Differential effects of intrauterine growth restriction and a hypersinsulinemic-isoglycemic clamp on metabolic pathways and insulin action in the fetal liver

Abstract

Intrauterine growth-restricted (IUGR) fetal sheep have increased hepatic glucose production (HGP) that is resistant to suppression during a hyperinsulinemic-isoglycemic clamp (insulin clamp). We hypothesized that the IUGR fetal liver would have activation of metabolic and signaling pathways that support HGP and inhibition of insulin-signaling pathways. To test this, we used transcriptomic profiling with liver samples from control (CON) and IUGR fetuses receiving saline or an insulin clamp. The IUGR liver had upregulation of genes associated with gluconeogenesis/glycolysis, transcription factor regulation, and cytokine responses and downregulation of genes associated with cholesterol synthesis, amino acid degradation, and detoxification pathways. During the insulin clamp, genes associated with cholesterol synthesis and innate immune response were upregulated in CON and IUGR. There were 20-fold more genes differentially expressed during the insulin clamp in IUGR versus CON. These genes were associated with proteasome activation and decreased amino acid and lipid catabolism. We found increased TRB3, JUN, MYC, and SGK1 expression and decreased PTPRD expression as molecular targets for increased HGP in IUGR. As candidate genes for resistance to insulin's suppression of HGP, expression of JUN, MYC, and SGK1 increased more during the insulin clamp in CON compared with IUGR. Metabolites were measured with ¹H-nuclear magnetic resonance and support increased amino acid concentrations, decreased mitochondria activity and energy state, and increased cell stress in the IUGR liver. These results demonstrate a robust response, beyond suppression of HGP, during the insulin clamp and coordinate responses in glucose, amino acid, and lipid metabolism in the IUGR fetus.

Keywords: developmental programming; fetus; gluconeogenesis; insulin resistance; liver.

Fig. 1.

Effect of intrauterine growth restriction (IUGR) and hyperinsulinemic-isoglycemic clamp (insulin clamp) on fetal hepatic transcriptome. A: gene expression data were analyzed by two-way ANOVA (4 experimental groups, n = 3 per group) resulting in groups of genes with a significant (P < 0.05) main effect for IUGR or clamp and those with a significant interaction (P < 0.05). The Venn diagram illustrates the groups of genes used for functional annotation. All genes with a significant interaction effect were grouped together (yellow circle). The remaining genes with significant main effect of IUGR or clamp were grouped together. B: genes with a significant main effect (P < 0.05) of IUGR and fold change >1.5 were loaded into a clustered heat map. C: genes with a significant main effect (P < 0.05) of clamp and fold change >1.5 were loaded into a clustered heat map. Genes with a significant IUGR × insulin clamp interaction were sorted into three categories based on biologically meaningful criteria using individual posttest as described in materials and methods. D and E: genes that were down- or upregulated only in the IUGR liver during the insulin clamp. F: genes resistant to insulin in the IUGR liver (IUGR + IR, yellow). In each heat map, genes that were downregulated or upregulated in are shown in blue or red, respectively. CON, control.

Fig. 2.

Regulation of metabolic genes and lipid synthesis by intrauterine growth restriction (IUGR) and hyperinsulinemic-isoglycemic clamp (insulin clamp) conditions in fetal liver. A: expression of metabolic genes arginase 2 (ARG2) and IDH1 in control (CON) and IUGR fetal livers during basal or insulin conditions. B: fetal hepatic triglyceride content. C: expression of genes in lipid biosynthesis pathway measured in control and IUGR fetal livers during basal or insulin clamp conditions. Significant effects from two-way ANOVA are indicated. Means ± SE are shown for each group in saline (n = 5 CON, 9 IUGR) and insulin (n = 11 CON, 13 IUGR). ACC1, acetyl-Co-A carboxylase 1; FASN, fatty acid synthase; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; HMGCS1, 3-hydroxy-3-methylglutaryl-CoA synthase 1; SREBP1c, sterol regulatory element binding transcription factor 1c.

Fig. 3.

Activation of proteins regulating protein synthesis pathway in the fetal liver in response to insulin clamp conditions. A: phosphorylated (p-) and total protein expression was measured by Western blotting in whole cell tissue lysates for mammalian target of rapamycin (mTOR) (S2448), ribosomal protein S6 kinase (S6K) (S421, T424), ribosomal protein S6 (S6) (S235/S236), 4EBP1 (T37/T46), and eIF2α (S51). Representative images are shown. B: results were quantified, and the ratio of phosphorylated to total protein was calculated. Means ± SE are shown for saline [n = 4 control (CON), 4 intrauterine growth restriction (IUGR)] and insulin (n = 6 CON, 6 IUGR). Significant effects from two-way ANOVA are indicated. 4EBP1, eukaryotic translation initiation factor 4E binding protein 1; eIF2α, eukaryotic translation initiation factor 2A; insulin clamp, hyperinsulinemic-isoglycemic clamp.

Fig. 4.

AMP-activated protein kinase (AMPK) and ERK signaling pathways in the intrauterine growth-restricted (IUGR) fetus and in response to the hyperinsulinemic-isoglycemic clamp (insulin clamp). Phosphorylated (p-) and total protein expression for ERK (T202, Y204) and AMPK (T172) measured by Western blotting in whole cell tissue lysates. Representative images are shown. Results were quantified and ratio of phosphorylated to total protein was calculated. Means ± SE are shown for saline [n = 4 control (CON), 4 IUGR) and insulin (n = 6 CON, 6 IUGR]. Significant effects from two-way ANOVA are indicated.

Fig. 5.

Candidate genes involved in regulating insulin sensitivity in intrauterine growth-restricted (IUGR) fetal liver. Expression of candidate genes for development of hepatic insulin resistance in IUGR are shown. The fold increase by insulin during the hyperinsulinemic-isoglycemic clamp (insulin clamp) within the control (CON) or IUGR group is indicated above the bars. Significant effects from two-way ANOVA are indicated. Means ± SE are shown for each group in saline (n = 5 CON, 9 IUGR) and insulin (n = 11 CON, 13 IUGR). APC, WNT signaling pathway regulator; JUN, AP-1 transcription factor subunit; MYC, c-MYC transcription factor; PPARG, peroxisome proliferator activated receptor-γ; PTPRD, protein tyrosine phosphatase receptor type D; SGK1, serum/glucocorticoid regulated kinase 1; TRIB3, tribbles pseudokinase 3.

Fig. 6.

Effect of intrauterine growth restriction (IUGR) and the hyperinsulinemic-isoglycemic clamp (insulin clamp) on fetal hepatic metabolites. Fetal liver samples were used for ¹H-nuclear magnetic resonance (¹H-NMR) profiling of hydrophilic metabolites (n = 4 per group). Partial least squares discriminate analysis (PLS-DA) was used to identify metabolites with changes in abundance that defined separation of samples between the groups, and the 2-dimensional blot is shown (A). Heat map of the 15 metabolites with the highest variable importance in projection score (VIP) scores between the groups (B). Each square is representative of the concentrations of that metabolite. Row values are normalized for each metabolitem and quantitative changes are color coded from blue (least) to red (greatest). Metabolite name and pathway category assignment is shown on left y-axis. CON, control; insulin clamp, hyperinsulinemic-isoglycemic clamp.