats Is Normalized by Megestrol Acetate but not by Insulin-Like Growth Factor-I^1,2

J. D. Browning^*, R. S. MacDonald, W. H. Thornton, and B. L. O'Dell^{*, 3}

^* Department of Biochemistry and  Department of Food Science and Nutrition, University of Missouri, Columbia, MO 65211

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Zinc deficiency in rats results in impaired growth accompanied by decreased and cyclic food intake. These signs are associated with decreased plasma insulin-like growth factor-I (IGF-I), a major mediator of growth. The purpose of this study was to determine the relationship between decreased plasma IGF-I and the impairment of appetite and growth in zinc deficiency. Immature male rats were fed free choice a low zinc (<1 mg/kg) diet (Zn) or a zinc adequate (100 mg/kg) control diet (+Zn). Plasma IGF-I concentrations were normalized in zinc-deficient rats by the following two methods: osmotic pump infusion of IGF-I (2.4 mg/kg body weight daily) and oral administration (50 mg/kg body weight twice daily) of the synthetic progestin, megestrol acetate (MA). Infusion of IGF-I for 8 d sustained plasma IGF-I concentrations in zinc-deficient rats at control levels but had no effect on either food intake or growth rate. MA administration for 8 d maintained the plasma IGF-I of deficient rats and significantly increased food intake. The early aspects of cyclic food intake were eliminated, and, after a few days, food intake of deficient rats given MA was not different than that of controls. MA increased food intake and fat deposition regardless of zinc status, but it had no effect on the growth rate of deficient rats. MA significantly decreased body weight of controls, uncoupling energy intake and gain. The results suggest that reduced food intake precedes the decreased plasma IGF-I concentration and that IGF-I is not responsible for the decreased growth and food intake of zinc-deficient rats. The appetite and growth impairment of zinc-deficient rats may arise from disrupted function of IGF-I receptors in the brain and peripheral tissues, but not from low circulating levels of IGF-I.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Growth failure is a cardinal sign of zinc deficiency in young animals, and it is closely associated with reduced food intake. Although it is obvious that growth cannot occur without food consumption, it has been suggested that the failure to grow precedes and causes the loss of appetite (Chesters 1989). After a few days, rats fed a severely zinc-deficient diet fail to gain, and essentially all of the food consumed is used for maintenance. Forced-feeding fails to stimulate growth and actually causes zinc-deficient rats to become ill and die sooner than otherwise (Chesters and Quarterman 1970, Flanagan, 1984). These observations have stimulated considerable research into the mechanism by which appetite is impaired by zinc deficiency.

Because growth hormone (GH)⁴ plays a key role in growth stimulation, its deficiency has been suspected as the basis of growth failure in zinc deficiency. Root et al. (1979) observed a decrease in serum GH, but this has not been confirmed (Dorup et al. 1991, Roth and Kirchgessner 1994). However, the concentration of growth hormone receptor mRNA is reduced in livers of zinc-deficient rats (McNall et al. 1995a). Growth hormone stimulates the synthesis and release of insulin-like growth factor-I (IGF-I), and the IGF-I concentration in the serum of zinc-deficient rats is markedly decreased (Dorup et al. 1991, McNall et al. 1995a, Ninh et al. 1995, Oner et al. 1984, Roth and Kirchgessner 1994). Treatment of zinc-deficient rats with growth hormone did not affect growth rate or serum IGF-I levels (McNall et al. 1995b). Serum IGF-I concentration is decreased by restriction of food intake as well as by zinc deficiency (McNall et al. 1995a, Ninh et al. 1995) so that it is unclear whether zinc deficiency has a direct or indirect effect on serum IGF-I.

View larger version (20K): [in this window] [in a new window]   Fig 3. Cumulative body weight gain of rats fed diets low or adequate in zinc and treated orally with megestrol acetate (MA) or vehicle for 8 d (Experiment 2). Rats were fed for 8 d either the low zinc (Zn) or control (+Zn) diets free choice and were given megestrol acetate (+MA; 50 mg/kg BW) or corn oil vehicle (MA) orally twice daily. ANOVA showed significant effects of MA and zinc after d 3 and MA × Zn interaction after d 4; n = 5; pooled SEM: d 4, 1.8; d 5, 2.0; d 6, 1.9; d 7, 2.0; d 8, 2.6. Significant (P < 0.05) daily differences as a result of MA treatment of controls (+Zn) are indicated by * and of Zn rats by .

The synthetic progestin, megestrol acetate (MA), stimulates food intake in rats and increases the concentration of neuropeptide Y in the hypothalamus (McCarthy et al. 1994). Treatment of human cancer patients with MA stimulates appetite and results in significantly elevated concentrations of plasma IGF-I (Frost et al. 1996). The mechanism of the MA effect is unclear, but MA treatment of neurones isolated from the rat hypothalamus inhibits a portion of the voltage-activated calcium current, suggesting that appetite enhancement relates to calcium channel function (Costa et al. 1995).

Although growth failure may initiate the loss of appetite, it is possible that both growth and appetite fail because the signal transduction required for both is impaired by zinc deficiency. The objective of this study was to determine if increasing the plasma concentration of IGF-I stimulates food intake and/or growth rate of zinc-deficient rats. A corollary objective was to determine whether zinc status or food intake is the primary regulator of plasma IGF-I concentration. The circulating concentration of IGF-I was increased experimentally by two methods, osmotic pump infusion of IGF-I and administration of MA. It appears that zinc deficiency impairs food intake and growth by separate mechanisms and that administration of MA by-passes the food intake defect.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diets.  Immature male rats (5-6 wk of age; 120-160 g) of Wistar origin and produced in the departmental colony were housed individually in suspended stainless steel cages in a room maintained at 22°C with a 12-h light:dark cycle (lights on 0700-1900 h). The rats were fed a low zinc diet (Zn; <1 mg Zn/kg) based on EDTA-treated soy protein (O'Dell et al. 1983) or a control diet (+Zn), which was the basal diet supplemented with 100 mg Zn/kg. Food and deionized water were supplied free choice. Rats were weighed and their food consumption measured daily. Four experiments were performed according to the protocols described below. The experimental protocols were approved by the University of Missouri, Columbia, Animal Care and Use Committee.

Implantation of osmotic pumps.  Osmotic pumps designed to deliver 10.8 µL/d were obtained from Alza (Palo Alto, CA). They were loaded with either a saline solution of rhIGF-I (Genentech, San Francisco, CA) or vehicle and inserted intraperitoneally at the abdominal midline while the rats were under pentobarbital anesthesia. The pumps were recovered at the end of the experiment and weighed to verify the quantity of IGF-I delivered. More than 90% of the IGF-I was displaced from all pumps during the experiment.

Determination of plasma zinc and IGF-I.  At the end of the experiments, rats were anesthetized with pentobarbital and blood collected in heparinized syringes from the exposed heart. Plasma was prepared by centrifugation (1000 g for 15 min) and zinc concentration determined by atomic absorption spectrophotometry. Plasma was extracted with acid-ethanol to remove binding proteins; IGF-I concentration of the extracts was determined by RIA by using an anti-human IGF-I antibody (Nichols Institute Diagnostics, San Juan Capistrano, CA) and ¹²⁵I-IGF-I (Amersham, Arlington Heights, IL) according to Clemmons et al. (1979).

Determination of body composition.  Body composition was estimated by measurement of total body electrical conductivity (TOBEC) using a Small Animal Body Composition Analyzer (Em-scan, Springfield, IL). Rats were anesthetized with pentobarbital (50 mg/kg) and electrical conductivity and crown-rump length measured in triplicate (Bracco et al. 1983, Cunningham et al. 1986). Calculation of fat-free mass (FFM) was made according to the equation: FFM (g) = 1.82 + 2.177 (E × CR)^1/2, where E is the conductivity reading and CR the crown-rump length in centimeters. The proportion of body fat was calculated as the difference between body weight (BW) and FFM divided by BW.

Experiment 1: IGF-I infusion.  Twenty rats were assigned to four groups of five so as to achieve equal mean BW. The four groups comprised a 2 × 2 factorial design, with two levels of IGF-I [0 or 2.4 mg/(kg BW·d)] and two levels of dietary zinc (0 and 100 mg/kg diet). All rats were acclimated to the zinc-supplemented diet for 7 d, at which time osmotic pumps were implanted under pentobarbital anesthesia. Two groups (10 rats) received pumps that contained IGF-I, and the remaining two groups received pumps containing vehicle. Rats were allowed to recover until their food intake returned to presurgery levels (3 d), and then groups were started on dietary treatments to complete the factorial design. Food intake and body weight were recorded daily. The experiment was terminated on d 8 after initiation of dietary treatment.

View larger version (20K): [in this window] [in a new window]   Fig 1. Mean cumulative body weight gain of rats fed diets low or adequate in zinc and infused with insulin-like growth factor-I (IGF) or vehicle (Experiment 1). Rats were fed either the low zinc diet (Zn) or control diet (+Zn) free choice for 8 d and were infused with IGF-I (+IGF; 2.4 mg/kg BW daily) or vehicle (IGF) via osmotic minipumps. ANOVA showed significant effects of zinc after d 4; n = 5; pooled SEM: d 5, 1.9; d 6, 1.7; d 7, 2.0; d 8, 1.8. *Indicates significant (P < 0.05) differences in controls due to IGF infusion.

Experiment 2: megestrol acetate (MA) treatment for 8 d.  Twenty rats were assigned to four groups as described above, in a 2 × 2 factorial design with two levels of oral MA [0 and 100 mg/(kg BW·d), in corn oil] and two levels of dietary zinc. All rats were fed the zinc-supplemented diet for 5 d and given an oral dose of corn oil, 1.0 mL/kg BW, each morning for the purpose of acclimation. Groups were then started on dietary treatments and received either MA (50 mg/kg BW, morning and evening) or corn oil. Food intake and body weight were recorded daily. The experiment was terminated on d 8 of dietary treatment.

Experiment 3: MA treatment for 6 d.  This experiment comprised four groups of five rats each, treated as in Experiment 2, except that the experiment was terminated on d 6 of dietary treatment. The experimental period was shortened to terminate at the time point when the food intake of the deficient rats was predicted to be at its lowest.

Experiment 4: MA and body composition.  In this experiment there were four groups of four rats each, treated as in Experiment 2, except that the period of dietary treatment was extended to 20 d. All rats were acclimated to the control diet for 5 d. On d 5, the first day of dietary treatment, one of each dietary treatment group received MA as in Experiment 2 and one received corn oil only. After 8 d, all rats were anesthetized and scanned (TOBEC) for body composition. At this point, the MA treatment was discontinued, and after 12 d the rats were scanned again for body composition. Food intake and body weight were recorded daily.

Statistical treatment of data.  Body weight gain and food intake data for all experiments were analyzed by day of treatment as 2 × 2 factorial designs, i.e., dietary zinc × drug administration (IGF-I for Experiment 1, MA for Experiments 2-4), by using the GLM procedure of SAS (Statistical Analysis System, SAS Institute, Cary, NC). Group least-square means were compared by using the LSMEANS component of GLM. Plasma zinc, plasma IGF-I, total gain and food intake for d 3-6 of Experiment 4 (Fig. 7), and body composition data (Fig. 8) were analyzed as 2 × 2 factorial designs using GLM and LSMEANS.

View larger version (53K): [in this window] [in a new window]   Fig 7. Daily food intake and weight gain for the 4-d period, d 3-6, of rats fed diets low or adequate in zinc and treated orally with megestrol acetate (MA) or vehicle for 6 d (Experiment 3). ANOVA results, food intake: zinc, P < 0.0001; MA, P < 0.0001; zinc × MA, P = 0.023; gain: zinc, P < 0.0001; MA, P = 0.0006; zinc × MA, P = 0.007. Means and SEM indicated by bars and extensions; n = 5. Bars with different letter designations are significantly different at P < 0.05.

View larger version (57K): [in this window] [in a new window]   Fig 8. Mean percentage body fat of rats fed diets low or adequate in zinc and treated orally with megestrol acetate (MA) or vehicle. Body composition was estimated after 8 d of megestrol acetate treatment and 12 d after withdrawal of megestrol acetate (Experiment 4). Values are means ± SEM, n = 4. Dietary zinc and megestrol acetate treatments were the same as described in Figure 3. ANOVA of 8-d data: zinc, P = 0.007; MA, P = 0.014; zinc × MA 0.554; after 12-d withdrawal: zinc, P = 0.007; MA, P = 0.458; zinc × MA, 0.508.

	RESULTS

Abstract Introduction Methods Results Discussion References

Although Experiments 1-3 were relatively short term, the plasma zinc concentrations of rats fed the low zinc diet were extremely low (Table 1). IGF-I infusion had no effect on the plasma zinc concentration of deficient or control rats. MA had no consistent effect on plasma zinc.

Both IGF-I infusion and administration of MA for 8 d maintained the circulating levels of IGF-I in zinc-deficient rats at control levels (Table 2). In Experiment 3, when rats were sampled on d 6, IGF-I levels were not lower in rats fed the low zinc diet, nor were they affected by MA treatment in either dietary group.

In Experiment 1, rats fed the low zinc diet failed to gain weight after d 4 (Fig. 1). The mean food intake, based on BW, began to drop on d 3 and reached a nadir on d 5 (Fig. 2). Even though IGF-I infusion into deficient rats maintained the plasma concentration at control levels, it did not affect their food intake or weight gain. IGF-I infusion had no effect on food intake of control rats but resulted in a significant increase in weight gain.

View larger version (21K): [in this window] [in a new window]   Fig 2. Daily food intake per 100 g body weight of rats fed diets low or adequate in zinc and infused with insulin-like growth factor-I (IGF) or vehicle (Experiment 1). Rats were fed either the low zinc diet (Zn) or control diet (+Zn) free choice for 8 d and were infused with IGF-I (+IGF; 2.4 mg/kg BW daily) or vehicle (IGF) via osmotic minipumps. ANOVA showed a significant effect of zinc after d 2; n = 5; pooled SEM: d 3, 0.68; d 4, 0.82; d 5, 0.44; d 6, 0.45; d 7, 0.39; d 8, 0.47.

Oral administration of MA (50 mg/kg body weight twice daily) for 8 d dramatically decreased body weight gain of the controls but had a smaller effect in deficient rats (Fig. 3). During the last 3 d of the trial, both dietary groups that received MA gained at approximately the same rate and at a rate similar to the untreated deficient group. MA administration increased food intake of rats fed the low zinc diet, largely by eliminating the typical cyclic intake. Intake of the untreated deficient group reached a low point on d 5 (Fig. 4). Food intakes of zinc-deficient and control rats given MA were essentially the same; on d 8, these intakes were not different from those of untreated controls.

View larger version (20K): [in this window] [in a new window]   Fig 4. Mean daily food intake per 100 g body weight of rats fed diets low or adequate in zinc and treated orally with megestrol acetate (MA) or vehicle for 8 d (Experiment 2). Rats were fed for 8 d either the low zinc (Zn) or control (+Zn) diets free choice and were given MA (+MA; 50 mg/kg BW) or corn oil vehicle (MA) orally twice daily. ANOVA showed significant effects of zinc and MA after d 3; n = 5; pooled SEM: d 4, 0.24; d 5, 0.51; d 6, 0.78; d 7, 0.40; d 8, 0.52. Significant (P < 0.05) differences due to MA treatment of controls (+Zn) are indicated by * and of Zn rats by .

Because MA treatment of the zinc-deficient rats largely eliminated the depressed appetite, including cyclic food intake, and maintained plasma IGF-I at normal levels, it was of interest to determine the time point in the cycle at which the effect on plasma IGF-I occurred. Experiment 3, a 6-d experiment, was designed to determine whether MA maintains normal plasma IGF-I levels at the time in the cycle when food intake in zinc-deficient rats is minimal. This occurred on d 5 of Experiments 1 and 2, but the magnitude of cyclic change varies among groups because the intake of all rats in a group may not be synchronous. In Experiment 3, weight gain was significantly less in rats fed the low zinc diet after d 4 (Fig. 5). The food intake nadir of the rats fed the low zinc diet likely occurred on d 6 (Fig. 6) at which time plasma was sampled for zinc and IGF-I determination. In contrast to the 8-d experiment (Experiment 2), the plasma IGF-I concentration was not depressed in the zinc-deficient group, and in fact it did not differ among the treatments (Table 2). As in the previous experiment, there was no depression of food intake in the deficient rats treated with MA. A summary of mean food intakes and weight gains over a 4-d period, d 3-6, is presented in Fig. 7. Relative to body weight, food intake was depressed by zinc deficiency and was increased in both groups by MA. However, zinc-deficient rats given MA did not gain more weight than those without MA, and controls given MA gained less than those without MA. The gain in body weight per unit of food consumed (g/g) in the zinc-deficient groups was unchanged by MA treatment (0.41 ± 0.04 vs. 0.37 ± 0.04), but that in the control groups decreased from 0.72 ± 0.09 to 0.49 ± 0.03 (P < 0.01).

View larger version (20K): [in this window] [in a new window]   Fig 5. Cumulative body weight gain of rats fed diets low or adequate in zinc and treated orally with megestrol acetate (MA) or vehicle for 6 d (Experiment 3). Protocol as for Experiment 2 except treatments were for 6 d. ANOVA showed a significant effect of MA after d 1 and of zinc after d 4; n = 5; pooled SEM: d 2, 4.6; d 3, 4.8; d 4, 5.2; d 5, 5.5; d 6, 5.5. Significant (P < 0.05) daily differences due to MA treatment of controls (+Zn) are indicated by * and of Zn rats by .

View larger version (20K): [in this window] [in a new window]   Fig 6. Daily food intake per 100 g body weight for rats fed diets low or adequate in zinc and treated orally with megestrol acetate (MA) or vehicle for 6 d (Experiment 3). ANOVA showed a significant effect of MA after d 3 and for zinc after d 5; n = 5; pooled SEM: d 4, 0.37; d 5, 0.50; d 6, 0.42. Significant (P < 0.05) daily differences due to MA treatment of controls (+Zn) are indicated by * and of Zn rats by .

The fact that MA-treated rats fed low zinc consumed more food yet gained the same weight as untreated deficient rats, and that controls given MA gained less weight than those without MA, raised the question as to disposition of the energy consumed. As shown in Figure 8, both zinc-deficient and control rats given MA had a higher percentage of body fat at d 8. Twelve days after discontinuing MA, the percentages of fat in the deficient groups were the same, but both deficient groups had less fat than controls.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The results of the short-term experiments described here confirm the initial portion of the cyclic feeding pattern observed in zinc-deficient rats exhibiting decreased appetite (Chesters and Quarterman 1970, O'Dell and Reeves 1989). They also confirm that zinc deficiency for a period of 8 d or longer decreases the plasma concentration of IGF-I. Although consumption of the low zinc diet for 6 d induced zinc deficiency as indicated by extremely low plasma zinc, 3.5 µmol/L, and failure of growth after d 4, there was no decrease in plasma IGF-I. It is not entirely clear why the 6-d depletion failed to lower IGF-I, but it likely relates to the short period of low food intake rather than to zinc status per se. In this regard, it is notable that the effect of zinc deficiency on hypothalamic neuropeptide Y (NPY) mRNA concentrations relates directly to food intake and that a sustained decrease in food intake is required to induce the effect of zinc deprivation (Selvais et al. 1997). In that study, there was no difference in hypothalamic NPY peptide or mRNA between the peak and trough periods of food intake. The effect of zinc deficiency was reversed by zinc repletion for 4 d. In the 6-d experiment of this study, there was no zinc effect on food intake until d 5, and blood was sampled for IGF-I analysis on d 6.

In rats fed the low zinc diet for 8 d, both infusion of IGF-I and oral administration of MA resulted in plasma IGF-I concentrations comparable to control levels. However, maintenance of plasma IGF-I levels by infusion did not restore food intake or growth rate of deficient rats, showing that these deficiency signs are not direct reflections of plasma IGF-I concentration. As suggested above, the low plasma IGF-I concentration in zinc deficiency likely results from low food intake rather than from zinc deficiency per se. This does not preclude the possibility of a direct effect of zinc status on IGF-I function, e.g., impairment of signal transduction at the IGF-I receptor.

Administration of MA increased the appetite of zinc-deficient rats and largely eliminated the typical cyclic feeding behavior, but it did not stimulate body weight gain. Although MA had a minimal effect on the growth rate of deficient rats, it uncoupled the physiologic processes of food intake and body weight gain in controls. The increase in appetite in response to MA may relate to the fact that MA administration increases NPY concentration in the rat hypothalamus (McCarthy et al. 1994). NPY is the most potent known stimulator of appetite (Dryden et al. 1994) and may be involved in the reduced food intake observed in zinc deficiency. In a recent study, Selvais et al. (1997) observed an increase in hypothalamic NPY mRNA in zinc-deprived rats but no change in NPY protein. These changes were accompanied by a decrease in the hypothalamic concentration of galanin mRNA. Similar concentrations of both messengers were found in pair-fed controls so that the effect was primarily the result of reduced food intake. Although the mechanism by which MA stimulates appetite in zinc-deficient rats requires clarification, it is not simply a matter of increasing plasma IGF-I. MA appears to stimulate appetite by a mechanism that by-passes the lesion, leading to reduced food intake in deficient rats. Regardless of the mechanism involved, it is of considerable interest that MA uncouples the long observed relationship between zinc deficiency and loss of appetite in rats.

MA suppressed body weight gain in control rats while increasing their food intake. This occurred, at least in part, because more of the ingested energy was stored as fat, as observed here. Impaired energy absorption or increased energy expenditure, including increased basal metabolism, could also be involved. Others have observed increased fat storage in response to MA administration (Gray and Wade 1981, McCarthy et al. 1994). The change in distribution of stored energy may relate to the fact that MA increases NPY in the hypothalamus (McCarthy et al. 1994), inasmuch as NPY increases energy balance in the form of body fat (Bing et al. 1996). The effect of MA on appetite and energy storage is likely mediated by way of NPY, stimulating food intake in zinc-deficient rats without promoting true growth. The reduction of gain in control rats given MA may relate to the fact that MA increases plasma levels of IGF-I binding protein 3 (IGF-BP3), a condition that would reduce the availability of IGF-I and thereby impair growth (Frost et al. 1996).

MA may act by impairment of calcium uptake of critical brain cells inasmuch as it decreases a fraction of the calcium channel current in neurons of the ventromedial hypothalamic nucleus (Costa et al. 1995). Calcium plays a critical second messenger role in the growth process. The mitogenic activity of IGF-I depends on the uptake of calcium (Kojima et al. 1988). If MA inhibits calcium channel function in the peripheral cells as it does in the hypothalamus, such cells would be less responsive to IGF-I and would be growth inhibited.

Plasma IGF-I concentration and appetite in zinc-deficient rats are restored to normal by MA (this paper and Frost et al. 1996), but growth is not stimulated. Because signal transduction at the IGF-I receptor requires calcium uptake, and zinc deficiency has been shown to impair calcium uptake in several tissues (Browning and O'Dell 1995, Xia and O'Dell 1995), it is possible that the IGF-I resistance observed in zinc deficiency is due to disruption of an IGF-I gated calcium channel. Because IGF-I may be involved in the release of NPY (Barnea and Cho, 1993), impaired IGF-I receptor function in zinc deficiency could lead to both decreased NPY release and impaired appetite. A similar IGF-I resistance in the peripheral tissues would cause decreased growth. Both MA and zinc deficiency inhibit growth, perhaps by the same or a similar mechanism, but they have opposite effects on appetite. Although MA and zinc deficiency may regulate appetite by affecting the same endpoint, MA acts later in the pathway, bypassing the effect of zinc deficiency.

	FOOTNOTES

Manuscript received 31 March 1997. Initial reviews completed 20 May 1997. Revision accepted 15 September 1997.

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