Inhibition Of Pyruvate Carboxylase Research Articles

Synthesis and Evaluation of 1,3-Disubstituted Imidazolidine-2,4,5-triones as Inhibitors of Pyruvate Carboxylase.

Substituted imidazolidinetriones (IZTs) have been identified as potent inhibitors of pyruvate carboxylase (PC) through an in silico screening approach. Alkyl 2-(2,4,5-trioxo-3-substituted imidazolidin-1-yl)acetates (6i-6r) are the most potent of the series, with IC50 values between 3 and 12 μM, and several IZTs demonstrate high passive permeability across an artificial membrane. IZTs are mixed-type inhibitors with respect to pyruvate and noncompetitive with respect to ATP. This class of inhibitors appears to be selective for PC. Inhibitors in the IZT series do not inhibit the metalloenzymes human carbonic anhydrase II and matrix metalloprotease-12, and they do not inhibit the related biotin-dependent enzyme, guanidine carboxylase. Altogether, IZTs offer promise as PC inhibitors with potential downstream applications in cellular and in vivo systems.

Anemoside B4, a new pyruvate carboxylase inhibitor, alleviates colitis by reprogramming macrophage function.

Colitis is a global disease usually accompanied by intestinal epithelial damage and intestinal inflammation, and an increasing number of studies have found natural products to be highly effective in treating colitis. Anemoside B4 (AB4), an abundant saponin isolated from Pulsatilla chinensis (Bunge), whichwas found to have strong anti-inflammatory activity. However, the exact molecular mechanisms and direct targets of AB4 in the treatment of colitis remain to be discovered. The anti-inflammatory activities of AB4 were verified in LPS-induced cell models and 2, 4, 6-trinitrobenzene sulfonic (TNBS) or dextran sulfate sodium (DSS)-induced colitis mice and rat models. The molecular target of AB4 was identified by affinity chromatography analysis using chemical probes derived from AB4. Experiments including proteomics, molecular docking, biotin pull-down, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA) were used to confirm the binding of AB4 to itsmolecular target. Overexpression of pyruvate carboxylase (PC) and PC agonist were used to study the effects of PC on the anti-inflammatory and metabolic regulation of AB4 in vitro and in vivo. AB4 not only significantly inhibited LPS-induced NF-κB activation and increased ROS levels in THP-1 cells, but also suppressed TNBS/DSS-induced colonic inflammation in mice and rats. The molecular target of AB4 was identified as PC, a key enzyme related to fatty acid, amino acid and tricarboxylic acid (TCA) cycle. We next demonstrated that AB4 specifically bound to the His879 site of PC and altered the protein's spatial conformation, thereby affecting the enzymatic activity of PC. LPS activated NF-κB pathway and increased PC activity, which caused metabolic reprogramming, while AB4 reversed this phenomenon by inhibiting the PC activity. In vivo studies showed that diisopropylamine dichloroacetate (DADA),a PC agonist, eliminated the therapeutic effects of AB4 by changing the metabolic rearrangement of intestinal tissues in colitis mice. We identified PC as a direct cellular target of AB4 in the modulation of inflammation, especially colitis. Moreover, PC/pyruvate metabolism/NF-κB is crucial for LPS-driven inflammation andoxidative stress. These findings shed more light on thepossibilities ofPC as a potential new target for treating colitis.

The presence of pyruvate carboxylase in the human brain and its role in the survival of cultured human astrocytes.

Pyruvate carboxylase (PC) is a mitochondrial, biotin-containing enzyme catalyzing the ATP-dependent synthesis of oxaloacetate from pyruvate and bicarbonate, with a critical anaplerotic role in sustaining the brain metabolism. Based on the studies performed on animal models, PC expression was assigned to be glia-specific. To study PC distribution among human neural cells, we probed the cultured human astrocytes and brain sections with antibodies against PC. Additionally, we tested the importance of PC for the viability of cultured human astrocytes by applying the PC inhibitor 3-chloropropane-1,2-diol (CPD). Our results establish the expression of PC in mitochondria of human astrocytes in culture and brain tissue and also into a subpopulation of the neurons in situ. CPD negatively affected the viability of astrocytes in culture, which could be partially reversed by supplementing media with malate, 2-oxoglutarate, citrate, or pyruvate. The provided data estimates PC expression in human astrocytes and neurons in human brain parenchyma. Furthermore, the enzymatic activity of PC is vital for sustaining the viability of cultured astrocytes.

Genome-Wide CRISPR/Cas9 Library Screening Revealed Dietary Restriction of Glutamine in Combination with Inhibition of Pyruvate Metabolism as Effective Liver Cancer Treatment.

Hepatocellular carcinoma (HCC) is the second most lethal cancer worldwide. Glutamine is an essential, extracellular nutrient which supports HCC growth. Dietary glutamine deficiency may be a potential therapeutic approach for HCC. HCC cells overcome metabolic challenges by rewiring their metabolic pathways for rapid adaptations. The efficiency of dietary glutamine deficiency as HCC treatment is examined and the adaptation machinery under glutamine depletion in HCC cells is unraveled. Using genome-wide CRISPR/Cas9 knockout library screening, this study identifies that pyruvate dehydrogenase α (PDHA), pyruvate dehydrogenase β (PDHB), and pyruvate carboxylase (PC) in pyruvate metabolism are crucial to the adaptation of glutamine depletion in HCC cells. Knockout of either PDHA, PDHB or PC induced metabolic reprogramming of the tricarboxylic acid (TCA) cycle, disrupts mitochondrial function, leading to the suppression of HCC cell proliferation under glutamine depletion. Surprisingly, dietary glutamine restriction improves therapeutic responses of HCC to PDH or PC inhibitor in mouse HCC models. Stable isotope carbon tracing confirms that PDH or PC inhibitors further disrupt the metabolic rewiring of the TCA cycle induced by dietary glutamine depletion in HCC. In summary, the results demonstrate that pyruvate metabolism acts as novel targetable metabolic vulnerabilities for HCC treatment in combination with a glutamine-deficient diet.

Pyruvate carboxylase promotes malignant transformation of papillary thyroid carcinoma and reduces iodine uptake

Previous studies have shown that pyruvate carboxylase (PC) plays a key role in the occurrence and progression of thyroid cancer (TC); however, the relationship between PC and iodine-refractory TC is unclear. Therefore, the present study aimed to investigate the molecular mechanism of PC in the malignant progression and loss of iodine uptake in papillary TC (PTC) and to explore the potential therapeutic effect of PC inhibitors in iodine-refractory PTC. PC increased cell proliferation, invasion, and metastasis, inhibited expression of the iodine metabolism-related genes TSHR, NIS, TPO, and TG, and decreased the iodine-uptake capacity by activating the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in PTC cell lines. Furthermore, the PC inhibitor ZY-444 effectively inhibited the activation of PC, reduced the malignant invasiveness, and restored the expression of iodine metabolism-related genes and the iodine-uptake capacity in PTC cells. These findings suggest that PC activation is involved in the progression of iodine-refractory TC and that PC inhibitors may represent a potentially novel targeted therapy for iodine-refractory TC.

Inhibition of Anaplerosis Prevented Mitochondrial Dysfunction and Vascular Remodeling in Pulmonary Hypertension

IntroductionPulmonary hypertension (PH) is a deadly disease of the lung vasculature. Recent studies reported mitochondrial dysfunction as a significant contributor to PH pathobiology, the prominent type of dysfunction is still unclear. Importantly, partial mitochondrial functioning is necessary to provide the metabolic intermediates for the proliferative growth of vascular cells in PH. Recently, we reported that in PH, the mitochondrial anabolic branch of metabolism ‐ anaplerosis plays a critical role in the upregulation of the tricarboxylic acid cycle in pulmonary vascular cells with mitochondrial dysfunction.HypothesisWe hypothesize that the inhibition of both anaplerosis targets ‐ pyruvate carboxylase (PC) and glutaminolysis ‐ glutamate dehydrogenase 1 (GLUD1) in the early stages of PH could effectively attenuate mitochondrial dysfunction and vascular remodeling.MethodsPH was induced in female Sprague Dawley rats by subcutaneous injection of sugen (50 mg/kg)/hypoxia (10% O2). The treatment groups received sugen/hypoxia (Su/Hx) and two‐weeks intraperitoneal injection of inhibitors (PC inhibitor, phenylacetic acid (PAA), 20 mg/kg/every other day and R162, an inhibitor of GLUD1 (30mg/kg/daily).ResultsHemodynamic analysis of five weeks of Su/Hx rats showed a progressive PH phenotype with increased right ventricular (RV) systolic pressure (111.08 ± 6.87 mmHg). Inhibiting anaplerosis at early stages of PH (2 weeks Su/Hx), decreased the RV hypertrophy (0.5 ± 0.02 to 0.42 ± 0.02, p<0.05), RV systolic pressure (71.88 ± 3.59 mmHg to 54.14 ± 2.48 mmHg, p<0.05), and Max dP/dt (3863.89 ± 180.27 mmHg/s to 2890.63 ± 154.52 mmHg/s, p<0.01). Su/Hx group showed an increased glycolytic flux with hexokinase‐I upregulation (p<0.05), leading to an upregulation of anaplerosis. This was followed by the impairment in mitochondrial function with a reduction of pyruvate dehydrogenase (PDH) expression (p<0.001) and activation of mitochondrial glycerol‐3‐phosphate‐dehydrogenase (GPD2) (p<0.001), an important mediator of the glycerophosphate shuttle. The increased glycolysis also fluxed into the pentose phosphate pathway (PPP) through myo‐inositol oxygenase (MIOX) (p<0.001), a novel source of reactive oxygen species (ROS) generation in PH. These metabolic alterations lead to the upregulation of proliferative signaling pathways Akt (p<0.01), STAT3 (p<0.001), and P38 (p<0.01). Importantly, the inhibition of the anaplerotic pathways by combination (PAA+R162) treatment in the Su/Hx PH model significantly resolved the activation of PPP, restored mitochondrial metabolism, and prevented the proliferative signaling leading to lung vascular remodeling.ConclusionThe results suggest that inhibition of the anaplerotic pathway effectively attenuated mitochondrial dysfunction and vascular remodeling in Su/HX animals. Therefore, our findings indicate that simultaneous targeting of both PC and glutamine‐mediated anaplerosis is a promising therapeutic target for the resolution of vascular remodeling in PH.

Expression of pyruvate carboxylase in cultured human astrocytoma, glioblastoma and neuroblastoma cells.

Pyruvate carboxylase (PC) is an enzyme catalyzing the conversion of pyruvate to oxaloacetate, which possesses anaplerotic role in cellular metabolism. The expression of PC was confirmed in cells of several cancer types, in which it ensures several cellular functions, such as growth and division. To investigate the expression of PC in human astrocytoma, glioblastoma and neuroblastoma cells we applied the immunodetection methods. The results of the Western blot analysis and immunocytochemical detection revealed the presence of PC in human astrocytoma, glioblastoma and neuroblastoma cells. Furthermore, application of PC inhibitor, 3-chloro-1,2-dihydroxypropane (CDP), negatively impacts the viability of astrocytoma cells. The cytotoxic effect of CDP could be partially reversed by application of citrate, 2-oxoglutarate and malate in incubation media. Our results revealed that astrocytoma, glioblastoma and neuroblastoma cells are equipped with PC, which might significantly contribute by its anaplerotic activity to sustain the metabolism of cancer cells.

Inhibition of Anaplerosis Attenuated Vascular Proliferation in Pulmonary Arterial Hypertension

Vascular remodeling is considered a key event in the pathogenesis of pulmonary arterial hypertension (PAH). However, mechanisms of gaining the proliferative phenotype by pulmonary vascular cells are still unresolved. Due to well-established pyruvate dehydrogenase (PDH) deficiency in PAH pathogenesis, we hypothesized that the activation of another branch of pyruvate metabolism, anaplerosis, via pyruvate carboxylase (PC) could be a key contributor to the metabolic reprogramming of the vasculature. In sugen/hypoxic PAH rats, vascular proliferation was found to be accompanied by increased activation of Akt signaling, which upregulated membrane Glut4 translocation and caused upregulation of hexokinase and pyruvate kinase-2, and an overall increase in the glycolytic flux. Decreased PDH activity and upregulation of PC shuttled more pyruvate to oxaloacetate. This results in the anaplerotic reprogramming of lung vascular cells and their subsequent proliferation. Treatment of sugen/hypoxia rats with the PC inhibitor, phenylacetic acid 20 mg/kg, starting after one week from disease induction, significantly attenuated right ventricular systolic pressure, Fulton index, and pulmonary vascular cell proliferation. PC inhibition reduced the glycolytic shift by attenuating Akt-signaling, glycolysis, and restored mitochondrial pyruvate oxidation. Our findings suggest that targeting PC mediated anaplerosis is a potential therapeutic intervention for the resolution of vascular remodeling in PAH.

Evaluation of α-hydroxycinnamic acids as pyruvate carboxylase inhibitors

Through a structure-based drug design project (SBDD), potent small molecule inhibitors of pyruvate carboxylase (PC) have been discovered. A series of α-keto acids (7) and α-hydroxycinnamic acids (8) were prepared and evaluated for inhibition of PC in two assays. The two most potent inhibitors were 3,3′-(1,4-phenylene)bis[2-hydroxy-2-propenoic acid] (8u) and 2-hydroxy-3-(quinoline-2-yl)propenoic acid (8v) with IC50 values of 3.0 ± 1.0 μM and 4.3 ± 1.5 μM respectively. Compound 8v is a competitive inhibitor with respect to pyruvate (Ki = 0.74 μM) and a mixed-type inhibitor with respect to ATP, indicating that it targets the unique carboxyltransferase (CT) domain of PC. Furthermore, compound 8v does not significantly inhibit human carbonic anhydrase II, matrix metalloproteinase-2, malate dehydrogenase or lactate dehydrogenase.

Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-13C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-13C]glucose to [13C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ∼50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.

Kaempferol ameliorates hyperglycemia through suppressing hepatic gluconeogenesis and enhancing hepatic insulin sensitivity in diet-induced obese mice

Obesity-associated insulin resistance (IR) is a major risk factor for developing type 2 diabetes and an array of other metabolic disorders. In particular, hepatic IR contributes to the increase in hepatic glucose production and consequently the development of fasting hyperglycemia. In this study, we explored whether kaempferol, a flavonoid isolated from Gink go biloba, is able to regulate hepatic gluconeogenesis and blood glucose homeostasis in high-fat diet-fed obese mice and further explored the underlying mechanism by which it elicits such effects. Oral administration of kaempferol (50 mg/kg/day), which is the human equivalent dose of 240 mg/day for an average 60 kg human, significantly improved blood glucose control in obese mice, which was associated with reduced hepatic glucose production and improved whole-body insulin sensitivity without altering body weight gain, food consumption or adiposity. In addition, kaempferol treatment increased Akt and hexokinase activity, but decreased pyruvate carboxylase (PC) and glucose-6 phosphatase activity in the liver without altering their protein expression. Consistently, kaempferol decreased PC activity and suppressed gluconeogenesis in HepG2 cells as well as primary hepatocytes isolated from the livers of obese mice. Furthermore, we found that kaempferol is a direct inhibitor of PC. These findings suggest that kaempferol may be a naturally occurring antidiabetic compound that acts by suppressing glucose production and improving insulin sensitivity. Kaempferol suppression of hepatic gluconeogenesis is due to its direct inhibitory action on the enzymatic activity of PC.

Discovery of Novel Small Molecule Inhibitors of Bacterial Pyruvate Carboxylase

Staphylococcus aureus and Listeria monocytogenes are pathogenic bacteria that can cause serious infections for immunocompromised individuals1, 2. Bacterial metabolism has been linked to pathogenicity and, in particular, the metabolic enzyme pyruvate carboxylase (PC) has been shown to be an important contributor to L. monocytogenes virulence1, 2. Small molecules that could inhibit bacterial PC activity would better reveal the role of this enzyme in a wide range of bacterial infections and could have the potential to be developed into antimicrobial agents. However, there are currently no known specific or potent small molecule inhibitors of PC activity3. We aim to find, develop, and characterize novel small molecule inhibitors of PC that will aid in understanding the structure, mechanism, and regulation of this enzyme. PC catalyzes the conversion of pyruvate to oxaloacetate (OAA) and a novel fixed‐time assay has been developed to detect OAA with a diazonium salt, Fast Violet B (FVB). The validated assay has been used to screen specific subsets of rationally designed inhibitors as well as compounds in an unbiased high‐throughput screen. Using both approaches, we have discovered novel small molecule inhibitors of bacterial PC that have the potential to be developed into valuable research tools and future antimicrobial agents.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression

Maintaining reductive-oxidative (redox) balance is an essential feature in breast cancer cell survival, with cellular metabolism playing an integral role in maintaining redox balance through its supply of reduced NADPH. In the present studies, the effect of 1,25-dihydroxyvitamin D (1,25(OH)2D) on redox balance was investigated in early stages of breast cancer. Treatment with 1,25(OH)2D promoted oxidative stress in MCF10A-ras and MCF10A-ErbB2 breast epithelial cells, as measured by the decreased ratios of NADPH/NADP+ and reduced to oxidized glutathione (GSH/GSSG). The mRNA and protein expression of the enzyme pyruvate carboxylase (PC) was downregulated with 1,25(OH)2D treatment, suggesting a potential mechanism. Genetic depletion of PC in MCF10A-ras cells resulted in a decreased ratio of NADPH/NADP+ and GSH/GSSG, with 1,25(OH)2D treatment having no further effect. Mutation analysis confirmed the presence and functionality of a vitamin D response element in the PC gene promoter region. Collectively, these results provide evidence that 1,25(OH)2D promotes oxidative stress in early breast cancer progression through transcriptional downregulation of PC.

Toxicity generated through inhibition of pyruvate carboxylase and carnitine palmitoyl transferase-1 is similar to high glucose/palmitate-induced glucolipotoxicity in INS-1 beta cells

This work was initiated to determine whether toxicity generated through inhibition of mitochondrial fuel metabolism is similar to high glucose/palmitate (HG/PA)-induced glucolipotoxicity. Influx of glucose and free fatty acids into the tricarboxylic acid (TCA) cycle was inhibited by treatment with the pyruvate carboxylase (PC) inhibitor phenylacetic acid (PAA) and carnitine palmitoyl transferase-1 (CPT-1) inhibitor etomoxir (Eto), or knockdown of PC and CPT-1. Treatment of PAA/Eto or knockdown of PC/CPT-1 induced apoptotic death in INS-1 beta cells. Similar to HG/PA treatment, PAA/Eto increased endoplasmic reticulum stress responses but decreased the Akt signal. JNK inhibitor or chemical chaperone was protective against both PAA/Eto- and HG/PA-induced cell death. All attempts to reduce [Ca2+]i, stimulate lipid metabolism, and increase the TCA cycle intermediate pool protected PAA/Eto-induced death as well as HG/PA-induced death. These data suggest that signals induced from impaired mitochondrial fuel metabolism play a critical role in HG/PA-induced glucolipotoxicity.

Activation and Inhibition of Pyruvate Carboxylase from Rhizobium etli

While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg(2+), and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg(2+) assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg(2+) vs pyruvate were nonlinear at low concentrations of Mg(2+) and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg(2+) also resulted in a decrease in the K(a) for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K(i) = 0.026 mM) and pyruvate carboxylation (K(i) = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg(2+) metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg(2+) are described.

Supplement of TCA cycle intermediates protects against high glucose/palmitate-induced INS-1 beta cell death

The aim of this study is to investigate the effect of mitochondrial metabolism on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death. Long-term treatment of INS-1 cells with HG/PA impaired energy-producing metabolism accompanying with depletion of TCA cycle intermediates. Whereas an inhibitor of carnitine palmitoyl transferase 1 augmented HG/PA-induced INS-1 cell death, stimulators of fatty acid oxidation protected the cells against the HG/PA-induced death. Furthermore, whereas mitochondrial pyruvate carboxylase inhibitor phenylacetic acid augmented HG/PA-induced INS-1 cell death, supplementation of TCA cycle metabolites including leucine/glutamine, methyl succinate/α-ketoisocaproic acid, dimethyl malate, and valeric acid or treatment with a glutamate dehydrogenase activator, aminobicyclo-heptane-2-carboxylic acid (BCH), significantly protected the cells against the HG/PA-induced death. In particular, the mitochondrial tricarboxylate carrier inhibitor, benzene tricarboxylate (BTA), also showed a strong protective effect on the HG/PA-induced INS-1 cell death. Knockdown of glutamate dehydrogenase or tricarboxylate carrier augmented or reduced the HG/PA-induced INS-1 cell death, respectively. Both BCH and BTA restored HG/PA-induced reduction of energy metabolism as well as depletion of TCA intermediates. These data suggest that depletion of the TCA cycle intermediate pool and impaired energy-producing metabolism may play a role in HG/PA-induced cytotoxicity to beta cells and thus, HG/PA-induced beta cell glucolipotoxicity can be protected by nutritional or pharmacological maneuver enhancing anaplerosis or reducing cataplerosis.

Inhibitors of Pyruvate Carboxylase~!2009-12-01~!2010-02-01~!2010-04-15~!

This review aims to discuss the varied types of inhibitors of biotin-dependent carboxylases, with an emphasis on the inhibitors of pyruvate carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of pyruvate carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin - biotin, FTP - ATP binding site, oxamate - pyruvate binding site, phosphonoacetate - binding site of the putative carboxyphosphate intermediate.

Substrate overload: Glucose oxidation in human myotubes conquers palmitate oxidation through anaplerosis

To date, two cardinal principles govern oxidation of glucose and fatty acids in skeletal muscle; exogenous fatty acid reduces glucose oxidation and glucose reduces fatty acid oxidation. Both glucose and palmitate (PA) oxidation was increased by increasing their concentration and inhibited by increasing concentrations of the other in human myotubes established from healthy, lean subjects exposed to acute stepwise increases in glucose and PA levels. At high substrate levels; PA oxidation was reduced while release of acid soluble metabolites was increased and, both glucose oxidation and release of citrate was increased which could be abolished by phenylacetic acid (inhibitor of pyruvate carboxylase (PC)). The present data challenges above preconceptions. Although they operate at low-moderate substrate levels additional two principles determine substrate oxidation at higher substrate concentrations; first, anaplerosis of the tricarboxylic cycle through PC promoting complete and incomplete glucose oxidation; second, inhibition of complete PA oxidation with increasing incomplete PA oxidation mediated by high glucose and PA levels, respectively.

Tight Coupling between Glucose and Mitochondrial Metabolism in Clonal β-Cells Is Required for Robust Insulin Secretion

The biochemical mechanisms underlying glucose-stimulated insulin secretion from pancreatic beta-cells are not completely understood. To identify metabolic disturbances in beta-cells that impair glucose-stimulated insulin secretion, we compared two INS-1-derived clonal beta-cell lines, which are glucose-responsive (832/13 cells) or glucose-unresponsive (832/2 cells). To this end, we analyzed a number of parameters in glycolytic and mitochondrial metabolism, including mRNA expression of genes involved in cellular energy metabolism. We found that despite a marked impairment of glucose-stimulated insulin secretion, 832/2 cells exhibited a higher rate of glycolysis. Still, no glucose-induced increases in respiratory rate, ATP production, or respiratory chain complex I, III, and IV activities were seen in the 832/2 cells. Instead, 832/2 cells, which expressed lactate dehydrogenase A, released lactate regardless of ambient glucose concentrations. In contrast, the glucose-responsive 832/13 line lacked lactate dehydrogenase and did not produce lactate. Accordingly, in 832/2 cells mRNA expression of genes for glycolytic enzymes were up-regulated, whereas mitochondria-related genes were down-regulated. This could account for a Warburg-like effect in the 832/2 cell clone, lacking in 832/13 cells as well as primary beta-cells. In human islets, mRNA expression of genes such as lactate dehydrogenase A and hexokinase I correlated positively with HbA(1c) levels, reflecting perturbed long term glucose homeostasis, whereas that of Slc2a2 (glucose transporter 2) correlated negatively with HbA(1c) and thus better metabolic control. We conclude that tight metabolic regulation enhancing mitochondrial metabolism and restricting glycolysis in 832/13 cells is required for clonal beta-cells to secrete insulin robustly in response to glucose. Moreover, a similar expression pattern of genes controlling glycolytic and mitochondrial metabolism in clonal beta-cells and human islets was observed, suggesting that a similar prioritization of mitochondrial metabolism is required in healthy human beta-cells. The 832 beta-cell lines may be helpful tools to resolve metabolic perturbations occurring in Type 2 diabetes.

Anaplerosis via pyruvate carboxylase is required for the fuel-induced rise in the ATP:ADP ratio in rat pancreatic islets

The molecular mechanisms of insulin release are only partially known. Among putative factors for coupling glucose metabolism to insulin secretion, anaplerosis has lately received strong support. The anaplerotic enzyme pyruvate carboxylase is highly expressed in beta cells, and anaplerosis influences insulin secretion in beta cells. By inhibiting pyruvate carboxylase in rat islets, we aimed to clarify the hitherto unknown metabolic events underlying anaplerotic regulation of insulin secretion. Phenylacetic acid (5 mmol/l) was used to inhibit pyruvate carboxylase in isolated rat islets, which were then assessed for insulin secretion, fuel oxidation, ATP:ADP ratio, respiration, mitochondrial membrane potential, exocytosis and ATP-sensitive K(+) channel (K(ATP)-channel) conductance. We found that the glucose-provoked rise in ATP:ADP ratio was suppressed by inhibition of pyruvate carboxylase. In contrast, fuel oxidation, respiration and mitochondrial membrane potential, as well as Ca(2+)-induced exocytosis and K(ATP)-channel conductance in single cells, were unaffected. Insulin secretion induced by alpha-ketoisocaproic acid was suppressed, whereas methyl-succinate-stimulated secretion remained unchanged. Perifusion of rat islets revealed that inhibition of anaplerosis decreased both the second phase of insulin secretion, during which K(ATP)-independent actions of fuel secretagogues are operational, as well as the first and K(ATP)-dependent phase. Our results are consistent with the concept that anaplerosis via pyruvate carboxylase determines pyruvate cycling, which has previously been shown to correlate with glucose responsiveness in clonal beta cells. These processes, controlled by pyruvate carboxylase, seem crucial for generation of an appropriate ATP:ADP ratio, which may regulate both phases of fuel-induced insulin secretion.