Peter A. Antinozzi

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

Mutations in the HNF4α 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 β-cells. Hepatocyte nuclear factor 4α (HNF4α) is a transcription factor critical for liver development and hepatocyte-specific gene expression. However, the role of HNF4α in the regulation of pancreatic β-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 HNF4α in INS-1 cells. The induction of WT-HNF4α resulted in a left shift in glucose-stimulated insulin secretion, whereas DN-HNF4α selectively impaired nutrient-stimulated insulin release. Induction of DN-HNF4α also caused defective mitochondrial function substantiated by reduced [14C]pyruvate oxidation, attenuated substrate-evoked mitochondrial membrane hyperpolarization, and blunted nutrient-generated cellular ATP production. Quantitative evaluation of HNF4α-regulated pancreatic β-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 HNF4α-regulated gene expression are strikingly similar to that of its downstream transcription factor HNF1α. Indeed, HNF4α changed the HNF1α mRNA levels and HNF1α promoter luciferase activity through altered HNF4α binding. These results demonstrate the importance of HNF4α in β-cell metabolism-secretion coupling.

Molecular or Pharmacologic Perturbation of the Link between Glucose and Lipid Metabolism Is without Effect on Glucose-stimulated Insulin Secretion A RE-EVALUATION OF THE LONG-CHAIN ACYL-CoA HYPOTHESIS

The mechanism by which glucose stimulates insulin secretion from the pancreatic islets of Langerhans is incompletely understood. It has been suggested that malonyl-CoA plays a regulatory role by inhibiting fatty acid oxidation and promoting accumulation of cytosolic long-chain acyl-CoA (LC-CoA). In the current study, we have re-evaluated this “long-chain acyl-CoA hypothesis” by using molecular and pharmacologic methods to perturb lipid metabolism in INS-1 insulinoma cells or rat islets during glucose stimulation. First, we constructed a recombinant adenovirus containing the cDNA encoding malonyl-CoA decarboxylase (AdCMV-MCD), an enzyme that decarboxylates malonyl-CoA to acetyl-CoA. INS-1 cells treated with AdCMV-MCD had dramatically lowered intracellular malonyl CoA levels compared with AdCMV-βGal-treated cells at both 3 and 20 mm glucose. Further, at 20 mm glucose, AdCMV-MCD-treated cells were less effective at suppressing [1-14C]palmitate oxidation and incorporated 43% less labeled palmitate and 50% less labeled glucose into cellular lipids than either AdCMV-βGAL-treated or untreated INS-1 cells. Despite the large metabolic changes caused by expression of MCD, insulin secretion in response to glucose was unaltered relative to controls. The alternative, pharmacologic approach for perturbing lipid metabolism was to use triacsin C to inhibit long-chain acyl-CoA synthetase. This agent caused potent attenuation of palmitate oxidation and glucose or palmitate incorporation into cellular lipids and also caused a 47% decrease in total LC-CoA. Despite this, the drug had no effect on glucose-stimulated insulin secretion in islets or INS-1 cells. We conclude that significant disruption of the link between glucose and lipid metabolism does not impair glucose-stimulated insulin secretion in pancreatic islets or INS-1 cells.

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.

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 HNF1α‐P291fsinsC also significantly inhibits expression of mitochondrial 2‐oxoglutarate dehydrogenase (OGDH) E1 subunit mRNA and protein. OGDH enzyme activity and [14C]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 HNF1α‐P291fsinsC causing β‐cell dysfunction.

Engineering of Glycerol-stimulated Insulin Secretion in Islet Beta Cells DIFFERENTIAL METABOLIC FATES OF GLUCOSE AND GLYCEROL PROVIDE INSIGHT INTO MECHANISMS OF STIMULUS-SECRETION COUPLING

Insulin secretion from beta cells in the islets of Langerhans can be stimulated by a number of metabolic fuels, including glucose and glyceraldehyde, and is thought to be mediated by metabolism of the secretagogues and an attendant increase in the ATP:ADP ratio. Curiously, glycerol fails to stimulate insulin secretion, even though it has been reported that islets contain abundant glycerol kinase activity and oxidize glycerol efficiently. We have reinvestigated this point and find that rat islets and the well differentiated insulinoma cell line INS-1 contain negligible glycerol kinase activity. A recombinant adenovirus containing the bacterial glycerol kinase gene (AdCMV-GlpK) was constructed and used to express the enzyme in islets and INS-1 cells, resulting in insulin secretion in response to glycerol. In AdCMV-GlpK-treated INS-1 cells a greater proportion of glycerol is converted to lactate and a lesser proportion is oxidized compared with glucose. The two fuels are equally potent as insulin secretagogues, despite the fact that oxidation of glycerol at its maximally effective dose (2–5 mm) occurs at a rate that is similar to the rate of glucose oxidation at its basal, nonstimulatory concentration (3 mm). We also investigated the possibility that glycerol may signal via expansion of the glycerol phosphate pool to allow enhanced fatty acid esterification and formation of complex lipids. Addition of Triacsin-C, an inhibitor of long-chain acyl-CoA synthetase, to AdCMV-GlpK-treated INS-1 cells did not inhibit glycerol-stimulated insulin secretion despite the fact that it blocked glycerol incorporation into cellular lipids. We conclude from these studies that glycerol kinase expression is sufficient to activate glycerol signaling in beta cells, showing that the failure of normal islets to respond to this substrate is due to a lack of this enzyme activity. Further, our studies show that glycerol signaling is not linked to esterification or oxidation of the substrate, but is likely mediated by its metabolism in the glycerol phosphate shuttle and/or the distal portion of the glycolytic pathway, either of which can lead to production of ATP and an increased ATP:ADP ratio.

Metabolomic profile associated with insulin resistance and conversion to diabetes in the Insulin Resistance Atherosclerosis Study.

CONTEXT Metabolomic profiling of amino acids and acylcarnitines has revealed consistent patterns associated with metabolic disease. OBJECTIVE This study used metabolomic profiling to identify analytes associated with insulin sensitivity (SI) and conversion to type 2 diabetes (T2D). DESIGN A multiethnic cohort from the Insulin Resistance Atherosclerosis Study. SETTING Community-based. PATIENTS A total of 196 subjects (European American, Hispanic, and African American) were selected to represent extremes of the SI distribution and conversion to T2D between baseline and followup exams. MAIN OUTCOME Mass spectrometry-based profiling of 69 metabolites. Subjects participated in a frequently sampled i.v. glucose tolerance test to measure SI and acute insulin response. T2D status was determined by a 2-hour oral glucose tolerance test. RESULTS Logistic regression analysis from 72 high and 75 low SI subjects revealed significantly decreased glycine and increased valine, leucine, phenylalanine, and combined glutamine and glutamate (P = .0079-7.7 × 10(-6)) in insulin-resistant subjects. Ethnic-stratified results were strongest in European Americans. Comparing amino acid profiles between subjects that converted to T2D (76 converters; 70 nonconverters) yielded a similar pattern of associations: decreased glycine and increased valine, leucine, and combined glutamine and glutamate (P = .016-.00010). Importantly, β-cell function as a covariate revealed a similar pattern of association. CONCLUSIONS A distinct pattern of differences in amino acids were observed when comparing subjects with high and low levels of SI. This pattern was associated with conversion to T2D, remaining significant when accounting for β-cell function, emphasizing a link between this metabolic profile and insulin resistance. These results demonstrate a consistent metabolic signature associated with insulin resistance and conversion to T2D, providing potential insight into underlying mechanisms of disease pathogenesis.

Lipotoxicity in Diabetic Nephropathy: The Potential Role of Fatty Acid Oxidation

Cellular toxicity mediated by lipids (lipotoxicity) has been implicated in the pathophysiology of metabolic syndrome and diabetes mellitus. Genetic analyses now implicate lipotoxicity in susceptibility to type 2 diabetes mellitus-associated nephropathy (T2DN), a pathway that had previously been unexplored. A genome-wide association study in Japanese patients identified a single nucleotide polymorphism in the acetyl-CoA carboxylase β (ACACB) gene associated with T2DN. Replication analyses suggest that this same polymorphism may be a diabetic nephropathy risk allele in other ethnic groups. The ACACB gene (also called ACC2 or acetyl-CoA carboxylase 2) plays a critical role in intracellular fatty acid (FA) oxidation. This manuscript reviews the physiology of FA metabolism and adverse cellular effects that can result from dysregulation of this process. It is hypothesized that glomerular and tubular dysfunction can be induced by increases in intracellular FA concentrations, a process that may be enabled by genetic risk variants. This novel glucolipotoxicity hypothesis in T2DN warrants further investigation.

Apolipoprotein L1 nephropathy risk variants associate with HDL subfraction concentration in African Americans

BACKGROUND Coding variants in the apolipoprotein L1 gene (APOL1) are strongly associated with non-diabetic nephropathy in African Americans. ApoL1 proteins associate with high-density lipoprotein (HDL) particles in the circulation. Plasma HDL particle subclass concentrations were compared in 73 African Americans based on APOL1 genotypes to detect differences potentially contributing to renal disease. METHODS HDL subclass concentrations were measured using nuclear magnetic resonance spectroscopy in African American first-degree relatives of patients with non-diabetic end-stage renal disease. Participants had estimated glomerular filtration rates (GFRs) > 80 mL/min and lacked albuminuria. Additive effects of the number of APOL1 risk variants on natural logarithm-transformed HDL subclass concentrations were computed. RESULTS Participants were 58.9% female with mean ± SD age 47.2 ± 13.3 years and GFR 92.4 ± 18.8 mL/min. The numbers with 2, 1 and 0 APOL1 nephropathy risk variants, respectively, were 36, 17 and 20. Mean ± SD medium-sized HDL concentrations were significantly lower for each additional APOL1 risk variant (2 versus 1 versus 0 risk variants: 9.0 ± 5.6 versus 10.1 ± 5.5 versus 13.1 ± 8.2 μmol/L, respectively; P = 0.0222 unadjusted; P = 0.0162 triglyceride- and ancestry adjusted). CONCLUSIONS Lower medium-sized HDL subclass concentrations are present in African Americans based on increasing numbers of APOL1 nephropathy risk variants. Potential mechanistic roles of altered medium HDL concentrations on APOL1-associated renal microvascular diseases should be evaluated.

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 INS1E 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

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 Ca2+ 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.