Maternal insulin resistance and transient hyperglycemia impact the metabolic and endocrine phenotypes of offspring

Abstract

Studies in both humans and rodents suggest that maternal diabetes leads to a higher risk of the fetus developing impaired glucose tolerance and obesity during adulthood. However, the impact of hyperinsulinemia in the mother on glucose homeostasis in the offspring has not been fully explored. We aimed to determine the consequences of maternal insulin resistance on offspring metabolism and endocrine pancreas development using the LIRKO mouse model, which exhibits sustained hyperinsulinemia and transient increase in blood glucose concentrations during pregnancy. We examined control offspring born to either LIRKO or control mothers on embryonic days 13.5, 15.5, and 17.5 and postpartum days 0, 4, and 10. Control offspring born to LIRKO mothers displayed low birth weights and subsequently rapidly gained weight, and their blood glucose and plasma insulin concentrations were higher than offspring born to control mothers in early postnatal life. In addition, concentrations of plasma leptin, glucagon, and active GLP-1 were higher in control pups from LIRKO mothers. Analyses of the endocrine pancreas revealed significantly reduced β-cell area in control offspring of LIRKO mothers shortly after birth. β-Cell proliferation and total islet number were also lower in control offspring of LIRKO mothers during early postnatal days. Together, these data indicate that maternal hyperinsulinemia and the transient hyperglycemia impair endocrine pancreas development in the control offspring and induce multiple metabolic alterations in early postnatal life. The relatively smaller β-cell mass/area and β-cell proliferation in these control offspring suggest cell-autonomous epigenetic mechanisms in the regulation of islet growth and development.

Keywords: endocrine pancreas development; hyperinsulinemia; intrauterine environment; maternal insulin resistance; offspring metabolism.

Fig. 1.

Breeding scheme. Female control mice were crossed to male control mice to produce control offspring exposed to a normal intrauterine environment (CC, control offspring from control mother). Female liver-specific insulin receptor knockout (LIRKO) mice were crossed to male control mice to produce control offspring exposed to an insulin-resistant intrauterine environment (CL, control offspring from LIRKO mother; LL, LIRKO offspring from LIRKO mother). All studies were focused on comparing control pups (CC vs. CL). IR, insulin receptor; alb, albumin.

Fig. 2.

Changes in body weight and metabolic and hormonal parameters in offspring. Changes in body weight (A and B), blood glucose concentrations (C and D), and plasma insulin concentrations (E and F) with age for male (A, C, and E) and female (B, D, F) offspring. Changes in plasma leptin, glucagon, and glucagon-like peptide-1 (GLP-1) concentrations of 4-day-old male (G) and female (H) offspring. Open bars, CC; black bars, CL. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. CC (Student's t-test). Data are expressed as means ± SE. Numbers (n) are included within each bar. E13.5, E15.5, and E17.5, embryonic days 13.5, 15.5, and 17.5, respectively; P0, newborn (postnatal day 0); P4 and P10, postnatal days 4 and 10, respectively.

Fig. 3.

Plasma C-peptide concentrations and C-peptide/insulin (C/I) ratios in the male offspring. Changes in plasma C-peptide concentrations of 4- (A) and 10-day-old male offspring (B). The C/I ratio was calculated as the C-peptide concentration divided by the insulin concentration for each individual pup. C/I ratios were given for 4- (C) and 10-day-old males (D). Open bars, CC; black bars, CL. Data are expressed as means ± SE; n = 3/group.

Fig. 4.

Representative hematoxylin and eosin staining of subcutaneous adipose tissue of 10-day-old male (A) and female (B) offspring. Scale bar, 100 μm. Mean adipocyte diameter (μm) for males (C) and females (D). Adipocyte cell size distribution for males (E) and females (F). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. CC (Student's t-test). Data are expressed as means ± SE; n = 4/group.

Fig. 5.

Expression of genes related to adipocyte differentiation by quantitative RT-PCR of RNA from white adipose tissue of 10-day-old males (A) and females (B). *P < 0.05 and ***P < 0.001 vs. CC (Student's t-test). Data are expressed as means ± SE; n = 4/group. Data are normalized to expression of TATA box-binding protein (TBP).

Fig. 6.

Changes in islet morphology and β-cell proliferation in the offspring. A: representative immunofluorescent images of islets from male offspring at different ages coimmunostained for insulin (red), glucagon (blue), and somatostatin (green). Top: CC. Bottom: CL. Scale bar, 200 μm. B and C: alterations in β-cell mass (B) and %β-cell area (C) with age in the male and female offspring. D: alterations in total no. of islets in the male and female offspring. E: coimmunostaining of pancreas sections for the proliferation marker Ki-67 (green) with insulin (red) and DAPI (blue). Arrows indicate proliferating β-cells. Top: CC. Bottom: CL. Scale bar, 200 μm. F: changes in %β-cell proliferation in the male and female offspring. Open bars, control offspring from control mother; black bars, control offspring from LIRKO mother. *P < 0.05 vs. CC (Student's t-test). Data are expressed as means ± SE; n = 3–4/group (2 sections per pancreas).

Fig. 6.

Changes in islet morphology and β-cell proliferation in the offspring. A: representative immunofluorescent images of islets from male offspring at different ages coimmunostained for insulin (red), glucagon (blue), and somatostatin (green). Top: CC. Bottom: CL. Scale bar, 200 μm. B and C: alterations in β-cell mass (B) and %β-cell area (C) with age in the male and female offspring. D: alterations in total no. of islets in the male and female offspring. E: coimmunostaining of pancreas sections for the proliferation marker Ki-67 (green) with insulin (red) and DAPI (blue). Arrows indicate proliferating β-cells. Top: CC. Bottom: CL. Scale bar, 200 μm. F: changes in %β-cell proliferation in the male and female offspring. Open bars, control offspring from control mother; black bars, control offspring from LIRKO mother. *P < 0.05 vs. CC (Student's t-test). Data are expressed as means ± SE; n = 3–4/group (2 sections per pancreas).

Fig. 7.

Changes in the number of islet clusters and α-cell area in the offspring. A: representative immunofluorescent images of newly formed islets from 10-day-old male offspring coimmunostained for insulin (Ins; red), glucagon (Glu; blue), and somatostatin (Ss; green). Arrows indicate small islet clusters. Left: CC. Right: CL. Scale bar, 100 μm. B: the number of small islet clusters was decreased significantly in 10-day-old CL vs. CC in both sexes. C and D: alterations in α-cell area (C) and α-cell mass (D) of male and female offspring at different ages. *P < 0.05 vs. CC (Student's t-test). Data are expressed as means ± SE; n = 3–4/group (2 sections per pancreas).