Binding of thiamin thiazolone pyrophosphate to mammalian pyruvate dehydrogenase and its effects of kinase and phosphatase activities.

The pyruvate dehydrogenase component of the bovine kidney pyruvate dehydrogenase complex has two thiamin-PP binding sites per α₂β₂ tetramer. Titration of these binding sites with the transition state analog, thiamin thiazolone pyrophosphate, strongly inhibits phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase and ATP. The analog has little effect, if any, on dephosphorylation of phosphorylated pyruvate dehydrogenase by pyruvate dehydrogenase phosphatase. Phosphorylation of pyruvate dehydrogenase inactivates the enzyme, but does not significantly affect the thiamin-PP binding sites. It appears that phosphorylation produces a conformational change in pyruvate dehydrogenase that displaces a catalytic group (or groups) at the active center.     

Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion

Metformin, a drug widely used in the treatment of type 2 diabetes, has recently been shown to act on skeletal muscle and liver in part through the activation of AMP-activated protein kinase (AMPK). Whether metformin or the satiety factor leptin, which also stimulates AMPK in muscle, regulates this enzyme in pancreatic islets is unknown. We have recently shown that forced increases in AMPK activity inhibit insulin secretion from MIN6 cells (da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, and Rutter GA. Biochem J 371: 761-774, 2003). Here, we explore whether 1) glucose, metformin, or leptin regulates AMPK activity in isolated islets from rodent and human and 2) whether changes in AMPK activity modulate insulin secretion from human islets. Increases in glucose concentration from 0 to 3 and from 3 to 17 mM inhibited AMPK activity in primary islets from mouse, rat, and human, confirming previous findings in insulinoma cells. Incubation with metformin (0.2-1 mM) activated AMPK in both human islets and MIN6 beta-cells in parallel with an inhibition of insulin secretion, whereas leptin (10-100 nM) was without effect in MIN6 cells. These studies demonstrate that AMPK activity is subject to regulation by both glucose and metformin in pancreatic islets and clonal beta-cells. The inhibitory effects of metformin on insulin secretion may therefore need to be considered with respect to the use of this drug for the treatment of type 2 diabetes.     

E1 component of pyruvate dehydrogenase complex does not regulate the expression of NADPH-ferredoxin reductase in Azotobacter vinelandii

In Azotobacter vinelandii, the E1 component of pyruvate dehydrogenase complex (PDHE1) is proposed to be a key regulatory protein in an oxidative stress management system that responds to superoxide. This proposal was tested by constructing an A. vinelandii mutant that had a disruption of aceE gene encoding PDHE1. This mutant exhibited wild-type exponential growth and a normal response to oxidative stress induced by paraquat. Electrophoretic mobility-shift assays revealed that a protein previously shown to bind to a paraquat-activatable DNA promoter was still present in the extract prepared from the mutant, implying that the protein cannot be PDHE1. These observations strongly contradict the previous claim that PDHE1 is a DNA-binding protein that is directly involved in the A. vinelandii oxidative stress-regulatory system.     

Knockdown of both mitochondrial isocitrate dehydrogenase enzymes in pancreatic beta cells inhibits insulin secretion

Background

There are three isocitrate dehydrogenases (IDHs) in the pancreatic insulin cell; IDH1 (cytosolic) and IDH2 (mitochondrial) use NADP(H). IDH3 is mitochondrial, uses NAD(H) and was believed to be the IDH that supports the citric acid cycle.

Methods

With shRNAs targeting mRNAs for these enzymes we generated cell lines from INS-1 832/13 cells with severe (80%–90%) knockdown of the mitochondrial IDHs separately and together in the same cell line.

Results

With knockdown of both mitochondrial IDH's mRNA, enzyme activity and protein level, (but not with knockdown of only one mitochondrial IDH) glucose- and BCH (an allosteric activator of glutamate dehydrogenase)-plus-glutamine-stimulated insulin release were inhibited. Cellular levels of citrate, α-ketoglutarate, malate and ATP were altered in patterns consistent with blockage at the mitochondrial IDH reactions. We were able to generate only 50% knockdown of Idh1 mRNA in multiple cell lines (without inhibition of insulin release) possibly because greater knockdown of IDH1 was not compatible with cell line survival.

Conclusions

The mitochondrial IDHs are redundant for insulin secretion. When both enzymes are severely knocked down, their low activities (possibly assisted by transport of IDH products and other metabolic intermediates from the cytosol into mitochondria) are sufficient for cell growth, but inadequate for insulin secretion when the requirement for intermediates is certainly more rapid. The results also indicate that IDH2 can support the citric acid cycle.

General significance

As almost all mammalian cells possess substantial amounts of all three IDH enzymes, the biological principles suggested by these results are probably extrapolatable to many tissues.     

Evidence for a 2-oxoglutarate dehydrogenase complex activity in mitochondria of Neurospora crassa and compared localization with the pyruvate dehydrogenase complex

A 2-oxoglutarate dehydrogenase complex activity is demonstrated in Neurospora crassa mitochondria. A submitochondrial fractionation by digitonin treatment followed by freeze-thawing enables measurement of a well preserved activity in the mitochondrial matrix. In contrast to other reports, the pyruvate dehydrogenase activity is also found to be localized in the matrix.     

Reduction of mitochondrial pyruvate dehydrogenase phosphatase activity in lactating rat mammary gland following starvation or insulin deprivation.

The initial rates of activation of inactivated pyruvate dehydrogenase from lactating rat mammary gland and from pig heart were employed to assay pyruvate dehydrogenase phosphatase activity in mammary gland mitochondrial extracts. 24 h-starvation or 3 h-deprivation of insulin diminished phosphatase activity compared to fed controls. Refeeding and insulin treatment of 24 h starved animals restored in 1 h control levels of phosphatase activity.     

Insulin resistance in the liver in fasting and diabetes mellitus: the failure of insulin to stimulate the release of a chemical modulator of pyruvate dehydrogenase

To further define the mechanism(s) of insulin resistance in the liver associated with diabetes and fasting, we evaluated the ability of insulin to release an activator of pyruvate dehydrogenase activity from a liver particulate fraction. Insulin reproduceably and significantly enhanced the release of mediator from the liver particulate fraction of control animals. The particulate fractions from fasted and diabetic animals were resistant to this effect of insulin. Refeeding and insulin treatment, respectively, restored responsiveness to insulin. These data support the concept that alterations at or near the plasma membrane can be responsible for or accompany the insulin resistance observed in the liver in fasting and diabetes mellitus.     

Glucose‐stimulated insulin release: Parallel perifusion studies of free and hydrogel encapsulated human pancreatic islets

To explore the effects immune‐isolating encapsulation has on the insulin secretion of pancreatic islets and to improve our ability to quantitatively describe the glucose‐stimulated insulin release (GSIR) of pancreatic islets, we conducted dynamic perifusion experiments with isolated human islets. Free (unencapsulated) and hydrogel encapsulated islets were perifused, in parallel, using an automated multi‐channel system that allows sample collection with high temporal resolution. Results indicated that free human islets secrete less insulin per unit mass or islet equivalent (IEQ) than murine islets and with a less pronounced first‐phase peak. While small microcapsules (d = 700 µm) caused only a slightly delayed and blunted first‐phase insulin response compared to unencapsulated islets, larger capsules (d = 1,800 µm) completely blunted the first‐phase peak and decreased the total amount of insulin released. Experimentally obtained insulin time‐profiles were fitted with our complex insulin secretion computational model. This allowed further fine‐tuning of the hormone‐release parameters of this model, which was implemented in COMSOL Multiphysics to couple hormone secretion and nutrient consumption kinetics with diffusive and convective transport. The results of these GSIR experiments, which were also supported by computational modeling, indicate that larger capsules unavoidably lead to dampening of the first‐phase insulin response and to a sustained‐release type insulin secretion that can only slowly respond to changes in glucose concentration. Bioartificial pancreas type devices can provide long‐term and physiologically desirable solutions only if immunoisolation and biocompatibility considerations are integrated with optimized nutrient diffusion and insulin release characteristics by design.     

Cortisone induces insulin resistance in C2C12 myotubes through activation of 11beta‐hydroxysteroid dehydrogenase 1 and autocrinal regulation

The enzyme 11β‐hydroxysteroid dehydrogenase 1 (11β‐HSD1) is known to catalyse inactive glucocorticoids into active forms, and its dysregulation in adipose and muscle tissues has been implicated in the development of metabolic syndrome. To delineate the molecular mechanism by which active cortisol has an antagonizing effect against insulin, we optimized the metabolic production of cortisol and its biological functions in myotubes (C2C12). Myotubes supplemented with cortisone actively catalysed its conversion into cortisol, which in turn abolished phosphorylation of Akt in response to insulin treatment. This led to diminished uptake of insulin‐induced glucose. This was corroborated by the application of 11β‐HSD1 inhibitor glycyrrhetinic acid and a glucocorticoid receptor antagonist RU‐486, which reversed completely the antagonizing effects of cortisol on insulin action. Therefore, development of specific inhibitors targeting 11β‐HSD1 might be a promising way to improve impaired insulin‐stimulated glucose uptake. Copyright © 2013 John Wiley & Sons, Ltd.     

Fructose induced deactivation of glucose-6-phosphate dehydrogenase activity and its prevention by pyruvate: Implications in cataract prevention

Glucose-6-phosphate dehydrogenase (G6PDH) is an important lens enzyme diverting about 14% of the tissue glucose to the hexose monophosphate shunt pathway. The main function of such a pronounced activity of the enzyme is to support reductive biosyntheses, as well as to maintain a reducing environment in the tissue so as to prevent oxy-radical induced damage and consequent cataract formation. Sugars are one of the well-known cataractogenic agents. Several reports suggest that the cataractogenic effect of the sugars in diabetes as well as in normal aging is initiated by the glycation of the proteins including the enzymes and subsequent formation of more complex and biologically inactive or harmful structures. In a diabetic lens the concentration of fructose exceeds significantly the concentration of glucose, suggesting that the contribution of fructosylation may be greater than that of glucosylation. These studies were undertaken to examine further the possibility that in addition to glycation, generation of oxygen free radicals by fructose and consequent oxidative modifications in certain enzymes may be an important participant in the cataractogenic process. This hypothesis was tested by using G6PDH. The enzyme was incubated with various levels of fructose (0–20 mM) and its activity determined as a function of time. This led to a significant loss of its activity, which was prevented by superoxide dismutase, catalase, mannitol and myoinositol. Most interestingly, pyruvate at levels between 0.2 and 1.0 mM also offered substantial protection. Hence, the results, while elucidating further the mechanism of enzyme deactivation by sugars such as fructose, also demonstrate the possibility of therapeutic prevention of cataracts by pyruvate and other such keto acids, in diabetes and other disabilities involving oxygen free radicals in the pathogenetic process.     

Metabolic reorganization in winter: Regulation of pyruvate dehydrogenase (PDH) during long-term freezing and anoxia

Wood frogs, Rana sylvatica, can undergo prolonged periods of whole body freezing during winter, locking as much as 65–70% of total body water into extracellular ice and imposing both anoxia and dehydration on their cells. Metabolic rate depression (MRD) is an adaptation used by R. sylvatica to survive these environmental stresses, where a finite amount of ATP generated through anaerobic metabolism is directed towards maintaining pro-survival functions, while most ATP-expensive cellular processes are temporarily reduced in function. Pyruvate dehydrogenase (PDH) is a vital metabolic enzyme that links anaerobic glycolysis to the aerobic TCA cycle and is an important regulatory site in MRD. PDH enzymatic activity is regulated via reversible protein phosphorylation in response to energetic demands of cells. This study explored the posttranslational regulation of PDH at three serine sites (S232, S293, S300) on the catalytic E1α subunit along with protein expression of four pyruvate dehydrogenase kinases (PDHK1-4) in response to 24 h Freezing, 8 h Thaw, 24 h Anoxia, and 4 h Recovery in the liver and skeletal muscle of R. sylvatica using Luminex multiplex technology and western immunoblotting. Overall, inhibitory regulation of PDH was evident during 24 h Freezing and 24 h Anoxia, which could indicate a notable reduction in glycoytic flux and carbon entry into the tricarboxylic acid cycle as part of MRD. Furthermore, the expression of PDHK1-4 and phosphorylation of PDH at S232, S293, and S300 were highly tissue and stress-specific, indicative of how different tissues respond differently to stress within the same organism.     

The effect of islet amyloid polypeptide (amylin) and calcitonin gene-related peptide on glucose removal in the anaesthetized rat and on insulin secretion from rat pancreatic isletsin vitro

The effect of intravenous infusion of islet amyloid polypeptide (IAPP/amylin) and calcitonin gene-related peptide (CGRP) on blood glucose and plasma insulin in the basal and glucose-stimulated state was investigated in the anaesthetized rat. Both peptides had no effect on basal blood glucose or plasma insulin but following an intravenous bolus of glucose, CGRP-treated rats were hyperglycaemic and hyperinsulinaemic compared with control animals which were similar to IAPP-treated rats. IAPP had no effect on glucose-stimulated islet insulin secretion. These results suggest that CGRP, but not IAPP, alters glucose removalin vivo.     

The effect of amino acids,monoamines and polyamines on pyruvate dehydrogenase activity in mitochondria from rat adipocytes

Summary The ability of polyamines and other cationic compounds including monoamines, amino acids, poly-L-arginine, poly-D-lysine and poly-L-lysine, to alter pyruvate dehydrogenase (PDH) activity in mitochondria from rat epididymal adipocytes was determined. PDH was assayed with the substrate [1-¹⁴C] pyruvate in the presence of 0.05 mM Ca²⁺ and Mg²⁺. Nine of the fourteen compounds tested at 0.1 mM caused a significant increase (procaine, 3-(-morpholinopropionyl) benzo[b]thiophene [VII], spermine, spermidine, putrescine, lysine and tryptophan) or decrease (poly-L-arginine, 3-(-piperidinopropionyl) benzo[b]thiophene) in PDH activity. None of these compounds nonenzymatically decarboxylated [1-¹⁴C] pyruvate to release ¹⁴CO₂. NaF, a PDH phosphatase inhibitor, suppressed the stimulatory effects of those compounds tested: procaine, tryptophan, VII, spermine and spermidine. These results imply that these five compounds activate PDH activity through stimulation of the PDH phosphatase. When the Mg²⁺ concentration was increased from 0.05 to 4.5 mM, the stimulatory effect of spermine was increased, consistent with the finding by others that spermine lowers the Km of the enzyme for Mg²⁺. However, at Mg²⁺ concentrations greater than 0.3 mM, the stimulatory effect of VII was unaltered, procaine failed to alter PDH activity, lysine inhibited PDH activity, and poly-L-lysine stimulated PDH activity. Therefore, polyamines and other positively charged small molecules may be physiologic regulators of PDH activity.     

Overexpression of regucalcin enhances glucose utilization and lipid production in cloned rat hepatoma H4-II-E cells: Involvement of insulin resistance

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of glucose utilization and lipid production was investigated using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin/pCXN2-transfected cells (transfectant) were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. Cells with subconfluency were cultured for 24 or 72 h in medium containing either vehicle or insulin (10(-8) or 10(-7) M) with or without supplementation of glucose (10, 25, or 50 mg/ml of medium) in the absence of insulin. The production of triglyceride and free fatty acid was significantly increased in transfectants cultured without insulin and glucose supplementation as compared with that of wild-type cells. The supplementation of glucose (10, 25, or 50 mg/ml) caused a remarkable increase in medium glucose consumption, triglyceride, and free fatty acid productions in transfectants cultured without insulin. The presence of insulin (10(-7) M) caused a significant increase in medium glucose consumption, triglyceride, and free fatty acid productions in wild-type cells cultured with glucose supplementation. These increases were significantly prevented in transfectants cultured for 72 h. The expression of acetyl-CoA carboxylase, HMG-CoA reductase, glucokinase, pyruvate kinase, and glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNAs in wild-type cells was not significantly changed by culture with or without glucose supplementation in the presence of insulin. These gene expressions were not significantly changed in transfectants. The expression of glucose transporter 2 mRNA was significantly increased in transfectants as compared with that of wild-type cells. Such an increase was not seen in transfectants cultured in the presence of insulin with or without glucose supplementation. This study demonstrates that overexpression of regucalcin enhances glucose utilization and lipid production in the cloned rat hepatoma H4-II-E cells, and that it regulates the effect of insulin.     

Localization and characterization of the mitochondrial isoform of the nucleoside diphosphate kinase in the pancreatic beta cell: evidence for its complexation with mitochondrial succinyl-CoA synthetase

Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of terminal phosphates from nucleoside triphosphates to nucleoside diphosphates to yield nucleotide triphosphates. The present study was undertaken to localize and characterize the mitochondrial isoform of NDPK (mNDPK) in the pancreatic beta cell since it could contribute to the generation of mitochondrial nucleotide triphosphates and, thereby, to the mitochondrial high-energy phosphate metabolism of the pancreatic beta cell. Mitochondrial fractions from the insulin-secreting beta cells were isolated by differential centrifugation. mNDPK activity was assayed as the amount of [(3)H]GTPgammaS formed from ATPgammaS and [(3)H]GDP. Incubation of isolated mitochondrial extracts with either [gamma-(32)P]ATP or GTP resulted in the formation [(32)P]NDPK, which could be immunoprecipitated by an anti-NDPK serum. mNDPK exhibited saturation kinetics with respect to its nucleoside diphosphate acceptors and nucleoside triphosphate donors and sensitivity to known inhibitors of NDPK (e.g., uridine diphosphate and cromoglycate). By Western blot analyses, at least three isoforms of NDPK were identified in various subcellular fractions of the beta cell. The nm23-H1 (NDPK-A) was predominantly soluble whereas nm23-H2 (NDPK-B) was associated with the soluble as well as membranous fractions. The mitochondrial isoform of NDPK, nm23-H4, was uniformly distributed in the beta cell mitochondrial subfractions. A significant amount of NDPK (as determined by the catalytic activity and immunological methods) was recovered in the immunoprecipitates of mitochondrial fraction precipitated with an antiserum directed against succinyl-CoA synthetase (SCS), suggesting that NDPK might remain complexed with SCS. We provide the first evidence for the localization of a mitochondrial isoform of the NDPK in the islet beta cell and thus offer a potential mechanism for the generation of intramitochondrial GTP which, unlike ATP, is not transported into mitochondria via the classical nucleotide translocase. Further work will be required to determine the importance of the NDPK/SCS complex to normal beta cell function in the secretion of insulin.     

Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.     

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins. J. Cell. Biochem. 68:139–150, 1998. © 1998 Wiley-Liss, Inc.