Pyruvate dehydrogenase kinase 1 controls mitochondrial metabolism and insulin secretion in INS-1 832/13 clonal β-cells

2010 ◽  
Vol 429 (1) ◽  
pp. 205-213 ◽  
Author(s):  
Ulrika Krus ◽  
Olga Kotova ◽  
Peter Spégel ◽  
Elna Hallgard ◽  
Vladimir V. Sharoyko ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Insulin Secretion ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Pyruvate Dehydrogenase Kinase ◽  
Β Cells ◽  
Acid Cycle ◽  
Metabolic Coupling ◽  
Pyruvate Dehydrogenase Kinase 1

Tight coupling between cytosolic and mitochondrial metabolism is key for GSIS (glucose-stimulated insulin secretion). In the present study we examined the regulatory contribution of PDH (pyruvate dehydrogenase) kinase 1, a negative regulator of PDH, to metabolic coupling in 832/13 clonal β-cells. Knockdown of PDH kinase 1 with siRNA (small interfering RNA) reduced its mRNA (>80%) and protein level (>40%) after 72 h. PDH activity, glucose-stimulated cellular oxygen consumption and pyruvate-stimulated mitochondrial oxygen consumption increased 1.7- (P<0.05), 1.6- (P<0.05) and 1.6-fold (P<0.05) respectively. Gas chromatography/MS revealed an altered metabolite profile upon silencing of PDH kinase 1, determined by increased levels of the tricarboxylic acid cycle intermediates malate, fumarate and α-ketoglutarate. These metabolic alterations were associated with exaggerated GSIS (5-fold compared with 3.1-fold in control cells; P<0.01). Insulin secretion, provoked by leucine and dimethylsuccinate, which feed into the tricarboxylic acid cycle bypassing PDH, was unaffected. The oxygen consumption and metabolic data strongly suggest that knockdown of PDH kinase 1 in β-cells permits increased metabolic flux of glucose-derived carbons into the tricarboxylic acid cycle via PDH. Enhanced insulin secretion is probably caused by increased generation of tricarboxylic acid cycle-derived reducing equivalents for mitochondrial electron transport to generate ATP and/or stimulatory metabolic intermediates. On the basis of these findings, we suggest that PDH kinase 1 is an important regulator of PDH in clonal β-cells and that PDH kinase 1 and PDH are important for efficient metabolic coupling. Maintaining low PDH kinase 1 expression/activity, keeping PDH in a dephosphorylated and active state, may be important for β-cells to achieve the metabolic flux rates necessary for maximal GSIS.

scholarly journals Bioenergetic Analysis of Single Pancreatic β-Cells Indicates an Impaired Metabolic Signature in Type 2 Diabetic Subjects

Endocrinology ◽  
2015 ◽  
Vol 156 (10) ◽  
pp. 3496-3503 ◽  
Author(s):  
Akos A. Gerencser
Keyword(s):  
Insulin Secretion ◽  
Membrane Potential ◽  
Energy Metabolism ◽  
Tricarboxylic Acid Cycle ◽  
Atp Synthesis ◽  
Tricarboxylic Acid ◽  
Β Cells ◽  
Acid Cycle ◽  
Type 2 Diabetic

Impaired activation of mitochondrial energy metabolism by glucose has been demonstrated in type 2 diabetic β-cells. The cause of this dysfunction is unknown. The aim of this study was to identify segments of energy metabolism with normal or with altered function in human type 2 diabetes mellitus. The mitochondrial membrane potential (ΔψM), and its response to glucose, is the main driver of mitochondrial ATP synthesis and is hence a central mediator of glucose-induced insulin secretion, but its quantitative determination in β-cells from human donors has not been attempted, due to limitations in assay technology. Here, novel fluorescence microscopic assays are exploited to quantify ΔψM and its response to glucose and other secretagogues in β-cells of dispersed pancreatic islet cells from 4 normal and 3 type 2 diabetic organ donors. Mitochondrial volume densities and the magnitude of ΔψM in low glucose were not consistently altered in diabetic β-cells. However, ΔψM was consistently less responsive to elevation of glucose concentration, whereas the decreased response was not observed with metabolizable secretagogue mixtures that feed directly into the tricarboxylic acid cycle. Single-cell analysis of the heterogeneous responses to metabolizable secretagogues indicated no dysfunction in relaying ΔψM hyperpolarization to plasma membrane potential depolarization in diabetic β-cells. ΔψM of diabetic β-cells was distinctly responsive to acute inhibition of ATP synthesis during glucose stimulation. It is concluded that the mechanistic deficit in glucose-induced insulin secretion and mitochondrial hyperpolarization of diabetic human β-cells is located upstream of the tricarboxylic acid cycle and manifests in dampening the control of ΔψM by glucose metabolism.

scholarly journals Fibre-type specific modification of the activity and regulation of skeletal muscle pyruvate dehydrogenase kinase (PDK) by prolonged starvation and refeeding is associated with targeted regulation of PDK isoenzyme 4 expression

2000 ◽  
Vol 346 (3) ◽  
pp. 651-657 ◽  
Author(s):  
Mary C. SUGDEN ◽  
Alexandra KRAUS ◽  
Robert A. HARRIS ◽  
Mark J. HOLNESS
Keyword(s):  
Skeletal Muscle ◽  
Protein Expression ◽  
Pyruvate Dehydrogenase ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Pyruvate Dehydrogenase Kinase ◽  
Acid Cycle ◽  
Slow Twitch ◽  
Fast Twitch ◽  
Anterior Tibialis

Using immunoblot analysis with antibodies raised against recombinant pyruvate dehydrogenase kinase (PDK) isoenzymes PDK2 and PDK4, we demonstrate selective changes in PDK isoenzyme expression in slow-twitch versus fast-twitch skeletal muscle types in response to prolonged (48 h) starvation and refeeding after starvation. Starvation increased PDK activity in both slow-twitch (soleus) and fast-twitch (anterior tibialis) skeletal muscle and was associated with loss of sensitivity of PDK to inhibition by pyruvate, with a greater effect in anterior tibialis. Starvation significantly increased PDK4 protein expression in both soleus and anterior tibialis, with a greater response in anterior tibialis. Starvation did not effect PDK2 protein expression in soleus, but modestly increased PDK2 expression in anterior tibialis. Refeeding for 4 h partially reversed the effect of 48-h starvation on PDK activity and PDK4 expression in both soleus and anterior tibialis, but the response was more marked in soleus than in anterior tibialis. Pyruvate sensitivity of PDK activity was also partially restored by refeeding, again with the greater response in soleus. It is concluded that targeted regulation of PDK4 isoenzyme expression in skeletal muscle in response to starvation and refeeding underlies the modulation of the regulatory characteristics of PDK in vivo. We propose that switching from a pyruvate-sensitive to a pyruvate-insensitive PDK isoenzyme in starvation (a) maintains a sufficiently high pyruvate concentration to ensure that the glucose → alanine → glucose cycle is not impaired, and (b) may ‘spare’ pyruvate for anaplerotic entry into the tricarboxylic acid cycle to support the entry of acetyl-CoA derived from fatty acid (FA) oxidation into the tricarboxylic acid cycle. We further speculate that FA oxidation by skeletal muscle is both forced and facilitated by upregulation of PDK4, which is perceived as an essential component of the operation of the glucose-FA cycle in starvation.

scholarly journals Enhancing Bioethanol Production by Deleting Phosphoenolpyruvate Synthase and ADP-Glucose Pyrophosphorylase, and Shunting Tricarboxylic Acid Cycle In Synechocystis Sp. PCC 6803

2021 ◽  
Author(s):  
E-Bin Gao ◽  
Penglin Ye ◽  
Haiyan Qiu ◽  
Junhua Wu ◽  
Huayou Chen
Keyword(s):  
Carbon Dioxide ◽  
Ethanol Production ◽  
Atmospheric Carbon Dioxide ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Acid Cycle ◽  
Photosynthetic Production ◽  
Engineered Strain ◽  
Pcc 6803

Abstract Background: The outstanding ability of directly assimilating carbon dioxide and sunlight to produce biofuels and chemicals impels photosynthetic cyanobacteria to become attractive organisms for the solution to the global warming crises and the world energy growth. The cyanobacteria-based method for ethanol production has been increasingly regarded as alternatives to food biomass-based fermentation and traditional petroleum-based production. Therefore, we engineered the model cyanobacterium Synechocystis sp. PCC 6803 to synthesize ethanol and optimized the biosynthetic pathways for improving ethanol production under photoautotrophic conditions.Results: In this study, we successfully achieved the photosynthetic production of ethanol from atmospheric carbon dioxide by an engineered mutant Synechocystis sp. PCC 6803 with over-expressing the heterologous genes encoding Zymomonas mobilis pyruvate decarboxylase (PDC) and Escherichia coli NADPH-dependent alcohol dehydrogenase (YqhD). The engineered strain was further optimized by an alternative engineering approach to improve cell growth, and increase the intracellular supply of the precursor pyruvate for ethanol production under photoautotrophic conditions. This approach includes blocking phosphoenolpyruvate synthetic pathway from pyruvate, removing glycogen storage, and shunting carbon metabolic flux of tricarboxylic acid cycle. Through redirecting and optimizing the metabolic carbon flux of Synechocystis, a high ethanol-producing efficiency was achieved (248 mg L-1 day-1) under photoautotrophic conditions with atmospheric CO2 as the sole carbon source. Conclusions: The engineered strain SYN009 (∆slr0301/pdc-yqhD, ∆slr1176/maeB) would become a valuable biosystem for photosynthetic production of ethanol and for expanding our knowledge of exploiting cyanobacteria to produce value chemicals directly from atmospheric CO2.

scholarly journals Metabolomic analyses reveal profound differences in glycolytic and tricarboxylic acid cycle metabolism in glucose-responsive and -unresponsive clonal β-cell lines

2011 ◽  
Vol 435 (1) ◽  
pp. 277-284 ◽  
Author(s):  
Peter Spégel ◽  
Siri Malmgren ◽  
Vladimir V. Sharoyko ◽  
Philip Newsholme ◽  
Thomas Koeck ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Cell Lines ◽  
Target Genes ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Gas Chromatography Mass Spectrometry ◽  
Glycolytic Metabolism ◽  
Β Cell ◽  
Acid Cycle ◽  
Stimulus Secretion Coupling

Insulin secretion from pancreatic β-cells is controlled by complex metabolic and energetic changes provoked by exposure to metabolic fuels. Perturbations in these processes lead to impaired insulin secretion, the ultimate cause of T2D (Type 2 diabetes). To increase our understanding of stimulus–secretion coupling and metabolic processes potentially involved in the pathogenesis of T2D, a comprehensive investigation of the metabolic response in the glucose-responsive INS-1 832/13 and glucose-unresponsive INS-1 832/2 β-cell lines was performed. For this metabolomics analysis, we used GC/MS (gas chromatography/mass spectrometry) combined with multivariate statistics. We found that perturbed secretion in the 832/2 line was characterized by disturbed coupling of glycolytic and TCA (tricarboxylic acid)-cycle metabolism. The importance of this metabolic coupling was reinforced by our observation that insulin secretion partially could be reinstated by stimulation of the cells with mitochondrial fuels which bypass glycolytic metabolism. Furthermore, metabolic and functional profiling of additional β-cell lines (INS-1, INS-1 832/1) confirmed the important role of coupled glycolytic and TCA-cycle metabolism in stimulus–secretion coupling. Dependence of the unresponsive clones on glycolytic metabolism was paralleled by increased stabilization of HIF-1α (hypoxia-inducible factor 1α). The relevance of a similar perturbation for human T2D was suggested by increased expression of HIF-1α target genes in islets from T2D patients.

scholarly journals Systems-Level Metabolic Flux Profiling Elucidates a Complete, Bifurcated Tricarboxylic Acid Cycle in Clostridium acetobutylicum

2011 ◽  
Vol 193 (23) ◽  
pp. 6805-6805
Author(s):  
D. Amador-Noguez ◽  
X.-J. Feng ◽  
J. Fan ◽  
N. Roquet ◽  
H. Rabitz ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Acid Cycle

scholarly journals Tricarboxylic acid cycle flux and enzyme activities in the isolated working rat heart

1981 ◽  
Vol 200 (3) ◽  
pp. 701-703 ◽  
Author(s):  
G J Cooney ◽  
H Taegtmeyer ◽  
E A Newsholme
Keyword(s):  
Oxygen Consumption ◽  
Enzyme Activities ◽  
Rat Heart ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Work Load ◽  
Tricarboxylic Acid ◽  
Acid Cycle ◽  
Oxoglutarate Dehydrogenase ◽  
Working Rat Heart

Flux through the tricarboxylic acid cycle was calculated from oxygen consumption in hearts perfused near the physiological work load. Activities of citrate synthase, 2-oxoglutarate dehydrogenase and succinate dehydrogenase were measured in the same hearts. Only the activities of 2-oxoglutarate dehydrogenase correlated with calculated fluxes through the cycle.

scholarly journals Die Wirkung von 6-Furfurylaminopurin auf die Atmung von Zellsüspensionen von Nicotiana tabacum

1963 ◽  
Vol 18 (11) ◽  
pp. 942-946 ◽  
Author(s):  
L. Bergmann
Keyword(s):  
Oxygen Consumption ◽  
Nicotiana Tabacum ◽  
Glucose Uptake ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Cell Suspensions ◽  
Tissue Cultures ◽  
Acid Cycle ◽  
Glucose Catabolism

6-Furfurylaminopurine (“kinetin”) has been found to reduce markedly the respiration of cell suspensions from tissue cultures of Nicotiana tabacum var. “Samsun” in glucose containing media. The observed reduction in the oxygen consumption is not caused by an inhibition of the glucose uptake by the cells. Pyruvate, succinate, and α-ketoglutarate can restore the respiration of cells inhibited by kinetin. The increased oxygen consumption following the addition of the acids is accompanied by an increased R.Q. It is concluded therefore, that kinetin does not inhibit the tricarboxylic acid cycle, and the suggestion is put forward that kinetin acts via the Embden- Meyerhof - pathway of the glucose catabolism.

Effect of Hypothermia on the Cellular Respiration of Ventricular Tissue

1957 ◽  
Vol 192 (1) ◽  
pp. 121-125 ◽  
Author(s):  
John P. Hannon ◽  
Benjamin G. Covino
Keyword(s):  
Oxygen Consumption ◽  
Rectal Temperature ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Cellular Respiration ◽  
Acid Cycle ◽  
Enzyme Systems ◽  
Ventricular Tissue ◽  
Significant Depression ◽  
Irreversible Effect

The respiration of ventricular slices and homogenates in the presence of various substrates was studied in control normothermic and experimental hypothermic rats. The Qoo2 of the slices prepared from rats cooled to a rectal temperature of 15°C was significantly higher than that of control normothermic animals. No difference in the rate of oxidation of ventricular homogenates was observed between the two groups except when no substrate was used or when pyruvate was added as the test substrate. In the absence of substrate, there was a tendency toward higher rates in the hypothermic group, whereas addition of pyruvate caused a significant depression in the oxygen consumption of this group. Addition of malate to the pyruvate containing medium produced a marked increase in respiratory rate. The data obtained indicate that hypothermia does not have any adverse irreversible effect on the function of the tricarboxylic acid cycle and its associated enzyme systems. The results do suggest a possible alteration in membrane permeability, an accumulation of reduced intracellular intermediates, or both as a result of the hypothermic episode.

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