The Journal of Nutrition

Nutrition and Disease

Maternal Zinc Deficiency in Rats AffectsGrowth and Glucose Metabolism inthe Offspring by Inducing InsulinResistance Postnatally1,2

Ming-Yu Jou,3 Anthony F. Philipps,4 and Bo Lonnerdal3*

3Department of Nutrition and 4Department of Pediatrics, University of California, Davis, CA 95616

Abstract

Interactions among zinc (Zn), insulin, and glucose metabolism are complex. Maternal Zn deficiency affects maternal

carbohydrate metabolism, but the mechanisms underlying changes in glucose homeostasis of offspring are not well

understood. Rats consumed Zn-deficient (ZnD; 7 mg/g) or control (ZnC; 25 mg/g) diets ad libitum from 3 wk preconception

to 21 d postparturition. Litters were culled to 7 pups/dam postnatally and pups were allowed to nurse their original

mothers; after weaning, pups were fed nonpurified diet. Insulin and glucose tolerance tests were performed on the pups

at wk 5 and 10. Although there was no difference in birth weight between groups, ZnD pups weighed significantly more

than controls by d 10 (+5%) and 20 (+10%). Both blood glucose and serum insulin-like growth factor (IGF-1)

concentrations at wk 3 were significantly higher in ZnD pups than in controls. Both male and female ZnD rats were less

sensitive to insulin and glucose stimulation than controls at wk 5 and 10. At wk 15, serum leptin concentrations were

higher in male ZnD rats than in controls. Phosphorylation of muscle Akt protein, an insulin receptor (IR) signaling

intermediate, was lower in female ZnD rats than in controls at wk 15, but they did not differ in phosphorylation of IR.

Maternal Zn deficiency resulted in greater serum IGF-1 concentrations and the excessive postnatal weight gain in their

offspring as well as impaired subsequent glucose sensitivity. It was associated with gender-specific alterations in the

serum leptin concentration and the insulin signaling pathway. These findings suggest that suboptimal maternal Zn status

induces long-term changes in the offspring related to abnormal glucose tolerance. J. Nutr. 140: 1621–1627, 2010.

Introduction

Zinc (Zn) is an essential micronutrient and is critical for normalgrowth and development in humans and other mammals (1–3).Increased Zn requirements during periods of rapid growth (2),such as infancy, adolescence, pregnancy, and lactation, may alsoincrease the risk for Zn deficiency. Zn deficiency is common indeveloping countries (4) due to inadequate dietary Zn intake andpoor Zn bioavailability (2). It is estimated that pregnant womenare at high risk for at least mild to moderate Zn deficiency inboth developing and developed countries (5,6).

Hales et al. (7) proposed the “developmental origin of healthand disease,” whereby inadequate nutrition during fetal life mayresult in permanent metabolic modifications and increased riskof diabetes and other diseases in adulthood. The prevalence ofmaternal Zn deficiency worldwide is difficult to estimate but isfairly prevalent. Thus, the potential effect of such deficiency onsubsequent development of adult diseases such as diabetes in the

offspring may be of great importance. To investigate the effect ofmaternal Zn deficiency on diabetes in the offspring, a study wasdesigned to explore potential links between maternal Zndeficiency and subsequent insulin resistance in the offspringand to study mechanisms by which this phenomenon mightoccur.

Several hundred metalloproteins require Zn for diversecellular functions, in which it either participates directly in chemicalcatalysis (enzymes) or is important for maintaining the structureand stability of proteins (8,9) [such as insulin and insulin re-ceptor (IR)5]. The interactions among Zn, insulin, and glucosehomeostasis are complex, and Zn deficiency might induce a stateof insulin deficiency by interfering with either insulin storage oractivation. A connection between Zn metabolism and diabetesmellitus is demonstrated by consistent symptoms of hyper-zincuria and hypozincemia in type 2 diabetic patients (10,11). Ina previous study conducted in weanling rats (12), we found thatsevere postnatal Zn deficiency in pups induced hyperglycemia,

1 Supported by the Children’s Miracle Network.2 Author disclosures: M. Jou, A. Philipps, and B. Lonnerdal, no conflicts of

interest.

* To whom correspondence should be addressed. E-mail: bllonnerdal@ucdavis.

edu.

5 Abbreviations used: GTT, glucose tolerance test; IGF-1, insulin-like growth

factor-1; IGI, insulinogenic index; IR, insulin receptor; ITT, insulin tolerance test;

ZnC, zinc control diet; ZnD, zinc-deficient diet.

ã 2010 American Society for Nutrition.

Manuscript received December 28, 2009. Initial review completed March 17, 2010. Revision accepted June 23, 2010. 1621First published online July 21, 2010; doi:10.3945/jn.109.119677.

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and mild Zn deficiency also altered glucose metabolism. Studieshave shown that transient mild maternal Zn deficiency duringthe last week of gestation affects maternal carbohydratemetabolism (13). Zn is also known to have insulinomimeticeffects (14), and increased phosphorylation of PI3K/Akt withZn exposure has been demonstrated (15), suggesting that Zndeficiency might stimulate a state of insulin resistance throughmodulating IR signaling cascades. However, the actual effects ofmaternal Zn deficiency during pregnancy on postnatal glucosehomeostasis and the mechanisms behind these effects are notwell understood.

Transient mild maternal Zn deficiency during the last week ofgestation has been shown to stimulate growth as well as increasethe fetal protein:DNA ratio (13). Insulin-like growth factor(IGF-1) was found to participate in the stimulation of DNA andprotein synthesis, which is mediated through its receptorsignaling pathway (16). The IGF-1 receptor requires Zn foroptimal configuration due to a molecular structure similar to theIR. Leptin and adiponectin are mainly produced by adiposetissue and play important roles in energy balance. Studies haveshown that rats fed a marginally Zn-deficient diet (3 mg/kg) haddecreased leptin expression, independent of changes in foodintake (17), and severe Zn deficiency suppressed not onlycirculating leptin concentrations but also leptin secretion byrat adipocytes (18). In the present study, we investigated theeffects of maternal Zn deficiency induced throughout pregnancyand lactation on postnatal growth, carbohydrate metabolism/insulin signaling cascades, and hormone concentrations in theoffspring.

Materials and Methods

Rats. The study was conducted under the auspices of Animal ResourceServices of the University of California, Davis, which is accredited by the

American Association for the Accreditation of Laboratory Animal Care.

Virgin female Sprague-Dawley rats (n = 10; 7–8 wk) were obtained from

Charles River. Rats were housed in solid plastic hanging cages underconstant conditions (temperature, 228C; humidity, 65%) with a 12-h-

dark:-light cycle and were allowed to consume food ad libitum. After a

3-d acclimatization period, rats were randomly assigned to 1 of the 2

experimental diets. The Zn-deficient pregnant rat model has beendeveloped previously in our laboratory (19). The Zn-adequate control

group (ZnC; n = 6) was fed a semipurified diet containing 25mg Zn/kg of

diet while the other group (ZnD; n = 6) received a diet marginallydeficient in Zn (7 mg Zn/kg of diet) (12,20). Rats were fed these diets

from 3 wk preconception to 21 d postparturition.

On postnatal d 2, pups were culled to 7 pups/litter and nursed by

their respective dams. Weight of pups was monitored and one-half of thepups were killed at wk 3 after 4 h of food deprivation. The remaining

pups were separated by gender, weaned, and fed a standard nonpurified

rodent diet (LabDiet 5001, PMI). The proximate composition of this

cereal-based diet is 28.5% protein, 13.5% lipid, and 58.0% carbohy-drate. For signaling experiments, the rats were injected intraperitoneally

with 8 units/kg of insulin (Humulin R, Eli Lilly & Co.,) after 12–14 h of

food deprivation. The rats were killed 10 min after injection and tissueswere removed and snap-frozen.

Insulin and glucose tolerance tests. An insulin tolerance test (ITT)

was performed after 4 h of food deprivation (between 1300 and 1600 h).Rats (n = 9 rats of each gender/group) were briefly removed from their

cages for tail blood sampling and glucose concentrations were measured

with a glucose meter (Easycheck, Home Aide Diagnostics). After

measuring fasting glucose, rats were injected intraperitoneally withinsulin. Dosages of 0.7 or 0.8 units/kg of Humulin R were used at wk 5

or 10, respectively. In this test, blood glucose from tail blood was

sampled at 0, 10, 20, 30, 45, 60, and 90 min after insulin injection. After

a 3-d acclimation period, glucose tolerance test (GTT) experiments were

performed on the same rats after 12–15 h of food deprivation. Rats were

injected intraperitoneally with 2 g/kg of glucose solution (Sigma) and

blood for glucose and insulin analyses was sampled at the same timepoints as ITT after glucose injection. The insulinogenic index (IGI) was

used to estimate insulin secretion ability and insulin reserves in the pan-

creas and the following equation was used for GTT calculations (21):

IGI ¼ ðIns10=302Ins0Þ=18ðGlc10=302Glc0Þ;

where Ins0 and Glc0 are the basal insulin and glucose concentrations at0 min (mU/L, mmol/L), respectively, and Ins10/30 and Glc10/30 are the

insulin and glucose concentrations 10 or 30 min after glucose injection.

Mineral analysis. Zn concentrations of diets and tissues were analyzedby flame atomic absorbance spectrometry as previously described (22)

using a SOLAAR M series atomic absorbance spectrometer (Thermo

Electron). Samples (;0.1 g) were wet ashed with 3 mL mineral analysis

grade 16 mol/L nitric acid (Fisher Scientific).

IGF-1, insulin, leptin, and adiponectin assays. Serum insulin and

leptin concentrations were measured with commercial RIA kits (Sensi-tive rat insulin RIA kit and rat leptin RIA kit, Linco Research). The

ELISA kit for rat adiponectin (Rat adiponectin ELISA kit) was from

Millipore. Serum IGF-I concentrations were measured with a commer-

cial kit (IGF-1 high sensitive ELISA kit; IDS).

Antibodies. Rabbit polyclonal antibody against IR was purchased from

Santa Cruz Biotechnology. Rabbit polyclonal phosphospecific antibodies

against Akt1/2/3 and ERK1/2 were from Santa Cruz Biotechnology andCell Signaling Technology, respectively. Monoclonal anti-phosphotyrosine

(4G10) antibody was from Upstate Biotechnology, Inc.

Immunoprecipitation and immunoblotting. Tissues were homoge-nized in a modified radioimmune precipitation assay buffer (50 mmol/L

Tris-Cl, pH 7.5, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxy-

cholate, 0.1% SDS, and 5 mmol/L EDTA) containing 2 mmol/L sodiumorthovanadate and a protease inhibitor cocktail (Sigma). The lysates

were clarified by centrifugation at 16,000 3 g for 30 min and protein

concentrations were measured using the Bradford assay (Bio-Rad). For

immunoprecipitation, lysates were incubated with appropriate anti-bodies at 48C for 3 h to overnight. Immune complexes were collected

onto protein G-Sepharose beads, washed extensively, resolved by SDS-

PAGE, transferred onto nitrocellulose membranes at 350 mA for 60 min,

and blocked in 10 mmol/L Tris-Cl, pH 7.4, 150 mmol/L NaCl, 0.05%Tween 20 with 5% bovine serum albumin. After incubation with

appropriate primary and secondary antibodies (used at the concentra-

tions recommended by their suppliers), specific bands were visualizedwith Super Signal Femto Chemiluminescent Reagent (Pierce) and

molecular masses of visualized proteins were assessed relative to

molecular weight markers (GE Healthcare).

Data analysis. Data are expressed as means 6 SD. Student’s t test was

performed to determine gender effects and data were pooled when there

were no differences between males and females. Student’s t test was also

performed to determine significant differences between ZnD and ZnCgroups. Statistical analysis was conducted using GraphPad Prism 3.02

and significance was accepted at P , 0.05.

Results

Food intake of the female dams fed ZnC or ZnD diets for 9 wkdid not differ. Weights of dams before pregnancy (ZnC, 233.5612.6 g; ZnD, 247.66 13.2 g; n = 5–6) and after lactation (ZnC,264.0 6 21.8 g; ZnD, 269.6 6 19.1 g; n = 5–6) did not differ,and there was no difference in litter size between the ZnC andZnD (13.91 6 1.92 pups/litter; n = 11) groups.

Growth of pups. Birth weight did not differ between the ZnCand ZnD groups or between males and females (5.96 0.8 g; n =

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132). The weight of pups at d 10 and 20 did not differ betweengenders, but pups from ZnD dams weighed more than ZnC pupsby postnatal d 10 (+5%; ZnC, 20.6 6 2.2 g; ZnD, 21.76 1.8 g;n = 35–42; P, 0.05) and d 20 (+10%; ZnC, 41.76 2.7 g; ZnD,44.3 6 3.2 g; n = 35–42; P , 0.05). Rats consumed ad libitumnonpurified rodent diet after weaning and male rats in the ZnC(154.6 6 12.3 g) and ZnD (157.0 6 10.4 g) groups weighedmore than female ZnC (133.8 6 17.7 g) and ZnD (130.6 69.6 g) rats at wk 5 (P , 0.05; n = 7–9). However, body weightdid not differ between dietary groups at wk 5, 10, and 15.

Zn status of pups. Serum Zn concentrations at d 2 and wk 3did not differ between dietary groups, but liver and muscle Znconcentrations were lower in ZnD pups compared with ZnCpups (Table 1). Interestingly, the proportion of liver to totalbody weight was significantly higher in ZnD pups comparedwith ZnC pups at wk 3 (data not shown), which resulted in nodifference in total liver Zn content (Table 1). Although serum Znconcentrations of female ZnD rats did not differ from ZnC ratsat wk 15 (data not shown), male ZnD rats had higher serum Znconcentrations (20.8 6 2.0 mmol/L; P , 0.05) than ZnC rats(18.2 6 2.3 mmol/L). Pancreas and muscle Zn concentrationsand total liver Zn contents did not differ between dietary groupsat wk 15 (data not shown).

Blood glucose and serum hormone concentrations. ZnDpups had higher basal blood glucose concentrations than ZnCpups, but basal serum insulin concentrations did not differ at wk3 (Table 2). At wk 15, basal glucose concentrations in male,but not female, ZnD rats tended to be higher than in ZnC rats(P = 0.07; Table 2). Insulin concentrations did not differ betweendietary groups in either males or females at 15 wk of age(Table 2).

To explore mechanisms behind the effects of maternal Zndeficiency on pup postnatal growth, serum IGF-1 concentrations

were measured in pups at wk 3. Serum IGF-1 concentrations ofZnD rat pups (62.7 6 6.8 nmol/L; n = 9) were higher than inZnC pups (49.5 6 6.6 nmol/L; n = 12; P , 0.05). Serumadiponectin concentrations did not differ between ZnD andZnC pups at 3 or 15 wk of age (Table 2). Serum leptinconcentrations were greater in male ZnD rats at wk 15 than inZnC rats, but there were no differences in female rats or in eithergender at 3 wk of age (Table 2).

Insulin sensitivity. Basal glucose and insulin concentrationsdid not differ between dietary groups at wk 5 and 10 in femaleor male rats. During the ITT, female ZnD rats exhibited asignificantly smaller decrease in blood glucose concentrations at10 and 20 min than ZnC rats at both wk 5 (Fig. 1B) and 10 (Fig.1D), and male ZnD rats had a significantly higher fold of basalblood glucose concentrations than ZnC rats only at 10 and 60min at wk 10 (Fig. 1C). During the GTT, male ZnD rats hadsignificantly greater blood glucose concentrations at 20 and30 min than ZnC rats at wk 5 (Fig. 2A). At wk 10, male ZnDrats had significantly higher blood glucose concentrations at 20,30, 45, and 60 min than ZnC rats (Fig. 2C) and female ZnD ratshad a significantly higher blood glucose concentration at 30 minthan ZnC rats at wk 10 (Fig. 2D).

IGI, the insulin secretion ability index, was calculated usingglucose and insulin concentrations from the GTT. At 10 min,but not at 30 min, the IGI was 29% lower in male ZnD ratsthan in controls (Fig. 3A). Females did not differ at either time(Fig. 3B).

Insulin signaling in muscle. Total IR, Akt1/2/3, and ERK1/2phosphorylation did not differ between dietary groups in muscleof male rats at wk 15 (data not shown). Phosphorylation ofAkt1/2/3 in female ZnD rats was significantly lower than inZnC rats, but phosphorylation of IR and ERK1/2 did not differ(Fig. 4A,B).

Discussion

Increased Zn requirements during pregnancy and infancy aresuspected to increase the risk for Zn deficiency among theyoung. Remarkably, it has been estimated that ~80% ofpregnant women worldwide consume less than the recommen-ded dietary allowance for Zn (6). To investigate the effects ofmild maternal Zn deficiency on early signs of diabetes in theoffspring, the present study was designed to provide some linksbetween moderate maternal Zn deficiency and abnormalities inpostnatal growth and insulin sensitivity and to explore mech-anisms by which these might occur.

TABLE 1 Effects of mild maternal Zn deficiency in rats onserum and tissue Zn in male and female offspring1

Age

d 2 wk 3

ZnC ZnD ZnC ZnD

Serum Zn, mmol/L 62.9 6 4.3 59.5 6 8.5 25.1 6 3.2 23.9 6 2.9

Liver Zn, mmol/g 2.08 6 0.26 1.31 6 0.20* 0.56 6 0.08 0.50 6 0.08*

Total liver Zn, mmol ___2 ___2 0.72 6 0.11 0.79 6 0.10

Muscle Zn, mmol/g ___2 ___2 0.20 6 0.02 0.17 6 0.03*

1 Data are means 6 SD, n = 20. *Different from ZnC, P , 0.05.2 Not measured.

TABLE 2 Effects of mild maternal Zn deficiency in rats on blood glucose and serum insulin, IGF-1, leptin,and adiponectin concentrations at 3 and 15 wk in the offspring1

wk 15

wk 3 Male Female

ZnC ZnD ZnC ZnD ZnC ZnD

n 24 24 9 9 9 9

Blood glucose, mmol/L 8.0 6 1.3 8.8 6 0.7* 5.7 6 0.5 6.1 6 0.4 6.2 6 0.8. 6.4 6 0.4

Insulin, pmol/L 76 6 9 81 6 13 301 6 143 320 6 167 288 6 189 289 6 143

Leptin, nmol/L 0.10 6 0.01 0.11 6 0.02 0.15 6 0.05 0.36 6 0.14* 0.13 6 0.04 0.14 6 0.02

Adiponectin, nmol/L 32.3 6 8.4 29.2 6 4.1 35.2 6 15.5 39.3 6 12.5 40.6. 6 16.6 51.1 6 18.1

1 Data are means 6 SD. *Different from ZnC, P , 0.05.

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Maternal Zn deficiency increased IGF-1 concentrationassociated with postnatal overgrowth of pups. In thepresent study, litter size and birth weight did not differ betweenZnD and ZnC rats, confirming previous results by others usingmodels of mild Zn deficiency (10 mg Zn/kg of diet) duringpregnancy (23) or preconception (24,25). These results suggestthat there are no adverse effects of mild to moderate maternalZn deficiency before and/or during pregnancy on perinatalgrowth. Mildly Zn-deficient rats had higher Zn turnover duringpregnancy, which can be interpreted as an attempt to maintain asupply of Zn from the maternal placenta to the growing fetus(24). The restricted fetal growth typical of severely Zn-deficient(1 mg Zn/kg of diet) rats was not found in our moderately Zn-deficient (7 mg Zn/kg of diet) rats, suggesting that regulation of

maternal Zn homeostasis might be able to spare Zn to meet theZn requirements for fetal growth in a state of moderate Znavailability. In contrast, although moderately Zn-deficient(5.8 mg Zn/kg of diet) rats had no depression of neonatalgrowth during lactation, body weights of these suckling pupswere slightly higher than ZnC rats at d 17 postpartum (26). OurZnD pups had significantly higher postnatal weights than ZnCpups at both d 10 and 20, in combination with elevated serumIGF-1 concentrations, suggesting that elevated IGF-1 concentra-tions in newborns of Zn-deficient dams might be closely asso-ciated with increased postnatal growth.

FIGURE 1 Changes in blood glucose concentrations during an ITT in

ZnC and ZnD male (A,C) and female (B,D) rat pups at wk 5 (A,B) and

10 (C,D) of age. Values are means6 SD, n = 6–8. *Different from ZnC

at that time, P , 0.05.FIGURE 2 Changes in blood glucose concentrations during a GTT in

ZnC and ZnD male (A,C) and female (B,D) rat pups at wk 5 (A,B) and

10 (C,D) of age. Values are means6 SD, n = 5–9. *Different from ZnC

at that time, P , 0.05.

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Maternal Zn deficiency induced a long-term effect oninsulin resistance in the offspring. The “developmentalorigin of health and disease” hypothesis proposed by Hales andBarker (7) links poor fetal and infant growth with subsequentdevelopment of diabetes and cardiovascular disease (7). Anassociation between prenatal exposure to famine and abnormalglucose tolerance in adults has been shown in humans (27). It issuggested that programming caused by early malnutrition mayresult in long-term or lifetime metabolic modifications, includ-ing insulin resistance and diabetes. Maternal mineral deficiencyhas been found to be associated with impaired glucose metab-olism in rats (28,29). However, a specific link between pre- andpostnatal exposure to Zn deficiency and glucose metabolism ismissing. In the present study, rat pups from mildly Zn-deficientdams had impaired glucose tolerance, suggesting that earlyexposure to Zn deficiency might be related to the development ofinsulin resistance and/or diabetes in later life.

Maternal Zn deficiency decreased phosphorylation ofinsulin signaling cascades in female pups. Zn hasinsulinomimetic effects, including stimulation of lipogenesisand glucose transport via activation of IR and intracellularsecond messenger signaling pathways, such as mitogen-activatedprotein kinase and protein kinase B (9,14). Phosphorylation ofAkt is activated in cells exposed to Zn and in Zn-deficient micegiven Zn (30,31), suggesting that Zn participates in activation ofthe Akt signaling pathway. We found significantly lowerphosphorylation of total muscular Akt in female rats frommildly Zn-deficient dams compared with controls after insulinstimulation, suggesting that early exposure to Zn deficiencymight depress Akt signaling cascades in female rats, which couldresult in impaired glucose metabolism in adulthood. However,further work is required to elucidate the regulatory mechanism

of Akt signaling cascades in the development of insulinresistance during maternal Zn deficiency.

Maternal Zn deficiency was associated with alterations ininsulin secretion and decreased leptin concentrations inmale rats. Leptin functions in the central nervous system byreducing food intake and increasing energy expenditure (32).Multiple other peripheral actions of leptin also have beensuggested with the identification of the leptin receptor-mediatedsignaling pathway, which includes suppression of biosynthesis ofpreproinsulin and inhibition of insulin secretion from its storagevesicles in pancreatic b-cells (33). Inhibition of insulin secretionvia leptin has been demonstrated in human and rodent pancre-atic islets and b-cell lines (34–36). In our study, insulinresistance, increased leptin concentrations, and a reduction inpancreatic insulin reserves were observed in male offspring ofZn-deficient dams and there was also a positive correlationbetween glucose concentration at 20 min during GTTand leptinconcentration in these rats (r2 = 0.83; P = 0.01; data not shown).These findings suggest that Zn deficiency induced changes inboth IR sensitivity and leptin-mediated inhibition of insulinbiosynthesis and secretion.

In addition, leptin resistance has been defined from obser-vations in obese patients and in animal models in which in-creased leptin concentrations seem to exert fewer effects in thehypothalamus than in pancreas (37). In turn, leptin resistancehas been found to be associated with insulin resistance (38).Circulating leptin concentrations are positively correlated withthe degree of adiposity (39), and we found increased leptinconcentrations and insulin insensitivity in male rats born to ZnDdams by postnatal wk 15. Although there were no significantdifferences in weight gain between groups at wk 15, thepercentage of the gain that was fat was not measured. Increasedleptin concentrations in pups of ZnD dams may result fromexcess fat deposition, but the actual cause and effect relationshipbetween leptin and insulin resistance is still unknown. Takentogether, our data demonstrate that mild maternal Zn deficiencyis associated with elevated leptin concentrations and insulinresistance in male offspring in adulthood, but the underlyingmechanisms still need be explored.

Maternal Zn deficiency impairs glucose metabolism inmale offspring. Epidemiologic studies have shown that men

FIGURE 3 The IGI at 10 and 30 min during a GTT in ZnC and ZnD

male (A) and female (B) rat pups at wk 10 of age. IGI were calculated

from glucose and insulin concentrations during the GTT (10 and 30 min

after glucose injection). Values are means 6 SD, n = 5–9. *Different

from ZnC at that time, P , 0.05.

FIGURE 4 Phosphorylation of muscle IR, Akt, and ERK after insulin

stimulation in ZnC and ZnD female rat pups at wk 15 of age. The

phosphorylation ratio was calculated as total:phosphorylated (T-, P-)

protein densities. Relative phosphorylation is expressed as fold of ZnC

(A). Representative western blots are shown in B. Values are means6SD, n = 3. *Different from ZnC, P , 0.05.

Maternal zinc deficiency and insulin resistance 1625

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have a higher risk of developing diabetes than women (40,41),but the mechanism underlying this gender difference is unclear.Although we observed similar effects of maternal Zn deficiencyon insulin resistance in male and female rats, different mecha-nisms appear to be involved. A sex-specific effect of Zndeprivation on growth has been found in other studies (42,43),and data also suggest that males are more susceptible tonutritional deprivation than females (44). We observed elevatedleptin concentrations only in males born to ZnD dams. Leptinexpression and secretion are regulated by sex hormones,including androgenic and estrogenic hormones (45), suggestingthat gender-specific regulation of leptin expression mightparticipate in the development of insulin resistance in offspringof Zn-deficient dams. Furthermore, Hamdi et al. (46) have shownthat marginally Zn-deficient rats have significantly lower se-rum testosterone concentrations without differences in follicle-stimulating hormone and luteinizing hormone compared withcontrols, indicating that interactions between Zn deficiency andserum leptin and testosterone concentrations might contributeto the gender-specific development of insulin resistance.

In conclusion, mild maternal Zn deficiency resulted in greaterserum IGF-1 concentrations and the excessive postnatal weightgain in their offspring and impaired subsequent glucose sensi-tivity. It was associated with gender-specific alterations in serumleptin concentrations and the insulin-signaling pathway. Thesefindings strongly suggest that suboptimal maternal Zn statusinduces long-term, gender-specific changes related to abnormalglucose metabolism in the offspring.

AcknowledgmentsAll authors contributed to the design of the study, and M.J.conducted the animal experiments and data analysis. Themanuscript was written by M.J. with significant input by theother authors. All authors had primary responsibility for finalcontent. All authors read and approved the final version of themanuscript.

Literature Cited

1. MacDonald RS. The role of zinc in growth and cell proliferation. J Nutr.2000;130:S1500–8.

2. Hambidge M. Human zinc deficiency. J Nutr. 2000;130:S1344–9.

3. Solomon A, Rosenblum G, Gonzales PE, et al. Pronounced diversity inelectronic and chemical properties between the catalytic zinc sites of tumornecrosis factor-alpha-converting enzyme and matrix metalloproteinasesdespite their high structural similarity. J Biol Chem. 2004;279:31646–54.

4. Sandstead HH. Is zinc deficiency a public health problem? Nutrition.1995;11:87–92.

5. Shah D, Sachdev HP. Zinc deficiency in pregnancy and fetal outcome.Nutr Rev. 2006;64:15–30.

6. Caulfield LE, Zavaleta N, Shankar AH, Merialdi M. Potential contri-bution of maternal zinc supplementation during pregnancy to maternaland child survival. Am J Clin Nutr. 1998;68:S499–508.

7. Hales CN, Barker DJ. Type 2 (non-insulin-dependent) diabetes mellitus:the thrifty phenotype hypothesis. Diabetologia. 1992;35:595–601.

8. Berg JM, Shi Y. The galvanization of biology: a growing appreciationfor the roles of zinc. Science. 1996;271:1081–5.

9. McCall KA, Huang C, Fierke CA. Function and mechanism of zincmetalloenzymes. J Nutr. 2000;130:S1437–46.

10. Aguilar MV, Saavedra P, Arrieta FJ, Mateos CJ, Gonzalez MJ, MeseguerI, Martinez-Para MC. Plasma mineral content in type-2 diabetic patientsand their association with the metabolic syndrome. Ann Nutr Metab.2007;51:402–6.

11. Kinlaw WB, Levine AS, Morley JE, Silvis SE, McClain CJ. Abnormalzinc metabolism in type II diabetes mellitus. Am J Med. 1983;75:273–7.

12. Hall AG, Kelleher SL, Lonnerdal B, Philipps AF. A graded model ofdietary zinc deficiency: effects on growth, insulin-like growth factor-I,and the glucose/insulin axis in weanling rats. J Pediatr GastroenterolNutr. 2005;41:72–80.

13. Southon S, Fairweather-Tait SJ, Williams CM. Fetal growth, glucosetolerance and plasma insulin concentration in rats given a marginal-zincdiet in the latter stages of pregnancy. Br J Nutr. 1988;59:315–22.

14. Tang X, Shay NF. Zinc has an insulin-like effect on glucose transportmediated by phosphoinositol-3-kinase and Akt in 3T3–L1 fibroblastsand adipocytes. J Nutr. 2001;131:1414–20.

15. Barthel A, Ostrakhovitch EA, Walter PL, Kampkotter A, Klotz LO.Stimulation of phosphoinositide 3-kinase/Akt signaling by copper andzinc ions: mechanisms and consequences. Arch Biochem Biophys. 2007;463:175–82.

16. Jones JI, Clemmons DR. Insulin-like growth factors and their bindingproteins: biological actions. Endocr Rev. 1995;16:3–34.

17. Kwun IS, Cho YE, Lomeda RA, Kwon ST, Kim Y, Beattie JH. Marginalzinc deficiency in rats decreases leptin expression independently of foodintake and corticotrophin-releasing hormone in relation to food intake.Br J Nutr. 2007;98:485–9.

18. Ott ES, Shay NF. Zinc deficiency reduces leptin gene expression and leptinsecretion in rat adipocytes. Exp Biol Med (Maywood). 2001;226:841–6.

19. Kelleher SL, Lonnerdal B. Long-term marginal intakes of zinc andretinol affect retinol homeostasis without compromising circulatinglevels during lactation in rats. J Nutr. 2001;131:3237–42.

20. Jou MY, Hall AG, Philipps AF, Kelleher SL, Lonnerdal B. Tissue-specificalterations in zinc transporter expression in intestine and liver reflect athreshold for homeostatic compensation during dietary zinc deficiencyin weanling rats. J Nutr. 2009;139:835–41.

21. Rhee SY, Chon S, Oh S, Kim SW, Kim JW, Kim YS, Woo JT. Insulinsecretion and insulin resistance in newly diagnosed, drug naiveprediabetes and type 2 diabetes patients with/without metabolicsyndrome. Diabetes Res Clin Pract. 2007;76:397–403.

22. Clegg MS, Lonnerdal B, Hurley LS, Keen CL. Analysis of whole bloodmanganese by flameless atomic absorption spectrophotometry and itsuse as an indicator of manganese status in animals. Anal Biochem.1986;157:12–8.

23. Fairweather-Tait SJ, Wright AJ, Cooke J, Franklin J. Studies of zincmetabolism in pregnant and lactating rats. Br J Nutr. 1985;54:401–13.

24. Lowe NM, Woodhouse LR, Wee J, King JC. Short-term zinc kinetics inpregnant rats fed marginal zinc diets. J Nutr. 1999;129:1020–5.

25. Chowanadisai W, Kelleher SL, Lonnerdal B. Maternal zinc deficiencyraises plasma prolactin levels in lactating rats. J Nutr. 2004;134:1314–9.

26. Hildebrandt KM, Luecke RW, Fraker PJ. Effect of maternal dietary zincon growth and mitogenic responsiveness in suckling mice. J Nutr. 1982;112:1921–8.

27. Ravelli AC, van der Meulen JH, Michels RP, Osmond C, Barker DJ,Hales CN, Bleker OP. Glucose tolerance in adults after prenatalexposure to famine. Lancet. 1998;351:173–7.

28. Venu L, Harishankar N, Krishna TP, Raghunath M. Does maternaldietary mineral restriction per se predispose the offspring to insulinresistance? Eur J Endocrinol. 2004;151:287–94.

29. Venu L, Kishore YD, Raghunath M. Maternal and perinatal magnesiumrestriction predisposes rat pups to insulin resistance and glucoseintolerance. J Nutr. 2005;135:1353–8.

30. Kim S, Jung Y, Kim D, Koh H, Chung J. Extracellular zinc activates p70S6 kinase through the phosphatidylinositol 3-kinase signaling pathway.J Biol Chem. 2000;275:25979–84.

31. McClung JP, Tarr TN, Barnes BR, Scrimgeour AG, Young AJ. Effect ofsupplemental dietary zinc on the mammalian target of rapamycin(mTOR) signaling pathway in skeletal muscle and liver from post-absorptive mice. Biol Trace Elem Res. 2007;118:65–76.

32. Speakman JR, Stubbs RJ, Mercer JG. Does body mass play a role in theregulation of food intake? Proc Nutr Soc. 2002;61:473–87.

33. Seufert J. Leptin effects on pancreatic beta-cell gene expression andfunction. Diabetes. 2004;53 Suppl 1:S152–8.

34. Seufert J, Kieffer TJ, Leech CA, Holz GG, Moritz W, Ricordi C,Habener JF. Leptin suppression of insulin secretion and gene expressionin human pancreatic islets: implications for the development ofadipogenic diabetes mellitus. J Clin Endocrinol Metab. 1999;84:670–6.

35. Pallett AL, Morton NM, Cawthorne MA, Emilsson V. Leptin inhibitsinsulin secretion and reduces insulin mRNA levels in rat isolatedpancreatic islets. Biochem Biophys Res Commun. 1997;238:267–70.

1626 Jou et al.

at UN

IV O

F P

RIN

CE

ED

WA

RD

ISLA

ND

on Decem

ber 17, 2014jn.nutrition.org

Dow

nloaded from

36. Ahren B, Havel PJ. Leptin inhibits insulin secretion induced by cellular

cAMP in a pancreatic B cell line (INS-1 cells). Am J Physiol. 1999;277:

R959–66.

37. Bjorbaek C, Elmquist JK, Frantz JD, Shoelson SE, Flier JS. Identification

of SOCS-3 as a potential mediator of central leptin resistance. Mol Cell.

1998;1:619–25.

38. Antuna-Puente B, Feve B, Fellahi S, Bastard JP. Adipokines: the missing

link between insulin resistance and obesity. Diabetes Metab. 2008;34:

2–11.

39. Ostlund RE Jr, Yang JW, Klein S, Gingerich R. Relation between plasma

leptin concentration and body fat, gender, diet, age, and metabolic

covariates. J Clin Endocrinol Metab. 1996;81:3909–13.

40. Phillips DI, Barker DJ, Hales CN, Hirst S, Osmond C. Thinness

at birth and insulin resistance in adult life. Diabetologia. 1994;37:

150–4.

41. Ferrannini E. Physiological and metabolic consequences of obesity.

Metabolism. 1995;44:15–7.

42. Hambidge KM, Walravens PA, Brown RM, Webster J, White S,

Anthony M, Roth ML. Zinc nutrition of preschool children in the

Denver Head Start program. Am J Clin Nutr. 1976;29:734–8.

43. Golub MS, Gershwin ME, Hurley LS, Saito WY, Hendrickx AG. Studies

of marginal zinc deprivation in rhesus monkeys. IV. Growth of infants

in the first year. Am J Clin Nutr. 1984;40:1192–202.

44. Hoyenga KB, Hoyenga KT. Gender and energy balance: sex differences

in adaptations for feast and famine. Physiol Behav. 1982;28:545–63.

45. Casabiell X, Pineiro V, Vega F, De La Cruz LF, Dieguez C, Casanueva

FF. Leptin, reproduction and sex steroids. Pituitary. 2001;4:93–9.

46. Hamdi SA, Nassif OI, Ardawi MS. Effect of marginal or severe dietary

zinc deficiency on testicular development and functions of the rat. Arch

Androl. 1997;38:243–53.

Maternal zinc deficiency and insulin resistance 1627

at UN

IV O

F P

RIN

CE

ED

WA

RD

ISLA

ND

on Decem

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nloaded from