Lotta E. Andersson

Time-resolved metabolomics analysis of β-cells implicates the pentose phosphate pathway in the control of insulin release

Insulin secretion is coupled with changes in β-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH and insulin secretion were measured within 15 min of stimulation of clonal INS-1 832/13 β-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the β-cell metabolic response. This was fulfilled by the NADPH/NADP+ ratio, which was elevated (8-fold; P<0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 β-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in β-cell stimulus-secretion coupling.

Genotype-based treatment of type 2 diabetes with an α2A-adrenergic receptor antagonist

The α2A-adrenergic receptor antagonist yohimbine improves insulin secretion in type 2 diabetics carrying the ADRA2A risk variant. Personalized Diabetes Therapy The sequencing of the human genome has been heralded as the advent of personalized medicine; however, translating an individual’s genetic variants to tailored therapy for a specific disease is a long road. Now, Tang et al. report that yohimbine, an antagonist for the α2A-adrenergic receptor, can improve insulin secretion in type 2 diabetics who carry a genetic variant in the encoding gene, ADRA2A. Type 2 diabetics with this variant overexpress the α2A-adrenergic receptor and have impaired insulin secretion. In a randomized, placebo-controlled study, yohimbine increased insulin secretion in type 2 diabetics with the risk variant to levels comparable to those without the risk variant. These results suggest that risk variant analysis can lead to individualized therapies that target patient-specific pathophysiology. The feasibility of exploiting genomic information for individualized treatment of polygenic diseases remains uncertain. A genetic variant in ADRA2A, which encodes the α2A-adrenergic receptor (α2AAR), was recently associated with type 2 diabetes. This variant causes receptor overexpression and impaired insulin secretion; thus, we hypothesized that blocking α2AAR pharmacologically could improve insulin secretion in patients with the risk genotype. A total of 50 type 2 diabetes patients were recruited on the basis of ADRA2A genotype for a randomized placebo-controlled intervention study with the α2AAR antagonist yohimbine. The patients received 0, 10, or 20 mg of yohimbine at three separate visits. The primary endpoint was insulin secretion at 30 min (Ins30) during an oral glucose tolerance test (OGTT). Patients with the risk variant had 25% lower Ins30 than those without risk genotype. After administration of 20 mg of yohimbine, Ins30 was enhanced by 29% in the risk group, making secretion similar to patients carrying the low-risk allele. The corrected insulin response and disposition index in individuals with the high-risk (but not low-risk) allele were improved by 59 ± 18% and 43 ± 14%, respectively. The beneficial effect of yohimbine was not a consequence of improved insulin sensitivity. In summary, the data show that the insulin secretion defect in patients carrying the ADRA2A risk genotype can be corrected by α2AAR antagonism. The findings show that knowledge of genetic risk variants can be used to guide therapeutic interventions that directly target the underlying pathophysiology and demonstrate the potential of individualized genotype-specific treatment of type 2 diabetes.

Coordinate Changes in Histone Modifications, mRNA Levels, and Metabolite Profiles in Clonal INS-1 832/13 β-Cells Accompany Functional Adaptations to Lipotoxicity

Background: Epigenetic regulation may mediate lipotoxic effects on β-cells in type 2 diabetes. Results: Lipotoxicity impairs insulin secretion, and alters metabolism, gene expression, and histone marks in INS-1 832/13 β-cells. Conclusion: Perturbed insulin secretion results from epigenetic, genetic, and metabolic adaptations to increased fatty acid metabolism in β-cells. Significance: Elucidation of the link between lipotoxicity and insulin secretion is crucial for understanding the pathogenesis of type 2 diabetes. Lipotoxicity is a presumed pathogenetic process whereby elevated circulating and stored lipids in type 2 diabetes cause pancreatic β-cell failure. To resolve the underlying molecular mechanisms, we exposed clonal INS-1 832/13 β-cells to palmitate for 48 h. We observed elevated basal insulin secretion but impaired glucose-stimulated insulin secretion in palmitate-exposed cells. Glucose utilization was unchanged, palmitate oxidation was increased, and oxygen consumption was impaired. Halting exposure of the clonal INS-1 832/13 β-cells to palmitate largely recovered all of the lipid-induced functional changes. Metabolite profiling revealed profound but reversible increases in cellular lipids. Glucose-induced increases in tricarboxylic acid cycle intermediates were attenuated by exposure to palmitate. Analysis of gene expression by microarray showed increased expression of 982 genes and decreased expression of 1032 genes after exposure to palmitate. Increases were seen in pathways for steroid biosynthesis, cell cycle, fatty acid metabolism, DNA replication, and biosynthesis of unsaturated fatty acids; decreases occurred in the aminoacyl-tRNA synthesis pathway. The activity of histone-modifying enzymes and histone modifications of differentially expressed genes were reversibly altered upon exposure to palmitate. Thus, Insig1, Lss, Peci, Idi1, Hmgcs1, and Casr were subject to epigenetic regulation. Our analyses demonstrate that coordinate changes in histone modifications, mRNA levels, and metabolite profiles accompanied functional adaptations of clonal β-cells to lipotoxicity. It is highly likely that these changes are pathogenetic, accounting for loss of glucose responsiveness and perturbed insulin secretion.

Characterization of Stimulus-Secretion Coupling in the Human Pancreatic EndoC-βH1 Beta Cell Line.

Aims/Hypothesis Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets. Methods Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets. Results Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells. Conclusions/Interpretation Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.

Chronic high glucose and pyruvate levels differentially affect mitochondrial bioenergetics and fuel-stimulated insulin secretion from clonal INS-1 832/13 cells.

Background: Elevated glucose may cause β-cell dysfunction in type 2 diabetes, i.e., glucotoxicity. Results: Elevated glucose, but not pyruvate, perturbed insulin secretion and content, plasma and mitochondrial membrane potentials, and proton leak, while increasing glycolytic metabolites in β-cells. Conclusion: Early metabolism of glucose exerts a toxic effect on clonal insulin-producing cells. Significance: Unraveling these mechanisms may provide protection of β-cells in diabetes. Glucotoxicity in pancreatic β-cells is a well established pathogenetic process in type 2 diabetes. It has been suggested that metabolism-derived reactive oxygen species perturb the β-cell transcriptional machinery. Less is known about altered mitochondrial function in this condition. We used INS-1 832/13 cells cultured for 48 h in 2.8 mm glucose (low-G), 16.7 mm glucose (high-G), or 2.8 mm glucose plus 13.9 mm pyruvate (high-P) to identify metabolic perturbations. High-G cells showed decreased responsiveness, relative to low-G cells, with respect to mitochondrial membrane hyperpolarization, plasma membrane depolarization, and insulin secretion, when stimulated acutely with 16.7 mm glucose or 10 mm pyruvate. In contrast, high-P cells were functionally unimpaired, eliminating chronic provision of saturating mitochondrial substrate as a cause of glucotoxicity. Although cellular insulin content was depleted in high-G cells, relative to low-G and high-P cells, cellular functions were largely recovered following a further 24-h culture in low-G medium. After 2 h at 2.8 mm glucose, high-G cells did not retain increased levels of glycolytic or TCA cycle intermediates but nevertheless displayed increased glycolysis, increased respiration, and an increased mitochondrial proton leak relative to low-G and high-P cells. This notwithstanding, titration of low-G cells with low protonophore concentrations, monitoring respiration and insulin secretion in parallel, showed that the perturbed insulin secretion of high-G cells could not be accounted for by increased proton leak. The present study supports the idea that glucose-induced disturbances of stimulus-secretion coupling by extramitochondrial metabolism upstream of pyruvate, rather than exhaustion from metabolic overload, underlie glucotoxicity in insulin-producing cells.

Unique and Shared Metabolic Regulation in Clonal β-Cells and Primary Islets Derived From Rat Revealed by Metabolomics Analysis

As models for β-cell metabolism, rat islets are, to some extent, a, heterogeneous cell population stressed by the islet isolation procedure, whereas rat-derived clonal β-cells exhibit a tumor-like phenotype. To describe to what extent either of these models reflect normal cellular metabolism, we compared metabolite profiles and gene expression in rat islets and the INS-1 832/13 line, a widely used clonal β-cell model. We found that insulin secretion and metabolic regulation provoked by glucose were qualitatively similar in these β-cell models. However, rat islets exhibited a more pronounced glucose-provoked increase of glutamate, glycerol-3-phosphate, succinate, and lactate levels, whereas INS-1 832/13 cells showed a higher glucose-elicited increase in glucose-6-phosphate, alanine, isocitrate, and α-ketoglutarate levels. Glucose induced a decrease in levels of γ-aminobutyrate (GABA) and aspartate in rat islets and INS-1 832/13 cells, respectively. Genes with cellular functions related to proliferation and the cell cycle were more highly expressed in the INS-1 832/13 cells. Most metabolic pathways that were differentially expressed included GABA metabolism, in line with altered glucose responsiveness of GABA. Also, lactate dehydrogenase A, which is normally expressed at low levels in mature β-cells, was more abundant in rat islets than in INS-1 832/13 cells, confirming the finding of elevated glucose-provoked lactate production in the rat islets. Overall, our results suggest that metabolism in rat islets and INS-1 832/13 cells is qualitatively similar, albeit with quantitative differences. Differences may be accounted for by cellular heterogeneity of islets and proliferation of the INS-1 832/13 cells.

Mitochondrial transcription factor B2 is essential for mitochondrial and cellular function in pancreatic β-cells

Objective Insulin release from pancreatic β-cells is controlled by plasma glucose levels via mitochondrial fuel metabolism. Therefore, insulin secretion is critically dependent on mitochondrial DNA (mtDNA) and the genes it encodes. Mitochondrial transcription factor B2 (TFB2M) controls transcription of mitochondrial-encoded genes. However, its precise role in mitochondrial metabolism in pancreatic β-cells and, consequently, in insulin secretion remains unknown. Methods To elucidate the role of TFB2M in mitochondrial function and insulin secretion in vitro and in vivo, mice with a β-cell specific homozygous or heterozygous knockout of Tfb2m and rat clonal insulin-producing cells in which the gene was silenced were examined with an array of metabolic and functional assays. Results There was an effect of gene dosage on Tfb2m expression and function. Loss of Tfb2m led to diabetes due to disrupted transcription of mitochondrial DNA (mtDNA) and reduced mtDNA content. The ensuing mitochondrial dysfunction activated compensatory mechanisms aiming to limit cellular dysfunction and damage of β-cells. These processes included the mitochondrial unfolded protein response, mitophagy, and autophagy. Ultimately, however, these cell-protective systems were overridden, leading to mitochondrial dysfunction and activation of mitochondrial-dependent apoptotic pathways. In this way, β-cell function and mass were reduced. Together, these perturbations resulted in impaired insulin secretion, progressive hyperglycemia, and, ultimately, development of diabetes. Conclusions Loss of Tfb2m in pancreatic β-cells results in progressive mitochondrial dysfunction. Consequently, insulin secretion in response to metabolic stimuli is impaired and β-cell mass reduced. Our findings indicate that TFB2M plays an important functional role in pancreatic β-cells. Perturbations of its actions may lead to loss of functional β-cell mass, a hallmark of T2D.

Inhibition of the malate–aspartate shuttle in mouse pancreatic islets abolishes glucagon secretion without affecting insulin secretion

Altered secretion of insulin as well as glucagon has been implicated in the pathogenesis of Type 2 diabetes (T2D), but the mechanisms controlling glucagon secretion from α-cells largely remain unresolved. Therefore, we studied the regulation of glucagon secretion from αTC1-6 (αTC1 clone 6) cells and compared it with insulin release from INS-1 832/13 cells. We found that INS-1 832/13 and αTC1-6 cells respectively secreted insulin and glucagon concentration-dependently in response to glucose. In contrast, tight coupling of glycolytic and mitochondrial metabolism was observed only in INS-1 832/13 cells. Although glycolytic metabolism was similar in the two cell lines, TCA (tricarboxylic acid) cycle metabolism, respiration and ATP levels were less glucose-responsive in αTC1-6 cells. Inhibition of the malate-aspartate shuttle, using phenyl succinate (PhS), abolished glucose-provoked ATP production and hormone secretion from αTC1-6 but not INS-1 832/13 cells. Blocking the malate-aspartate shuttle increased levels of glycerol 3-phosphate only in INS-1 832/13 cells. Accordingly, relative expression of constituents in the glycerol phosphate shuttle compared with malate-aspartate shuttle was lower in αTC1-6 cells. Our data suggest that the glycerol phosphate shuttle augments the malate-aspartate shuttle in INS-1 832/13 but not αTC1-6 cells. These results were confirmed in mouse islets, where PhS abrogated secretion of glucagon but not insulin. Furthermore, expression of the rate-limiting enzyme of the glycerol phosphate shuttle was higher in sorted primary β- than in α-cells. Thus, suppressed glycerol phosphate shuttle activity in the α-cell may prevent a high rate of glycolysis and consequently glucagon secretion in response to glucose. Accordingly, pyruvate- and lactate-elicited glucagon secretion remains unaffected since their signalling is independent of mitochondrial shuttles.

Glycogen metabolism in the glucose‐sensing and supply‐driven β‐cell

Glycogen metabolism in β‐cells may affect downstream metabolic pathways controlling insulin release. We examined glycogen metabolism in human islets and in the rodent‐derived INS‐1 832/13 β‐cells and found them to express the same isoforms of key enzymes required for glycogen metabolism. Our findings indicate that glycogenesis is insulin‐independent but influenced by extracellular glucose concentrations. Levels of glycogen synthase decrease with increasing glucose concentrations, paralleling accumulation of glycogen. We did not find cAMP‐elicited glycogenolysis and insulin secretion to be causally related. In conclusion, our results reveal regulated glycogen metabolism in human islets and insulin‐secreting cells. Whether glycogen metabolism affects insulin secretion under physiological conditions remains to be determined.

Glutamine-Elicited Secretion of Glucagon-Like Peptide 1 Is Governed by an Activated Glutamate Dehydrogenase

Glucagon-like peptide 1 (GLP-1), secreted from intestinal L cells, glucose dependently stimulates insulin secretion from β-cells. This glucose dependence prevents hypoglycemia, rendering GLP-1 analogs a useful and safe treatment modality in type 2 diabetes. Although the amino acid glutamine is a potent elicitor of GLP-1 secretion, the responsible mechanism remains unclear. We investigated how GLP-1 secretion is metabolically coupled in L cells (GLUTag) and in vivo in mice using the insulin-secreting cell line INS-1 832/13 as reference. A membrane-permeable glutamate analog (dimethylglutamate [DMG]), acting downstream of electrogenic transporters, elicited similar alterations in metabolism as glutamine in both cell lines. Both DMG and glutamine alone elicited GLP-1 secretion in GLUTag cells and in vivo, whereas activation of glutamate dehydrogenase (GDH) was required to stimulate insulin secretion from INS-1 832/13 cells. Pharmacological inhibition in vivo of GDH blocked secretion of GLP-1 in response to DMG. In conclusion, our results suggest that nonelectrogenic nutrient uptake and metabolism play an important role in L cell stimulus-secretion coupling. Metabolism of glutamine and related analogs by GDH in the L cell may explain why GLP-1 secretion, but not that of insulin, is activated by these secretagogues in vivo.