A physiological increase in insulin suppresses muscle-specific ubiquitin ligase gene activation in fetal sheep with sustained hypoglycemia.

Decreased glucose transfer to the fetus is characteristic of pregnancies complicated by maternal under nutrition and placental insufficiency. Chronic experimental restriction of glucose transfer to the sheep fetus for the final 40% of gestation with a maternal insulin infusion (HG fetuses) results in fetal hypoglycemia, hypoinsulinemia, and decreased rates of fetal growth and protein accretion compared to controls (CON). Lower rates of fetal protein accretion are due to increased fetal protein breakdown and not decreased protein synthesis. However, the specific skeletal muscle pathways responsible for increased protein breakdown have not been determined. Nor has it been determined if low fetal glucose or insulin concentrations are more important for regulating these skeletal muscle protein breakdown pathways. We tested whether chronic restriction of glucose transfer to the fetus increased the ubiquitin-proteosome pathway or autophagy-lysosome pathway in fetal sheep skeletal muscle and found no evidence for an increase in the autophagy-lysosome pathway. However, HG fetuses had increase mRNA expression of MaFBx1 (twofold, P < 0.01) and a trend for increased mRNA expression of MuRF1 (P = 0.08) compared to CON. A subset of chronically hypoglycemic fetuses received an isoglycemic insulin infusion for the final 7 days of the maternal insulin infusion (HG + INS fetuses) and had MaFBx1 and MuRF1 mRNA concentrations similar to CON fetuses. These results demonstrate that fetuses exposed to sustained hypoglycemia have decreased protein accretion due to activation of the skeletal muscle ubiquitin-proteosome pathway and that a fetal hyperinsulinemic clamp can suppress this pathway even in the context of continued hypoglycemia.

Changes in fetal mannose and other carbohydrates induced by a maternal insulin infusion in pregnant sheep.

BACKGROUND: The importance of non-glucose carbohydrates, especially mannose and inositol, for normal development is increasingly recognized. Whether pregnancies complicated by abnormal glucose transfer to the fetus also affect the regulation of non-glucose carbohydrates is unknown. In pregnant sheep, maternal insulin infusions were used to reduce glucose supply to the fetus for both short (2-wk) and long (8-wk) durations to test the hypothesis that a maternal insulin infusion would suppress fetal mannose and inositol concentrations. We also used direct fetal insulin infusions (1-wk hyperinsulinemic-isoglycemic clamp) to determine the relative importance of fetal glucose and insulin for regulating non-glucose carbohydrates. RESULTS: A maternal insulin infusion resulted in lower maternal (50%, P < 0.01) and fetal (35-45%, P < 0.01) mannose concentrations, which were highly correlated (r(2) = 0.69, P < 0.01). A fetal insulin infusion resulted in a 50% reduction of fetal mannose (P < 0.05). Neither maternal nor fetal plasma inositol changed with exogenous insulin infusions. Additionally, maternal insulin infusion resulted in lower fetal sorbitol and fructose (P < 0.01). CONCLUSIONS: Chronically decreased glucose supply to the fetus as well as fetal hyperinsulinemia both reduce fetal non-glucose carbohydrates. Given the role of these carbohydrates in protein glycosylation and lipid production, more research on their metabolism in pregnancies complicated by abnormal glucose metabolism is clearly warranted.

Increased fetal insulin concentrations for one week fail to improve insulin secretion or ß-cell mass in fetal sheep with chronically reduced glucose supply.

Maternal undernutrition during pregnancy and placental insufficiency are characterized by impaired development of fetal pancreatic ß-cells. Prolonged reduced glucose supply to the fetus is a feature of both. It is unknown if reduced glucose supply, independent of other complications of maternal undernutrition and placental insufficiency, would cause similar ß-cell defects. Therefore, we measured fetal insulin secretion and ß-cell mass following prolonged reduced fetal glucose supply in sheep. We also tested whether restoring physiological insulin concentrations would correct any ß-cell defects. Pregnant sheep received either a direct saline infusion (CON = control, n = 5) or an insulin infusion (HG = hypoglycemic, n = 5) for 8 wk in late gestation (75 to 134 days) to decrease maternal glucose concentrations and reduce fetal glucose supply. A separate group of HG fetuses also received a direct fetal insulin infusion for the final week of the study with a dextrose infusion to prevent a further fall in glucose concentration [hypoglycemic + insulin (HG+I), n = 4]. Maximum glucose-stimulated insulin concentrations were 45% lower in HG fetuses compared with CON fetuses. ß-Cell, pancreatic, and fetal mass were 50%, 37%, and 40% lower in HG compared with CON fetuses, respectively (P < 0.05). Insulin secretion and ß-cell mass did not improve in the HG+I fetuses. These results indicate that chronically reduced fetal glucose supply is sufficient to reduce pancreatic insulin secretion in response to glucose, primarily due to reduced pancreatic and ß-cell mass, and is not correctable with insulin.

Increased amino acid supply potentiates glucose-stimulated insulin secretion but does not increase ß-cell mass in fetal sheep.

Amino acids and glucose acutely stimulate fetal insulin secretion. In isolated adult pancreatic islets, amino acids potentiate glucose-stimulated insulin secretion (GSIS), but whether amino acids have this same effect in the fetus is unknown. Therefore, we tested the effects of increased fetal amino acid supply on GSIS and morphology of the pancreas. We hypothesized that increasing fetal amino acid supply would potentiate GSIS. Singleton fetal sheep received a direct intravenous infusion of an amino acid mixture (AA) or saline (CON) for 10-14 days during late gestation to target a 25-50% increase in fetal branched-chain amino acids (BCAA). Early-phase GSIS increased 150% in the AA group (P < 0.01), and this difference was sustained for the duration of the hyperglycemic clamp (105 min) (P < 0.05). Glucose-potentiated arginine-stimulated insulin secretion (ASIS), pancreatic insulin content, and pancreatic glucagon content were similar between groups. ß-Cell mass and area were unchanged between groups. Baseline and arginine-stimulated glucagon concentrations were increased in the AA group (P < 0.05). Pancreatic α-cell mass and area were unchanged. Fetal and pancreatic weights were similar. We conclude that a sustained increase of amino acid supply to the normally growing late-gestation fetus potentiated fetal GSIS but did not affect the morphology or insulin content of the pancreas. We speculate that increased ß-cell responsiveness (insulin secretion) following increased amino acid supply may be due to increased generation of secondary messengers in the ß-cell. This may be enhanced by the paracrine action of glucagon on the ß-cell.

A physiological increase in insulin suppresses gluconeogenic gene activation in fetal sheep with sustained hypoglycemia.

Reduced maternal glucose supply to the fetus and resulting fetal hypoglycemia and hypoinsulinemia activate fetal glucose production as a means to maintain cellular glucose uptake. However, this early activation of fetal glucose production may be accompanied by hepatic insulin resistance. We tested the capacity of a physiological increase in insulin to suppress fetal hepatic gluconeogenic gene activation following sustained hypoglycemia to determine whether hepatic insulin sensitivity is maintained. Control fetuses (CON), hypoglycemic fetuses induced by maternal insulin infusion for 8 wk (HG), and 8 wk HG fetuses that received an isoglycemic insulin infusion for the final 7 days (HG+INS) were studied. Glucose and insulin concentrations were 60% lower in HG compared with CON fetuses. Insulin was 50% higher in HG+INS compared with CON and four-fold higher compared with HG fetuses. Expression of the hepatic gluconeogenic genes, PCK1, G6PC, FBP1, GLUT2, and PGC1A was increased in the HG and reduced in the HG+INS liver. Expression of the insulin-regulated glycolytic and lipogenic genes, PFKL and FAS, was increased in the HG+INS liver. Total FOXO1 protein expression, a gluconeogenic activator, was 60% higher in the HG liver. Despite low glucose, insulin, and IGF1 concentrations, phosphorylation of AKT and ERK was higher in the HG liver. Thus, a physiological increase in fetal insulin is sufficient for suppression of gluconeogenic genes and activation of glycolytic and lipogenic genes in the HG fetal liver. These results demonstrate that fetuses exposed to sustained hypoglycemia have maintained hepatic insulin action in contrast to fetuses exposed to placental insufficiency.