Prenatal and perinatal zinc restriction: effects on ... - Wiley Online Library

761

Exp Physiol 94.6 pp 761–769

Experimental Physiology – Research Paper

Prenatal and perinatal zinc restriction: effects on body composition, glucose tolerance and insulin response in rat offspring Inagadapa J. N. Padmavathi1 , Yedla Durga Kishore1 , Lagishetty Venu1 , Manisha Ganeshan1 , Nemani Harishankar2 , N. V. Giridharan2 and Manchala Raghunath1 1

Division of Endocrinology and Metabolism and 2 National Centre for Laboratory Animal Sciences, National Institute of Nutrition, Hyderabad – 500 007, India

Maternal undernutrition increases the risk of adult chronic diseases, such as obesity and type 2 diabetes. This study evaluated the effect of maternal zinc restriction in predisposing the offspring to adiposity and altered insulin response in later life. Seventy-day-old female Wistar/NIN rats received a control (ZnC) or zinc-restricted (ZnR) diet for 2 weeks. Following mating with control males, a subgroup of the ZnR dams were rehabilitated with ZnC diet from parturition. Half the offspring born to the remaining ZnR dams were weaned onto the ZnC diet and the other half continued on the ZnR diet throughout their life. Body composition, glucose tolerance, insulin response and plasma lipid profile were assessed in male and female offspring at 3 and 6 months of age. The ZnR offspring weighed less than control offspring at birth and weaning and continued so until 6 months of age. Rehabilitation regimens corrected the body weights of male but not female offspring. Maternal zinc restriction increased the percentage of body fat and decreased lean mass, fat-free mass and fasting plasma insulin levels in both male and female offspring at 6 months of age. Also, glucose-induced insulin secretion was decreased in female but not male offspring. Despite the differences in fasting insulin and the area under the curve for insulin, the fasting glucose and the area under the curve for glucose were in general comparable among offspring of different groups. Rehabilitation from parturition or weaning partly corrected the changes in the percentage of body fat but had no such effect on other parameters. Changes in plasma lipid profile were inconsistent among the offspring of different groups. Thus chronic maternal zinc restriction altered the body composition and impaired the glucose-induced insulin secretion in the offspring. (Received 13 November 2008; accepted after revision 12 February 2009; first published online 27 February 2009) Corresponding author M. Raghunath: Division of Endocrinology and Metabolism, National Institute of Nutrition, Jamai Osmania P.O., Hyderabad – 500 007, India. Email: [email protected]

An adverse intra-uterine environment is associated with long-term metabolic consequences, in particular obesity, insulin resistance and type 2 diabetes (Barker, 2004, 2006). Data from epidemiological and animal studies have given rise to the concept of developmental programming, which proposes that challenges during an organism’s intrauterine development evoke a persistent physiological response in adult life (Ozanne et al. 1996; Garofano

I. J. N. Padmavathi and Y. Durga Kishore contributed equally to this work. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

et al. 1997; Nyirenda et al. 2001; Simmons et al. 2001; Reusens & Remacle, 2001). Although the effect of maternal macronutrients on adult chronic diseases in the offspring has been the topic of many studies, the effect of micronutrients (especially trace elements) is poorly understood. Using maternal micronutrient restriction models, we have previously shown that chronic 50% mineral or vitamin restriction in Wistar/NIN (WNIN) rat dams increased the percentage of body fat and decreased glucose-stimulated insulin secretion in their offspring (Venu et al. 2004a,b, 2007). We also reported recently that maternal and perinatal magnesium restriction increased the percentage of body fat, decreased the percentage of DOI: 10.1113/expphysiol.2008.045856

762

I. J. N. Padmavathi and others

lean body mass (LBM) and fat-free mass (FFM) and appeared to predispose the offspring to insulin resistance and glucose intolerance (Venu et al. 2005, 2008). Zinc is an essential trace mineral directly involved in the physiology and action of insulin. Insulin is stored as Insulin-zinc crystals in the β-cells of the pancreas (Taylor, 2005). Abnormal zinc metabolism may play a role in the pathogenesis of diabetes and some of its complications (Chausmer, 1998). Zinc performs a range of functions in the body and is a cofactor for the synthesis of a number of enzymes, DNA and RNA (MacDonald, 2000). Zinc deficiency is associated with complications of pregnancy and delivery, as well as growth retardation and congenital abnormalities in the fetus (Shah & Sachdev, 2006). During pregnancy, there is a progressive decline in circulating zinc, possibly due to decreased zinc binding and increased transfer from the mother to the fetus (Caulfield et al. 1998; King, 2000). The adverse outcomes of zinc deficiency are well documented in experimental animals but the effects, if any, of maternal zinc restriction in the aetiology of insulin resistance syndrome in the offspring have not yet been explored. Despite difficulties in measuring zinc status validly, it has been estimated using the probability approach that about 82% of all pregnant women worldwide are likely to suffer zinc deficiency (Caulfield et al. 1998). In view of the foregone literature, the effect of dietary zinc restriction during pregnancy and lactation in female WNIN rats was assessed on the body composition, glucose tolerance and insulin secretion of the offspring. Gender specificity of the effects, if any, and the dependence of the effects on the stage of development at which zinc restriction occurs, viz. pregnancy alone or also during lactation, were also evaluated.

Figure 1. Schematic representation of the feeding protocol of different groups of mothers and the offspring Abbreviations: ZnC, control diet throughout; ZnR, zinc-restricted diet throughout; ZnRP, ZnR mothers rehabilitated on control diet from parturition and offspring of such mothers on control diet from weaning; ZnRW, zinc-restricted offspring weaned onto control diet. n = 8 male/female offspring in each group from weaning.


Methods Animals, diet and experimental design

The experiment was carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996) and with the approval of the ‘Institute’s ethical committee on animal experiments’ at National Institute of Nutrition, Hyderabad, India. Seventy-day-old female WNIN rats (n = 24) were obtained from National Centre for Laboratory Animal Sciences, National Institute of Nutrition, Hyderabad, India. They were divided into two groups of six and 18, housed individually in polypropylene cages (dimensions: length, 39 cm; width, 28 cm; and depth, 14 cm) with rice husk bedding (autoclaved) of 20 mm thickness, in standard lighting conditions (12 h–12 h light–dark cycle). Temperature and relative humidity were kept constant at 22 ± 2◦ C and 55 ± 10%, respectively. For 2 weeks, the group of 18 rats was fed an egg albumin-based 20% protein diet containing mineral mixture without zinc (zinc, 10 mg kg−1 diet; ZnR) and the other group (n = 6) was maintained on a control diet (zinc, 35 mg kg−1 diet; ZnC). After ensuring that ZnR rats were zinc deficient as indicated by their plasma zinc levels, they were mated with breeding stock males (two females to one male) and maintained on their respective diets throughout gestation. At parturition, six dams from the restricted group were shifted to control diet (ZnRP), while the remaining ZnR dams continued on restricted diet till weaning. During lactation, a uniform litter size was maintained in all groups from postnatal day 3 by adjusting the number of offspring per litter to eight (equal number of male and female pups for most mothers). The excess pups from each litter were killed by decapitation. At weaning (postnatal day 22) half the offspring born to the remaining ZnR dams were weaned onto control diet (ZnRW) and the other half continued on restricted diet (ZnR). Offspring born to control (ZnC) dams were maintained throughout on ZnC diet. From weaning, the pups were group housed (four females or four males per cage), with each cage housing two pairs of siblings obtained from two different litters. From postnatal day 60, the pairs of littermates were separated and each pair housed in a cage. From weaning, eight male and eight female pups (derived from four or five mothers of the corresponding group) were maintained in each group until postnatal day 180, pups in excess of the numbers kept for weaning were sacrificed. Experimental diets (deionized water and food) were available ad libitum to the dams and their offspring. Their health status and diet intake were monitored daily, and the animals were weighed weekly. All test animals were killed on postnatal day 180 by CO 2 inhalation. The dams were killed on postnatal day 29, i.e. one week after their pups were weaned. The feeding protocol used in this experiment is depicted schematically in Fig. 1. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

Zinc and developmental programming


763

Table 1. Diet intake, physical and biochemical parameters in WNIN female rats fed control and zinc-restricted diets for 2 weeks before mating Parameter

Control diet (n = 6)

Zinc-restricted diet (n = 18)

11.7 ± 0.24 180 ± 4.66 13.2 ± 0.85 1.26 ± 0.18 4.27 ± 0.21 201 ± 29.7 11.81 ± 1.24 1.33 ± 0.15 0.59 ± 0.009

9.89 ± 0.55∗ 165 ± 2.12∗ 12.8 ± 0.77 1.09 ± 0.24 3.97 ± 0.10 102 ± 32.2 11.56 ± 1.11 1.34 ± 0.26 0.47 ± 0.15

day−1 )

Food intake (g Body weight (g) Haemoglobin (g dl−1 ) Plasma zinc (μg ml−1 ) Fasting glucose (mmol l−1 ) Fasting insulin (pmol l−1 ) Glucose AUC (mmol l−1 h−1 ) Total cholesterol (mmol l−1 ) Triglycerides (mmol l−1 )

Values are means ± S.E.M. ∗ P < 0.05 by Student’s unpaired t test.

Body composition of the offspring

Statistical analysis

Body composition of the offspring was determined on postnatal days 90 and 180 using TOBEC (Total Body Electrical Conductivity) small animal body composition analysis system (model SA-3000 multi-detector, EMSCAN Inc., Springfield, IL, USA), as described by us previously (Venu et al. 2004a,b, 2005). The body composition parameters, lean body mass (LBM), total body fat percentage and fat-free mass (FFM) were obtained mathematically according to the methods of Morbach & Brans (1992).

All values are presented as means ± S.E.M. Differences between the control and restricted groups (maternal and neonatal data) were analysed using Student’s unpaired t test. Data from the offspring after weaning were analysed by one-way analysis of variance (ANOVA) followed by the post hoc least significance difference method. Wherever heterogeneity of variance was observed, differences between groups were tested by non-parametric Mann– Whitney U test. Differences were considered significant if P < 0.05.

Glucose tolerance test. An

Results

intraperitoneal glucose tolerance test was performed in six offspring of each of the four groups on postnatal days 90 and 180. After an overnight fast, a blood sample was collected from the supra-orbital sinus under light ether anaesthesia. The animals were then administered glucose (250 g l−1 ) intraperitoneally as a bolus, at a dose of 1 g (kg body weight)−1 , and blood samples were collected from the tail vein at 15, 30, 60 and 120 min under light ether anaesthesia, for the determination of plasma glucose and insulin. Glucose and insulin responses during the glucose tolerance test were computed from the total area under the glucose and insulin curves, respectively, using the trapezoidal method (Matthews et al. 1990). Biochemical analyses in offspring. Plasma zinc concentration was measured by atomic absorption spectroscopy. Total cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides were measured in fasting plasma samples using kits from Biosystems (Barcelona, Spain), whereas free fatty acids were measured using an enzymatic kit (Randox Laboratories Ltd, Crumlin, County Antrim, UK). Glucose and insulin were determined in different plasma samples (fasting and at different time points during the glucose tolerance test) using a kit from Biosystems and a radioimmunoassay kit from the Board of Radiation and isotope Technology (Mumbai, India), respectively. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

Effects in WNIN female rats (mothers to be)

Dietary zinc restriction for 2 weeks significantly decreased (P < 0.05) the diet intake of WNIN female rats (Table 1), but the decrease in plasma zinc levels in ZnR rats was not statistically significant, indicating moderate zinc deficiency. The fasting plasma glucose and lipid profile were comparable between ZnC and ZnR rats before mating (Table 1). Although fasting plasma insulin levels were lower in ZnR than ZnC rats, the difference was not statistically significant. Weight gain during pregnancy was significantly lower in ZnR than in ZnC dams (P < 0.001). Although there were significant increases (P < 0.001) in the rate of abortion and stillbirths in ZnR compared with ZnC dams, the litter size was reduced only slightly (Table 2). Parameters in the offspring Growth characteristics. Body weight at birth and weaning were significantly (P < 0.001) lower in ZnR than in ZnC offspring of both sexes (Table 2). The male ZnR offspring continued to weigh less until postnatal day 180, but ZnRP and ZnRW offspring caught up with control offspring as early as postnatal day 90 and continued so thereafter (Table 3). Female ZnR offspring also weighed significantly less than ZnC offspring on postnatal day 180, and neither

764



Table 2. Reproductive performance of WNIN female rats fed control or zinc-restricted diets for 2 weeks before mating Parameter Percentage conceived Weight gain during pregnancy (g) Percentage aborted Litter size Still births (%) Mean birth weight (g) Deaths of offspring during lactation (%) Weaning weight (g)

Control diet (n = 6)

Zinc-restricted diet (n = 18)

100% 86.62 ± 9.25 Nil 6–10 1 5.72 ± 0.14 Nil 33.5 ± 0.42

100% 55.15 ± 4.1∗∗ 16.6%∗∗∗ 2–9 16.6∗∗∗ 4.59 ± 0.16∗∗∗ 10 19.91 ± 1.12∗∗∗ (ZnRP 30.85 ± 0.91∗ )

Values are mean ± S.E.M. ∗ P < 0.05 and ∗∗∗ P < 0.001 by Student’s unpaired t test.

Table 3. Body weights (in grams) of different groups of offspring at 3 and 6 months of age Sex

Age (months)

ZnC

ZnR

ZnRP

ZnRW

Male offspring

3 6

5.56a

259 ± 323 ± 3.20a

7.53b

236 ± 294 ± 12.6b

6.48a

249 ± 318 ± 10.0a

261 ± 7.23a 307 ± 3.37a

Female offspring

3 6

182 ± 3.65 204 ± 3.28a

176 ± 2.94 183 ± 3.12b

169 ± 4.90 185 ± 5.17b

182 ± 7.40 188 ± 3.69b

Values are means ± S.E.M. (n = 6). Means without a common letter are significantly different at P < 0.05 by ANOVA/post hoc least significance difference.

of the rehabilitation regimens corrected the body weight of female ZnR offspring by postnatal day 180 (Table 3). Body composition of the offspring. Maternal zinc restriction significantly increased (P < 0.05) the percentage of body fat in the offspring of both sexes at 180 days of age (Fig. 2A and D). While both the rehabilitation regimens corrected the changes in body fat percentage in male offspring, in females only ZnRP but not ZnRW corrected the change. Though LBM and FFM were comparable in general among the four groups at 90 days, they were significantly decreased (P < 0.05) in ZnR offspring of both sexes on postnatal day 180 (Fig. 2B, C, E and F), and rehabilitation in general did not correct the changes in LBM and FFM (Fig. 2). Fasting glucose and insulin levels. On postnatal day 90, fasting plasma insulin levels were comparable among the male ZnC, ZnR and ZnRP offspring, while ZnRW appeared to have decreased levels. On postnatal day 180, male ZnR offspring had significantly lower fasting plasma insulin than ZnC and, interestingly, both ZnRP and ZnRW did not correct the change (Fig. 3B). Not withstanding these differences in the fasting plasma insulin levels, there were no significant differences among the male offspring of different groups in their fasting plasma glucose (Fig. 3A) on both postnatal day 90 and postnatal day 180. Similar to the male offspring, fasting plasma insulin levels were comparable among female offspring of different groups on postnatal day 90, while on day 180 the ZnR female offspring had significantly lower levels

than ZnC. Also, both ZnRP and ZnRW did not correct the change (Fig. 3D). Here again, fasting plasma glucose levels were in general comparable among the offspring of four groups at both 3 and 6 months of age (except that ZnRP had lower levels on postnatal day 90 but not 180; Fig. 3C). Glucose tolerance and insulin response. Impairment, if any, in glucose tolerance was assessed by an intraperitoneal glucose tolerance test in the offspring. On postnatal days 90 and 180, there were no significant differences among the male offspring of different groups in the area under the curve (AUC) for glucose or insulin (Fig. 4A and B). Although the AUC for insulin was comparable among the female offspring on postnatal day 90, on postnatal day 180 the ZnR had significantly lower AUC for insulin than ZnC, and both the rehabilitation regimens appeared unable to mitigate the change. Despite these changes in AUC for insulin, AUC for glucose was comparable among the female offspring at both the time points studied (Fig. 4C and D). Plasma lipid profile. Plasma total cholesterol levels were

significantly lower (P < 0.05) in ZnR offspring of both sexes on postnatal day 90 but not on postnatal day 180. Only ZnRP corrected the change in males, whereas both rehabilitation regimens corrected the changes in female offspring (Table 4). At 3 months of age, HDL cholesterol levels were comparable among the four groups of offspring (both in males and females), whereas at 6 months, the levels in ZnRP and ZnRW but not ZnR were significantly C 2009 The Authors. Journal compilation C 2009 The Physiological Society



765

Figure 2. Body fat percentage (top panels), lean body mass (middle panels) and fat-free mass (bottom panels) of the male (A–C) and female offspring (D–F) on postnatal days 90 and 180, as determined by TOBEC Each bar represents a mean + S.E.M. of observations in six rats. Means with different superscripts are significantly different by one-way ANOVA followed by post hoc least significant difference test.

lower compared with ZnC offspring. At 3 months of age, plasma triglycerides were significantly (P < 0.01) lower in ZnR, ZnRP and ZnRW compared with ZnC but only in male offspring. By 6 months of age they were

comparable among the different groups (both male and female offspring; Table 4). Plasma free fatty acids were significantly (P < 0.05) lower in female ZnR, ZnRP and ZnRW than in ZnC offspring at 3 months, whereas in

Figure 3. Fasting plasma glucose (top panels) and insulin levels (bottom panels) of the male (A–B) and female offspring (C–D) on postnatal days 90 and 180 Each bar represents a mean + S.E.M. of observations in six rats. Means with different superscripts are significantly different by one-way ANOVA followed by post hoc least significant difference test. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

766



Table 4. Lipid profile (in mmol l−1 ) of different groups of offspring at 3 and 6 months of age Parameter

Age (months)

ZnC

ZnR

ZnRP

ZnRW

3 6

1.74 ± 0.140a 1.62 ± 0.138

1.44 ± 0.080b 1.69 ± 0.046

1.62 ± 0.059a 1.48 ± 0.071

1.3 ± 0.026b 1.55 ± 0.076

HDL cholesterol

3 6

0.92 ± 0.052 1.34 ± 0.083a

0.810 ± 0.042 1.34 ± 0.035a

0.82 ± 0.082 1.04 ± 0.088b

0.91 ± 0.029 1.19 ± 0.049a

Tryglycerides

3 6

0.71 ± 0.033a 0.69 ± 0.049

0.512 ± 0.020b 0.61 ± 0.060

0.547 ± 0.062b 0.79 ± 0.097

0.57 ± 0.048b 1.01 ± 0.227

Free fatty acids

3 6

1.07 ± 0.081a 0.62 ± 0.059

1.27 ± 0.124a 0.61 ± 0.047

1.60 ± 0.052b 0.45 ± 0.065

1.04 ± 0.053a 0.65 ± 0.076

3 6

1.99 ± 0.149a 1.94 ± 0.094

1.49 ± 0.043b 1.72 ± 0.115

1.88 ± 0.129a 1.74 ± 0.019

1.93 ± 0.111a 1.82 ± 0.074

HDL cholesterol

3 6

1.60 ± 0.153 1.69 ± 0.189a

1.40 ± 0.060 1.36 ± 0.096a

1.18 ± 0.250 1.23 ± 0.088b

1.83 ± 0.074 1.26 ± 0.057b

Tryglycerides

3 6

0.64 ± 0.050 0.59 ± 0.076

0.52 ± 0.056 0.41 ± 0.031

0.52 ± 0.013 0.54±0.042

0.52 ± 0.046 0.52 ± 0.077

Free fatty acids

3 6

1.71 ± 0.051a 0.93 ± 0.141

1.16 ± 0.058b 0.98 ± 0.100

1.37 ± 0.036b 1.09 ± 0.129

1.16 ± 0.034b 0.83 ± 0.075

Male offspring Total cholesterol

Female offspring Total cholesterol

Values are means ± S.E.M. (n = 6). Means without a common letter are significantly different at P < 0.05 by ANOVA/post hoc least significance difference.

male offspring the levels were significantly higher only in ZnRP at this time point. However, the plasma free fatty acid levels were comparable among the offspring (both males and females) of different groups at 6 months of age (Table 4).

Discussion In humans and experimental animals, zinc deficiency modulates insulin sensitivity and may be associated with impaired insulin secretion (Chausmer, 1998; Taylor, 2005). Bearing in mind the significant prevalence of zinc

Figure 4. Area under the curve of plasma glucose (top panels) and insulin (bottom panels) in the male (A–B) and female offspring (C–D) on postnatal days 90 and 180 Each bar represents a mean + S.E.M. of observations in six rats. Means with different superscripts are significantly different by one-way ANOVA followed by post hoc least significant difference test. C 2009 The Authors. Journal compilation C 2009 The Physiological Society



deficiency in pregnant/lactating women in developing countries, such as India (Yasodhara et al. 1994), we assessed the effects of maternal zinc restriction on body composition, insulin response, glucose tolerance and plasma lipid profile in the offspring. The results demonstrate, for the first time to the best of our knowledge, that maternal zinc restriction during pregnancy and lactation alters body composition, insulin secretion and lipid profile in the offspring. Feeding a zinc-restricted diet for 2 weeks lowered plasma zinc levels only slightly, indicating a marginal drop in zinc status of the rat dams. As reported previously (Lee et al. 2003), food intake and body weight gain were lower in the zinc-restricted dams, reiterating the importance of zinc nutrition for proper growth and development. Despite decreased diet intake and body weight, it was interesting that insulin response, plasma glucose and lipid profile were unaffected in zinc-restricted dams. This is probably due to the moderate zinc deficiency developed and/or short duration of the deficiency in these rats. Nevertheless, the finding that zinc restriction impaired the reproductive performance of WNIN female rats and decreased the birth weight of the offspring is in line with earlier reports of a similar nature (Herman et al. 1985). Furthermore, the divergence observed in the effects of zinc restriction on different parameters could be due to their varied sensitivities to zinc deficiency. Several studies have demonstrated that lower zinc levels during pregnancy result in low-birth-weight offspring (Castillo-Duran & Weisstaub, 2003;Pathak & Kapil, 2004), and our results are consistent with these reports. Further zinc restriction, when continued through lactation and weaning, decreased the body weight of the offspring at weaning and thereafter, reiterating the importance of neonatal zinc nutrition in the development of rat offspring (Herman et al. 1985; Keen et al. 2003). Increased body adiposity and/or altered lipid metabolism are the earliest changes, seen well before tissue insulin resistance manifests, and insulin resistance has indeed been hypothesized to originate in impaired adipogenesis and/or lipid metabolism (Smith et al. 1999; Smith, 2002; Yajnik, 2002). In line with our earlier reports that maternal multiple mineral or vitamin or magnesium restriction in rat dams increased the body fat percentage in the offspring (Venu et al. 2004a,b, 2005, 2007, 2008), chronic maternal zinc restriction also increased the percentage of body fat in ZnR offspring. While the changes were reversible in male offspring by rehabilitation from parturition or weaning, in females ZnRP but not ZnRW was effective. These findings not only indicate the importance of maternal zinc status during pregnancy and lactation in determining the body fat percentage in the offspring but also the gender differences in the effects. In addition, the finding that a significant decrease was observed in LBM and FFM of ZnR offspring C 2009 The Authors. Journal compilation C 2009 The Physiological Society

767

of both sexes at 6 months of age indicates that maternal zinc deficiency may also decrease muscle (and bone) mass in the offspring. That the changes in LBM and FFM were not corrected by rehabilitation not only indicates their irreversible nature but also suggests that maternal zinc restriction might have programmed the body fat, lean and fat-free mass in the offspring. Thus the ZnR offspring with decreased body weights, LBM and FFM but higher body fat percentage are akin to the ‘thin-fat babies’ in India, an abnormal condition attributed to maternal malnutrition (Yajnik et al. 2003). It looks reasonable that the increased body fat in these offspring could be in compensation for the decreased lean mass and fat-free mass seen in them. The observed changes suggest the relevance of maternal zinc restriction in programming the body composition of the offspring. Our observation that fasting insulin levels were significantly lower in both male and female ZnR offspring at 180 days stresses that maternal zinc nutrition may modulate insulin synthesis in the offspring and is in line with other reports (Hall et al. 2005; Dunn, 2005). However, the finding that neither ZnRP nor ZnRW could correct this change not only stresses the importance of maternal zinc nutrition in programming insulin biosynthesis in the offspring but also stresses that the programming may be irreversible by rehabilitation from as early as the birth of the offspring. Since insulin is required for normal growth and development, lower insulin secretion in ZnR might be one of the reasons for their impaired growth and altered body composition. In addition, it was of interest that the AUC for insulin was significantly lower in female ZnR than in ZnC offspring during the glucose tolerance test, indicating that maternal zinc restriction not only induced fasting hypo-insulinaemia in the offspring but also impaired their insulin response to a glucose challenge. The finding that ZnRP but not ZnRW was effective in partly correcting the change stresses the importance of zinc nutrition during lactation in programming the insulin secretion capacity in the offspring. However, the finding that maternal zinc restriction did not affect this parameter in male offspring probably indicates the sex-specific nature of the effect. In general, these findings are on similar lines to our earlier findings in the offspring of mineral- and magnesium-restricted rat dams (Venu et al. 2004b, 2005, 2007, 2008), in that in all these studies the offspring appeared predisposed to impaired insulin response to a glucose challenge. Whether the fasting hypoinsulinaemia and impaired insulin response to glucose challenge are due to a decrease in the number, size and/or exhaustion of the β-cells in ZnR offspring remains to be elucidated. Notwithstanding the observed changes in fasting and postglucose insulin levels, the finding that glucose tolerance was not affected in the ZnR offspring probably suggests a higher insulin sensitivity and/or better glucose tolerance

768


in these animals. These findings are in line with similar reports of heightened insulin sensitivity in the early life of the offspring of rat dams subjected to protein restriction during pregnancy (Langely et al. 1994; Holness 1996; Gluckman & Harding, 1997; Petry et al. 1997). Few studies (Lucas et al. 1996; Lewis et al. 2001) have reported altered lipid metabolism in the offspring of rat dams subjected to protein malnutrition or iron deficiency during pregnancy. Although some changes, e.g. low levels of total cholesterol, triglycerides and free fatty acids, were observed in ZnR offspring at 90 days of age, similar changes were not observed at 180 days, indicating the transient nature of the changes. It is, however, of interest that at 180 days the HDL cholesterol levels were lower in ZnRP and ZnRW offspring, although ZnR had levels comparable to ZnC. Although the reasons for the decreased levels of these parameters are not clear at present, they could be due to increased uptake by tissues and/or reduced rates of production. These results are in line with an earlier report that maternal iron restriction decreased plasma triglycerides in 3-month-old offspring (Lewis et al. 2001) and our similar reports in the offspring of mineral- or magnesium-restricted rat dams (Venu et al. 2004b, 2005). It is reported that intestinal absorption of lipids in general is impaired in zinc-deficient rats (Koo et al. 1986; Kim et al. 1998). Whether the changes in cholesterol profile in this study are due to similar effects on intestinal lipid absorption remain to be deciphered. Also, whether the altered lipid profile is the cause or consequence of the increased body fat percentage and if it has any role in modulating insulin response (fasting and/or in response to a challenge of glucose) of the offspring remains to be deciphered. Nevertheless, the observed changes in lipid profile (except the decreased levels of HDL cholesterol) appear to suggest or to be in line with a state of heightened insulin sensitivity of these offspring. In conclusion, zinc restriction significantly affected reproductive performance of WNIN female rats. In addition, chronic maternal zinc restriction increased the percentage of body fat and decreased the lean/fatfree mass in the offspring, which suggests a decrease in bone and muscle mass. The finding that changes in LBM and FFM were irreversible while those in body fat percentage were mitigated by rehabilitation probably suggests their differential sensitivity to the effects of maternal zinc restriction. Fasting insulin levels were decreased in offspring of both sexes, but insulin response to a glucose challenge was impaired in female offspring only. The finding that these changes were mostly irreversible by rehabilitation indicates the importance of maternal zinc status during pregnancy and lactation in modulating/programming insulin secretion in the offspring. However, their fasting glucose and glucose tolerance were comparable to control animals, probably suggesting their greater insulin sensitivity. Overall, the


results stress the importance of maternal rat zinc status (restriction) in modulating the body composition and insulin secretion of the offspring. References Barker DJ (2004). The developmental origins of adult disease. J Am Coll Nutr 23, 588S–595S. Barker DJ (2006). Adult consequences of fetal growth restriction. Clin Obstet Gynecol 49, 270–283. Castillo-Durán C & Weisstaub G (2003). Zinc supplementation and growth of the fetus and low birth weight infant. J Nutr 133, 1494S–1497S. Caulfield LE, Zavaleta N, Shankar AH & Merialdi M (1998). Potential contribution of maternal zinc supplementation during pregnancy to maternal and child survival. Am J Clin Nutr 68, 449S–508S. Chausmer AB (1998). Zinc, insulin and diabetes. J Am Coll Nutr 17, 109–115. Dunn MF (2005). Zinc–ligand interactions modulate assembly and stability of the insulin hexamer – a review. Biometals 18, 295–303. Garofano A, Czernichow P & Bréant B (1997). In utero undernutrition impairs rat beta-cell development. Diabetologia 40, 1231–1234. Gluckman PD & Harding JE (1997). Fetal growth retardation: underlying endocrine mechanisms and postnatal consequences. Acta Paediatr Suppl 422, 69–72. Hall AG, Kelleher SL, Lönnerdal B & Philipps AF (2005). A graded model of dietary zinc deficiency: effects on growth, insulin-like growth factor-I, and the glucose/insulin axis in weanling rats. J Pediatr Gastroenterol Nutr 41, 72–80. Herman Z, Greeley S & King JC (1985). Placenta and maternal effects of marginal zinc deficiency during gestation in rats. Nutr Res 5, 211–219. Holness MJ (1996). Impact of early growth retardation on glucoregulatory control and insulin action in mature rats. Am J Physiol Endocrinol Metab 270, E946–E954. Keen CL, Hanna LA, Lanoue L, Uriu-Adams JY, Rucker RB & Clegg MS (2003). Developmental consequences of trace mineral deficiencies in rodents: acute and long-term effects. J Nutr 133, 1477S–1480S. Kim ES, Noh SK & Koo SI (1998). Marginal zinc deficiency lowers the lymphatic absorption of α−tocopherol in rats. J Nutr 128, 265–270. King JC (2000). Determinants of maternal zinc status during pregnancy. Am J Clin Nutr 71, 1334S–1343S. Koo SI, Norvell JE, Algilani K & Chow JJ (1986). Effect of marginal zinc deficiency on the lymphatic absorption of [14 C]cholesterol. J Nutr 116, 2363–2371. Langley SC, Browne RF & Jackson AA (1994). Altered glucose tolerance in rats exposed to maternal low protein diets in utero. Comp Biochem Physiol A Physiol 109, 223–229. Lee SL, Kwak EH, Kim YH, Choi JY, Kwon ST, Beattie JH & Kwun IS (2003). Leptin gene expression and serum leptin levels in zinc deficiency: implications for appetite regulation in rats. J Med Food 6, 281–289. C 2009 The Authors. Journal compilation C 2009 The Physiological Society



Lewis RM, Petry CJ, Ozanne SE & Hales CN (2001). Effects of maternal iron restriction in the rat on blood pressure, glucose tolerance, and serum lipids in the 3-month-old offspring. Metabolism 50, 562–567. Lucas A, Baker BA, Desai M & Hales CN (1996). Nutrition in pregnant or lactating rats programs lipid metabolism in the offspring. Br J Nutr 76, 605–612. MacDonald RS (2000). The role of zinc in growth and cell proliferation. J Nutr 130, 1500S–1508S. Matthews JN, Altman DG, Campbell MJ & Royston P (1990). Analysis of serial measurements in medical research. BMJ 300, 230–235. Morbach CA & Brans YW (1992). Determination of body composition in growing rats by total body electrical conductivity. J Pediatr Gastroenterol Nutr 14, 283–292. Nyirenda MJ, Welberg LA & Seckl JR (2001). Programming hyperglycaemia in the rat through prenatal exposure to glucocorticoids-fetal effect or maternal influence? J Endocrinol 170, 653–660. Ozanne SE, Wang CL, Coleman N & Smith GD (1996). Altered muscle insulin sensitivity in the male offspring of protein-malnourished rats. Am J Physiol Endocrinol Metab 271, E1128–E1134. Pathak P & Kapil U (2004). Role of trace elements zinc, copper and magnesium during pregnancy and its outcome. Indian J Pediatr 71, 1003–1005. Petry CJ, Desai M, Ozanne SE & Hales CN (1997). Early and late nutritional windows for diabetes susceptibility. Proc Nutr Soc 56, 233–242. Reusens B & Remacle C (2001). Intergenerational effect of an adverse intrauterine environment on perturbation of glucose metabolism. Twin Res 4, 406–411. Shah D & Sachdev HP (2006). Zinc deficiency in pregnancy and fetal outcome. Nutr Rev 64, 15–30. Simmons RA, Templeton LJ & Gertz SJ (2001). Intrauterine growth retardation leads to the development of type 2 diabetes in the rat. Diabetes 50, 2279–2286. Smith U (2002). Impaired (‘diabetic’) insulin signaling and action occur in fat cells long before glucose intolerance—is insulin resistance initiated in the adipose tissue? Int J Obes Relat Metab Disord 26, 897–904. Smith U, Axelsen M, Carvalho E, Eliasson B, Jansson PA & Wesslau C (1999). Insulin signaling and action in fat cells: associations with insulin resistance and type 2 diabetes. Ann N Y Acad Sci 892, 119–126. Taylor CG (2005). Zinc, the pancreas, and diabetes: insights from rodent studies and future directions. Biometals 18, 305–312.

C 2009 The Authors. Journal compilation C 2009 The Physiological Society

769

Venu L, Durga Kishore Y & Raghunath M (2005). Maternal and perinatal magnesium restriction predisposes the rat pups to insulin resistance and glucose intolerance. J Nutr 135, 1353–1358. Venu L, Harishankar N, Prasanna Krishna T & Raghunath M (2004a). Maternal dietary vitamin restriction increases body fat content but not insulin resistance in WNIN rat offspring up to 6 months of age. Diabetologia 47, 1493–1501. Venu L, Harishankar N, Prasanna Krishna T & Raghunath M (2004b). Does maternal dietary mineral restriction per se predispose the offspring to insulin resistance? Eur J Endocrinol 151, 287–294. Venu L, Padmavathi IJN, Kishore YD, Vijaya Bhanu N, Rajender Rao K, Sainath PB, Ganeshan M & Raghunath M (2008). Long-term effects of maternal magnesium restriction on adiposity and insulin resistance in rat pups. Obesity 16, 1270–1276. Venu L, Vijaya Bhanu N, Rajender Rao K & Raghunath M (2007). Effect of maternal vitamin and mineral restrictions on the body fat content and adipocytokine levels of WNIN rat offspring. Nutr Metab (Lond) 4, 21–25. Yajnik CS (2002). The lifecycle effects of nutrition and body size on adult adiposity, diabetes and cardiovascular disease. Obes Rev 3, 217–224. Yajnik CS, Fall CH, Coyaji KJ, Hirve SS, Rao S, Barker DJ, Joglekar C & Kellingray S (2003). Neonatal anthropometry: the thin-fat Indian baby. The Pune Maternal Nutrition Study. Int J Obes Relat Metab Disord 27, 173–180. Yasodhara P, Ramaraju LA & Raman L (1994). Trace minerals in pregnancy 1. Copper and zinc. Nutr Res 11, 15–21.

Acknowledgements This work was supported by a research grant to M.R. from the Department of Biotechnology, Government of India, New Delhi, India (project no. BT/PR2832/Med/14/390/2001). The authors acknowledge Council for Scientific and Industrial Research, Indian Council of Medical Research and University Grants Commission for awarding research fellowships to L.V., Y.D.K., I.J.N.P. and M.G.

Author’s present address L. Venu: Department of Orthopaedic Surgery, David Geffen School of Medicine UCLA, Los Angeles, CA 90095, USA.