Xing-Hai Yao

Hepatic insulin resistance induced by prenatal alcohol exposure is associated with reduced PTEN and TRB3 acetylation in adult rat offspring.

Prenatal alcohol exposure (EtOH) results in insulin resistance in rats of both sexes with increased expression of hepatic gluconeogenic genes and glucose production. To investigate whether hepatic insulin signaling is defective, we studied 3-mo-old female offspring of dams that were given EtOH during pregnancy compared with those from pair-fed and control dams. We performed an intraperitoneal pyruvate tolerance test, determined the phosphorylation status of hepatic phosphoinositide-dependent protein kinase-1 (PDK1), Akt, and PKCzeta before and after intravenous insulin bolus, and measured mRNA and in vivo acetylation of TRB3 (tribbles 3) and PTEN (phosphatase and tensin homolog deleted on chromosome ten) as well as the expression of the histone acetylase (HAT) PCAF (p300/CREB-binding protein-associated factor), histone deacetylase-1 (HDAC1), and HAT and HDAC activities. In EtOH compared with pair-fed and control offspring, basal and pyruvate-induced blood glucose was increased, insulin-induced PDK1, Akt, and PKCzeta phosphorylation was reduced, and expression of PTEN and TRB3 was increased while their acetylation status was decreased in association with increased HDAC and decreased HAT activities. Thus female adult rats prenatally exposed to EtOH have increased gluconeogenesis, reduced insulin signaling, and increased PTEN and TRB3 expression in the liver. In addition, PTEN and TRB3 are hypoacetylated, which can contribute to Akt-inhibiting activity. These results suggest that hepatic insulin resistance in rats prenatally exposed to EtOH is explained, at least in part, by increased PTEN and TRB3 activity due to both increased gene expression and reduced acetylation.

Prenatal Ethanol Exposure Causes Glucose Intolerance with Increased Hepatic Gluconeogenesis and Histone Deacetylases in Adult Rat Offspring: Reversal by Tauroursodeoxycholic Acid

Prenatal ethanol exposure results in increased glucose production in adult rat offspring and this may involve modulation of protein acetylation by cellular stress. We used adult male offspring of dams given ethanol during gestation days 1–7 (early), 8–14 (mid) and 15–21 (late) compared with those from control dams. A group of ethanol offspring was treated with tauroursodeoxycholic acid (TUDCA) for 3 weeks. We determined gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase, hepatic free radicals, histone deacetylases (HDAC), acetylated foxo1, acetylated PEPCK, and C/EBP homologous protein as a marker of endoplasmic reticulum stress. Prenatal ethanol during either of the 3 weeks of pregnancy increased gluconeogenesis, gluconeogenic genes, oxidative and endoplasmic reticulum stresses, sirtuin-2 and HDAC3, 4, 5, and 7 in adult offspring. Conversely, prenatal ethanol reduced acetylation of foxo1 and PEPCK. Treatment of adult ethanol offspring with TUDCA reversed all these abnormalities. Thus, prenatal exposure of rats to ethanol results in long lasting oxidative and endoplasmic reticulum stresses explaining increased expression of gluconeogenic genes and HDAC proteins which, by deacetylating foxo1 and PEPCK, contribute to increased gluconeogenesis. These anomalies occurred regardless of the time of ethanol exposure during pregnancy, including early embryogenesis. As these anomalies were reversed by treatment of the adult offspring with TUDCA, this compound has therapeutic potentials in the treatment of glucose intolerance associated with prenatal ethanol exposure.

Prenatal alcohol exposure alters phosphorylation and glycosylation of proteins in rat offspring liver

To gain more insights into the translational and PTM that occur in rat offspring exposed to alcohol in utero, 2‐D PAGE with total, phospho‐ and glycoprotein staining and MALDI‐MS/MS and database searching were conducted. The results, based on fold‐change expression, revealed a down‐regulation of total protein expression by prenatal alcohol exposure in 7‐day‐old and 3‐month‐old rats. There was an up‐regulation of protein phosphorylation but a down‐regulation of glycosylation by prenatal alcohol exposure in both age groups. Of 31 protein spots examined per group, differentially expressed proteins were identified as ferritin light chain, aldo‐keto reductase, tumor rejection antigen gp96, fructose‐1,6‐bisphosphatase, glycerol‐3‐phosphate dehydrogenase, malate dehydrogenase, and γ‐actin. Increased phosphorylation was observed in proteins such as calmodulin, gluthatione S‐transferase, glucose regulated protein 58, α‐enolase, eukaryotic translation elongation factor 1 β‐2, riboprotein large P2, agmatinase, ornithine carbamoyltransferase, quinolinate phosphoribosyltransferase, formimidoyltransferase cyclodeaminase, and actin. In addition, glycosylation of adenosine kinase, adenosylhomocysteine hydrolase, and 3‐hydroxyanthranilate dioxygenase was reduced. Pathways affected by these protein alterations include cell signaling, cellular stress, protein synthesis, cytoskeleton, as well as glucose, aminoacid, adenosine and energy metabolism. The activity of the gluconeogenic enzyme fructose‐1,6‐bisphosphatase was elevated by prenatal alcohol. The observations may have important physiological implications.

Human IGF Binding Protein-3 Overexpression Impairs Glucose Regulation in Mice via an Inhibition of Insulin Secretion

Human IGF binding protein-3 (hIGFBP-3) overexpression in mice causes hyperglycemia, but its effect on β-cell function is unknown. We compared wild-type mice with mice overexpressing hIGFBP-3 [phoshoglycerate kinase (PGK)BP3] and mutant (Gly⁵⁶/Gly⁸⁰/Gly⁸¹)hIGFBP-3 devoid of IGF binding affinity (PGKmBP3). Intraperitoneal glucose and insulin tolerance tests were performed, and glucose, IGFBP-3, IGF-I, and insulin were determined. Pancreatic sections were used for islet histomorphometry and stained with antibodies against insulin, glucagon, and hIGFBP-3. Pancreatic islets were isolated to determine the expression of IGFBP-3, and glucose-stimulated insulin secretion was measured using both islet batch incubation and perifusion. IGFBP-3 was expressed in β-cells but not in other islet cell types. Fasting glucose concentration was elevated in PGKBP3 mice (6.27 ± 0.31 mm) compared with PGKmBP3 mice (3.98 ± 0.36 mm) and wild-type mice (4.84 ± 0.07 mm). During glucose tolerance test, glucose declined more slowly in PGKBP3 and PGKmBP3 mice than in wild-type mice, and insulin secretion was impaired in PGKBP3 mice. During insulin tolerance test, insulin declined more slowly in both transgenic mice compared with wild-type mice. Insulin secretion in islets incubated with 3.3 mm glucose was similar among groups, but islet insulin response to 16.7 mm glucose alone, or with carbachol and cAMP enhancers, was reduced in PGKBP3 and PGKmBP3 mice compared with wild-type controls. ATP content, Akt phosphorylation, and phosphoglucose isomerase activity were reduced in islets from both transgenic mice. Thus, overexpression of hIGFBP-3 in mice delays in vivo insulin clearance and reduces glucose-stimulated insulin secretion in pancreatic islets by both IGF-dependent and IGF-independent mechanisms.

Glucose intolerance in aging male IGFBP-3 transgenic mice: differential effects of human IGFBP-3 and its mutant IGFBP-3 devoid of IGF binding ability.

We have reported a reduction of insulin secretion and glucose intolerance in young mice overexpressing human IGFBP-3 (phosphoglycerate kinase [PGK]BP3) or its mutant Gly56/Gly80/Gly81-IGFBP-3 (PGKmutBP3) under the PGK promoter. Here, we investigated changes in glucose and lipid homeostasis with age in PGKBP3 and PGKmutBP3 mice compared with wild-type mice. Body weight, glucose tolerance, insulin tolerance, visceral fat, interscapular brown adipose tissue (BAT), serum lipids, and pancreas histology were examined at age 3, 6, and 12 months. Murine IGFBP-3 was similar in all mouse genotypes and decreased with age in parallel with total IGF-1. Visceral fat and BAT masses increased in PGKmutBP3 mice, but not in PGKBP3 mice. Glucose tolerance was impaired in both PGKBP3 and PGKmutBP3 mice. However, PGKBP3 mice had increased expression of uncoupling protein-1 in BAT and reduced adiposity, and continued to have smaller pancreatic β-cell mass and reduced insulin secretion through age 12 months. In contrast, PGKmutBP3 mice developed insulin resistance with age in association with pancreatic β-cell hyperplasia, impaired expression of uncoupling protein-1 in BAT, and increased adiposity. In addition, both PGKBP3 and PGKmutBP3 mice had elevated glycerol in the circulation, but only PGKBP3 mice had elevated free fatty acids and only PGKmutBP3 mice had elevated triglycerides. Estimated free IGF-1 did not increase with age in transgenic mice, as it did in wild-type mice. Thus, overexpression of human IGFBP-3 or its mutant devoid of IGF binding ability leads to glucose intolerance with, however, different effects on insulin secretion, insulin sensitivity, and lipid homeostasis in aging mice.

Reversal of glucose intolerance in rat offspring exposed to ethanol before birth through reduction of nuclear skeletal muscle HDAC expression by the bile acid TUDCA

Prenatal ethanol exposure causes cellular stress, insulin resistance, and glucose intolerance in adult offspring, with increased gluconeogenesis and reduced muscle glucose transporter‐4 (glut4) expression. Impaired insulin activation of Akt and nuclear translocation of histone deacetylases (HDACs) in the liver partly explain increased gluconeogenesis. The mechanism for the reduced glut4 is unknown. Pregnant rats were gavaged with ethanol over the last week of gestation and adult female offspring were studied. Some ethanol exposed offspring was treated with tauroursodeoxycholic acid (TUDCA) for 3 weeks. All these rats underwent intraperitoneal glucose tolerance and insulin tolerance tests. The expression of glut4, HDACs, and markers of endoplasmic reticulum (ER) unfolded protein response (XBP1, CHOP, ATF6) was examined in the gastrocnemius muscle fractions, and in C2C12 muscle cells cultured with ethanol, TUDCA, and HDAC inhibitors. Non‐TUDCA‐treated rats exposed to prenatal ethanol were insulin resistant and glucose intolerant with reduced muscle glut4 expression, increased ER marker expression, and increased nuclear HDACs, whereas TUDCA‐treated rats had normal insulin sensitivity and glucose tolerance with normal glut4 expression, ER marker expression, and HDAC levels. In C2C12 cells, ethanol reduced glut4 expression, but increased ER makers. While TUDCA restored glut4 and ER markers to control levels and HDAC inhibition rescued glut4 expression, HDAC inhibition had no effect on ER markers. The increase in nuclear HDAC levels consequent to prenatal ethanol exposure reduces glut4 expression in adult rat offspring, and this HDAC effect is independent of ER unfolded protein response. HDAC inhibition by TUDCA restores glut4 expression, with improvement in insulin sensitivity and glucose tolerance.