We have developed a model of gestational diabetes in the rat to determine whether an altered metabolic intrauterine milieu is directly linked to the development of diabetes later in life. Uteroplacental insufficiency is induced in the pregnant rat on day 19 of gestation. Sham-operated animals serve as controls. Offspring are growth retarded at birth; however, they catch up by 5–7 weeks of age. At ∼8 weeks of age, they are bred to normal males. During pregnancy, these animals develop progressive hyperglycemia and hyperinsulinemia accompanied by impaired glucose tolerance and insulin resistance. Offspring, designated as infants of a diabetic mother (IDMs), are heavier at birth and remain heavy throughout life. IDMs are insulin resistant very early in life, and glucose homeostasis is progressively impaired. Defects in insulin secretion are detectable as early as 5 weeks of age. By 26 weeks of age, IDMs are overtly diabetic. These data demonstrate that the altered metabolic milieu of the diabetic pregnancy causes permanent defects in glucose homeostasis in the offspring that lead to the development of diabetes later in life.

In humans, diabetic pregnancy induces marked abnormalities in glucose homeostasis and insulin secretion in the fetus that result in aberrant fetal growth (1). Population-based studies have also demonstrated long-term consequences for the offspring of gestational diabetic mothers. These progeny have an increased risk for obesity, glucose intolerance, and type 2 diabetes in later childhood and as adults (25). A genetic contribution to the development of diabetes is commonly assumed, but it is difficult to know the extent to which intrauterine metabolic perturbations rather than inherited genotype contribute to diabetic transmission via the mother. Animal models of diabetes during pregnancy can help pinpoint the specific effects of exposure to an abnormal intrauterine metabolic milieu, independent of inherited traits. Three experimental approaches have been widely used to generate hyperglycemia during pregnancy. High-dose streptozotocin (STZ) given during gestation induces marked maternal and fetal hyperglycemia. However, unlike human infants of gestational diabetic mothers, these rat fetuses and newborns are growth retarded and have low pancreatic insulin content and low plasma levels of insulin (69). In contrast to high-dosage effects of STZ, low dosage of the drug induces fetal macrosomia, and birth weights are increased. However, the effects on fetal growth, pancreatic insulin content, and plasma levels of insulin are highly variable (811). The third method was introduced by Picon’s group in France. Glucose infusions were used to induce hyperglycemia during the last days of gestation in the rat (1215). The offspring are macrosomic, and in adulthood, they have very mildly elevated levels of glucose and insulin in the fed state (1215).

Despite the different techniques used to induce hyperglycemia during pregnancy and the variability in outcome of these models, the offspring all develop either defects in insulin secretion or insulin action later in life (8,9,1119). These studies unequivocally demonstrate that there are long-term and persistent effects of gestational diabetes on glucose homeostasis in the offspring.

In previous studies, we developed a model of uteroplacental insufficiency (intrauterine growth retardation) in the rat that produces diabetes in mature offspring (20). Intrauterine growth-retarded (IUGR) rats develop diabetes in middle age (5–6 months), with a phenotype remarkably similar to that observed in humans with type 2 diabetes: progressive dysfunction in insulin secretion and insulin action. Interestingly, pregnancy induces diabetes at a much earlier age. This model of gestational diabetes (onset of diabetes during pregnancy) circumvents many of the difficulties with the animal models outlined above and produces overgrown offspring that develop β-cell secretory defects and insulin resistance early in life. We hypothesize that the abnormal milieu of diabetes in pregnancy, in the face of a normal genetic background, leads to the development of diabetes in the offspring. We propose that the use of this model will elucidate mechanisms involved in the pathogenesis of altered glucose homeostasis in individuals who were subjected to gestational diabetes as fetuses.

Animals.

We have described our surgical methods previously (20,2123). In brief, time-dated Spraque-Dawley pregnant rats (Charles River) were individually housed under standard conditions and allowed free access to standard rat chow and water. On day 19 of gestation (term is 21.5 days), the maternal rats were anesthetized with intraperitoneal Xylazine (8 mg/kg) and Ketamine (40 mg/kg) and both uterine arteries were ligated (IUGR rats). Control animals underwent the identical anesthetic and surgical procedure except for ligation (sham-operated rats). Rats recovered within a few hours and had ad libitum access to food and water. The pregnant rats were allowed to deliver spontaneously (n = 3 for each group), and the litter size was randomly reduced to eight at birth to assure uniformity of litter size between IUGR and control litters. The pups were fostered to unoperated normal female rats and remained with their foster mothers until they were weaned. At 2 months of age, 16 IUGR and 16 control females were randomly picked from the original litters and were bred to normal unoperated male rats. The resultant litters were culled to eight at birth and designated as infants of a diabetic mother (IDMs). Offspring from normal pregnant rats were used as controls.

Assays.

Blood glucose concentrations were determined in duplicate using the Freestyle glucose meter (Angelholm, Sweden), which only requires 3 μl blood. Plasma insulin concentrations were measured in duplicate by radioimmunoassays using rat insulin as the standard (Linco, St. Louis, MO). The within- and between-assay coefficients of variation for the insulin assay were 4 and 10%, respectively. For fetal samples, blood was pooled from one litter. Triglyceride concentrations were determined using a Sigma kit (Sigma, St. Louis, MO).

Glucose and insulin tolerance tests.

Glucose tolerance was serially investigated in IUGR and control pregnant rats, and their offspring (IDMs and controls). Glucose (2 g/kg) was injected intraperitoneally in awake fasted rats. Blood samples were collected via tail vein sequentially before and 60, 120, and 180 min after injection. Insulin tolerance tests were also performed serially in IUGR and control pregnant rats and their offspring. Next, 1 unit/kg insulin (Humulin R; Eli Lilly, Indianapolis, IN) was injected subcutaneously. Blood samples were collected via tail vein at 0, 20, 40, and 60 min.

Glucose uptake.

Glucose uptake was measured in epitrochlearis muscles of IDM and control animals using the glucose analog 2-deoxyglucose (2-DG) and the procedure of Young et al. (24).

Western blot analysis.

Membrane proteins were prepared from muscle tissue (25) of IDMs and controls. Protein concentration was determined by the method of Lowry et al. (26). Protein from muscle (50 μg) was loaded in duplicate onto a discontinuous 12% polyacrylamide gel and size fractionated. Colorimetric detection of GLUT was performed using a double-antibody system and the Immuno-Blot assay kit (Biorad) or the Amersham biotinylated detection system (Arlington Heights, IL). The primary antibody (GLUT1 and -4; East Acres Biologicals, Southbridge, MA) was diluted 1:5,000. The second antibody was diluted 1:3,000 before use. Resulting signals were quantitated by densitometry.

β-Cell mass.

For immunohistochemistry, five animals from each group were analyzed at 1, 7, 15, and 26 weeks of age. Pancreas was harvested, weighed, and fixed in 4% paraformaldehyde before embedding in paraffin. Histological sections of pancreas (5 μm) were cut from paraffin blocks and mounted on glass microscope slides. Each block was serially sectioned throughout its length to avoid any bias due to regional change in islet distribution and cell composition. Immunohistochemistry was performed on insulin within islets by a modified avidin-biotin peroxidase method. Sections were incubated with primary antibody (polyclonal guinea pig anti-porcine insulin antibody) at 1:300 dilution for 1 h at room temperature. Then, sections were immunoperoxidase labeled with the Vectastain system (Vector Labs, Burlingame, CA), developed with 3,3′ diaminobenzidine (DAB; Amersham, Arlington Heights, IL), and counterstained with hematoxylin. Additional sections were also stained with a cocktail of antibodies to non-β-cell hormones, somatostatin, glucagon, and pancreatic polypeptide (Vector Labs). At least 200 fields were counted per animal. Using point-counting morphometrics (27), the relative volumes of both islets and β-cells were quantitated. The islet and β-cell masses were then calculated by multiplying the relative volume times the pancreatic weight and expressed in milligrams.

β-Cell proliferation.

Rats (n = 7 per age, per treatment) at 1, 7, and 15 weeks of age were injected with 100 mg/kg body wt i.p. 5-bromo-2′deoxyuridine (BrdU; Sigma) 6 h before killing. BrdU is a thymidine analog that is incorporated in newly synthesized DNA. After killing, the pancreas was excised, fixed in Bouin’s solution, and embedded in paraffin. Sections (5 μm) were double stained with immunoperoxidase for BrdU (Amersham cell-proliferation kit; Amersham, U.K.) and for the endocrine non-β-cells of the islets. BrdU-positive and -negative cells were counted using an Olympus BH-2 microscope. In each section, 1,200 β-cells were counted, and the results were expressed as a percentage of BrdU-positive β-cells. A total of 20 sections were counted per age (27).

Islet studies.

Islets were isolated from IDMs and controls at 1, 7, and 15 weeks of age by collagenase digestion (28). A total of 200 islets were subjected to static incubation experiments under the following conditions: 3.3 mmol/l glucose, 3.3 mmol/l glucose + 10 mmol/l arginine, 16.7 mmol/l glucose, and 16.7 mmol/l glucose + 10 mmol/l arginine. After 90 min of incubation, insulin was measured in the incubation medium and the results expressed per DNA. Islet insulin content was determined by radioimmunoassay, and islet DNA content was determined by fluorometric assay (29).

Statistical analysis.

Statistical analyses were performed using ANOVA and the Student’s unpaired t test (30). These studies were approved by the animal care committee of Children’s Hospital of Philadelphia and the University of Pennsylvania.

Induction of gestational diabetes.

The body weights of IUGR animals were significantly lower than controls until ∼7 weeks of age, when catch-up growth was attained (Table 1). At the time of breeding, there was no difference in weight between IUGR and control females. However, during pregnancy IUGR females gained significantly more weight than controls (Table 1).

Blood glucose and plasma insulin and triglyceride levels progressively increased during pregnancy in both IUGR and controls, but they were significantly higher in IUGR animals (Table 2). In contrast to Wistar rats, glucose levels in normal pregnant Sprague-Dawley rats increase with gestation. By 14 days of gestation, IUGR pregnant animals developed diabetes characterized by elevated fasting glucose and insulin levels and glucosuria (Table 2). On day 19 of gestation, intraperitoneal glucose tolerance tests demonstrated impaired glucose tolerance in IUGR pregnant rats. A 2-g/kg injection of glucose in IUGR pregnant females caused a rapid and sustained increase in blood glucose concentration, to a maximum of 350 mg/dl, decreasing slowly to 270 mg/dl at 120 min (Fig. 1A). In contrast, an intraperitoneal glucose bolus given to control pregnant rats elicited a rise in glucose levels to only 160 mg/dl, which decreased back to baseline by 120 min after injection (Fig. 1A).

To determine whether pregnant IUGR rats were insulin resistant, we measured fasting serum insulin levels and determined insulin sensitivity in vivo. At the time of breeding (day 0 of gestation), insulin levels in IUGR and control pregnant rats were not significantly different. However, by day 19 of gestation, fasting insulin levels were twofold higher in IUGR mothers compared with controls (P < 0.05) (Table 2) and insulin-tolerance tests showed a significantly blunted glycemic response to exogenous insulin (Fig. 1B).

IDM offspring of diabetic and control animals.

Male and female offspring (IDM) of diabetic animals were significantly heavier at birth and remained heavier than offspring of control animals (Fig. 2 shows data for males). Fostering the offspring to normal mothers did not significantly alter weight gain during the suckling period, even though fat content in the breast milk was 35% higher (P < 0.05 vs. controls) in diabetic mothers.

Fasting blood glucose, plasma insulin, and triglyceride levels were sequentially measured at various ages in IDM and control rats (Table 3). IDM fetal rats, like human fetuses exposed to the diabetic intrauterine milieu, were hyperglycemic and hyperinsulinemic. At birth, glucose levels did not differ between IDMs and controls. However, insulin levels remained mildly elevated in IDMs until 1 week of age, when no significant differences could be detected. By 15 weeks of age, the IDMs developed mild hyperglycemia, hyperinsulinemia, and hyperlipidemia. At 26 weeks of age, glucose, insulin, and triglyceride levels were markedly elevated in IDMs compared with controls.

Glucose tolerance.

We determined the ability of IDM rats to dispose of a glucose load before the onset of diabetes. Glucose tolerance was measured by intraperitoneal injection of glucose (2 g/kg) in the fed state in 1-week-old animals and after an overnight fast in 7- and 15-week-old animals. After challenge with a glucose load, 1-week-old IDM rats had lower 30-min blood glucose levels than controls (Fig. 3); however, at 60 and 120 min, glucose levels were no different from controls. In contrast, 7-week-old IDM animals displayed mild glucose intolerance, which progressively worsened as the IDM animals aged. Even in the prediabetic stage (7–15 weeks), glucose levels remained elevated 180 min after injection of glucose.

β-Cell secretion of insulin.

We assessed β-cell function by static islet studies in IDM and control rats at 1, 7, and 15 weeks of age. Insulin secretion at low and high glucose concentrations was significantly more robust in 1-week-old IDMs than in controls (Table 4). However, as the animals aged, insulin secretion in response to low and high glucose was markedly attenuated in IDMs compared with controls (Table 4).

Arginine stimulates insulin release by mechanisms independent of those used by glucose. This secretagogue induces an increase in the intracellular concentration of Ca2+, which results in the depolarization of the β-cell membrane. To determine whether this pathway is adversely affected, we measured insulin release in the presence of arginine. There was no difference in insulin output in response to arginine between IDMs and controls at any age (data not shown).

Morphometry of the pancreas and β-cell proliferation.

Immunohistochemical studies on the pancreas of IDM and control animals were carried out at sequential ages. At 1 week of age, β-cell mass was slightly increased in IDM newborns. However, by 15 weeks of age, β-cell mass was significantly lower in IDMs than in controls (Table 5).

Despite the increase in β-cell mass at 1 week of life, the proliferation rate of preexisting β-cells was not significantly different between IDM and controls at 1 week of life. However, by 7 weeks of age, the rate of β-cell proliferation was significantly reduced in IDM animals and continued to decline as the animals aged (Fig. 4).

Insulin resistance.

To determine insulin sensitivity, we performed sequential insulin tolerance tests. Remarkably, even at 1 week of age, insulin tolerance tests showed a significantly blunted glycemic response to exogenous insulin in IDM rats (Fig. 5A). Insulin sensitivity further deteriorated with age, and by 15 weeks, there was only a small drop in glucose levels, compared with a 50% decrease in blood glucose in controls, after insulin was administered (Fig. 5B).

To gain some insight into the mechanisms underlying the blunted insulin sensitivity that we observed in IDM animals, we measured the insulin responsiveness of glucose transport in isolated epitrochlearis muscle of 5-week-old IDM and control rats. The optimal age in which to determine glucose uptake in isolated muscle strips is 5 weeks (24). Before this age it is difficult to isolate high-quality muscle strips, and after this age obesity complicates the accurate measurement of glucose uptake.

In IDM rats, basal 2-DG transport was similar to that of controls. However, maximal glucose transport in response to insulin (1,000 μU/ml) only increased 45% in IDMs compared with a more than fourfold increase in glucose transport in response to insulin in controls (Fig. 6).

Insulin resistance in some models of diabetes has been linked to decreased GLUT expression. To determine whether this situation occurs in IDM rats, we measured GLUT1 protein levels in muscle of newborn rats and GLUT4 protein levels in muscle of adult IDM and control rats. In the newborn period, GLUT1 protein levels were significantly lower in IDMs than in controls. Correspondingly, GLUT4 protein levels were significantly lower in IDM rats than in controls at 15 weeks of age (Fig. 7).

The extent to which an abnormal intrauterine milieu contributes to the development of type 2 diabetes in offspring is difficult to determine because genetic factors play such an important role in the development of the disease. Animal models of gestational diabetes during pregnancy are vitally important in determining the specific metabolic effects of exposure to an abnormal intrauterine milieu against a homogeneous genetic background. A number of animal models have been used to determine the effects of hyperglycemia during pregnancy on the adult offspring (810,1217,31,32). To extend these experimental studies of gestational diabetes, we have developed a new model of gestational diabetes that produces offspring that eventually develop diabetes later in life.

In previous studies, we have characterized a model of uteroplacental insufficiency in the pregnant rat that causes fetal and neonatal growth retardation (20). These animals eventually develop a phenotype that is consistent with type 2 diabetes in the human: progressive deterioration in insulin secretion and insulin action. By age 6 months, the IUGR animals are overtly diabetic (20). The results of the current studies demonstrate that pregnancy prematurely precipitates the development of diabetes in the IUGR rat. By day 14 of gestation, pregnant IUGR rats demonstrated glucose intolerance and insulin resistance.

Normal pregnancy is associated with hyperinsulinemia and a progressive decline in insulin sensitivity (33,34). In response to the increased demand for insulin, the maternal pancreas adapts by increasing insulin synthesis, secretion, and β-cell proliferation (3537). In the rat, these adaptations peak around day 14 of pregnancy, and by the end of gestation, the maternal β-cell mass has nearly doubled (38,39). In the IUGR rat, the β-cell does not function normally, and proliferation is impaired (20). The additional stress of pregnancy may result in a further deterioration of these processes in the IUGR rat, leading to the onset of diabetes in pregnancy.

The results of our studies show that the abnormal intrauterine metabolic milieu of the diabetic pregnancy in IUGR rats has profound affects on the offspring. It is well established that the altered metabolic parameters of maternal diabetes result in fetal hyperglycemia and hyperinsulinemia (1,40). This leads to the development of macrosomia. Similar to the human IDM, IDM rats were significantly heavier at birth than controls and remained so throughout life. Of note was our finding that even though IDM animals were fostered to normal mothers, they still remained heavy throughout life.

Early in life, insulin output of islets in IDM rats was increased in the presence of high glucose concentrations, indicating that β-cells of IDMs have increased responsiveness to glucose stimulation. However, as the IDM animal matures, β-cell dysfunction is manifested by decreased insulin secretion in response to glucose. Impaired insulin secretion was specific to glucose because arginine-stimulated insulin release was similar in IDM and control rats, indicating an intact secretory apparatus. The mechanisms responsible for β-cell malfunction are unknown. One possibility may be that fetal IDM β-cells are chronically overstimulated, which eventually leads to β-cell exhaustion. Alternatively, progressive hyperglycemia may exacerbate β-cell abnormalities through a process known as glucose toxicity (4143). Chronic exposure to high glucose concentrations decreases insulin gene promoter activity and binding of PDX-1 (pancreatic/duodenal homeobox-1) to the insulin promoter, leading to defects in insulin secretion (43).

Like humans with type 2 diabetes and animal models of diabetes in pregnancy, the β-cell of the IDM fetus exposed to short-term elevations in glucose concentrations responds by proliferating (44). Once the fetus is removed from the hyperglycemic intrauterine milieu, β-cell mass very quickly involutes and total pancreatic β-cell mass progressively declines, resulting in a deterioration of glucose homeostasis. These defects result in chronic hyperglycemia, which stimulates a cascade of events culminating in the development of overt diabetes.

In humans and animals predestined to develop type 2 diabetes, after prolonged exposure to hyperglycemia, β-cell proliferation is inhibited and β-cell death is increased (4547). In animals, this is associated with the loss of expression (43,48,49) of a number of β-cell/islet transcription factors that are important for the development of the endocrine pancreas (5052), such as PDX-1, RIPE-3b1 activator, Nkx 6.1, β2/Neuro D, Pax 6, HNF1a, HNF4a1, and HNF3b. A decreased expression of any of these transcription factors will profoundly inhibit further proliferation and neogenesis of the β-cell, making it impossible for the β-cell to compensate for progressive worsening of defects in insulin secretion and insulin action. It remains to be determined whether any of these mechanisms are responsible for the decline in β-cell mass observed in IDM animals.

Insulin resistance was apparent at a very early age in IDMs. Insulin tolerance tests were abnormal at 1 week, the earliest age that we could reliably test the animals. In an attempt to determine the mechanisms underlying defects in insulin action, we measured glucose transport in isolated muscle of IDMs and controls at different ages. Glucose transport in skeletal muscle is widely considered the rate-limiting step for whole-body glucose disposal under most conditions (53,54). We found that insulin-stimulated glucose uptake was significantly attenuated in 5-week-old IDM rats compared with controls. We measured protein levels of GLUT1 and -4, the transporters responsible for insulin-stimulated glucose transport in newborns and adults, respectively. GLUT1 protein levels were significantly diminished in muscle of IDMs compared with controls, and, correspondingly, GLUT4 protein levels were decreased in adult IDM rats. Further studies will be necessary to determine whether other steps in the pathway of insulin-stimulated glucose transport are altered.

The mechanisms underlying the observed downregulation of GLUT1 and -4 in the newborn and adult IDM rat may be related to high fetal glucose concentrations resulting from maternal hyperglycemia. High circulating levels of glucose induce a substantial downregulation of glucose transporters in muscle under chronic diabetic conditions (5557). Downregulation of glucose transporters may be an adaptive mechanism by which the fetus protects itself against the deleterious effects of high cellular glucose concentrations. Of interest is our observation that once glucose levels are normalized after birth, glucose transporter expression remains permanently reduced in IDM animals.

In conclusion, we have developed a model of diabetes in pregnancy with a phenotype that is similar to that observed in humans with gestational diabetes. This model results in the onset of diabetes in the offspring once they reach adulthood. The development of diabetes appears to be related to defects in both insulin secretion and insulin action. The data presented here support the hypothesis that an abnormal intrauterine milieu can induce permanent changes in glucose homeostasis after birth. The fetus adapts to an altered environment in utero that may enhance its short-term survival probability at the expense of a long-term capacity for normal growth and development, resulting in the development of diabetes.

FIG. 1.

A: Blood glucose levels during an intraperitoneal glucose tolerance test at 19 days of pregnancy in IUGR and control (sham) rats. Values are the means ± SE from five animals from each group. *P < 0.05 vs. control. B: Blood glucose levels during a subcutaneous injection of insulin at 19 days of pregnancy in IUGR and control (sham) rats. Values are the means ± SE from five animals from each group. *P < 0.05 vs. control.

FIG. 1.

A: Blood glucose levels during an intraperitoneal glucose tolerance test at 19 days of pregnancy in IUGR and control (sham) rats. Values are the means ± SE from five animals from each group. *P < 0.05 vs. control. B: Blood glucose levels during a subcutaneous injection of insulin at 19 days of pregnancy in IUGR and control (sham) rats. Values are the means ± SE from five animals from each group. *P < 0.05 vs. control.

Close modal
FIG. 2.

Weights of IDM and control rats from birth until 26 weeks of age. Values are the means ± SE from 10 animals from each group followed sequentially from birth until 26 weeks of age. *P < 0.05 vs. control.

FIG. 2.

Weights of IDM and control rats from birth until 26 weeks of age. Values are the means ± SE from 10 animals from each group followed sequentially from birth until 26 weeks of age. *P < 0.05 vs. control.

Close modal
FIG. 3.

AC: Blood glucose levels during an intraperitoneal glucose tolerance test performed sequentially at 1 week (A), 7 weeks (B), and 15 weeks (C) of age in IDM and control rats. Values are the means ± SE from eight animals from each group. *P < 0.05 vs. control.

FIG. 3.

AC: Blood glucose levels during an intraperitoneal glucose tolerance test performed sequentially at 1 week (A), 7 weeks (B), and 15 weeks (C) of age in IDM and control rats. Values are the means ± SE from eight animals from each group. *P < 0.05 vs. control.

Close modal
FIG. 4.

Rats (n = 7 per age, per group) were injected with BrdU 6 h before killing. After killing, the pancreas was excised, fixed in Bouin’s solution, and embedded in paraffin. Sections (5 μm) were double stained with immunoperoxidase for BrdU and for the endocrine non-β- cells of the islets. A total of 20 sections were counted per age. Using the methods of Scaglia et al. (27), we calculated the proliferation rate. Data are expressed as the percent control. *P < 0.05 vs. control.

FIG. 4.

Rats (n = 7 per age, per group) were injected with BrdU 6 h before killing. After killing, the pancreas was excised, fixed in Bouin’s solution, and embedded in paraffin. Sections (5 μm) were double stained with immunoperoxidase for BrdU and for the endocrine non-β- cells of the islets. A total of 20 sections were counted per age. Using the methods of Scaglia et al. (27), we calculated the proliferation rate. Data are expressed as the percent control. *P < 0.05 vs. control.

Close modal
FIG. 5.

Blood glucose levels during an intraperitoneal injection of insulin performed sequentially at 1 week (A) and 15 weeks (B) of age in IDM and control rats. Values are the means ± SE of eight animals from each group at each age. *P < 0.05 vs. control.

FIG. 5.

Blood glucose levels during an intraperitoneal injection of insulin performed sequentially at 1 week (A) and 15 weeks (B) of age in IDM and control rats. Values are the means ± SE of eight animals from each group at each age. *P < 0.05 vs. control.

Close modal
FIG. 6.

Glucose uptake in isolated epitrochlearis muscle from IDMs and controls at 5 weeks of age. [cjs2108], glucose uptake under basal conditions; ▪, glucose uptake in response to 2 mU/ml insulin. Values are the means ± SE from five animals from each group. *P < 0.05 vs. control.

FIG. 6.

Glucose uptake in isolated epitrochlearis muscle from IDMs and controls at 5 weeks of age. [cjs2108], glucose uptake under basal conditions; ▪, glucose uptake in response to 2 mU/ml insulin. Values are the means ± SE from five animals from each group. *P < 0.05 vs. control.

Close modal
FIG. 7.

GLUT protein levels as determined by Western blot analyses. Data are expressed as percent control. Values are the means ± SE from five animals from each group. C, control. *P < 0.05 vs. control.

FIG. 7.

GLUT protein levels as determined by Western blot analyses. Data are expressed as percent control. Values are the means ± SE from five animals from each group. C, control. *P < 0.05 vs. control.

Close modal
TABLE 1

Weights (in grams) of IUGR and control animals

IUGR
Control
MeansnMeansn
Birthweight 5.26 ± 0.17* 6 liters 6.76 ± 0.1 6 liters 
Weight at breeding 250 ± 10 16 248 ± 12 16 
Midgestation weight 350 ± 21* 16 290 ± 13 16 
End gestation weight 370 ± 19* 16 315 ± 21 16 
IUGR
Control
MeansnMeansn
Birthweight 5.26 ± 0.17* 6 liters 6.76 ± 0.1 6 liters 
Weight at breeding 250 ± 10 16 248 ± 12 16 
Midgestation weight 350 ± 21* 16 290 ± 13 16 
End gestation weight 370 ± 19* 16 315 ± 21 16 

Data are means ± SD or n.

*

P < 0.05 vs. control.

TABLE 2

Glucose, insulin, and triglyceride concentrations in pregnant IUGR (n = 16) and control (n = 16) animals

Day 0Day 14Day 19
Glucose (mg/dl)    
 IUGR 90.2 ± 8.4 149.1 ± 10.2* 250.2 ± 16.6* 
 Control 89.3 ± 9.3 100.5 ± 9.6 103.7 ± 8.7 
Insulin (μU/ml)    
 IUGR 50.1 ± 4.8 87.4 ± 8.9* 97.7 ± 9.6* 
 Control 45.2 ± 8.3 44.5 ± 8.6 50.2 ± 7.3 
Triglyceride mg/dl)    
 IUGR 93.8 ± 6.9 127.4 ± 8.9 325.7 ± 9.6* 
 Control 101.3 ± 6.9 104.5 ± 8.6 100.2 ± 7.3 
Day 0Day 14Day 19
Glucose (mg/dl)    
 IUGR 90.2 ± 8.4 149.1 ± 10.2* 250.2 ± 16.6* 
 Control 89.3 ± 9.3 100.5 ± 9.6 103.7 ± 8.7 
Insulin (μU/ml)    
 IUGR 50.1 ± 4.8 87.4 ± 8.9* 97.7 ± 9.6* 
 Control 45.2 ± 8.3 44.5 ± 8.6 50.2 ± 7.3 
Triglyceride mg/dl)    
 IUGR 93.8 ± 6.9 127.4 ± 8.9 325.7 ± 9.6* 
 Control 101.3 ± 6.9 104.5 ± 8.6 100.2 ± 7.3 

Data are means ± SD.

*

P < 0.05 vs. control.

TABLE 3

Glucose, insulin, and triglyceride concentrations in IDM and control animals

Fetal (day 20)1 week15 weeks26 weeks
n 6 litters 40 30 30 
Glucose (mg/dl)     
 IDM 140.2 ± 5.8* 87 ± 7.2 199.1 ± 20.2* 310.2 ± 26.5* 
 Control 102 ± 6.5 91.1 ± 9.2 130.4 ± 11.6 143.6 ± 10.7 
Insulin (μU/ml)     
 IDM 320.2 ± 29.1* 49.2 ± 8.2 77.4 ± 8.9* 87.7 ± 9.6* 
 Control 150.1 ± 12.4 37.9 ± 6.3 49.5 ± 8.9 55.2 ± 6.2 
Triglyceride (mg/dl)     
 IDM NA NA 287.2 ± 28.4* 337.6 ± 29.5* 
 Control NA NA 142.5 ± 12.9 152.5 ± 16.4 
Fetal (day 20)1 week15 weeks26 weeks
n 6 litters 40 30 30 
Glucose (mg/dl)     
 IDM 140.2 ± 5.8* 87 ± 7.2 199.1 ± 20.2* 310.2 ± 26.5* 
 Control 102 ± 6.5 91.1 ± 9.2 130.4 ± 11.6 143.6 ± 10.7 
Insulin (μU/ml)     
 IDM 320.2 ± 29.1* 49.2 ± 8.2 77.4 ± 8.9* 87.7 ± 9.6* 
 Control 150.1 ± 12.4 37.9 ± 6.3 49.5 ± 8.9 55.2 ± 6.2 
Triglyceride (mg/dl)     
 IDM NA NA 287.2 ± 28.4* 337.6 ± 29.5* 
 Control NA NA 142.5 ± 12.9 152.5 ± 16.4 

Data are means ± SD.

*

P < 0.05 vs. control.

TABLE 4

Glucose-induced insulin release from noncultured islets

1 Week
7 Weeks
15 Weeks
IDMControlIDMControlIDMControl
n 5 litters 5 litters 10 10 
Glucose (mmol/l)       
 3.3 0.85 ± 0.01* 0.25 ± 0.02 0.35 ± 0.02* 0.99 ± 0.1 0.12 ± 0.02* 0.81 ± 0.05 
 16.7 6.01 ± 0.24* 1.92 ± 0.32 0.71 ± 0.08* 6.23 ± 0.67 0.69 ± 0.32* 4.10 ± 0.67 
1 Week
7 Weeks
15 Weeks
IDMControlIDMControlIDMControl
n 5 litters 5 litters 10 10 
Glucose (mmol/l)       
 3.3 0.85 ± 0.01* 0.25 ± 0.02 0.35 ± 0.02* 0.99 ± 0.1 0.12 ± 0.02* 0.81 ± 0.05 
 16.7 6.01 ± 0.24* 1.92 ± 0.32 0.71 ± 0.08* 6.23 ± 0.67 0.69 ± 0.32* 4.10 ± 0.67 

Results are means ± SE and expressed as microunits of insulin released per nanogram of islet DNA per hour.

*

P < 0.05 vs. control.

TABLE 5

β-Cell mass

Age (weeks)IDM (μg/body wt)Control (μg/body wt)
n 10 10 
 1 55.4 ± 13.2 53.3 ± 15.2 
 7 50.2 ± 8.2 62.5 ± 8.2 
 15 40.1 ± 15.2 60.5 ± 10.1 
 26 24.3 ± 2.2* 50.1 ± 7.2 
Age (weeks)IDM (μg/body wt)Control (μg/body wt)
n 10 10 
 1 55.4 ± 13.2 53.3 ± 15.2 
 7 50.2 ± 8.2 62.5 ± 8.2 
 15 40.1 ± 15.2 60.5 ± 10.1 
 26 24.3 ± 2.2* 50.1 ± 7.2 

Data are means ± SD.

This research was supported by National Institutes of Health Grant DK55704 (to R.A.S.), the American Diabetes Association, and the Diabetes Center at the University of Pennsylvania.

We thank Dr. Richard McCourt for his editorial assistance.

1.
Freinkel N, Metzger BE: Pregnancy as a tissue culture experience: the critical implications of maternal metabolism for fetal development. In
Pregnancy, Metabolism, Diabetes, and the Fetus
. Amsterdam, Excepta Med.,
1979
, p.
2
–23 (COBA Foundation Symposium no. 63)
2.
Pettitt DJ, Baird HR, Aleck KA, Knowler WC: Excessive obesity in offspring of Pima Indian women with diabetes during pregnancy.
N Engl J Med
308
:
242
–245,
1983
3.
Pettitt DJ, Aleck KA, Baird HR, Carraher MJ, Bennett PH, Knowler WC: Congenital susceptibility to NIDDM: role of the intrauterine environment.
Diabetes
37
:
622
–628,
1988
4.
Martin AO, Simpson JL, Ober C, Freinkel N: Frequency of diabetes mellitus in mothers of probands with gestational diabetes: possible maternal influence on the predisposition to gestational diabetes.
Am J Obstet Gynecol
151
:
471
–475,
1985
5.
Silverman B, Metzger BE, Cho NH, Loeb CA: Impaired glucose tolerance in adolescent offspring of diabetic mothers: relationship to fetal hyperinsulinism.
Diabetes Care
18
:
611
–617,
1995
6.
Ericksson U, Andersson A, Efendic S, Elde R, Hellerstrom C: Diabetes in pregnancy: effects on the foetal and newborn rat with particular regard to body weight, serum insulin concentration and pancreatic contents of insulin, glucagon, and somatostatin.
Acta Endocrinologica
94
:
354
–364,
1980
7.
Cueza JM, Burkett ES, Kerr DS, Rodman HV, Patel MS: The newborn of diabetic rat: hormonal and metabolic changes in the postnatal period.
Pediatr Res
16
:
632
–637,
1982
8.
Aerts L, Sodoyez-Goffaux F, Sodoyez JC, Malaisse WJ, Van Assche FA: The diabetic intrauterine milieu has a long-lasting effect on insulin secretion by β-cells and on insulin uptake by target tissues.
Am J Obstet Gynecol
159
:
1287
–1292,
1988
9.
Kervran A, Guillaume M, Jost A: The endocrine pancreas of the fetus from diabetic pregnant rat.
Diabetologia
15
:
387
–393,
1978
10.
Oh W, Gelardi NL, Cha CJ: Maternal hyperglycemia in pregnant rats: its effect on growth and carbohydrate metabolism in the offspring.
Metabolism
37
:
1146
–1151,
1988
11.
Oh W, Gelardi NL, Cha CM: The cross-generation effect of neonatal macrosomia in rat pups of streptozotocin-induced diabetes.
Pediatr Res
29
:
606
–610,
1991
12.
Bihoreau MT, Ktorza A, Kinebanyan MF, Picon L: Impaired glucose homeostasis in adult rats from hyperglycemic mothers.
Diabetes
35
:
979
–984,
1986
13.
Ktorza A, Girard JR, Kinebanyan MF, Picon L: Hyperglycaemia induced by glucose infusion in the unrestrained pregnant rat during the last three days of gestation: metabolic and hormonal changes in the mother and the fetuses.
Diabetologia
21
:
569
–574,
1981
14.
Nurjhan N, Ktorza A, Ferre P, Girard JR, Picon L: Effects of gestational hyperglycemia on glucose metabolism and its hormonal control in the fasted, newborn rat during the early postnatal period.
Diabetes
34
:
995
–1001,
1985
15.
Gauguier D, Bihoreau MT, Ktorza A, Berthault MF, Picon L: Inheritance of diabetes mellitus as consequence of gestational hyperglycemia in rats.
Diabetes
39
:
734
–739,
1990
16.
Holemans K, Aerts L, Van Ashe FA: Evidence for an insulin resistance in the adult offspring of pregnant streptozotocin-diabetic rats.
Diabetologia
34
:
81
–85,
1991
17.
Holemans K, Van Bree R, Verhaeghe J, Aerts L, Van Assche FA: In vivo glucose utilization by individual tissues in virgin and pregnant offspring of severely diabetic rats.
Diabetes
42
:
530
–536,
1993
18.
Gelardi NL, Cha CJ, Oh W: Evaluation of insulin sensitivity in obese offspring of diabetic rats by hyperinsulinemic-euglycemic clamp technique.
Pediatr Res
30
:
40
–44,
1991
19.
Gelardi NL, Cha CJ, Oh W: Glucose metabolism in adipocytes of obese offspring of mild hyperglycemic rats.
Pediatr Res
28
:
641
–645,
1990
20.
Simmons RA, Templeton L J, Gertz SJ: Intrauterine growth retardation leads to the development of type 2 diabetes in the rat.
Diabetes
50
:
2279
–2286,
2001
21.
Ogata Es, Bussey M, Finley S: Altered gas exchange, limited glucose, branched chain amino acids, and hyperinsulinism retard fetal growth in the rat.
Metabolism
35
:
950
–977,
1986
22.
Simmons RA, Gounis AS, Bangalore SA, Ogata ES: Intrauterine growth retardation: fetal glucose transport is diminished in lung but spared in brain.
Pediatr Res
31
:
59
–63,
1991
23.
Unterman T, Lascon R, Gotway M, Oehler D, Gounis A, Simmons RA, Ogata ES: Circulating levels of insulin-like growth factor binding protein-1 (IGFBP-1) and hepatic mRNA are increased in the small for gestational age fetal rat.
Endocrinology
127
:
2035
–2037,
1990
24.
Young DA, Uhl JJ, Cartee GD, Holloszy JO: Glucose transport in muscle by prolonged exposure to insulin.
J Biol Chem
261
:
16049
–16053,
1986
25.
Strout HV, Vicario PP, Biswas C, Saparstein R, Brady EJ, Pilch PF: Vanadate treatment of streptozotocin diabetic rats restores expression of the insulin-responsive glucose transporter in skeletal muscle.
Endocrinology
126
:
2728
–2732,
1990
26.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent.
J Biol Chem
93
:
265
–275,
1951
27.
Scaglia L, Cahill CJ, Finegood DT, Bonner-Weir S: Apoptosis participates in the remodeling of the endocrine pancreas in the neonatal period.
Endocrinology
138
:
1736
–1741,
1997
28.
Scharp DW, Kemp CB. Knight MJ, Ballinger WF, Lacy PE: The use of ficoll in the preparation of viable islets of Langerhans from the rat pancreas.
Transplantation
16
:
686
–689,
1973
29.
Setaro F, Morley C: A modified fluorometric method for the determination of microgram quantities of DNA from cell or tissue cultures.
Anal Biochem
71
:
313
–317,
1976
30.
Zar JH: In
Biostatistical Analysis
. 2nd ed. Zar JH, Ed. Englewood Cliffs, New Jersey, Prentice Hall,
1984
, p.
150
–160
31.
Abdel-Halim SM, Guenifi A, Luthman H, Grill V, Efendic S, Ostenson CG: Impact of diabetic inheritance on glucose tolerance and insulin secretion in spontaneously diabetic GK-Wistar rats.
Diabetes
43
:
281
–288,
1994
32.
Ryan EA, Tobin BW, Tang J, Finegood DT: A new model for the study of mild diabetes during pregnancy: syngeneic islet-transplanted STZ-induced diabetic rats.
Diabetes
42
:
316
–323,
1993
33.
Catalano PM, Tyzbir ED, Roman NM, Amini SB, Sims EA: Longitudinal changes in insulin resistance and insulin release in non-obese pregnant women.
Am J Obstet Gynecol
165
:
1667
–1671,
1991
34.
Catalano PM, Tyzbir ED, Wolfe RR, Calles J, Roman NM, Amini SB, Sims EA: Carbohydrate metabolism during pregnancy in control subjects and women with gestational diabetes.
Am J Physiol
264
:
E60
–E67,
1993
35.
Parsons JA, Brelje TC, Sorenson RL: Adaptation of islets of Langerhans to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion.
Endocrinology
130
:
1459
–1466,
1992
36.
Bone A, Taylor K: Metabolic adaptation to pregnancy shown by increased biosynthesis of insulin in islets of Langerhans isolated from pregnant rats.
Nature
262
:
501
–502,
1976
37.
Kwai M, Kishi K: In vitro studies of the stimulation of insulin secretion and β-cell proliferation by rat placental lactogen-II during pregnancy in rats.
J Reprod Fertility
109
:
145
–152,
1997
38.
Nieuwenhuizen A, Schuiling G, Moes H, Koiter T: Role of increased insulin demand in the adaptation of the endocrine pancreas to pregnancy.
Acta Physiol Scand
159
:
303
–312,
1997
39.
Marynissen G, Aerts L, Assche FV: The endocrine pancreas during pregnancy and lactation.
J Dev Physiol
5
:
373
–381,
1983
40.
Cornblath M, Schwartz R:
Disorders of Carbohydrate Metabolism in Infancy. Major Problems in Clinical Pediatrics
. Vol 
3
. Philadelphia, Saunders,
1966
, p.
57
–81
41.
Robertson RP, Olson LK, Zhang H-J: Differentiating glucose toxicity from glucose desensitization: a new message from the insulin gene.
Diabetes
43
:
1085
–1089,
1994
42.
Yki-Jarvinen H: Glucose toxicity.
Endocrine Rev
13
:
413
–431,
1992
43.
Harmon JS, Gleason CE, Tanaka Y, Oseid EA, Hunter-Berger KK, Robertson RP: In vivo prevention of hyperglycemia also prevents glucotoxic effects on PDX-1 and insulin gene expression.
Diabetes
48
:
1995
–2000,
1999
44.
Aerts I, Van Assche FA: Rat foetal endocrine pancreas in experimental diabetes.
J Endocrinol
73
:
399
–346,
1977
45.
Saito K, Yaginuma N, Takahashi T: Differential volumetry of a, β, and d cells in the pancreatic islets of diabetic and non-diabetic subjects.
Tohoku J Exp Med
129
:
273
–283,
1979
46.
Kloppel G, Lohr M, Hablich K, Oberhilzer M, Heinz PU: Islet pathology and pathogenesis of type I and type II diabetes revisited.
Surv Synth Path Res
4
:
110
–125,
1985
47.
Clark A, Wells CA, Buley ID, Cruickshank JK, Vanhegan RI, Matthews DR, Cooper GJ, Holman RR, Turner RC: Islet amyloid, increased a-cells, reduced β-cells and exocrine fibrosis: quantitative changes in the pancreas of type 2 diabetes.
Diabetes Res
9
:
151
–159,
1988
48.
Kilpatrick ED, Robertson RP: Differentiation between glucose-induced desensitization of insulin secretion and β-cell exhaustion in the HIT-T15 cell line.
Diabetes
47
:
606
–611,
1998
49.
Sharma A, Olson LK, Robertson RP, Stein R: The reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to high glucose concentrations is associated with the loss of RIPE3b1 and STF-1 transcription factor expression.
Mol Endocrinol
9
:
1127
–1134,
1995
50.
Sander M, German MS: The beta cell transcription factors and development of the pancreas.
J Mol Med
75
:
327
–340.
1997
51.
Madsen OD, Jensen J, Petersen HV, Pedersen EE, Oster A, Andersen FG, Jorgensen MC, Jensen PB, Larsson LI, Serup P: Transcription factors contributing to the pancreatic beta-cell phenotype.
Horm Metab Res
29
:
265
–270,
1997
52.
Jonas JC, Sharma A, Hasenkamp W, Ilkova H, Patane G, Laybutt R, Bonner-Weir S, Weir GC: Chronic hyperglycemia triggers loss of pancreatic β-cell differentiation in an animal model of diabetes.
J Biol Chem
274
:
14112
–14121,
1999
53.
Bonadonna RC, Saccomani MP, Seely L, Zych KS, Ferrannini E, Cobelli C, DeFronzo RA: Glucose transport in human skeletal muscle: the in vivo response to insulin.
Diabetes
42
:
191
–198,
1993
54.
Ziel FH, Venkatesan N, Davidson MB: Glucose transport is rate limiting for skeletal muscle in normal and STZ-induced diabetic rats.
Diabetes
37
:
855
–890,
1988
55.
Dimitrakousdis D, Ramlal T, Rastogi S, Vranic M, Klip A: Glycemia regulates the glucose transporter number in the plasma membrane of rat skeletal muscle.
Biochem J
284
:
341
–348,
1992
56.
Kahn BB: Lilly Lecture 1995: Glucose transport: pivotal step in insulin action.
Diabetes
45
:
1644
–1645,
1996
57.
Richardson JM, Balon TW, Treadway JL, Pessin JE: Differential regulation of glucose transporter activity and expression in red and white skeletal muscle.
J Biol Chem
266
:
12690
–12694,
1991

Address correspondence and reprint requests to Rebecca Simmons, MD, University Pennsylvania, BRB II/III 13th Floor, 421 Curie Blvd., Philadelphia, PA 19104. E-mail: rsimmons@mail.med.upenn.edu.

Received for publication 15 May 2001 and accepted in revised form 7 February 2002.

BrdU, 5-bromo-2′deoxyuridine; 2-DG, 2-deoxyglucose; IDM, infant of a diabetic mother; IUGR, intrauterine growth-retarded; STZ, streptozotocin.