Nancy L. Gelardi

Maternal hyperglycemia in pregnant rats: Its effect on growth and carbohydrate metabolism in the offspring☆

Mild hyperglycemia during the last half of pregnancy was achieved by administration of streptozotocin to pregnant rats on the fifth day of gestation. Citrate buffer (vehicle for streptozotocin) was administered to control rats also on the fifth day of gestation. The pups born to the streptozotocin-treated mothers had higher birth weight, pancreatic insulin content, plasma insulin, and C-peptide concentrations compared with pups born to control mothers. The plasma glucose concentrations of the pups were similar between the two groups. The pups who were identified as macrosomic (birth weight greater than 1.7 SD than the mean of the control pups) maintained an accelerated postnatal growth through the first 10 weeks of age in female rats and in the first 3, and at 5 and 6 weeks of age for the male rats. The accelerated growth in the female rats was associated with higher perirenal-ovarian and salpingeal fat weight at 6 weeks. At 10 weeks of age, higher plasma insulin and glucose concentrations were observed following oral glucose challenge in both male and female macrosomic rats than in the control rats. At 12 weeks of age, only the female macrosomic rats showed abnormal glucose response due to peripheral insulin resistance. In the male rats at 12 weeks of age, a higher plasma insulin concentration in the macrosomic group was associated with a normal plasma glucose response to oral glucose challenge. We conclude that mild maternal hyperglycemia in rats resulted in fetal hyperinsulinemia and accelerated fetal growth.(ABSTRACT TRUNCATED AT 250 WORDS)

The cross-generation effect of neonatal macrosomia in rat pups of streptozotocin-induced diabetes.

ABSTRACT: To study the cross-generation effect of enhanced growth in macrosomic newborn rats, we induced mild hyperglycemia in 15 pregnant Sprague Dawley rats by intraperitoneal injection of streptozotocin, 35 mg/kg body weight, on the 5th d of gestation. As reported previously, we produced hyperinsulinemia and accelerated growth in the fetuses of hyperglycemic dams. We also showed that the macrosomic female pups (second generation) continued to have a higher growth rate through the first 12 wk of life. In this study, the second-generation female rats were mated with macrosomic second-generation males; they demonstrated glucose intolerance during late pregnancy and delivered pups (third generation) with higher birth weight and plasma insulin levels than the pups from control second-generation rats. When the macrosomic third-generation pups were raised under identical nutritional and environmental conditions as controls, the macrosomic rats showed accelerated growth and higher fat tissue weight during the first 12 wk of life. Furthermore, the macrosomia was associated with glucose intolerance and higher insulin to glucose ratios compared to controls. We also mated the offspring of second-generation streptozotocin-injected nonmacrosomic as well as the offspring of macrosomic pups of buffer-injected dams; none of the pups from these matings were significantly macrosomic. Therefore, we conclude that the perpetuation of obesity and possibly glucose intolerance across generations in this rat model is predominantly a result of abnormal intrauterine metabolic environment rather than genetic factor driven.

Glucose metabolism in adipocytes of obese offspring of mild hyperglycemic rats.

ABSTRACT: Six pregnant rats were made mildly hyperglycemic by intraperitoneal injection of streptozotocin on d 5 of gestation. Four control rats were injected with citrate buffer. Thirty pups born to experimental dams who had increased birth weight (birth weight >1.7 SD of mean birth weight of pups from control dams) maintained accelerated growth through 10 wk of age. At 10 wk, oral glucose tolerance tests showed higher glucose and insulin levels than the controls (n = 37). In addition to the higher body weight, the experimental rats also had higher fat weight to body weight ratios. Adipocytes of epididymal fat from obese males and periovarian fat from obese females had higher lipid content with significantly larger cell size than the adipocytes of the controls. The adipocytes of macrosomic rats showed attenuated response to insulin-stimulated glucose conversion to total lipid and fatty acid when compared with the responses seen in the adipocytes of the control rats. Interestingly, although the insulin-stimulated glucose conversion to CO2 was similar in macrosomic and control males, the response in the macrosomic female was blunted when compared with that of the control females. Insulin receptor binding capacities of the macrosomic rats were lower than those of the controls, which is consistent with a phenomenon of down-regulation. However, the receptor affinities were higher in the experimental animals than in controls. Therefore, a postreceptor defect may account for the abnormality in glucose metabolism in the obese rats. In conclusion, the abnormal response to oral glucose loading in these experimental obese, hyperinsulinemic rats is due to peripheral tissue insulin resistance that is probably postreceptor in nature.

Evaluation of Insulin Sensitivity in Obese Offspring of Diabetic Rats by Hyperinsulinemic-Euglycemic Clamp Technique

ABSTRACT: Young adult macrosomic offspring of streptozotocin-induced mildly hyperglycemic rats exhibit accelerated growth through the first 10 wk of age. At 10 wk, oral glucose loading resulted in elevated plasma insulin and glucose concentrations compared to controls. To assess the mechanism of the abnormal glucose tolerance in vivo, hyperinsulinemic-euglycemic clamp studies were performed. Ten-wk-old rats were fasted overnight, and porcine insulin was infused (2.4 mU.kg-1.min-1). Glucose was infused concurrently at varying rates to maintain euglycemia for 40–60 min. Insulin levels were raised from a baseline value of 163 ± 57 pmol/L (23 ± 8 μU/mL) (SD) to 476 ± 57 pmol/L (67 ± 8 μU/mL) at steady state for males and from 178 ± 43 pmol/L (25 ± 6 μU/mL) to 454 ± 43 pmol/L (64 ± 6 μU/mL) for females. The results showed that the macrosomic male and female animals were significantly less sensitive to the effects of insulin than were their respective controls; this was evident by a lower increment in glucose disposal rate per unit increase in insulin (0.04 ± 0.01 versus 0.11 ± 0.03 for males and 0.05 ± 0.03 versus 0.18 ± 0.07 mg.kg-1 per μU/mL for females). The endogenous glucose production by the liver in the basal (fasted) state in the macrosomic group compared to controls was higher, suggesting possible hepatic insulin resistance. However, endogenous glucose production was suppressed to the same degree between the experimental and control groups during the hyperinsulinemic period, suggesting that the hepatic insulin resistance can be overcome by high insulin levels. Thus, the reduced sensitivity to the effect of insulin on glucose disposal in the macrosomic animals provides additional in vivo evidence for peripheral insulin resistance as a contributing factor for the development of glucose intolerance in obese offspring of mildly hyperglycemic dams.

Insulin resistance and glucose transporter expression during the euglycemic hyperinsulinemic clamp in the lamb

Three- to six-day-old lambs infused with 100 mU ⋅ kg-1 ⋅ min-1insulin required greater amounts of glucose to maintain euglycemia during a euglycemic hyperinsulinemic clamp compared with 31- to 35-day-old insulin-infused lambs (15.87 ± 3.47 vs. 4.30 ± 1.11 mg ⋅ kg-1 ⋅ min-1, P < 0.05, respectively). Endogenous glucose production persisted in both groups; however, the percent decrease compared with age-matched lambs receiving no insulin was greater in the younger group compared with the older group (53%, P < 0.001, vs. 34%, P < 0.01). The younger animals showed greater glucose utilization compared with the older animals (215 vs. 96%, respectively, P < 0.01). No effect of insulin was noted on GLUT-4 protein expression in either group. GLUT-2 expression was increased in older vs. younger lambs. Older insulin-infused lambs showed lower GLUT-2 expression than older 0 insulin-infused lambs [0.94 ± 0.07 vs. 1.64 ± 0.10 (OD) units, P < 0.005]. Increased sensitivity to insulin in the younger animals was not related to acute changes in GLUT-4 expression. Increased GLUT-2 expression with age, as well as decreased expression with hyperinsulinemia, is consistent with the development of an insulin-resistant state in the adult.Three- to six-day-old lambs infused with 100 mU x kg(-1) x min(-1) insulin required greater amounts of glucose to maintain euglycemia during a euglycemic hyperinsulinemic clamp compared with 31- to 35-day-old insulin-infused lambs (15.87 +/- 3.47 vs. 4.30 +/- 1.11 mg x kg(-1) x min(-1), P < 0.05, respectively). Endogenous glucose production persisted in both groups; however, the percent decrease compared with age-matched lambs receiving no insulin was greater in the younger group compared with the older group (53%, P < 0.001, vs. 34%, P < 0.01). The younger animals showed greater glucose utilization compared with the older animals (215 vs. 96%, respectively, P < 0.01). No effect of insulin was noted on GLUT-4 protein expression in either group. GLUT-2 expression was increased in older vs. younger lambs. Older insulin-infused lambs showed lower GLUT-2 expression than older 0 insulin-infused lambs [0.94 +/- 0.07 vs. 1.64 +/- 0.10 (OD) units, P < 0.005]. Increased sensitivity to insulin in the younger animals was not related to acute changes in GLUT-4 expression. Increased GLUT-2 expression with age, as well as decreased expression with hyperinsulinemia, is consistent with the development of an insulin-resistant state in the adult.

Dose-Effect Relationship of Bilirubin on Striatal Synaptosomes in Rats

The uptake of tyrosine as precursor of dopamine in striatal nerve endings was studied in rat striatal synaptosomes to evaluate the role of different bilirubin concentrations on this mechanism of synaptic neurotransmission. Freshly prepared striatal synaptosomes obtained from adult male Sprague-Dawley rats (200-300 g) were homogenized in about 25 vol of 0.32 M sucrose and 5 nM Hepes at pH 7.4 and centrifugated at 12,000-16,000 rpm for 20 min. 14C tyrosine was evaporated under N2 and resuspended in tyrosine 100 microliters/ml. Different concentrations of crystalline bilirubin ranging from 70 to 140 microM at 10-min intervals were prepared. Eighty microliters of synaptosomes were added to 5 microliters of the different bilirubin concentrations and 20 microliters of 14C tyrosine. The results of this study show a statistically significant correlation between bilirubin levels and tyrosine uptake in rat synaptosomes (p < 0.05) supporting the hypothesis that the effect of bilirubin on neuronal excitability is dose-dependent.

Accelerated growth and abnormal glucose tolerance in young female rats exposed to fetal hyperinsulinemia.

ABSTRACT: This study was designed to evaluate the relationship between fetal macrosomia and postnatal growth as well as the glucose homeostasis in young female rats. We produced fetal macrosomia by fetal insulin injection at 20 Vi days of gestation. The control subjects were injected with saline. The weights were recorded weekly from birth up to 12 wk. Only the female rats were studied. At 4, 6, 10, and 12 wk of age, oral glucose tolerance tests were performed. Also, at wk 6, 10, and 12, peri-renalovarian- salpingeal fat weights, the RNA, DNA, and protein contents of the abdominal muscle were determined. Onehundred seventeen control and 78 macrosomic rats were studied. The macrosomic rats showed a higher body weight (10-12%) than the control rats from birth up to 8 wk, but at 10 and 12 wk their weights were similar. The fat weights reflected the body weights, i.e. a higher fat weight in the macrosomic rats during the period of accelerated growth (from birth up to 8 wk), and a similar fat weight when the body weight of the two groups were similar at 10 and 12 wk. At 4 and 6 wk of age, the plasma glucose level measured in response to the oral glucose loading were similar in both groups. However, at 10 and 12 wk of age, the macrosomic rats had significantly higher fasting plasma glucose levels and exhibited consistently higher plasma glucose levels for the 3.5-h period of postglucose administration compared to the control rats. The plasma insulin levels rose significantly following glucose challenge. However, the values were similar in both groups at 10 and 12 wk. We conclude that the primary hyperinsulinemiainduced fetal macrosomia is associated with an increased fat deposition resulting in an increased weight gain during young adulthood. The increased fat deposition may manifest peripheral insulin resistance exhibiting the glucose intolerance at a later age. The mechanism involved in this development remains to be investigated.

Insulin counterregulatory hormones are ineffective in neonatal hyperinsulinemic hypoglycemia.

Insulin counterregulatory hormones play a major role in the maintenance of glucose homeostasis. To evaluate the hypothesis that the reported imprecise control of glucose production by insulin is mirrored by a corresponding lack of response to the various insulin counterregulatory hormones, 30 spontaneously delivered mixed-breed term lambs weighing 4.9 +/- 0.5 kg (mean +/- SD) were studied at 5.0 +/- 0.7 days after birth following administration of 100 microCi D-[6-(3)H2]glucose in 0.9% NaCl by the primed-constant infusion technique to measure glucose kinetics. Infusion of 2.0 mU kg(-1) x min(-1) insulin produced hyperinsulinemic hypoglycemia and was combined with 1.0 mg x kg(-1) x min(-1) somatostatin (SRIF) to block insulin, glucagon, and growth hormone release. Infusion of 2 ng x mg(-1) x min(-1) glucagon or 10 microg x kg(-1) x h(-1) growth hormone with SRIF and insulin isolated the glucagon or growth hormone effect, respectively. The addition of metyrapone blocked cortisol release. Controls received only the isotope. In toto, the data can be interpreted to suggest that insulin has a greater effect on glucose uptake than on glucose production, and that neither glucagon, growth hormone, nor cortisol appreciably influenced the endogenous glucose production rate (Rp) during hyperinsulinemic hypoglycemia. The imprecise effect of these insulin counterregulatory hormones on neonatal glucose production mirrors the previously documented imprecise control by insulin.

The contribution of glucose to neonatal glucose homeostasis in the lamb

A multiplicity of substrates and hormones interact to influence neonatal glucose homeostasis. Based on prior studies in our laboratory, we hypothesized that glucose alone does not tightly control neonatal glucose homeostasis. Fifteen spontaneously delivered, mixed-breed term lambs, weighing 4.7 +/- 0.9 kg (mean +/- SD) were studied at 4.7 +/- 0.6 days after birth following administration of 100 microCi D[6,6-(3)H2] glucose in 0.9% NaCl by the prime plus constant infusion technique to measure glucose production. After a baseline period, five lambs received 8.5 mg x kg(-1) x min(-1) glucose in combination with the following to isolate the contribution of glucose: somatostatin to block insulin, glucagon, and growth hormone release; metyrapone to block cortisol release; phentolamine to block alpha-adrenergic release; and propranolol to block beta-adrenergic release (glucose + blockade). Five lambs received the above without the glucose infusion (blockade). Five lambs continued to receive 0.9% NaCl alone as the diluent for the isotopic tracer throughout the study (control). The glucose + blockade group had a significant increase in plasma glucose (P < .0001) and a significant increase in total glucose appearance (P < .0001) compared with both the blockade and control groups. Under conditions of glucose infusion at a rate 49% greater than the basal rate, the endogenous glucose production rate persisted such that there was only an evanescent decrease compared with that of the control group, which was not statistically different over time. As a substrate, glucose does not tightly control neonatal glucose homeostasis.

Cerebral metabolic response to hypoglycemia in severe intrauterine growth-retarded rat pups

This study was to examine the effects of hypoglycemia on the brain metabolism of severe intrauterine growth retardation (IUGR) rat pups. IUGR pups were produced by ligating one of the uterine arteries of the dams. The pups on the opposite uterine horn were used as the control. They were delivered by cesarean section on day 21 and received regular insulin 5 units/kg or equivalent volume of normal saline at 40 min of age. The plasma glucose, lactate, blood gas values and brain glucose, lactate, ATP contents were determined at 5 and 100 min of age. The IUGR pups had higher plasma and brain lactate concentrations than the control throughout the study period. They had lower plasma glucose, oxygen and pH values, brain glucose and ATP contents than control at 5 min of age. Despite insulin-induced hypoglycemia, brain ATP contents of the IUGR recovered to normal levels at 100 min of age when the oxygenation and pH improved. These data indicate that the brain energy metabolism of IUGR rat pups was suppressed by asphyxia and hypoglycemia. However, even in the continuing presence of hypoglycemia, brain energy metabolism returned to normal. The recovery is probably related to better oxygenation and utilization of alternative energy fuels, such as lactate.