Maternal low-protein diet on the last week of pregnancy contributes to insulin resistance and β-cell dysfunction in the mouse offspring

Abstract

Maternal low-protein diet (LP) throughout gestation affects pancreatic β-cell fraction of the offspring at birth, thus increasing their susceptibility to metabolic dysfunction and type 2 diabetes in adulthood. The present study sought to strictly examine the effects of LP during the last week of gestation (LP12.5) alone as a developmental window for β-cell programming and metabolic dysfunction in adulthood. Islet morphology analysis revealed normal β-cell fraction in LP12.5 newborns. Normal glucose tolerance was observed in 6- to 8-wk-old male and female LP12.5 offspring. However, male LP12.5 offspring displayed glucose intolerance and reduced insulin sensitivity associated with β-cell dysfunction with aging. High-fat diet exposure of metabolically normal 12-wk-old male LP12.5 induced glucose intolerance due to increased body weight, insulin resistance, and insufficient β-cell mass adaptation despite higher insulin secretion. Assessment of epigenetic mechanisms through microRNAs (miRs) by a real-time PCR-based microarray in islets revealed elevation in miRs that regulate insulin secretion (miRs 342, 143), insulin resistance (miR143), and obesity (miR219). In the islets, overexpression of miR143 reduced insulin secretion in response to glucose. In contrast to the model of LP exposure throughout pregnancy, islet protein levels of mTOR and pancreatic and duodenal homeobox 1 were normal in LP12.5 islets. Collectively, these data suggest that LP diet during the last week of pregnancy is critical and sufficient to induce specific and distinct developmental programming effects of tissues that control glucose homeostasis, thus causing permanent changes in specific set of microRNAs that may contribute to the overall vulnerability of the offspring to obesity, insulin resistance, and type 2 diabetes.

Keywords: diabetes; fetal nutrition disorders; fetal programming; islet biology; low-protein diet; β-cell function.

Fig. 1.

Maternal low-protein (LP) diet on the last week of pregnancy and phenotype of newborn offspring. A: pregnant C57BL/6 mice were exposed to diets [control (Ctrl, 23% protein) or LP during the last week of gestation (LP12.5, isocaloric and 9% protein)] during the last week of pregnancy. After delivery, LP12.5 and Ctrl dams and offspring were introduced to Ctrl diet. B: maternal body weight during the adaptation period [days (D) 0–30], during pregnancy [embryonic day (e) 05–16.5], and after pregnancy [postnatal day (P) 1–15]. C: litter size on P21 between LP12.5 and Ctrl dams. D–F: newborn body weight (D), blood glucose (E), body length (F) of LP12.5 and Ctrl offspring. G–J: newborn circulating insulin levels (G), β-cell area (H), average number of cells per islet (I), and β-cell proliferation, as measured through Ki-67 staining (J), were assessed in LP12.5 and Ctrl offspring. n = 4 Dams were used for maternal body weight assessment (B) and litter size (C). n = 16–27 Offspring were used for D–J and from n = 4 litter. Analyzed by Student’s t test, 2 tailed and unpaired, *P < 0.05 versus Ctrl.

Fig. 2.

Adult offspring administered low protein during the last week of gestation (LP12.5) exhibit glucose intolerance, but only females display insulin resistance. Intraperitoneal glucose tolerance tests were performed in 6- and 50-wk-old male [A and B, area under the curve (AUC), P = 0.026] and 6- and 48-wk-old female (C and D, AUC, P = 0.056) LP12.5 and control (Ctrl) offspring. Intraperitoneal insulin tolerance tests (ITT) were performed in 10- and 54-wk-old male (E and F, AUC, P = 0.787) and 8- and 51-wk-old female (G and H, AUC, P = 0.01) LP12.5 and Ctrl mice. *P < 0.05 vs. Ctrl. Analyzed by Student’s t test, 2 tailed and unpaired, *P < 0.05 vs. Ctrl, n = 4–9 animals in each group.

Fig. 3.

Reduced glucose-stimulated insulin secretion in aged male mice administered low protein (LP) during the last week of gestation (LP12.5). A and B: in vivo intraperitoneal glucose tolerance tests and glucose-stimulated insulin secretion (GSIS) were performed on 48-wk-old male LP12.5 and control (Ctrl) offspring [area under the curve (AUC), P = 0.2]. C: GSIS in isolated islets from adult LP12.5 and Ctrl male mice, in response to 2, 25 mM glucose and 30 mM KCl for 30 min. β-Cell mass of 48-wk-old male (D) and 51-wk-old female (E) LP12.5 and Ctrl mice. Ctrl, n = 4–5 mice. F: summary of altered microRNA level in islets of adult male LP12.5 mice. Only microRNAs passing the stringent threshold for significant change are shown. Islets were harvested from 12-wk-old LP12.5 and Ctrl mice. RNA was isolated by the MirVana Kit for OpenArray microarray. Quality of RNA was assessed by an Agilent Bioanalyzer (n = 5 mice). G: insulin secretion in primary mouse pancreatic β-cells from 12-wk-old male LP12.5 and Ctrl islets treated with or without miR143. LG, low glucose; HG, high glucose. H: representative Western blot for mTOR, pErk, total Erk, pancreatic and duodenal homeobox 1 (Pdx1), and Cdk5 regulatory-associated protein 1-like 1 (CDKAL1). Analyzed by Student’s t test, 2 tailed and unpaired, *P < 0.05 vs. Ctrl, n = 3–5.

Fig. 4.

Increased weight gain in adult male mice administered low protein during the last week of gestation (LP12.5) under high-fat (HF) diet-induced obesity. Body weight (A) and weight gain (B) over the course of HF diet in male LP12.5 and control (Ctrl) mice. Fed (C) and fasting (D) glucose over the course of HF diet in male LP12.5 and Ctrl mice. Fed (E) and fasting (F) insulin post-16-wk in HF diet of LP12.5 and Ctrl mice. Analyzed by Student’s t test, 2 tailed and unpaired, *P < 0.05 versus Ctrl, n = 6.

Fig. 5.

Adult male offspring administered low protein (LP) during the last week of gestation (LP12.5) exhibit glucose intolerance and insulin resistance in high-fat (HF) diet. Intraperitoneal glucose tolerance tests at 6 (A), 10 (B), and 14 (C) wk in HF [A and B: area under the curve (AUC), P = 0.05; C: AUC, P = 0.0063] and insulin tolerance tests at 16 wk in HF (D, AUC, P = 0.0238) of male LP12.5 and control (Ctrl) mice. In vivo glucose stimulated-insulin secretion (GSIS, E) and β-cell mass (F). Representative islets in male LP12.5 and Ctrl mice at 18 wk in HF diet (G, scale 100 μM). Analyzed by Student’s t test, 2-tailed and unpaired, *P < 0.05 vs. Ctrl, n = 4–6.

Fig. 6.

Normal level of miR143 in adipose tissue of male offspring administered low protein (LP) during the last week of gestation (LP12.5). Quantitative PCR (qPCR) of miR143 (A) and miR375 (B) epididymal white adipose tissue (eWAT) from LP12.5 and control (Ctrl) mice fed high-fat (HF) diet. Representative Western blot (C) and quantification (relative to vinculin or total-Akt, normalized to Ctrl average) of eWAT in HF diet from LP12.5 and Ctrl mice: phospho-Akt S473 (D), phospho-Akt T308 (E), and phospho-S6 S240 (F). G: representative immunohistochemistry (IHC) images of phosphorylated S6 (S240) in eWAT adipose tissue of male (48 wk of age) and female (51 wk of age) mice. Student’s t test, 2-tailed and unpaired, n = 4 mice.