Katsumi Iizuka

Hepatic FoxO1 integrates glucose utilization and lipid synthesis through regulation of Chrebp O-glycosylation.

In liver, glucose utilization and lipid synthesis are inextricably intertwined. When glucose availability exceeds its utilization, lipogenesis increases, leading to increased intrahepatic lipid content and lipoprotein secretion. Although the fate of three-carbon metabolites is largely determined by flux rate through the relevant enzymes, insulin plays a permissive role in this process. But the mechanism integrating insulin receptor signaling to glucose utilization with lipogenesis is unknown. Forkhead box O1 (FoxO1), a downstream effector of insulin signaling, plays a central role in hepatic glucose metabolism through the regulation of hepatic glucose production. In this study, we investigated the mechanism by which FoxO1 integrates hepatic glucose utilization with lipid synthesis. We show that FoxO1 overexpression in hepatocytes reduces activity of carbohydrate response element binding protein (Chrebp), a key regulator of lipogenesis, by suppressing O-linked glycosylation and reducing the protein stability. FoxO1 inhibits high glucose- or O-GlcNAc transferase (OGT)-induced liver-pyruvate kinase (L-PK) promoter activity by decreasing Chrebp recruitment to the L-PK promoter. Conversely, FoxO1 ablation in liver leads to the enhanced O-glycosylation and increased protein level of Chrebp owing to decreased its ubiquitination. We propose that FoxO1 regulation of Chrebp O-glycosylation is a mechanism linking hepatic glucose utilization with lipid synthesis.

Krüppel-like factor-10 is directly regulated by carbohydrate response element-binding protein in rat primary hepatocytes.

Krüppel-like factor (KLF)-10, is a circadian transcriptional regulator, which links the molecular clock to energy metabolism in the liver. Recently, it was reported that Klf-10 expression is induced by glucose stimulation in mouse hepatocytes. We previously reported that carbohydrate response element-binding protein (ChREBP) plays an important role in the regulation of hepatic lipogenic gene expression. Here, we investigate whether ChREBP, a glucose-activated transcription factor, directly regulates Klf-10 mRNA expression in rat primary hepatocytes. We found that both glucose stimulation and adenoviral overexpression of ChREBP induce Klf-10 mRNA expression in rat primary hepatocytes in a dose-dependent manner. Conversely, overexpression of dominant-negative Max-like protein inhibits glucose-induction expression of Klf-10 mRNA. Deletion analysis using rat Klf-10 promoter in the pGL3 vector combined with a chromatin immunoprecipitation assay against the anti-ChREBP antibody demonstrated that the carbohydrate response element is located between -125 bp and -109 bp in the rat Klf-10 promoter. Conversely, adenoviral overexpression of KLF-10 partly inhibits glucose induction of ChREBP target genes in primary hepatocytes. In conclusion, these data suggest that crosstalk between ChREBP and KLF-10 is involved in the regulation of the lipogenic pathway.

Rat glucagon receptor mRNA is directly regulated by glucose through transactivation of the carbohydrate response element binding protein

The glucagon receptor (Gcgr) is essential for maintaining glucose homeostasis in the liver and for stimulating insulin secretion in pancreatic β-cells. Glucose induces rat Gcgr mRNA expression; however, the precise mechanism remains unknown. We previously have studied the role of the carbohydrate response element binding protein (ChREBP), a glucose-activated transcription factor, in the regulation of glucose-stimulated gene expression. The G-box has previously been reported to be responsible for glucose regulation of Gcgr mRNA expression. The G-box comprises two E-boxes separated by 3bp, which distinguishes it from the carbohydrate response element (ChoRE), which has 5-bp spacing between the two E-boxes. In the rat Gcgr promoter, a putative ChoRE (-554bp/-538bp) is localized near the G-box (-543bp/-529bp). In rat INS-1E insulinoma cells, deletion studies of the rat Gcgr promoter show that ChoRE is a minimal glucose response element. Moreover, reporter assays using a pGL3 promoter vector, which harbors ChoRE and chromatin immunoprecipitation assays reveal that ChoRE is a functional glucose response element in the rat Gcgr promoter. Furthermore, In contrast, glucagon partly suppresses glucose-induced expression of Gcgr mRNA. Thus, ChREBP directly regulates rat Gcgr expression in INS-1E cells. In addition, negative feedback looping between ChREBP and GCGR may further contribute to the regulation of glucose-induced gene expression.

Synergistic effect of α‐glucosidase inhibitors and dipeptidyl peptidase 4 inhibitor treatment

Monotherapy of α‐glucosidase inhibitor (α‐GI) or dipeptidyl peptidase 4 (DPP4) inhibitor does not sufficiently minimize glucose fluctuations in the diabetic state. In the present study, we evaluated the combined effects of various of α‐GI inhibitors (acarbose, voglibose or miglitol) and sitagliptin, a DPP4 inhibitor, on blood glucose fluctuation, insulin and active glucagon‐like peptide‐1 (GLP‐1) levels after nutriment loading in mice. Miglitol and sitagliptin elicited a 47% reduction (P < 0.05) of the area under the curve of blood glucose levels for up to 2 h after maltose‐loading, a 60% reduction (P < 0.05) in the range of blood glucose fluctuation, and a 32% decrease in plasma insulin compared with the control group. All three of the combinations elicited a 2.5–4.9‐fold synergistic increase in active GLP‐1 (P < 0.05 vs control). Thus, combined treatment with the α‐GI miglitol, which more strongly inhibits the early phase of postprandial hyperglycemia, and DPP4 inhibitor yields both complementary and synergistic effects, and might represent a superior anti‐hyperglycemic therapy. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00081.x, 2011)

Association of genetic variants of the incretin-related genes with quantitative traits and occurrence of type 2 diabetes in Japanese

Background None of the high frequency variants of the incretin-related genes has been found by genome-wide association study (GWAS) for association with occurrence of type 2 diabetes in Japanese. However, low frequency and rare and/or high frequency variants affecting glucose metabolic traits remain to be investigated. Method We screened all exons of the incretin-related genes (GCG, GLP1R, DPP4, PCSK1, GIP, and GIPR) in 96 patients with type 2 diabetes and investigated for association of genetic variants of these genes with quantitative metabolic traits upon test meal with 38 young healthy volunteers and with the occurrence of type 2 diabetes in Japanese subjects comprising 1303 patients with type 2 diabetes and 1014 controls. Result Two mutations of GIPR, p.Thr3Alafsx21 and Arg183Gln, were found only in patients with type 2 diabetes, and both of them were treated with insulin. Of ten tagSNPs, we found that risk allele C of SNP393 (rs6235) of PCSK1 was nominally associated with higher fasting insulin and HOMA-R (P = 0.034 and P = 0.030), but not with proinsulin level, incretin level or BMI. The variant showed significant association with occurrence of type 2 diabetes after adjustment for age, sex, and BMI (P = 0.0043). Conclusion Rare variants of GIPR may contribute to the development of type 2 diabetes, possibly through insulin secretory defects. Furthermore, the genetic variant of PCSK1 might influence glucose homeostasis by altered insulin resistance independently of BMI, incretin level or proinsulin conversion, and may be associated with the occurrence of type 2 diabetes in Japanese.

ChREBP-Knockout Mice Show Sucrose Intolerance and Fructose Malabsorption

We have previously reported that 60% sucrose diet-fed ChREBP knockout mice (KO) showed body weight loss resulting in lethality. We aimed to elucidate whether sucrose and fructose metabolism are impaired in KO. Wild-type mice (WT) and KO were fed a diet containing 30% sucrose with/without 0.08% miglitol, an α-glucosidase inhibitor, and these effects on phenotypes were tested. Furthermore, we compared metabolic changes of oral and peritoneal fructose injection. A thirty percent sucrose diet feeding did not affect phenotypes in KO. However, miglitol induced lethality in 30% sucrose-fed KO. Thirty percent sucrose plus miglitol diet-fed KO showed increased cecal contents, increased fecal lactate contents, increased growth of lactobacillales and Bifidobacterium and decreased growth of clostridium cluster XIVa. ChREBP gene deletion suppressed the mRNA levels of sucrose and fructose related genes. Next, oral fructose injection did not affect plasma glucose levels and liver fructose contents; however, intestinal sucrose and fructose related mRNA levels were increased only in WT. In contrast, peritoneal fructose injection increased plasma glucose levels in both mice; however, the hepatic fructose content in KO was much higher owing to decreased hepatic Khk mRNA expression. Taken together, KO showed sucrose intolerance and fructose malabsorption owing to decreased gene expression.

ChREBP Rather Than SHP Regulates Hepatic VLDL Secretion

The regulation of hepatic very-low-density lipoprotein (VLDL) secretion plays an important role in the pathogenesis of dyslipidemia and fatty liver diseases. VLDL is controlled by hepatic microsomal triglyceride transfer protein (MTTP). Mttp is regulated by carbohydrate response element binding protein (ChREBP) and small heterodimer partner (SHP). However, it is unclear whether both coordinately regulate Mttp expression and VLDL secretion. Here, adenoviral overexpression of ChREBP and SHP in rat primary hepatocytes induced and suppressed Mttp mRNA, respectively. However, Mttp induction by ChREBP was much more potent than suppression by SHP. Promoter assays of Mttp and the liver type pyruvate kinase gene revealed that SHP and ChREBP did not affect the transcriptional activity of each other. Mttp mRNA and protein levels of Shp−/− mice were similar to those of wild-types; however, those of Chrebp−/−Shp−/− and Chrebp−/− mice were significantly much lower. Consistent with this, the VLDL particle number and VLDL secretion rates in Shp−/− mice were similar to wild-types but were much lower in Chrebp−/− and Chrebp−/−Shp−/− mice. These findings suggest that ChREBP, rather than SHP, regulates VLDL secretion under normal conditions and that ChREBP and SHP do not affect the transcriptional activities of each other.