Human adult diseases such as cardiovascular disease, hypertension, and type 2 diabetes have been epidemiologically linked to poor fetal growth and development. Male offspring of rat dams fed a low-protein (LP) diet during pregnancy and lactation develop diabetes with concomitant alterations in their insulin-signaling mechanisms. Such associations have not been studied in female offspring. The aim of this study was to determine whether female LP offspring develop diabetes in later life. Control and LP female offspring groups were obtained from rat dams fed a control (20% protein) or an isocaloric (8% protein) diet, respectively, throughout pregnancy and lactation. Both groups were weaned and maintained on 20% normal laboratory chow until 21 mo of age when they underwent intravenous glucose tolerance testing (IVGTT). Fasting glucose was comparable between the two groups; however, LP fasting insulin was approximately twofold that of controls (P < 0.02). Glucose tolerance during IVGTT was comparable between the two groups; however, LP peak plasma insulin at 4 min was approximately threefold higher than in controls (P < 0.001). LP plasma insulin area under the curve was 1.9-fold higher than controls (P < 0.02). In Western blots, both muscle protein kinase C-ζ expression and p110β-associated p85α in abdominal fat were reduced (P < 0.05) in LPs. Hyperinsulinemia in response to glucose challenge coupled with attenuation of certain insulin-signaling molecules imply the development of insulin resistance in LP muscle and fat. These observations suggest that intrauterine protein restriction leads to insulin resistance in females in old age and, hence, an increased risk of type 2 diabetes.
- early growth restriction
- protein kinase C-ζ
many epidemiological studies have revealed links between poor early human growth and susceptibility to type 2 diabetes, insulin resistance, and other features of the metabolic syndrome in adulthood (6). The mechanistic basis of this relationship is not known and the relative importance of genetic and environmental factors remains the subject of much debate. Rare mutations in the glucokinase gene have been shown to be associated to low birth weight and maturity-onset diabetes of the young 2 (10). However, a recent study of polymorphisms known to be associated with insulin resistance revealed no association with birth weight (14). The importance of the environment has been suggested in studies of individuals who were in utero during a period of famine (25). Further evidence for the importance of the environment has come from studies of twins (23, 2). The study of twins in Denmark showed that when there was discordance for type 2 diabetes, the diabetic twin had a significantly lower birth weight than the nondiabetic co-twin (23). A recent systematic review by Newsome et al. (15) concluded that the published literature supports the association between low birth weight and subsequent adverse effects on adult glucose and insulin metabolism, which related mainly to higher insulin resistance.
Several animal models have been established to investigate the possible role of the fetal environment in determining future susceptibility to adult disease and the underlying mechanisms. These include maternal protein restriction (19), maternal calorie restriction (3), maternal anemia (13), and intrauterine artery ligation (27) in the rat. The model of protein restriction is one of the most extensively studied, and the outcomes for offspring bear striking similarities to human diabetes both at the whole body and molecular level (9).
In animal studies, 20-wk-old male offspring of low-protein-fed rat dams (LP group) were found to be relatively insulin resistant and hyperinsulinemic compared with control animals, although the female offspring of the same age were not similarly affected (30). Fifteen-mo-old male offspring of LP-fed rat dams were shown to have impaired glucose tolerance compared with control offspring and increased insulin concentrations during the glucose tolerance test (18). In contrast, female LP-fed offspring of the same age had a similar glucose tolerance to the female control offspring and demonstrated no significant difference in insulin concentrations during the glucose tolerance test (7). Further studies in this laboratory showed that 17-mo-old male LP rats developed frank diabetes (21). As female rats live longer than males (7), it is conceivable that glucose intolerance develops much later in female LP rats. Therefore, we chose to study them at an age close to their normal life span, which is between 22 and 24 mo.
Therefore, the aim of the present study was to determine whether female LP offspring develop diabetes at 21 mo of age. Several molecular aberrations have been reported for the insulin-signaling pathway of male LP rats. For example, protein kinase C-ζ (PKC-ζ) is reduced in muscle of 15-mo-old LP animals (18) and p85α-associated p110β levels were markedly reduced in epididymal fat of LP males (17). Therefore, we measured these parameters in the female at old age (21 mo). In addition to this, we also investigated whether any changes in protein expression that we observed for the 21-mo-old female LP rats were present at an early age (21 days).
MATERIALS AND METHODS
Female Wistar rats were bred locally at a designated animal unit of the University of Cambridge (Cambridge, UK). Adult females weighing between 235 and 250 g were mated and assumed to be pregnant when a vaginal plug was expelled. They were then fed ad libitum either a control diet [containing 20% (wt/vol) protein] or an isocaloric low protein [8% (wt/vol)protein] diet (Hope Farms BV, Woerden, The Netherlands) during gestation and lactation [see Petry et al. (21) for composition of the diets]. Two days after birth, litter sizes were standardized to eight pups, four males and four females in a completely random manner. At 21 days of age, the offspring were weaned onto a standard rat diet (LAD1; from Special Diet Services, Witham, UK). The offspring, termed control and low-protein (LP) rats, according to the diet that their mothers received, remained on the LAD1 diet for the remainder of the study. All animal procedures were carried out in compliance with the United Kingdom Animal (Scientific Procedures) Act 1986.
Twenty-one-month-old female rats were anesthetized with halothane (Fluothane; Zeneca, Macclesfield, UK) (4% halothane in oxygen for inducing and 2% for maintaining anesthesia). Sterile catheters (Esco Rubber, 0.5 mm bore, Bibby Sterilin, Stone, UK) were placed bilaterally into the jugular veins. The distal ends of the catheters were tunneled subcutaneously and exteriorized at the nape of the neck. Each catheter was back-filled with heparinized saline (20 U/ml) and then plugged. To maintain patency, the heparin block was first aspirated off, the line flushed with saline, and then the heparin block reinstalled daily. The animals were allowed to recover until they appeared to have normal feeding, drinking, and grooming behavior. The intravenous glucose tolerance test (IVGTT) was performed 4 days after surgery, only on animals that had not lost more than 5% of their initial body weight postsurgery.
Glucose tolerance tests were performed on 11 control and 12 LP fully conscious, unrestrained rats at 21 mo of age. Food was removed from their cages overnight. The following day, 200 μl of a fasting sample of blood was collected into heparinized tubes for measurements of fasting glucose and insulin. A dose of 1 g/kg body wt of glucose was then immediately infused [as a 50% (wt/vol) solution] into the dosing catheter over a period of 30 s followed by flushing with 500 μl saline. Subsequently, 200-μl blood samples were taken and stored on ice at 1, 2, 4, 6, 8, 10, 13, 18, 24, 30, 45, 60, and 90 min after the glucose infusion. At 25 min, the samples from the first 10 time points were centrifuged, and the plasma was removed and flash-frozen for later analyses. The remaining red blood cells were mixed with an equal volume of saline and infused into the rat via the catheter at 46 min to avoid potential effects of hypovolemia or anemia. The blood samples from the later time points were also centrifuged, and the plasma was collected and frozen.
Peak insulin is defined as the mean highest concentration of insulin observed throughout the course of the IVGTT (means ± SE). Insulin area under the curve (AUC) is defined as the total area under the insulin curve for the entire duration of the IVGTT (expressed in min·nmol/l) (means ± SE). Glucose AUC is defined as the total area under the glucose curve for the entire duration of the IVGTT (expressed in min·mmol/l) (means ± SE). Insulin:glucose ratio is calculated by dividing total insulin AUC by the total glucose AUC.
Western Blotting and Immunoprecipitation
The 21-mo-old animals were killed by anaesthetic overdose immediately after the glucose tolerance test. Of the 11 control and 12 LP rats that underwent IVGTT, 8 rats from each group were selected randomly at postmortem for collection of soleus muscle, intra-abdominal fat, and liver samples for the Western blotting and immunoprecipitation procedures. Twenty-one-day-old females (n = 8) were killed by CO2 asphyxiation, and soleus muscle, intra-abdominal fat, and liver were also collected for Western blotting. Tissue samples were flash-frozen in liquid nitrogen and stored at −80°C. Total protein was extracted from each tissue sample by homogenizing in lysis buffer [50 mM HEPES pH 8, containing 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 30 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM EDTA with a protease inhibitor cocktail from Calbiochem (Calbiochem Novabiochem Biosciences, Nottingham, UK) to a final concentration of 0.5 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), 0.4 μM aprotinin, 25 μM bestatin, 1.5 μM E-64, 10 μM leupeptin and 5 μM pepstatin A]. The homogenate was centrifuged at 13,000 rpm in a microcentrifuge at 4°C, and the clear supernatant was removed for analysis. Protein content was measured using a bicinchoninic acid kit for protein determination from Sigma Chemical (Poole, Dorset, UK). The cleared protein lysates from each animal were then used either in immunoprecipitations (see below) or were standardized to a final concentration of 2 mg/ml in Laemmli’s sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.02% bromophenol blue, and 150 mM DTT). The procedure was repeated for each tissue analyzed. The protein samples were boiled for 5 min, and then 20 μg of the protein samples were loaded onto 10% SDS polyacrylamide gels for separation by electrophoresis overnight. The next day, the separated proteins were transferred onto polyvinylidene difluoride membrane (Immobilon-P, Millipore, Billerica, MA), according to the manufacturer’s protocol, and Western blotting was carried out as previously described (16). The antibodies used in this study were to insulin receptor (IR)-β subunit [sc-711 Santa Cruz Biotechnologies (Santa Cruz, CA)] phosphatidylinositol (PI) 3-kinase p110-β subunit [Santa Cruz for blotting and Upstate Biotech (Lake Placid, NY) for immunoprecipitations], (PI) 3-kinase p85α subunit (Upstate Biotech), GLUT 4 (Abcam Ltd, Cambridgeshire, UK), and PKC-ζ (Santa Cruz). Horseradish peroxidase-conjugated secondary antibodies to mouse and rabbit were obtained from Amersham Biosciences (Bucks, UK). Antibody binding was detected using the enhanced chemiluminescence kit from Amersham.
Measurement of p85α Association with p110β
Two-hundred fifty micrograms of protein was immunoprecipitated with 2.5 μl of PI 3-kinase p110β antiserum (Upstate Biotechnology) in a total of 250 μl overnight with mixing at 4°C. The following day, 40 μl of prewashed protein A sepharose beads in a slurry (Sigma Chemicals, Poole, Dorset, UK) was added. The captured antibody-p110β complex was then washed four times in radioimmunoprecipitation (RIPA) buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 158 mM NaCl, 1 mM EGTA, and 10 mM Tris, pH 7.2, with inhibitor cocktail) before a final resuspension in 50 μl 2× SDS-loading buffer. The mixture was then boiled and centrifuged. Immunoprecipitated and solubilized proteins were then subjected to SDS-PAGE and blotted using anti-p85α antibodies to detect p110β-associated p85α protein.
Plasma glucose was measured on a Dimension auto-analyser RxL-HM from Dade-Behring as part of routine assays carried out at Addenbrookes Hospital, Cambridge, using kits from Roche Diagnostics (GmBH, Germany). Plasma insulin concentrations were measured using a rat insulin ELISA kit (Mercodia Ultra-sensitive Rat Insulin ELISA, Mercodia AB, Uppsala, Sweden). All samples were assayed in duplicate and an intra-assay coefficient of variation of up to 5% was accepted. Autoradiographs of Western blots were imaged on the image analysis software AlphaImager 1,220 v 5.5, and the optical density of the immunoreactive protein bands was measured using AlphaEase gs 3.3b.
Plasma glucose and insulin measurements are presented as means ± SE, with n = 11 for control and n = 12 for LP rats in the glucose tolerance tests. In the Western blot analysis, data were obtained from eight control and eight LP randomly selected rat samples, which were run on the same gel. AUC measurements were made using Prism 4 graphing and statistical software (Statistical Solutions, Fulton, NY). Comparisons between groups were assessed by unpaired two-tailed t-tests using GraphPad Instat (Statistical Solutions). P values of <0.05 were considered statistically significant.
At birth, body weights of LP females were 81.5% that of control animals (P < 0.0001; Table 1). Before surgery, the body weights of the LP rats were significantly lower than the controls by 11.9% (P < 0.05; see Table 1). The LP group were lighter than the controls by 11.6% before fasting and 11.9% immediately before the IVGTT (see Table 1; all P < 0.05).
Fasting Plasma and Glucose Tolerance Data
Figures 1, A and B shows the glucose and insulin curves from the glucose tolerance tests, respectively. There was no significant difference in the fasting plasma glucose concentrations between the control and LP groups (Fig. 1A and Table 1), and their glucose curves are almost superimposable (Fig. 1A). Glucose AUC was also similar in both groups (Table 2).
Fasting levels of insulin were overall significantly higher in the LP group compared with the controls (Fig. 1B and Table 1) and remained consistently two- to threefold higher between 1 and 8 min after glucose (Fig. 1B; P < 0.05 at these time points). A broad shallow insulin peak for controls was observed between 1 and 10 min postglucose, whereas a sharp high peak was noted for the LP rats at 4 min. Insulin AUC and the amount of insulin secreted in response to glucose challenge (reported as insulin:glucose ratio AUC) were also significantly higher in the LP group (Table 2).
Protein Expression of Insulin-Signaling Molecules
Western blot analysis revealed that there were no significant differences between 21-mo-old control and LP groups in their muscle protein expression of the GLUT 4 transporter [52,021 ± 5,134 vs. 52,245 ± 3,713 arbitrary units (au) for control and LP groups, respectively], IRβ subunit (3,556 ± 245 vs. 3,237 ± 267 au for control and LP groups, respectively), or p85α subunit of PI 3-kinase (9,163 ± 1,062 vs. 7,282 ± 1,150 au for control and LP groups, respectively) (see Fig. 2A). However, LP muscle expressed less PKC-ζ protein compared with the controls (64,472 ± 2,350 vs. 59,755 ± 2,213 au for control and LP groups, respectively) (P = 0.046) (Fig. 2A). This difference was not present at 21 days of age (33,260 ± 1,109 vs. 30,406 ± 1,970 au for control and LP groups, respectively). Representative blots are shown in Fig. 2B.
IR protein expression was similar in the 21-mo-old control and LP groups (5,025 ± 544 vs. 4,914 ± 488 au for control and LP groups, respectively). GLUT 4 protein expression was also comparable between the control and LP groups (64,615 ± 5,634 vs. 58,516 ± 3,673 au, respectively) as was PKC-ζ expression (15,559 ± 1,465 vs. 17,622 ± 3,074 au for control and LP groups, respectively). The protein expression of the p85α subunit of PI 3-kinase was likewise similar for the control and LP groups (29,829 ± 4,145 vs. 33,233 ± 2,405 au for control and LP groups, respectively). The protein levels of the p110β subunit were also comparable in both groups (60,809 ± 4,963 vs. 60,169 ± 2,703 au for the control and LP groups, respectively). However, when we investigated the amount of p85α associated with p110β by immunoprecipitation, we observed a significant reduction in p85α bound to p110β in LP fat samples compared with controls (3,132 ± 389 vs. 1,512 ± 327 au for control and LP groups, respectively; P = 0.003). This reduction was not observed at 21 days of age (8,257 ± 974 vs. 9,245 ± 1,410 au for control and LP groups, respectively). Representative blots are shown in Fig. 3.
IRβ protein levels were significantly increased in the livers of the LP group at 21 mo of age, although the magnitude of the difference was very small (13,695 ± 226 compared with 14,784 ± 399 au in the control and LP groups, respectively; P = 0.032). PKC-ζ expression was comparable between the two groups (12,509 ± 808 vs. 13,841 ± 728 au in the control and LP groups, respectively). The protein expression of the p85α subunit of PI 3-kinase was not different between the two groups (42,378 ± 1,312 vs. 43,007 ± 1,672 au in the control and LP groups, respectively) while the protein expression of the p110β subunit showed a tendency to be reduced in the LP group (7717 ± 761 vs. 5,942 ± 803 au in the control and LP groups, respectively), although this was not significant (P = 0.13). The amount of p85α associated with p110β was not different between the two groups (10,351 ± 1,119 vs. 12,523 ± 1,664 au in the control and LP groups, respectively). Representative blots are shown in Fig. 4.
Early growth restriction by maternal protein restriction during pregnancy and lactation in the rat is well documented as a model of the effects of early growth restriction on type 2 diabetes and metabolic disease in the human situation (6, 15). In young adult life, LP offspring have a slightly improved glucose tolerance. By 20 wk of age, they have a similar glucose tolerance to controls (30); however, male LP rats undergo a greater age-dependent loss of glucose tolerance than controls. Female LP rats, in contrast, seem to maintain a similar fasting glucose and insulin concentrations to controls at 20 wk of age (30), and even up to 15 mo of age (7). In the current study, we have shown that at 21 mo of age, the female LP offspring still had a comparable glucose tolerance with female control offspring. However, the aged LPs demonstrated substantial (at least double) hyperinsulinemia in response to a glucose challenge. In addition, they expressed lower levels of PKC-ζ in their muscle, which implies a loss of glucose uptake and points to developing insulin resistance in this tissue (18). Although expression of p110β was not altered in abdominal fat, p110β association with its p85α regulatory subunit was reduced by half. This observed reduction in p110β-associated p85α could therefore also contribute to the observed insulin resistance.
In humans, low birth weight is associated with subsequent impaired glucose tolerance in both men and women of older age (22). Studies to date show that the LP model of early growth restriction results in a phenotype that mirrors the epidemiological association between low birth weight and subsequent development of impaired glucose tolerance and type 2 diabetes in males. However, the phenotype is not so severe at a comparable age in the female rats, such that at around their average age of death, only hyperinsulinemia is apparent. Nevertheless, the underlying molecular basis of the insulin resistance in female LP offspring may be the same as that observed for males. As in human insulin resistance, the molecular defect appears to lie downstream of the IR in peripheral tissues (11, 24). There was no difference in expression of the IR between control and LP offspring in either muscle or adipose tissue. GLUT 4, the major insulin-responsive glucose transporter, was also expressed at similar levels in muscle from control and LP offspring. This is again consistent with findings in human insulin-resistant/diabetic muscle, which report no difference in total expression of GLUT 4 (20, 4). There may, however, be a difference in its subcellular distribution (5, 32). We recently reported a reduction in the protein expression of PKC-ζ in muscle from male LP offspring (18). We report here that expression levels of this protein were also reduced in the female LP offspring. PKC-ζ is a member of the atypical family of PKC, which is thought to play an important role in mediating the action of insulin to stimulate glucose uptake (1, 28). PKC-ζ expression is also reduced in muscle from persons suffering from type 2 diabetes (12, 31). Hence, reduced expression of PKC-ζ is a common feature between the LP rat model and human diabetes. In the female offspring, this reduction in expression was observed in the absence of glucose intolerance, suggesting that this reduction is not a secondary consequence of hyperglycemia but may well predict increased susceptibility to diabetes. However, it may be a consequence of hyperinsulinemia as no difference was observed in PKC-ζ expression in young LP offspring (before hyperinsulinemia).
We have shown previously that p85α-associated p110β levels of PI-3-kinase are markedly reduced in epididymal fat from male LP offspring (17). In the current study, we showed that the amount of p85α-associated p110β protein in intra-abdominal fat of 21-mo-old female offspring was reduced by half. The association of p85α regulatory subunit with the p110β catalytic subunit of PI 3-kinase has been shown to be crucial for many of the metabolic activities of insulin (26). Impaired PI 3-kinase activity has been reported in the muscle of diabetic patients and their first-degree relatives, who were prediabetic (29), which suggests that it may be a potential marker for insulin resistance. As for PKC-ζ in soleus, this reduction was also not observed in the young LP females at 21 days of age. Likewise, this suggests that the reduction in these components of PI 3-kinase follows an age-related pattern. This study therefore demonstrates that maternal protein restriction in the rat is associated with hyperinsulinemia and reduced expression of specific insulin-signaling proteins in female offspring. Hence, the association of the reduction of these proteins with low birth weight suggests that they may be useful markers of diabetes risk.
This study was supported by the National Institutes of Health (grant AG-20608–02), the Parthenon Trust, the Wellcome Trust, and the British Heart Foundation.
We thank A. Flack and D. Hutt for expert technical assistance and staff at the Department of Clinical Biochemistry, Addenbrookes National Health Service Trust, Cambridge, for performing the glucose and lipid analyses.
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