Chronic late-gestation hypoglycemia upregulates hepatic PEPCK associated with increased PGC1alpha mRNA and phosphorylated CREB in fetal sheep

Abstract

Hepatic glucose production is normally activated at birth but has been observed in response to experimental hypoglycemia in fetal sheep. The cellular basis for this process remains unknown. We determined the impact of 2 wk of fetal hypoglycemia during late gestation on enzymes responsible for hepatic gluconeogenesis, focusing on the insulin-signaling pathway, transcription factors, and coactivators that regulate gluconeogenesis. Hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNA increased 12-fold and 7-fold, respectively, following chronic hypoglycemia with no change in hepatic glycogen. Chronic hypoglycemia decreased fetal plasma insulin with no change in glucagon but increased plasma cortisol 3.5-fold. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA and phosphorylation of cAMP response element binding protein at Ser(133) were both increased, with no change in Akt, forkhead transcription factor FoxO1, hepatocyte nuclear factor-4alpha, or CCAAT enhancer binding protein-beta. These results demonstrate that chronic fetal hypoglycemia triggers signals that can activate gluconeogenesis in the fetal liver.

Figure 1. Hepatic mRNA concentrations

PEPCK (A), G6Pase (B), and PGC1α (C) mRNA concentrations were determined in livers from Control and Hypoglycemic fetuses by real-time quantitative PCR. Data was normalized to ribosomal protein s15 and is presented as fold change relative to control fetuses with SEM bars. Treatment groups are listed on the x-axis. The * denotes a higher amount of PEPCK (p<0.05) and G6Pase (p<0.0005) and PGC1α (p<0.05) in Hypoglycemic livers compared to Control livers. All statistics are from the Mann-Whitney test for nonparametric analysis.

Figure 2. Hepatic Glycogen

Glycogen content (milligram per gram of tissue) with SEM bars was determined for Control and Hypoglycemic fetuses. No differences were found between treatment groups, which are listed on the x-axis.

Figure 3. Hepatic Protein Concentrations

(A) Representative Western Blots for Insulin Receptor β, CREB, ser133 phosphorylated CREB, and β-actin. (B-D) Results of Western Blot analysis for Insulin Receptor β normalized to β-actin (B), CREB normalized to β-actin (C), and ser133 phosphorylated CREB normalized to total CREB (D). Treatment groups are listed on the x-axis. The * denotes a higher amount of Insulin Receptor β (p<0.05) and a higher ratio of ser133 phosphorylated CREB to total CREB (p<0.01) in Hypoglycemic livers compared to Control livers, and a lower amount of CREB in Hypoglycemic livers compared to Control livers (p<0.001). Statistics are from either Student's t test (parametric) or the Mann-Whitney test (nonparametric).