An ACACB variant implicated in diabetic nephropathy associates with body mass index and gene expression in obese subjects

Acetyl coenzyme A carboxylase B gene (ACACB) single nucleotide polymorphism (SNP) rs2268388 is reproducibly associated with type 2 diabetes (T2DM)-associated nephropathy (DN). ACACB knock-out mice are also protected from obesity. This study assessed relationships between rs2268388, body mass index (BMI) and gene expression in multiple populations, with and without T2DM. Among subjects without T2DM, rs2268388 DN risk allele (T) associated with higher BMI in Pima Indian children (n = 2021; p-additive = 0.029) and African Americans (AAs) (n = 177; p-additive = 0.05), with a trend in European Americans (EAs) (n = 512; p-additive = 0.09), but not Germans (n = 858; p-additive = 0.765). Association with BMI was seen in a meta-analysis including all non-T2DM subjects (n = 3568; p-additive = 0.02). Among subjects with T2DM, rs2268388 was not associated with BMI in Japanese (n = 2912) or EAs (n = 1149); however, the T allele associated with higher BMI in the subset with BMI≥30 kg/m(2) (n = 568 EAs; p-additive = 0.049, n = 196 Japanese; p-additive = 0.049). Association with BMI was strengthened in a T2DM meta-analysis that included an additional 756 AAs (p-additive = 0.080) and 48 Hong Kong Chinese (p-additive = 0.81) with BMI≥30 kg/m(2) (n = 1575; p-additive = 0.0033). The effect of rs2268388 on gene expression revealed that the T risk allele associated with higher ACACB messenger levels in adipose tissue (41 EAs and 20 AAs with BMI\u3e30 kg/m(2); p-additive = 0.018) and ACACB protein levels in the liver tissue (mixed model p-additive = 0.03, in 25 EA bariatric surgery patients with BMI\u3e30 kg/m(2) for 75 exams). The T allele also associated with higher hepatic triglyceride levels. These data support a role for ACACB in obesity and potential roles for altered lipid metabolism in susceptibility to DN

The Effect of ACACB cis-Variants on Gene Expression and Metabolic Traits

Acetyl Coenzyme A carboxylase β (ACACB) is the rate-limiting enzyme in fatty acid oxidation, and continuous fatty acid oxidation in Acacb knock-out mice increases insulin sensitivity. Systematic human studies have not been performed to evaluate whether ACACB variants regulate gene expression and insulin sensitivity in skeletal muscle and adipose tissues. We sought to determine whether ACACB transcribed variants were associated with ACACB gene expression and insulin sensitivity in non-diabetic African American (AA) and European American (EA) adults.ACACB transcribed single nucleotide polymorphisms (SNPs) were genotyped in 105 EAs and 46 AAs whose body mass index (BMI), lipid profiles and ACACB gene expression in subcutaneous adipose and skeletal muscle had been measured. Allelic expression imbalance (AEI) was assessed in lymphoblast cell lines from heterozygous subjects in an additional EA sample (n = 95). Selected SNPs were further examined for association with insulin sensitivity in a cohort of 417 EAs and 153 AAs.ACACB transcribed SNP rs2075260 (A/G) was associated with adipose ACACB messenger RNA expression in EAs and AAs (p = 3.8×10(-5), dominant model in meta-analysis, Stouffer method), with the (A) allele representing lower gene expression in adipose and higher insulin sensitivity in EAs (p = 0.04). In EAs, adipose ACACB expression was negatively associated with age and sex-adjusted BMI (r = -0.35, p = 0.0002).Common variants within the ACACB locus appear to regulate adipose gene expression in humans. Body fat (represented by BMI) may further regulate adipose ACACB gene expression in the EA population

Modulation of glutamate generation in mitochondria affects hormone secretion in INS-1E beta cells

The mitochondria play a pivotal role in regulating glucose-induced insulin secretion in the pancreatic beta cell. We have recently demonstrated that glutamate derived from mitochondria participates directly in the stimulation of insulin exocytosis. In the present study, mitochondria isolated from the beta cell line INS-1E generated glutamate when incubated with the tricarboxylic acid cycle intermediate succinate. The generation of glutamate correlated with stimulated mitochondrial activity monitored as oxygen consumption and was inhibited by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Glutamate is formed by the mitochondrial enzyme glutamate dehydrogenase from alpha-ketoglutarate. Transient overexpression of glutamate dehydrogenase in INS-1E cells resulted in potentiation of glucose-stimulated hormone secretion without affecting basal release. These results further point to glutamate as an intracellular messenger playing a key role in the control of insulin exocytosis

Mitochondrial metabolism sets the maximal limit of fuel-stimulated insulin secretion in a model pancreatic beta cell: a survey of four fuel secretagogues

The precise metabolic steps that couple glucose catabolism to insulin secretion in the pancreatic beta cell are incompletely understood. ATP generated from glycolytic metabolism in the cytosol, from mitochondrial metabolism, and/or from the hydrogen shuttles operating between cytosolic and mitochondrial compartments has been implicated as an important coupling factor. To identify the importance of each of these metabolic pathways, we have compared the fates of four fuel secretagogues (glucose, pyruvate, dihydroxyacetone, and glycerol) in the INS1-E beta cell line. Two of these fuels, dihydroxyacetone and glycerol, are normally ineffective as secretagogues but are enabled by adenovirus-mediated expression of glycerol kinase. Comparison of these two particular fuels allows the effect of redox state on insulin secretion to be evaluated since the phosphorylated products dihydroxyacetone phosphate and glycerol phosphate lie on opposite sides of the NADH-consuming glycerophosphate dehydrogenase reaction. Based upon measurements of glycolytic metabolites, mitochondrial oxidation, mitochondrial matrix calcium, and mitochondrial membrane potential, we find that insulin secretion most tightly correlates with mitochondrial metabolism for each of the four fuels. In the case of glucose stimulation, the high control strength of glucose phosphorylation sets the pace of glucose metabolism and thus the rate of insulin secretion. However, bypassing this reaction with pyruvate, dihydroxyacetone, or glycerol uncovers constraints imposed by mitochondrial metabolism, each of which attains a similar maximal limit of insulin secretion. More specifically, we found that the hyperpolarization of the mitochondrial membrane, related to the proton export from the mitochondrial matrix, correlates well with insulin secretion. Based on these findings, we propose that fuel-stimulated secretion is in fact limited by the inherent thermodynamic constraints of proton gradient formation

Nov/Ccn3, a novel transcriptional target of FoxO1, impairs pancreatic β-cell function.

Type 2 diabetes is characterized by both insulin resistance and progressive deterioration of β-cell function. The forkhead transcription factor FoxO1 is a prominent mediator of insulin signaling in β-cells. We reasoned that identification of FoxO1 target genes in β-cells could reveal mechanisms linking β-cell dysfunction to insulin resistance. In this study, we report the characterization of Nov/Ccn3 as a novel transcriptional target of FoxO1 in pancreatic β-cells. FoxO1 binds to an evolutionarily conserved response element in the Ccn3 promoter to regulate its expression. Accordingly, CCN3 levels are elevated in pancreatic islets of mice with overexpression of a constitutively active form of FoxO1 or insulin resistance. Our functional studies reveal that CCN3 impairs β-cell proliferation concomitantly with a reduction in cAMP levels. Moreover, CCN3 decreases glucose oxidation, which translates into inhibition of glucose-stimulated Ca(2+) entry and insulin secretion. Our results identify CCN3, a novel transcriptional target of FoxO1 in pancreatic β-cells, as a potential target for therapeutic intervention in the treatment of diabetes

Hepatocyte nuclear factor 4alpha regulates the expression of pancreatic beta -cell genes implicated in glucose metabolism and nutrient-induced insulin secretion

Mutations in the HNF4alpha gene are associated with the subtype 1 of maturity-onset diabetes of the young (MODY1), which is characterized by impaired insulin secretory response to glucose in pancreatic beta-cells. Hepatocyte nuclear factor 4alpha (HNF4alpha) is a transcription factor critical for liver development and hepatocyte-specific gene expression. However, the role of HNF4alpha in the regulation of pancreatic beta-cell gene expression and its correlation with metabolism secretion coupling have not been previously investigated. The tetracycline-inducible system was employed to achieve tightly controlled expression of both wild type (WT) and dominant-negative mutant (DN) of HNF4alpha in INS-1 cells. The induction of WT-HNF4alpha resulted in a left shift in glucose-stimulated insulin secretion, whereas DN-HNF4alpha selectively impaired nutrient-stimulated insulin release. Induction of DN-HNF4alpha also caused defective mitochondrial function substantiated by reduced [(14)C]pyruvate oxidation, attenuated substrate-evoked mitochondrial membrane hyperpolarization, and blunted nutrient-generated cellular ATP production. Quantitative evaluation of HNF4alpha-regulated pancreatic beta-cell gene expression revealed altered mRNA levels of insulin, glucose transporter-2, L-pyruvate kinase, aldolase B, 2-oxoglutarate dehydrogenase E1 subunit, and mitochondrial uncoupling protein-2. The patterns of HNF4alpha-regulated gene expression are strikingly similar to that of its downstream transcription factor HNF1alpha. Indeed, HNF4alpha changed the HNF1alpha mRNA levels and HNF1alpha promoter luciferase activity through altered HNF4alpha binding. These results demonstrate the importance of HNF4alpha in beta-cell metabolism-secretion coupling

Molecular targets of a human HNF1α mutation responsible for pancreatic β-cell dysfunction

The reverse tetracycline-dependent transactivator system was employed in insulinoma INS-1 cells to achieve controlled inducible expression of hepatocyte nuclear factor-1α (HNF1α)-P291fsinsC, the most common mutation associated with subtype 3 of maturity-onset diabetes of the young (MODY3). Nuclear localized HNF1α-P291fsinsC protein exerts its dominant-negative effects by competing with endogenous HNF1α for the cognate DNA-binding site. HNF1α controls multiple genes implicated in pancreatic β-cell function and notably in metabolism– secretion coupling. In addition to reduced expression of the genes encoding insulin, glucose transporter-2, l-pyruvate kinase, aldolase B and 3-hydroxy-3-methylglutaryl coenzyme A reductase, induction of HNF1αP291fsinsC also significantly inhibits expression of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) E1 subunit mRNA and protein. OGDH enzyme activity and [(14)C]pyruvate oxidation were also reduced. In contrast, the mRNA and protein levels of mitochondrial uncoupling protein-2 were dramatically increased by HNF1α-P291fsinsC induction. As predicted from this altered gene expression profile, HNF1α-P291fsinsC also inhibits insulin secretory responses to glucose and leucine, correlated with impaired nutrient-evoked mitochondrial ATP production and mitochondrial membrane hyperpolarization. These unprecedented results suggest the molecular mechanism of HNF1αP291fsinsC causing β-cell dysfunction

<i>Ccn3</i> is a transcriptional target of FoxO1 in β-cells.

<p>A) <i>Ccn3</i> expression by quantitative real-time qPCR in INS832/13 cells transduced with either Ad-β-Gal or Ad-CN-FoxO1 and cultured for 24 h. Results are means +/– SEM of 4 separate experiments. B) <i>Ccn3 </i>mRNA levels in INS832/13 cells treated with or without 10% serum and LY294002 (50 µM) for 4 h. Results represent means +/– SEM of 3 separate experiments. C) <i>Ccn3</i> expression in isolated islets from WT and transgenic mice with CAFoxO1 overexpression in their β-cells (called “305 mice”) (n = 5 for each). *, p<0.05.</p

CCN3 modifies the expression of several genes involved in the control of cell cycle progression.

<p>A) mRNA levels of cells treated with or without CCN3 for 24 h. B) Reported changes in gene expression following CCN3 treatment. Results represent means +/– SEM of 3 separate experiments. *, p<0.05; **, p<0.01; ns  =  not significant.</p