Objective:

We investigated the long-term effects of maternal/postnatal magnesium (Mg) restriction on adiposity, glucose tolerance, and insulin secretion in the offspring and the probable biochemical mechanisms associated with them.

Methods and Procedures:

Female weanling Wistar/NIN (WNIN) rats received a control diet or 70% Mg-restricted (MgR) diet for 9 weeks and mated with control males. A third of the restricted dams were shifted to control diet from parturition. Half of the pups born to the remaining restricted dams were weaned on to control diet, while the other half continued on MgR diet. Various parameters were determined in the offspring at 18 months of age.

Results:

The percentage of body fat increased, lean body mass (LBM) and fat free mass (FFM) decreased in restricted offspring and were irreversible by rehabilitation. While glucose tolerance and insulin resistance (IR) were comparable among groups, glucose-stimulated insulin secretion and basal glucose uptake by the diaphragm were significantly decreased in restricted offspring and not corrected by rehabilitation. Plasma leptin was lower, and tumor necrosis factor- (TNF-) was higher in restricted offspring, whereas expression of fatty acid synthase (FAS) and fatty acyl transport protein 1 (FATP 1) was higher in liver and adipose tissue. While changes in FAS and FATP 1 were not correctible by rehabilitation, those in leptin and TNF- were corrected by rehabilitation from parturition but not from weaning. Tissue oxidative stress and antioxidant status were comparable among groups.

Discussion:

Results indicate that maternal and postnatal Mg status is important in the long-term programming of body adiposity and insulin secretion in rat offspring.

Introduction

Maternal undernutrition and the consequent low birth weight predispose the offspring to various diseases, including insulin resistance (IR) syndrome in adult life. We reported earlier that maternal vitamin/mineral restriction altered the percentage of body fat, plasma lipid, and insulin levels (1,2), suggesting their predisposal to IR syndrome. This was associated with modulations in oxidative stress and adipocytokine levels in the offspring. We also reported that maternal mineral restriction-induced alterations in the percentage of body fat and fat/glucose metabolism in the offspring were mostly irreversible, whereas the alterations due to vitamin restriction were reversible, at least partially, by rehabilitation from parturition but not from weaning (1,2). Therefore it was considered pertinent to identify the mineral(s) important in the fetal programming for adiposity and IR in the offspring.

Deficiencies of minerals such as iron, calcium, and trace elements are common among our population (3). Despite universal supplementation of iron and folate during pregnancy, the incidence of low birth weight continues to be high (33% ) among Indians (4), and the prevalence of high body adiposity, IR and its associated diseases is on the rise in India (5). Therefore maternal mineral deficiency was considered to be a factor responsible for this. Hence, we initiated studies to identify the causative minerals and assess the effect of maternal mineral deficiency on the fetal programming for adiposity/IR.

Magnesium (Mg), an essential micromineral, is an important player in the structure and metabolism of the organism. Disturbances in Mg metabolism are widely recognized in IR and diabetes (6,7). Indeed, hypomagnesaemia and IR have been shown to be causally related both ways (8). Also, it is well documented that Mg modulates the synthesis, storage, and conformational integrity of insulin (9). Moreover, incidence of hypomagnesaemia in diabetes mellitus is reported to be 25–39% and Mg deficiency in Indian pregnant women is reported to be 45% (6,10). Recently, Takaya et al. (11,12) showed that intracellular Mg in cord blood platelets was lower in small for gestational age group than in those appropriate for gestational age and suggested that intrauterine Mg deficiency may result in intrauterine growth retardation and program the offspring for metabolic syndrome after birth. Therefore, we assessed whether chronic maternal dietary Mg restriction programs the offspring for adiposity and IR in later life.

Abundant literature indicates that increased adiposity precedes the development of IR and associated diseases, including type 2 diabetes (13,14,15). In line with these reports, we reported earlier that maternal Mg restriction increased the percentage of body fat in Wistar/NIN (WNIN) rat offspring at 3 months of age whereas IR was not manifest until 6 months of age (16). Whether these early changes will persist and/or precipitate type 2 diabetes in the offspring at later time points was assessed in this study. Also, the probable biochemical mechanism(s) underlying/associated with these changes were delineated in the offspring at 18 months of age.

Methods and Procedures

Animals: feeding, maintenance, and breeding

The animal experiments were conducted in adherence to the "principles of laboratory animal care" (National Institutes of Health publication no. 85-23, revised 1985) and with the approval of the Institute's ethical committee on animal experiments at National Institute of Nutrition, Hyderabad, India.

Female, albino, weanling WNIN rats (n = 28) were obtained from National Center for Laboratory Animal Sciences, National Institute of Nutrition (Hyderabad, India). They were divided into two groups of 7 and 21, housed individually in polypropylene cages with wire mesh bottom and maintained at 22 2 °C, under standard lighting conditions (12-h light/dark cycle). The group of 21 rats was fed a basal diet (AIN–93G) (17) containing 165 mg magnesium/kg diet (Mg restricted—MgR) for 9 weeks. The other group of seven rats received the same diet as above but containing 650 mg magnesium/kg diet (Mg control—MgC). Rats were mated with control males and maintained on their respective diets throughout pregnancy. Mg restriction in the diet did not influence food intake or reproductive performance (16). A subgroup (n = 7) of MgR rats was shifted to the control diet on the day of parturition and their offspring from weaning and these rats are termed as MgSP (Mg supplemented from parturition). The other group of MgR dams (n = 14) was continued on the same diet throughout lactation. The male offspring of these dams were either weaned on to control (MgSW—Mg supplemented from weaning) or MgR diets. Figure 1 gives the schematic representation of the feeding protocol used. Each group comprised eight male offspring from at least four separate litters from weaning and the various physical, physiological, and biochemical parameters were studied when they were 18 months old. Weekly body weights were recorded in the offspring, and at the end of 18 months, body length was measured as the length between the tip of nose and the center of the anus. BMI was calculated from the formula: BMI = body weight in kg/body length in m².

Figure 1.

Schematic representation of the feeding protocol of different groups of mothers and the offspring. Mg, magnesium; MgC, control diet throughout; MgR, Mg restriction throughout; MgSP, Mg rehabilitation to restricted dams from parturition and pups from weaning; MgSW, Mg-restricted pups weaned onto control diet; WNIN, Wistar/NIN.

Full figure and legend (10K)

The animals were killed by carbon dioxide inhalation. Liver and fat depots were dissected out immediately, washed thoroughly with ice-cold phosphate-buffered saline, frozen in liquid nitrogen and stored at –80 °C until the analysis. Glucose uptake was measured in freshly collected diaphragms as described below.

Body composition

Body composition of the offspring was determined using total body electrical conductivity small animal body composition analysis system (Model SA–3000 Multidetector; EM-SCAN, Springfield, IL), as described by us earlier (1,2). Body composition parameters (percentage of body fat, lean body mass (LBM), and fat free mass (FFM)) were computed mathematically according to Morbach and Brans (18).

Plasma lipid profile and Mg levels

Total cholesterol, high-density lipoprotein cholesterol, and triglycerides were measured in fasting plasma using enzymatic kits (Biosystems, Barcelona, Spain), whereas plasma-free fatty acids were measured using an enzymatic kit (Randox Laboratories, Antrim, UK). Mg concentrations were determined in fasting plasma by atomic absorption spectroscopy.

Plasma adipokine levels

Concentrations of leptin and tumor necrosis factor- (TNF-) were determined in fasting plasma using rat-specific enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN). Plasma adiponectin was determined by rat-specific RIA kit (Linco Research, St. Louis, MO). The lower limit of detection was <22 pg/ml for leptin, 5 pg/ml for TNF-, and 1 ng/ml for adiponectin. The plasma samples were appropriately diluted in order to measure the samples within the range of the standard curve.

Expression of fatty acid synthase and fatty acyl transport protein 1 in liver and adipose tissue

Approximately 1 g liver was weighed out, minced, and homogenized (10% wt/vol) in 50 mmol/l phosphate buffer (pH = 7.0). The homogenate was centrifuged at 1,000 g for 15 min at 4 °C, and the supernatant was further centrifuged at 20,000 g for 20 min at 4 °C to obtain the post mitochondrial supernatant. This supernatant was centrifuged at 120,000 g for 60 min to obtain cytosol. Whereas the adipose tissue lysate was prepared by incubating 500 mg of tissue in 2 ml lysis buffer (20 mmol/l Tris–HCl pH 7.5, 150 mmol/l NaCl, 1 mmol/l Na₂EDTA, 1 mmol/l ethylene glycol tetraacetic acid, 1% Triton, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l -glycerophosphate, 1 mmol/l sodium orthovanadate, 1 g/ml leupeptin, and 1 mmol/l phenylmethylsulphonyl fluoride), followed by centrifugation at 12,000 g for 15 min at 4 °C, the protein content of the liver cytosol and the adipose tissue lysate was determined by the bicinchoninic acid method (19).

Fatty acid synthase (FAS) and fatty acyl transport protein 1 (FATP 1) levels were determined in the liver cytosol and the adipose tissue lysate by western blotting using anti-FAS and anti-FATP 1 goat antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Following incubation with anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA), the signals were quantified in a gel documentation system using Quantity One software (Bio-Rad Laboratories, Hercules, CA). The concentrations were normalized with those of -actin (Sigma, St. Louis, MO).

IR and glucose tolerance

An intraperitoneal glucose tolerance test was performed in the overnight fasted offspring by administering glucose (250 g/l) intraperitoneally as a bolus, at a dose of 1 g/kg body weight. Blood samples were obtained from supraorbital sinus at 0, 15, 30, 60, and 120 min for the determination of plasma glucose and insulin concentrations. Glucose was estimated using an enzymatic kit (Biosystems), and plasma insulin was measured using a radioimmunoassay kit (Board of Radiation and Isotope Technology (BRIT), Mumbai, India).

IR was assessed from fasting glucose and insulin concentrations by computing the homeostasis model assessment (HOMA)-IR value as: HOMA-IR = (fasting insulin (U/ml) fasting glucose (mmol/l))/22.5. To assess the animals' insulin response to a challenge of glucose, the area under the curve (AUC) for insulin and glucose during the glucose tolerance test was computed and the ratio of AUC glucose:AUC insulin was calculated.

Glucose uptake by diaphragm

Glucose uptake was measured using the nonmetabolizable glucose analogue 3-O-methylglucose, 3-OMG (20,21). After rinsing the freshly dissected hemidiaphragms in Krebs Ringer bicarbonate buffer, they were incubated with (or without) 10 mol/l insulin for 30 min by shaking at 30 °C in 4 ml Krebs Ringer bicarbonate buffer, containing 0.1% bovine serum albumin and 2 mmol/l pyruvate. The hemidiaphragms were then blotted on filter paper and incubated in 3 ml Krebs Ringer bicarbonate buffer, containing 8 mmol/l (³H) 3-OMG for 15 min, during which period the 3-OMG accumulation rate was linear. Following incubation, the hemidiaphragms were blotted on filter paper dampened with incubation medium, trimmed, and frozen in liquid N₂. The muscle samples were weighed, homogenized in 10% trichloroacetic acid, and centrifuged at 1,000g. Aliquots of the muscle extracts were counted with channels preset for ³H counting. The amount of isotope present in the samples was determined and the concentration of 3-OMG was calculated.

Oxidative stress and antioxidant status

Lipid peroxidation (malondialdehyde levels), protein carbonyls, reduced/oxidized glutathione (GSH/GSSG), and activity of catalase were determined in the postmitochondrial supernatant (20,000g) of the liver, whereas the activities of superoxide dismutase and glutathione peroxidase were determined in the cytosol (120,000g supernatant) fraction, as described by us earlier (1,2).

Statistical analysis

Data were subjected to statistical analysis using SPSS package (version 10.0; SPSS, Chicago, IL). All values are presented as mean s.e.m. Data were analyzed using one-way ANOVA followed by Post hoc least significant difference tests. Wherever the heterogeneity was observed in the variance, differences between groups were tested by the nonparametric Mann–Whitney U-test. The differences were considered significant only if P < 0.05.

Results

Growth characteristics of the offspring

MgR offspring weighed lower than MgC at 18 months of age and the offspring of MgR mothers shifted to control diet from parturition (MgSP) or MgR pups weaned on to control diet (MgSW) caught up with controls (Table 1). Body length was comparable among the four groups. Accordingly, BMI was significantly lower (P < 0.05) in MgR offspring than in MgC and both the rehabilitation regimens corrected the changes in BMI (Table 1). As expected, MgR offspring had significantly lower plasma Mg levels (P < 0.05) at 18 months of age, compared to controls, while MgSP and MgSW caught up with controls (Table 1).

Table 1 - Body weights, BMI, and magnesium levels of 18-month-old rat offspring of different groups.

Full table (27K)

Body composition of the offspring

MgR offspring had a significantly higher (P < 0.001) percentage of body fat than MgC at 18 months of age, while their LBM and FFM (Figure 2) were significantly lower (P < 0.001). MgSP, but not MgSW, could mitigate changes in the percentage of body fat, LBM, and FFM, albeit partially (Figure 2). In line with total body electrical conductivity measurements, MgR, MgSP, and MgSW offspring had significantly higher (P < 0.01) wet weights of all three major visceral fat deposits (epidydimal, retroperitoneal, and mesenteric) compared to controls (Table 2). As a result, the adiposity index (AI = (sum of the weights of the three fat deposits/body weight) 100) was also significantly higher (P < 0.001) in these offspring compared to controls (Table 2).

Figure 2.

(a) Percentage of body fat, (b) lean body mass (LBM), and (c) fat free mass (FFM) of the offspring at 18 months of age as determined by total body electrical conductivity. Each bar represents a mean s.e.m. (n = 6). Means with different superscripts are significantly different by one-way ANOVA at P < 0.001.

Full figure and legend (14K)

Table 2 - Wet weights of different fat deposits and adiposity index in the rat offspring of different groups at 18 months of age.

Full table (31K)

Plasma lipid profile

Plasma total cholesterol, high-density lipoprotein cholesterol, free fatty acids, and triglycerides were comparable among the offspring of different groups at 18 months of age (data not shown).

Plasma adipokine levels

Although MgSW had the lowest adiponectin levels (Figure 3a), the values were comparable among the groups. Leptin levels were significantly (P < 0.05) lower in MgR than in MgC (Figure 3b). MgSP, but not MgSW, corrected the changes in leptin levels. On the other hand, plasma TNF- levels were higher in MgR than in MgC, although the difference was not significant (Figure 3c). Interestingly, MgSW, but not MgSP, significantly increased the levels further (Figure 3c).

Figure 3.

(a) Plasma adiponectin, (b) leptin, and (c) tumor necrosis factor- (TNF-) (adipocyte lysate) levels in the rat offspring of different groups in 18-month-old offspring. Each bar represents a mean s.e.m. (n = 6). Means with different superscripts are significantly different by one-way ANOVA at P < 0.05.

Full figure and legend (10K)

Expression of FAS and FATP 1 in liver and adipose tissue

Chronic maternal Mg restriction resulted in a significant (P < 0.05), 150–200% increase in FAS expression in liver cytosol (Figure 4a) and a similar (150% ) increase in the adipose tissue (Figure 4b). Both the rehabilitation regimes could not mitigate the changes in FAS expression in both the tissues (Figure 4a,b). In line with these results, expression of FATP 1 was significantly increased in adipose tissue of MgR offspring and both the rehabilitation regimes could not correct the changes (Figure 4c).

Figure 4.

Representative blots showing (a) fatty acid synthase (FAS) in liver, (b) FAS in adipose, and (c) fatty acyl transport protein 1 (FATP 1) expression in adipose tissue of 18-month-old offspring. -Actin was used as loading control protein. In the graph, results represent mean s.e. (n = 4) of ratios of specific protein levels to -actin (arbitrary unit(s)). Means with different superscripts are significantly different by one-way ANOVA at P < 0.05.

Full figure and legend (42K)

IR, glucose tolerance, and glucose-stimulatedinsulin secretion

Although fasting plasma insulin levels and HOMA-IR were higher in MgSP and MgSW than in MgC and MgR offspring (Table 3), the differences were not significant. In line with fasting glucose values, glucose AUC during glucose tolerance test was comparable among the four groups. However, insulin AUC during glucose tolerance test was significantly lower (P < 0.05) in MgR than in MgC, and the two rehabilitation regimes had little effect on this parameter. Although not significant, insulin AUC was further decreased in MgSW compared to MgR and MgSP offspring. As a consequence, the ratio of glucose AUC to insulin AUC was significantly higher (P < 0.05) in MgR than in MgC at 18 months of age. While MgSP did not affect this parameter, MgSW further increased it albeit not significantly (Table 3).

Table 3 - Glucose tolerance, insulin resistance (IR), and glucose-induced insulin secretion in 18-month-old rat offspring of different groups.

Full table (42K)

Glucose uptake by diaphragm

The basal glucose uptake was significantly (P < 0.01) decreased (by 63% ) in MgR compared to MgC offspring, and the two rehabilitation regimes had no corrective effect (Table 4). Absolute values for insulin-stimulated glucose uptake were comparable between MgC and MgSW offspring. Although lower than MgC, the value for MgSP, but not MgR, was statistically significant (P < 0.01). Despite considerable variation, the percent increase in insulin-stimulated glucose uptake was not significantly different among the groups (Table 4) probably due to the small sample size (n = 4).

Table 4 - Glucose uptake in diaphragm of 18-month-old offspring.

Full table (28K)

Oxidative stress and antioxidant defense

Neither maternal Mg restriction nor rehabilitation affected malondialdehyde, protein carbonyls, and glutathione (reduced and oxidized) levels in the liver homogenate of the offspring (Table 5). In line with these observations, activities of the antioxidant enzymes: catalase, superoxide dismutase, and glutathione peroxidase were comparable among the groups (Table 5).

Table 5 - Oxidative stress and antioxidant status in liver homogenate of offspring of different groups at 18 months of age.

Full table (48K)

Discussion

Mg deficiency modulates muscle development/contraction (22), bone density (23), and insulin sensitivity and is associated with impaired insulin secretion (6,7,8,9). We reported earlier altered body composition and plasma triglycerides in MgR offspring at 3 months of age and IR at 6 months of age (16). Long-term consequences of maternal MgR in the offspring and the associated biochemical mechanism(s) are reported here.

IR is hypothesized to originate in impaired adipogenesis and/or lipid metabolism (13,24), which precede it (13,14,15). In line with our earlier findings (16), we now report that high body fat content persisted in MgR offspring at 18 months of age, indicating the importance of maternal Mg status in long-term modulation of adiposity in the offspring. Together with the persistent decrease in LBM and FFM (representing muscle and bone mass), these results corroborate previous reports that maternal Mg deficiency modulates fetal growth and its body composition (22,23) and indicate that it programs adipose, muscle (and bone) mass of the offspring. These changes in body fat content, but not LBM and FFM, could be mitigated, albeit partially, by MgSP but not MgSW, suggesting the importance of Mg nutrition during lactation in programming the body composition of the offspring. The increased wet weight of the visceral fat deposits in MgR offspring appears to be a reason for their increased body fat content. The low body weight with high body fat, specially the visceral fat, seen in MgR offspring is akin to those in "thin fat babies" in India, an abnormal condition ascribed to maternal malnutrition (25), specially gestational protein and iron deficiencies (26). Despite increasing body fat content, maternal MgR did not affect plasma lipid profile in the offspring and reasons for this discrepancy are not clear.

Adiponectin, leptin, and TNF- modulate lipid metabolism and transport and are associated with pathogenesis of obesity and IR (27,28,29). Plasma adiponectin levels were comparable among the groups. Plasma TNF- levels were not different between MgR and MgC offspring, but MgSW significantly increased them. The finding that plasma TNF- levels followed a similar trend as earlier reports (28), taken together with comparable adiponectin levels probably suggests that TNF- and not adiponectin may be involved in maternal MgR-induced changes in body fat content of the offspring. The hypoleptinemia seen in MgR offspring, which had high body fat content is at variance with the reported positive correlation between leptin and body fat content (27). However, our findings agree with leptin deficiency reported in genetically obese rodent models (30,31). The restoration of plasma leptin levels, but not body fat content, by MgSP probably suggests that both hypoleptinemia and leptin resistance may be involved in maternal MgR-induced changes in body fat content of the offspring. Further studies are clearly needed to resolve the role of adipokines in this regard.

In the presence of NADPH, FAS mediates long-chain fatty acid synthesis (32,33,34), which provide energy to most organisms. FATPs facilitate long-chain fatty acid transport through plasma membrane (35,36), and FATP 1 is expressed maximally in muscle and adipose tissue (37). Blocking FATP 1 function helps control obesity, which can cause diabetes and hypertension (35,37,38). Significant increase in FAS and FATP 1 levels in liver and adipose tissue of MgR offspring indicates the probable involvement of increased fatty acid synthesis and transport in maternal MgR-induced increase in central adiposity and body fat content in the offspring. That both the rehabilitation regimes could not mitigate these changes indicates their irreversibility and also corroborates the above inference.

The increased fasting insulin and HOMA-IR seen in MgR offspring at 6 months of age (16) did not persist at 18 months, indicating the transient nature of IR induced in the offspring by maternal MgR and its probable inability to sustain IR in later life. However, low insulin AUC persisted in MgR offspring at 18 months of age and neither rehabilitation regime could mitigate the change. These results perhaps indicate that maternal MgR and its postnatal continuation irreversibly impair the offspring's insulin response to a glucose challenge. That MgSW offspring had the lowest insulin AUC probably corroborates the importance of Mg nutrition during lactation in modulating this function. Typical of type 2 diabetes (39), these observations are in line with reported effects of Mg deficiency on glucose-stimulated insulin secretion (40).

Basal glucose uptake by diaphragm was decreased irreversibly in MgR offspring. There were no differences among the groups in the percent increase of glucose uptake by insulin over basal uptake, but the small sample size (n = 4) does not appear to justify any decisive inference. Nonetheless, the persistent, irreversible decrease in LBM and FFM in MgR, MgSP, and MgSW offspring, together with decreased basal glucose uptake by diaphragm, may suggest that maternal MgR irreversibly decreased muscle mass and function (glucose uptake) in the offspring.

Oxidative stress is important in the etiology of adiposity and IR (41,42) and many trace elements influence oxidative stress/antioxidant status in experimental animals (4344,45). In line with our earlier observations (1), oxidative stress and antioxidant status were comparable among offspring of different groups, suggesting neither their association nor involvement in maternal MgR-induced changes in the offspring.

We have shown that maternal Mg restriction irreversibly increased the body fat content of the offspring and this was associated with increased visceral adiposity, synthesis, and transport of fatty acids. It may also irreversibly decrease muscle mass and basal glucose uptake by muscle. Although plasma TNF- and leptin levels were modulated, their role in maternal MgR-induced changes in the body fat of the offspring needs to be deciphered. It modulated fasting insulin levels in the offspring transiently but irreversibly impaired their insulin response to a glucose challenge. That rehabilitation from parturition, but not weaning, could mitigate some changes highlight the importance of Mg nutrition during lactation in modulating these changes. Overall, changes induced in the offspring by maternal MgR could result in hyperglycemia.

We thank Dr Giridharan and Dr Harishankar for help in body composition measurement by total body electrical conductivity; Dr Prasanna Krishna for statistical analysis; Mrs Indira Ravindranath, Mr Ch Narasimha Rao, and Mr Krishnakanth for technical help. 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). L.V. was supported by a senior research fellowship from the Council for Scientific and Industrial Research, India. This was presented, in part, in abstract form at the 3rd International Congress of Developmental Origins of Health and Disease, November 2005 at Toronto, Canada (Abstract No P3-104 Pediatric Research 58(5): 1127) and at the XXXVIII Annual Meeting of the Nutrition Society of India, November 2006 at Kolkata, India.

Disclosure

The authors declared no conflict of interest.

References

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