A Chronic Fetal Leucine Infusion Potentiates Fetal Insulin Secretion and Increases Pancreatic Islet Size, Vascularity, and β Cells in Late-Gestation Sheep

Abstract

Background: Infusion of a complete amino acid mixture into normal late-gestation fetal sheep potentiates glucose-stimulated insulin secretion (GSIS). Leucine acutely stimulates insulin secretion in late-gestation fetal sheep and isolated fetal sheep islets in vitro.

Objectives: We hypothesized that a 9-d leucine infusion would potentiate GSIS in fetal sheep.

Methods: Columbia-Rambouillet fetal sheep at 126 days of gestation received a 9-d leucine infusion to achieve a 50%-100% increase in leucine concentrations or a control infusion. At the end of the infusion we measured GSIS, pancreatic morphology, and expression of pancreatic mRNAs. Pancreatic islet endothelial cells (ECs) were isolated from fetal sheep and incubated with supplemental leucine or vascular endothelial growth factor A (VEGFA) followed by collection of mRNA. Data measured at multiple time points were compared with a repeated-measures 2-factor ANOVA. Data measured at 1 time point were compared using Student's t test or the Mann-Whitney test.

Results: Glucose-stimulated insulin concentrations were 80% higher in leucine-infused (LEU) fetuses than in controls (P < 0.05). In the pancreas, LEU fetuses had a higher proportion of islets >5000 μm2 than controls (75% more islets >5000 μm2; P < 0.05) and a larger proportion of the pancreas that stained for β cells (12% greater; P < 0.05). Pancreatic and pancreatic islet vascularity were both 25% greater in LEU fetuses (P < 0.05). Pancreatic VEGFA and hepatocyte growth factor (HGF) mRNA expressions were 38% and 200% greater in LEU fetuses than in controls (P < 0.05), respectively. In isolated islet ECs, HGF mRNA was 20% and 50% higher after incubation in supplemental leucine (P < 0.05) or VEGFA (P < 0.01), respectively.

Conclusions: A 9-d leucine infusion potentiates fetal GSIS, demonstrating that glucose and leucine act synergistically to stimulate insulin secretion in fetal sheep. A greater proportion of the pancreas being comprised of β cells and higher pancreatic vascularity contributed to the higher GSIS.

Keywords: VEGFA; fetus; hepatocyte growth factor; insulin; islet; leucine; pancreas; pregnancy; vascularity; β cell.

FIGURE 1

Fetal leucine infusion rates (A) and plasma leucine concentrations (B) in CON and LEU sheep. (A) Values are mean ± SE (n = 9) fetal leucine infusion rates in the LEU group. (B) Values are mean ± SE fetal arterial plasma leucine concentrations in CON (n = 8) and LEU (n = 9) animals. *Different from day 0 in the leucine group: P < 0.05. CON, control; LEU, leucine-infused.

FIGURE 2

Fetal GSIS (A, B) and arginine-stimulated insulin (C) and glucagon (D) secretion in CON and LEU sheep. Fetal GSIS was measured with a fetal hyperglycemic clamp beginning at time 0. Values are means ± SEs in CON (n = 8 for insulin and glucose, and n = 6 for glucagon) and LEU (n = 9) fetuses. Fetal plasma glucose (A) and insulin (B) concentrations during measurement of GSIS. Fetal arginine-stimulated (C) insulin and (D) glucagon concentrations. *Difference between CON and LEU fetuses: P < 0.05. CON, control; GSIS, glucose-stimulated insulin secretion; LEU, leucine-infused.

FIGURE 3

Chronic leucine infusions in fetal sheep resulted in fetal pancreases with larger islets (A), larger insulin⁺ areas of the pancreas (B) and pancreatic islets (C), and more vascularization (D). (A) Values are means ± SEs in CON (n = 8) and LEU (n = 9) fetuses. The frequency, as a percentage of total islet number, of islets 500–5000, 5000–20,000, and >20,000 μm² is shown for CON and chronically LEU fetuses. There was a higher proportion of larger islets and lower proportion of smaller islets in the LEU fetuses (P < 0.05). (B–D) Individual animal data with group means ± SEs are shown for CON (n = 8) and LEU (n = 9) fetuses. (B) Insulin⁺ and glucagon⁺ areas of the pancreas, (C) insulin⁺, glucagon⁺, and somatostatin⁺-pancreatic polypeptide⁺ areas of the islet, and (D) pancreatic and pancreatic islet vascularity. ^*,**Difference between CON and LEU fetuses: *P < 0.05; **P < 0.01. CON, control; LEU, leucine-infused.

FIGURE 4

Chronic leucine infusions in fetal sheep resulted in more pancreatic mRNA expression of key metabolic and paracrine signaling genes. Values are means ± SEs in CON (n = 8) and LEU (n = 9) fetuses. Expression in LEU pancreases is shown relative to expression in CON pancreases. ^*,**Significant difference between CON and LEU fetuses: *P < 0.05; **P < 0.01. ^#P = 0.066. CON, control; LEU, leucine-infused.

FIGURE 5

Supplemental leucine and VEGFA increase HGF mRNA expression in isolated fetal sheep islet ECs. Isolated fetal islet ECs were incubated with supplemental leucine (n = 4) or VEGFA (n = 3). HGF mRNA expression was then normalized to expression in control media. Values are means ± SEs. ^*,**Significant differences from nonsupplemented conditions: *P < 0.05; **P < 0.01. CON, control; EC, endothelial cell; GCG, glucagon; GCK, glucokinase; HGF, hepatocyte growth factor; INS, insulin; PDX1, pancreatic and duodenal homeobox-1; SLC2A2, glucose transporter-2; VEGFA, vascular endothelial growth factor A.