Preconception Alcohol Exposure Increases the Susceptibility to Diabetes in the Offspring

Abstract

Heavy alcohol drinking alters glucose metabolism, but the inheritability of this effect of alcohol is not well understood. We used an animal model of preconception alcohol exposure in which adult female rats were given free access to 6.7% alcohol in a liquid diet and water for about 4 weeks, went without alcohol for 3 weeks, and then were bred to generate male and female offspring. Control animals were either ad lib-fed rat chow or pair-fed an isocaloric liquid diet during the time of alcohol-feeding in the experimental animals. Our results show that the female rats fed with alcohol in the liquid diet, but not with the isocaloric liquid diet, prior to conception had an altered stress gene network involving glucose metabolism in oocytes when compared with those in ad lib-fed chow diet controls. The offspring born from preconception alcohol-fed mothers showed significant hyperglycemia and hypoinsulinemia when they were adults. These rats also showed increased levels of inflammatory cytokines and cellular apoptosis in the pancreas, altered insulin production and actions in the liver, and a reduced number of proopiomelanocortin neurons in the hypothalamus. Replenishment of proopiomelanocortin neurons in these animals normalized the abnormal glucose to restore homeostasis. These data suggest that preconception alcohol exposures alter glucose homeostasis by inducing proopiomelanocortin neuronal functional abnormalities. Our findings provide a novel insight into the impact of high doses of alcohol on the female gamete that may cause inheritance of an increased susceptibility to diabetes.

Keywords: alcohol; diabetes; hyperglycemia; hypoinsulinemia; preconception; proopiomelanocortin.

Figure 1.

Showing the amount of daily liquid diet intake by alcohol-fed (AF) and pair-fed (PF) rats (a), changes in body weight of AF, PF, and ad lib chow-fed (AD) rats (b), and the changes in blood concentrations of ethanol in AF rats (c) used in generating offspring for preconception alcohol exposure (PCAE) study. Data shown are mean ± SEM (n = 8).

Figure 2.

Preconception alcohol exposure (PCAE) alters oocyte transcriptomes in adult female rats as determined by RNA-seq analysis. (a-c) Expression of genes significantly downregulated (green) or upregulated (red) in oocytes as identified by RNA-seq analysis (cut-off log₂ fold change ≥ 2; vertical lines) of alcohol-fed (AF) animal oocytes vs ad libitum–fed normal rat chow (AD) animal oocytes (a), AF vs pair-fed liquid diet (PF) animal oocytes (b), or PF vs AD oocytes (c) as plotted against an FDR value < 0.01 (horizontal lines). Each dot represents the transcriptome from 3 biological replicates per group. [d] Venn view of significant downregulated and upregulated genes as determined by RNA-seq analysis in both AF vs AD and AF vs PF. (e) Ingenuity Pathway Analysis (IPA) identified the significantly affected canonical pathways including glucose metabolism (bars in red), immune functions (bars in orange), and stress regulation (bar in green) in AF animals.

Figure 3.

PCAE induces abnormalities in glucose homeostasis of female and male rat litters during the adult period. Female (F) and male (M) litters of AD (AD-F, AD-M), PF (PF-F, PF-M), and AF (AF-F, AF-M) mothers were tested for glucose and insulin status during the adult period. Shown in this figure are fasting glucose (a, b), fasting insulin (c, d), fasting glucagon (e, f), fasting leptin (g, h), blood glucose level after oral glucose tolerance test (OGTT) (i, k), area under the curve of blood glucose during OGTT (j, l), glucose-stimulated insulin secretion (GSIS) (m, o), area under the curve of blood insulin during GSIS (n, p), blood glucose during intraperitoneal insulin tolerance test (IPITT) (q,s), and area under the curve of blood glucose during IPITT (r, t). Data are presented as mean ± SEM (n = 7). Data were compared by 1-way analysis of variance (ANOVA) and then the Newman-Keuls posttest. Differences between control and other treatment groups are shown by lines with P values (*, P < 0.05 and **, P < 0.01) above the bar graphs.

Figure 4.

PCAE induces abnormalities in pancreatic cell functions in the litter. Shown here are the immunohistochemical characterizations of the single-labeled hormone insulin (a), glucagon (c), inflammatory markers IL-6 (e), COX-2 (g), IFN-γ (i), and CD3 (k), apoptotic marker Caspase 3 (m), and cell proliferation marker Ki-67 (0) in the pancreas of PCAE and control exposed female litters. Pictures shown are representative photographs. “-” denotes 100 µM. The intensity of protein staining in each section (10 areas/section) was quantitated and presented as mean ± SEM (n = 7) in histograms on the right for insulin (b), glucagon (d), IL-6 (f), COX-2 (h), IFN-γ (j), CD3 (i), Caspase 3 (n), and Ki-67 (p). Data were compared by 1-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between control and other treatment groups are shown by lines with P values above the bar graphs.

Figure 5.

PCAE reduces the level of insulin receptors and its signaling molecules in the liver of the litter. Histograms showing the data of insulin receptor proteins (a), insulin receptor mRNA (b), GLUT2 mRNA (c), Akt mRNA (d), glucokinase mRNA (e), FOXO1 mRNA (f), PEPCK mRNA (g), glucose-6-pase mRNA (h), PDK1 (i), and PTEN (j) in AD, PF, and AF hepatic tissues. Data are mean ± SEM (n = 6) and were compared by ANOVA and the Newman-Keuls posttest. Differences between control and other treatment groups are shown by lines with P values above the bar graphs. [k] Schematic diagram illustrating how the observed changes in insulin receptor function affect glucose homeostasis in PCAE offspring. (<-- activation; |-- inhibition).

Figure 6.

PCAE increases the susceptibility to develop diabetes in the litter. Histograms show the effects of PCAE on the level of blood glucose (a), blood insulin (b), and various inflammatory cytokines shown as blots (c) or as mean ± SEM (n = 5–7) actin ratios of IL-1β (d), IL-6 (e), or TNF-α (f) in the pancreas after 2 weeks of 40% HFD followed by a single injection of 40mg/kg of STZ treatment. *P < 0.05 and **P < 0.01.

Figure 7.

Third ventricular injection of c-AMP-delivering nanospheres increases β-endorphin cell numbers in the hypothalamus and prevents PCAE effects on fasting blood glucose and insulin in the litter. Percentage changes in β-endorphin neuronal number in the arcuate nucleus of AF-F and PF-F rats as compared to AD-F rats (a). Representative stainings of β-endorphin neurons (in green) are shown in d, f, and h. Illustrations showing the site where nanospheres containing cAMP or vehicle are administered (b). Immunofluorescence pictures of β-endorphin neuronal cells in the hypothalamus of AD (d), PF (f), and AF (h) litters not treated, treated with nanospheres containing vehicle (Nano-V-F), or treated with nanospheres containing cAMP (Nano-cAMP-F). Pictures shown are representative photographs. “-” denotes 100 µM. The number of β-endorphin neurons in the hypothalamus of each group was quantitated and the percentage of these neurons in AD-F was calculated and presented as mean ± SEM in histograms on the right (c, e, g). Effects of nanospheres containing cAMP on fasting blood glucose in AD (i), PF (j), and AF animals (k), on fasting blood insulin in AD (l), PF (m), and AF animals (n), fasting blood glucagon in AD (o), PF (p), and AF animals (q) and fasting blood leptin in AD (r), PF (s), and AF animals (t). Data are mean ± SEM (n = 6) and were compared by ANOVA and the Newman-Keuls posttest. Differences between control and other treatment groups are shown by lines with P values above the bar graphs.

Figure 8.

Third ventricular injection of cAMP-delivering nanospheres did not affect changes in the body weight. Changes in body weight following treatment with nanospheres containing vehicle (Nano-V-F), nanospheres containing cAMP (Nano-cAMP-F) or none in AD (a), PF (b) and AF (c). Data are mean ± SEM (n = 6) and were compared by ANOVA and the Newman-Keuls posttest. No treatment difference was found between groups.

Figure 9.

β-endorphin neuronal supplementation via third ventricular injection of cAMP-delivering nanospheres normalizes glucose tolerance (OGTT) and insulin response to glucose (IPITT) and reduces the susceptibility to develop type 2 diabetes in the PCAE litters. OGTT responses in the offspring without treatment, or treated with nanospheres containing vehicle (Nano-V-F), or treated with nanospheres containing cAMP (Nano-cAMP-F) in AD (a), PF (b), and AF (c) groups. The glucose response during the study period was calculated as the area under the curve (AUC) and presented in histograms for AD (d), PF (e), and AF (f) groups. IPITT responses in the offspring without treatment, or treated with Nano-V-F, or treated with Nano-cAMP-F in AD (g), PF (h), and AF (i) groups. The insulin response during the study period was calculated as AUC and presented in histograms for AD (j), PF (k), and AF (l) groups. [m-r] Show the effects of β-endorphin neuronal supplementation on the HFD- and STZ-induced levels of blood glucose and blood insulin in AD (m, p), PF (n, q), and AF (o, r) animals. Data are mean ± SEM (n = 5-7) and were compared by ANOVA and the Newman-Keuls posttest. Differences between control and other treatment groups are shown by lines with P values above the bar graphs.