Regulation Of Pyruvate Kinase Research Articles

Purification and Regulation of Pyruvate Kinase from the Foot Muscle of the Anoxia and Freeze Tolerant Marine Snail, Littorina littorea.

The intertidal marine snail, Littorina littorea, has evolved to survive bouts of anoxia and extracellular freezing brought about by changing tides and subsequent exposure to harsh environmental conditions. Survival in these anoxic conditions depends on the animals entering a state of metabolic rate depression in order to maintain an appropriate energy production-consumption balance during periods of limited oxygen availability. This study investigated the kinetic, physical, and regulatory properties of pyruvate kinase (PK), which catalyzes the final reaction of aerobic glycolysis, from foot muscle of L. littorea to determine if the enzyme is differentially regulated in response to anoxia and freezing exposure. PK purified from foot muscle of anoxic animals exhibited a lower affinity for its substrate phosphoenolpyruvate than PK from control and frozen animals. PK from anoxic animals was also more sensitive to a number of allosteric regulators, including alanine and aspartate, which are key anaerobic metabolites in L. littorea. Furthermore, PK purified from anoxic and frozen animals exhibited greater stability compared to the non-stressed control animals, determined through high-temperature incubation studies. Phosphorylation of threonine and tyrosine residues was also assessed and demonstrated that levels of threonine phosphorylation of PK from anoxic animals were significantly higher than those of PK from control and frozen animals, suggesting a potential mechanism for regulating PK activity. Taken together, these results suggest that PK plays a role in suppressing metabolic rate in these animals during environmental anoxia exposure.

Characterizing the regulation of pyruvate kinase in response to hibernation in ground squirrel liver (Urocitellus richardsonii).

The Richardson's ground squirrel (Urocitellus richardsonii) undergoes numerous changes to its core physiological and metabolic processes over the months it spends hibernating during the winter. Winter torpor is characterized by an overall reduction in metabolic rate, a lowering of core body temperature, and a switch to preferential consumption of lipids instead of carbohydrates. The alterations in central metabolic pathways are often accomplished by the regulation of key enzymes within the glycolytic pathway. The regulation of one such enzyme, pyruvate kinase (PK), was characterized in the present study in the liver of torpid ground squirrels. PK was purified from liver tissue of euthermic and hibernating U. richardsonii and subsequently assayed to determine the kinetic parameters of the enzyme at 22° and 5°C. Additional studies assessed the relative degree of post-translational modifications in PK from control and hibernating ground squirrels. The results from this study demonstrated significantly lowered maximal activity in the hibernating form of the enzyme and decreased sensitivity to the activator FBP when compared to the control. Immunoblotting demonstrated increased relative serine and threonine phosphorylation (~3 fold) in the hibernating PK. Taken together these results suggest that phosphorylation of liver PK is an important step in inhibiting glycolytic activity in the liver of the Richardson's ground squirrel during torpor.

The Absence of Pyruvate Kinase Affects Glucose-Dependent Carbon Catabolite Repression in Bacillus subtilis.

Pyruvate is a key intermediate of diverse metabolic pathways of central carbon metabolism. In addition to being the end product of glycolysis, pyruvate is an essential carbon distribution point to oxidative metabolism, amino acid and fatty acid syntheses, and overflow metabolite production. Hence, a tight regulation of pyruvate kinase (Pyk) activity is of great importance. This study aimed to analyze targeted metabolites from several pathways and possible changes in Bacillus subtilis lacking Pyk. Wild type and Δpyk cells were cultivated in chemically defined medium with glucose and pyruvate as carbon sources, and the extracted metabolites were analyzed by 1H-NMR, GC-MS, HPLC-MS, and LC-MS/MS. The results showed that the perturbation created in the pyruvate node drove an adaptation to new conditions by altering the nutritional compounds’ consumption. In Δpyk, pyruvate, which is subject to glucose-dependent carbon catabolite repression, did not comply with the hierarchy in carbon source utilization. Other metabolic alterations were observed such as the higher secretion of the overflow metabolites acetoin and 2,3-butanediol by Δpyk. Our results help to elucidate the regulatory transport of glucose and pyruvate in B. subtilis and possible metabolic reroute to alternative pathways in the absence of Pyk.

Nutritional regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase at the enzymatic and molecular levels in cobia Rachycentron canadum.

Despite being a carnivorous fish species, cobia (Rachycentron canadum) can utilize high levels of dietary carbohydrate (up to 360gkg-1). By contrast, rainbow trout (also carnivorous) cannot, due to the absence of molecular induction of glycolytic enzyme and inhibition of gluconeogenic enzyme gene expressions such as pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK). We hypothesized that this phenomenon is species-specific and will not be observed in cobia. Our results show that, at the molecular level, the mRNA abundance of the important glycolytic (PK) and gluconeogenic (PEPCK) enzymes in cobia liver are regulated by dietary carbohydrate-to-lipid (CHO:L) ratios and nutritional status (fed, unfed, and refed). Significantly upregulated hepatic PK and depressed PEPCK gene expressions were observed when the fish were fed with an increasing CHO/L-ratio diet or were refed. However, in contrast to gene expression, there was no significant effect of dietary CHO/L ratios on PK enzyme activity. The decrease in PEPCK activity was significantly found between low CHO/L ratio and high CHO/L ratio diets, whereas the moderate CHO/L ratio group showed intermediate values. But PEPCK activity appeared to be independent of nutritional status. These results suggest that nutritional regulation is obvious, at least at the molecular level, in the key hepatic enzymes (PK and PEPCK) of the glucose metabolism pathway, in response to different dietary CHO/L ratios and to the transition from being starved to fed. Determining whether other key enzymes involved in hepatic glucose metabolism contribute to glucose tolerance in cobia is necessary for further investigation of this phenomenon at the enzymatic and molecular levels.

Allosteric regulation of pyruvate kinase from Mycobacterium tuberculosis by metabolites

Mycobacterium tuberculosis (Mtb) causes both acute tuberculosis and latent, symptom-free infection that affects roughly one-third of the world's population. It is a globally important pathogen that poses multiple dangers. Mtb reprograms its metabolism in response to the host niche, and this adaptation contributes to its pathogenicity. Knowledge of the metabolic regulation mechanisms in Mtb is still limited. Pyruvate kinase, involved in the late stage of glycolysis, helps link various metabolic routes together. Here, we demonstrate that Mtb pyruvate kinase (Mtb PYK) predominantly catalyzes the reaction leading to the production of pyruvate, but its activity is influenced by multiple metabolites from closely interlinked pathways that act as allosteric regulators (activators and inhibitors). We identified allosteric activators and inhibitors of Mtb PYK originating from glycolysis, citrate cycle, nucleotide/nucleoside inter-conversion related pathways that had not been described so far. Enzyme was found to be activated by fructose-1,6-bisphosphate, ribose-5-phosphate, adenine, adenosine, hypoxanthine, inosine, L-2-phosphoglycerate, l-aspartate, glycerol-2-phosphate, glycerol-3-phosphate. On the other hand thiamine pyrophosphate, glyceraldehyde-3-phosphate and L-malate were identified as inhibitors of Mtb PYK. The detailed kinetic analysis indicated a morpheein model of Mtb PYK allosteric control which is strictly dependent on Mg2+ and substantially increased by the co-presence of Mg2+ and K+.

A Peptide Encoded by a Putative lncRNA HOXB-AS3 Suppresses Colon Cancer Growth

A substantial fraction of eukaryotic transcripts areconsidered long non-coding RNAs (lncRNAs), which regulate various hallmarks of cancer. Here, we discovered that the lncRNA HOXB-AS3 encodes a conserved 53-aa peptide. The HOXB-AS3 peptide, not lncRNA, suppresses colon cancer (CRC) growth. Mechanistically, the HOXB-AS3 peptide competitively binds to the ariginine residues in RGG motif of hnRNP A1 and antagonizes the hnRNP A1-mediated regulation of pyruvate kinase M (PKM) splicing by blocking the binding of the ariginine residues in RGG motif of hnRNP A1 to the sequences flanking PKM exon 9, ensuring the formation of lower PKM2 and suppressing glucose metabolism reprogramming. CRC patients with low levels of HOXB-AS3 peptide have poorer prognoses. Our study indicates that the loss of HOXB-AS3 peptide is a critical oncogenic event in CRC metabolic reprogramming. Our findings uncover a complex regulatory mechanism of cancer metabolism reprogramming orchestrated by a peptide encoded by an lncRNA.

Purification and characterization of skeletal muscle pyruvate kinase from the hibernating ground squirrel, Urocitellus richardsonii: potential regulation by posttranslational modification during torpor.

Ground squirrel torpor during winter hibernation is characterized by numerous physiological and biochemical changes, including alterations to fuel metabolism. During torpor, many tissues switch from carbohydrate to lipid catabolism, often by regulating key enzymes within glycolytic and lipolytic pathways. This study investigates the potential regulation of pyruvate kinase (PK), a key member of the glycolytic pathway, within the skeletal muscle of hibernating ground squirrels. PK was purified from the skeletal muscle of control and torpid Richardson's ground squirrels, and PK kinetics, structural stability, and posttranslational modifications were subsequently assessed. Torpid PK displayed a nearly threefold increase in K m PEP as compared to control PK when assayed at 5°C. ProQ Diamond phosphoprotein staining as well as phospho-specific western blots indicated that torpid PK was significantly more phosphorylated than the euthermic control. PK from the torpid condition was also shown to possess nearly twofold acetyl content as compared to control PK. In conclusion, skeletal muscle PK from the Richardson's ground squirrel may be regulated posttranslationally between the euthermic and torpid states, and this may inhibit PK functioning during torpor in accordance with the decrease in glycolytic rate during dormancy.

Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica

The wood frog (Rana sylvatica) can survive the winter in a frozen state, in which the frog’s tissues are also exposed to dehydration, ischemia, and anoxia. Critical to wood frog survival under these conditions is a global metabolic rate depression, the accumulation of glucose as a cryoprotectant, and a reliance on anaerobic glycolysis for energy production. Pyruvate kinase (PK) catalyzes the final reaction of aerobic glycolysis, generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. This study investigated the effect of each stress condition experienced by R. sylvatica during freezing, including dehydration and anoxia, on PK regulation. PK from muscle of frozen and dehydrated frogs exhibited a lower affinity for PEP (Km = 0.098 ± 0.003 and Km = 0.092 ± 0.008) than PK from control and anoxic conditions (Km = 0.065 ± 0.003 and Km = 0.073 ± 0.002). Immunoblotting showed greater serine phosphorylation on muscle PK from frozen and dehydrated frogs relative to control and anoxic states, suggesting a reversible phosphorylation regulatory mechanism for PK activity during freezing stress. Furthermore, PK from frozen animals exhibited greater stability under thermal and urea-induced denaturing conditions than PK from control animals. Phosphorylation of PK during freezing may contribute to mediating energy conservation and maintaining intracellular cryoprotectant levels, as well as increase enzyme stability during stress.

Regulation of pyruvate kinase from muscle of Freeze tolerant wood frog, Rana sylvatica

Regulation of pyruvate kinase from muscle of Freeze tolerant wood frog, Rana sylvatica

WITHDRAWN: Regulation of Pyruvate Kinase M Switch towards PKM1 by LKB1-AMPK Axis Tolerates Hypoglycemic Stress in Cancer Cells.

This article has been withdrawn by the authors. The PKM2 immunoblot in Fig 2E was reused as part of the Caspase-3 immunoblot in Fig 9C. The PKM2 immunoblot from 5 mM Glu, fractions 1-10 was reused as the PKM2 immunoblot from 1 mM Glu, fractions 1-10. The actin immunoblot from A549 cells from Fig 5A was reused as the actin blot from Fig 7C.

Transcript abundance, enzyme activity and metabolite concentration regulates differential carboxylate efflux in soybean under low phosphorus stress

Low phosphorus (P) availability in soil owing to adsorption, precipitation and conversion of organic forms drastically limits plant growth and yield. Among several strategies adopted by plants to cope with P starvation, research on root carboxylate exudation to release fixed P in soil is gaining impetus. Since the rate of exudation is a function of carboxylate synthesis and expression of efflux transporters, our objective was to analyse the expression and activity of genes influencing carboxylate exudation in soybean and the effect of P starvation on biomass and P acquisition efficiency. The relative expression levels and enzyme activity of phosphoenolpyruvate carboxylase, malate dehydrogenase and citrate synthase in soybean roots increased during low-P stress, particularly in genotypes exhibiting higher carboxylate efflux. With the exception of two genotypes (G-2656 and HIMSO-1521), the relative expression of efflux transporters for malate and citrate did not correspond to carboxylate exudation. Carboxylates in root apices were higher in NRC-7, HIMSO-1521 and EC-232019 under low-P as compared to sufficient P. P-efficient soybean genotypes (EC-232019 and HIMSO-1521) exhibiting higher carboxylate exudation showed better P acquisition and biomass under low-P stress. Down regulation of pyruvate kinase at the transcript- and enzyme-level was observed under low-P stress in P-efficient genotypes suggesting an effective glycolytic bypass mechanism to facilitate continued carbon flux from glycolytic pathway to the tricarboxylic acid cycle. Hence, transcript abundance, enzyme activity and internal carboxylate concentration in the root tissues govern carboxylate efflux in soybean, in turn, influencing biomass and P uptake.

Correction: Organism-Adapted Specificity of the Allosteric Regulation of Pyruvate Kinase in Lactic Acid Bacteria

Correction: Organism-Adapted Specificity of the Allosteric Regulation of Pyruvate Kinase in Lactic Acid Bacteria

Phosphorylation of pyruvate kinase A by protein kinase J leads to the altered growth and differential rate of intracellular survival of mycobacteria.

PknJ (Rv2088) is a serine/threonine protein kinase of mycobacteria which is present in Mycobacterium tuberculosis (MTB), but its gene is absent in Mycobacterium smegmatis (MS); a fast grower and nonpathogenic species of mycobacteria. The heterologous expression of MTB-specific PknJ in MS altered the growth of recombinant mycobacteria highlighting one of the characteristics of this protein. This nature of the protein was further confirmed when Mycobacterium bovis BCG (BCG) containing antisense copy of pknJ resulted in the increased growth of BCG. The real-time RNA quantification analysis pointed out toward increased expression of this protein during infection of THP-1 macrophage cells which further emphasized that the protein is essential for the intracellular survival of mycobacteria. The differential in gel electrophoresis (DIGE) data followed by mass spectroscopy suggested that PknJ is involved in regulation of pyruvate kinase A (Rv1617). Since pyruvate kinase (PK) A is one of the key enzymes which controls glycolytic cycle in mycobacteria, we looked for its interaction with PknJ during extracellular and intracellular growth of mycobacteria. In order to identify the specific residue(s) involved in post-translational modification, the phospho-null mutants of PK were generated, and their substrate specificities in response to PknJ were assessed through kinase assay. The findings thus underlined that the PK activity is predominantly dependent on the threonine residue at the 94(th) position and further suggested that this site may be plausible in intracellular survival of mycobacteria upon phosphorylation with PknJ.

Changes in intracellular metabolite pools during growth of adherent MDCK cells in two different media

In bioprocess engineering, the growth of continuous cell lines is mainly studied with respect to the changes in cell concentration, the resulting demand for substrates, and the accumulation of extracellular metabolites. The underlying metabolic process rests upon intracellular metabolite pools and their interaction with enzymes in the form of substrates, products, or allosteric effectors. Here, we quantitatively analyze time courses of 29 intracellular metabolites of adherent Madin-Darby canine kidney cells during cultivation in a serum-containing medium and a serum-free medium. The cells, which originated from the same pre-culture, showed similar overall growth behavior and only slight differences in their demand for the substrates glucose (GLC), glutamine (GLN), and glutamate (GLU). Analysis of intracellular metabolites, which mainly cover the glycolytic pathway, the citric acid cycle, and the nucleotide pools, revealed surprisingly similar dynamics for both cultivation conditions. Instead of a strong influence of the medium, we rather observed a growth phase-specific behavior in glycolysis and in the lower citric acid cycle. Furthermore, analysis of the lower part of glycolysis suggests the well-known regulation of pyruvate kinase by fructose 1,6-bisphosphate. The upper citric acid cycle (citrate, cis-aconitate, and isocitrate) is apparently uncoupled from the lower part (α-ketoglutarate, succinate, fumarate, and malate), which is in line with the characteristics of a truncated cycle. Decreased adenosine triphosphate and guanosine triphosphate pools, as well as a relatively low energy charge soon after inoculation of cells, indicate a high demand for cellular energy and the consumption of nucleotides for biosynthesis. We finally conclude that, with sufficient availability of substrates, the dynamics of GLC and GLN/GLU metabolism is influenced mainly by the cellular growth regime and regulatory function of key enzymes.

3-Phosphoglycerate Is an Allosteric Activator of Pyruvate Kinase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum

Pyruvate kinase (PK) is a highly regulated enzyme that catalyzes the final step of glycolysis. PK from the hyperthermophilic archaeon Pyrobaculum aerophilum (PaPK) is distinguished from most PK enzymes of eukarya and bacteria by not responding to any known allosteric effectors and apparently exhibiting only cooperative regulation. We determined the crystal structure of PaPK to 2.2 Å resolution and, in a manner consistent with the lack of a response to conventional effectors, observed that the canonical allosteric site is occluded by a tyrosine. Unexpectedly, though, a bound sulfate was observed at a position equivalent to the 6'-phosphate of sugar effectors, suggesting an allosteric site, but for an unknown effector and sharing only the phosphate position. A search of three-carbon intermediates of glycolysis revealed 3-phosphoglycerate (3PG) as a potent allosteric activator of PaPK. The response was abolished by mutation of residues that contact the sulfate and of an arginine proposed to interact with the 3PG carboxylate group. Regulation of PK by 3PG is consistent with the ancestral glycolysis of hyperthermophilic archaea in which this intermediate is produced by an irreversible enzyme, glyceraldehyde 3-phosphate ferredoxin oxidoreductase. Coordinated regulation within the lower half of glycolysis contrasts sharply with conventional glycolysis in which 3PG is produced reversibly and PK is regulated by fructose 1,6-bisphosphate, the product of phosphofructokinase, an irreversible enzyme in the upper half of the pathway. Regulation of PaPK by a carboxylate molecule rather than a sugar phosphate may reflect a step in the evolution of glycolysis that predates the dominance of sugars in metabolism.

Organism-adapted specificity of the allosteric regulation of pyruvate kinase in lactic acid bacteria.

Pyruvate kinase (PYK) is a critical allosterically regulated enzyme that links glycolysis, the primary energy metabolism, to cellular metabolism. Lactic acid bacteria rely almost exclusively on glycolysis for their energy production under anaerobic conditions, which reinforces the key role of PYK in their metabolism. These organisms are closely related, but have adapted to a huge variety of native environments. They include food-fermenting organisms, important symbionts in the human gut, and antibiotic-resistant pathogens. In contrast to the rather conserved inhibition of PYK by inorganic phosphate, the activation of PYK shows high variability in the type of activating compound between different lactic acid bacteria. System-wide comparative studies of the metabolism of lactic acid bacteria are required to understand the reasons for the diversity of these closely related microorganisms. These require knowledge of the identities of the enzyme modifiers. Here, we predict potential allosteric activators of PYKs from three lactic acid bacteria which are adapted to different native environments. We used protein structure-based molecular modeling and enzyme kinetic modeling to predict and validate potential activators of PYK. Specifically, we compared the electrostatic potential and the binding of phosphate moieties at the allosteric binding sites, and predicted potential allosteric activators by docking. We then made a kinetic model of Lactococcus lactis PYK to relate the activator predictions to the intracellular sugar-phosphate conditions in lactic acid bacteria. This strategy enabled us to predict fructose 1,6-bisphosphate as the sole activator of the Enterococcus faecalis PYK, and to predict that the PYKs from Streptococcus pyogenes and Lactobacillus plantarum show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role in vivo. These differences in the specificity of allosteric activation may reflect adaptation to different environments with different concentrations of activating compounds. The combined computational approach employed can readily be applied to other enzymes.

Cellular Bioenergetics in Lymphoid Neoplasia

Abstract SCI-25Many cancer cells metabolize glucose by aerobic glycolysis, a phenomenon characterized by increased glycolysis with lactate production and decreased oxidative phosphorylation. We have argued that alterations in cell metabolism associated with cancer may be selected by cancer cells to meet the distinct metabolic needs of proliferation. Unlike metabolism in differentiated cells, which is geared toward efficient ATP generation, the metabolism in cancer cells must be adapted to facilitate the accumulation of biomass. Cancer cells divert a larger fraction of their nutrient metabolism to pathways other than mitochondrial respiration regardless of oxygen availability. Nevertheless, oxygen levels still influence how nutrients are metabolized. We have found that the source of carbon used in various anabolic processes varies based on oxygen levels. Furthermore, the enzymes used to metabolize nutrients can also differ based on the cellular context. This includes regulation of isocitrate dehydrogenase, an enzyme that is mutated in some cancers. There is also strong selection for use of the M2 isoform of pyruvate kinase (PK-M2) to metabolize glucose in cancer cell lines. However, evidence from mouse models suggests that PK-M2 is dispensable for glucose metabolism by many tumors in vivo, suggesting an alternate pathway to convert phosphoenolpyruvate to pyruvate can be used to metabolize glucose. This regulation of pyruvate kinase also plays an important role in hematopoietic stem cell biology. Together, these findings argue that distinct metabolic phenotypes exist among proliferating cells, and both environmental and genetic factors influence how metabolism is regulated to support cell growth. Disclosures:Vander Heiden:Agios Pharmaceuticals: Consultancy, Equity Ownership.

Exon-centric regulation of pyruvate kinase M alternative splicing via mutually exclusive exons

Alternative splicing of the pyruvate kinase M gene (PK-M) can generate the M2 isoform and promote aerobic glycolysis and tumor growth. However, the cancer-specific alternative splicing regulation of PK-M is not completely understood. Here, we demonstrate that PK-M is regulated by reciprocal effects on the mutually exclusive exons 9 and 10, such that exon 9 is repressed and exon 10 is activated in cancer cells. Strikingly, exonic, rather than intronic, cis-elements are key determinants of PK-M splicing isoform ratios. Using a systematic sub-exonic duplication approach, we identify a potent exonic splicing enhancer in exon 10, which differs from its homologous counterpart in exon 9 by only two nucleotides. We identify SRSF3 as one of the cognate factors, and show that this serine/arginine-rich protein activates exon 10 and mediates changes in glucose metabolism. These findings provide mechanistic insights into the complex regulation of alternative splicing of a key regulator of the Warburg effect, and also have implications for other genes with a similar pattern of alternative splicing.

Acetylation Targets the M2 Isoform of Pyruvate Kinase for Degradation through Chaperone-Mediated Autophagy and Promotes Tumor Growth

Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between lysine acetylation and CMA.

Insulinotropic effect of cinnamaldehyde on transcriptional regulation of pyruvate kinase, phosphoenolpyruvate carboxykinase, and GLUT4 translocation in experimental diabetic rats

Diabetes mellitus is a chronic metabolic disorder affecting about 6% of population worldwide with its complications and is rapidly reaching epidemic scale. Cinnamomum zeylanicum is widely used in alternative system of medicine for treatment of diabetes. In the present study, we have performed bioassay guided fractionation of chloroform extract of C. zeylaniucm and identified cinnamaldehyde (CND) as an active principle against diabetes. In continuation to it, a detailed study was undertaken to elucidate its mode of antidiabetic action in STZ induced diabetic rats. Oral administration of CND (20 mg/kg bw) to diabetic rats for 2 months showed significant improvement ( p < 0.001) in muscle and hepatic glycogen content. In vitro incubation of pancreatic islets with CND enhanced the insulin release compared to glibenclamide. The insulinotropic effect of CND was found to increase the glucose uptake through glucose transporter (GLUT4) translocation in peripheral tissues. The treatment also showed a significant improvement in altered enzyme activities of pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) and their mRNA expression levels. Furthermore, the median lethal dose (LD 50) of CND could not be obtained even at 20 times (0.4 g/kg bw) of its effective dose. With the high margin of safety of CND, it can be developed as a potential therapeutic candidate for the treatment of diabetes.