Overload of Glucose Metabolism as Initiating Factor in Diabetic Embryopathy and Prevention by Glyoxalase 1 Inducer Dietary Supplement

Abstract

Hyperglycemia in early-stage embryogenesis is linked to diabetic embryopathy. High-glucose-concentration-induced accumulation of hexokinase-2 (HK2) may initiate metabolic dysfunction that contributes to diabetic embryopathy, including increased formation of methylglyoxal (MG). In this study, we evaluated changes in HK2 protein levels and embryo dysmorphogenesis in an experimental model of diabetic embryopathy. Rat embryos were cultured with high glucose concentrations, and the effects of glyoxalase 1 (Glo1) inducer, trans-resveratrol and hesperetin (tRES + HESP) were evaluated. Rat embryos, on gestational day 9, were cultured for 48 h in low and high glucose concentrations with or without tRES + HESP. Embryo crown-rump length, somite number, malformation score, concentrations of HK2 and Glo1 protein, rates of glucose consumption, and MG formation were assessed. Under low-glucose conditions, embryos exhibited normal morphogenesis. In contrast, high-glucose conditions led to reduced crown-rump length and somite number, and an increased malformation score. The addition of 10 μM tRES + HESP reversed these high glucose-induced changes by 60%, 49%, and 47%, respectively. Embryos cultured in high glucose showed increases in HK2 concentration (42%), glucose consumption (75%), and MG formation (27%), normalized to embryo volume. These elevated HK2 levels were normalized by treatment with 10 μM tRES + HESP. Thus, high-glucose-induced metabolic dysfunction and embryopathy may both be initiated by HK2 accumulation and may be preventable with tRES + HESP treatment.

Keywords: diabetic embryopathy; glycation; hyperglycemia; metabolic dysfunction; teratogenesis.

Figure 1

Assessment of development in rat embryos after exposure to 10 or 30 mM glucose in whole-embryo culture. (a) Normal embryo, fully rotated, with crown–rump length 3.6 mm, 28 somites and malformation score 0 after 48 h culture in 10 mM glucose. Note assessment of crown–rump length and somites. Rat embryo morphology viewed in stereo microscope at magnification of 10–20×. (b) Normal embryo, fully rotated with crown–rump length 3.5 mm, 29 somites and malformation score 0 after culture in 10 mM glucose. Rat embryo morphology viewed in stereo microscope at magnification of 10–20×. (c). Malformed embryos, malrotated with open neural tube and maldeveloped heart after 48 h culture in 30 mM glucose. Estimated crown–rump length 1.9 mm, 20 somites and malformation score 10. Rat embryo morphology viewed in stereo microscope at magnification of 10–20×. (d) Electron micrographs of 3 embryos after culture in 30 mM glucose. Left: normal embryo (crown–rump length 3.5 mm, 29 somites, malformation score 0. Center: embryo with one malformation, an open neural pore, crown–rump length of 3.4 mm, 29 somites, and malformation score of 1. Right: embryo with multiple malformations, open neural tube and tail malrotation, crown–rump length of 1.6 mm, 13 somites, and malformation score of 10.

Figure 2

Effect of glucose and Glyoxalase 1 inducer on embryogenesis in vitro. (a) Crown–rump length. (b) Somite number. (c) Malformation score. Key: 10 G, +10 mM glucose (n = 9); 10 G + 5tRES + HESP, +10 mM glucose and 5 µM tRES + HESP (n = 4); 10 G + 20tRES + HESP, +10 mM glucose and 20 µM tRES + HESP (n = 4); 30 G, +30 mM glucose (n = 9); 30 G + 5tRES + HESP, +30 mM glucose and 5 µM tRES + HESP (n = 2); 30 G + 10tRES + HESP, +30 mM glucose and 10 µM tRES + HESP (n = 16); and 30 G + 20tRES + HESP, +30 mM glucose and 20 µM tRES + HESP (n = 8). Data are mean ± SEM except mean only where n = 2. Significance: crown–rump length, p < 0.001, somite number, p < 0.01 and malformation score, p < 0.001 (one-way ANOVA for all 7 groups); *, **, and ***, p < 0.05, p < 0.01, and p < 0.001 with respect to low-glucose-concentration (10 mM) control; and o and oo, p < 0.05 and p < 0.01 with respect to high-glucose-concentration (30 mM) control (Student’s t-test).

Figure 3

Effect of glucose and Glyoxalase 1 inducer on hexokinase-2 and glyoxalase 1 concentration in embryogenesis in vitro. (a) Hexokinase 1 protein. (b) Glyoxalase 1 protein. The sample key is the same as given for Figure 2: Replicates: for HK2 protein and Glo1 protein measurements, 10 G (n = 6), 10 G + 5tRES + HESP and 10 G + 20tRES + HESP (n = 3), 30 G (n = 6), 30 G + 5tRES + HESP (n = 2), and 30 G + 10tRES + HESP and 30 G + 10tRES + HESP (n = 3). Data are mean ± SEM (n) except mean only where n = 2 and corrected for the decrease in embryo volume, assuming the change in embryo volume is proportional to the change in crown–rump length (Figure 1). Significance: HK2 and Glo1, p < 0.001 (one-way ANOVA for all 7 groups); ** and ***, p < 0.01, and p < 0.001 with respect to low-glucose-concentration (10 mM) control; and o and oo, p < 0.05 and p < 0.01 with respect to high-glucose-concentration (30 mM) control (Student’s t-test). (c) Western Blotting images used for densitometry showing immunoblotting bands for hexokinase 2 (HK2), glyoxalase 1 (Glo1), and β-actin housekeeping protein (β-Act).

Figure 4

Dysregulation of glycolytic enzymes and metabolic dysfunction in hexokinase-2-linked glycolytic overload hypothesis with impact of effector pathways of metabolic dysfunction and embryo dysmorphogenesis in diabetic embryopathy. Key: Black arrows—metabolism of glucose through early-stage glycolysis; Yellow highlight—HK2 detachment from mitochondria and steps of glycolysis of increased metabolic flux in high glucose concentration; Pink highlighted text—HK2 and metabolites increased in unscheduled glycolysis; and Red arrows—mechanisms of pathogenesis activated in high glucose concentration. Abbreviations: 13BPG, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetonephosphate; F6P, fructose-6-phosphate; F16BP, fructose-1,6-bisphosphate; G6P, glucose-6-phosphate; GA3P, glyceraldehyde-3-phosphate; GA3PD, glyceraldehyde-3-phosphate dehydrogenase; GPI, glucose-6-phosphate isomerase; HK1, hexokinase-1; HK2, hexokinase-2; MCT, monocarboxylate transporter; MG, methylglyoxal; PFK-1, phosphofructokinase-1; ROS, reactive oxygen species; TPI, triosephosphate isomerase; VDAC, voltage-dependent anion channel.