Modulation of pyruvate dehydrogenase kinase activity in cultured hepatocytes by glucagon and n-octanoate.
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ABSTRACT:
SUBMITTER: Fatania HR
PROVIDER: S-EPMC1146552 | biostudies-other | 1986 Feb
REPOSITORIES: biostudies-other
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Project description:The activities of pyruvate dehydrogenase (PDH) kinase and of PDH kinase activator protein (KAP) were increased 2-2.4-fold during 25 h of culture of hepatocytes from fed rats with glucagon plus n-octanoate. PDH kinase activity in hepatocytes from starved rats (initially 2.2 x fed control) fell during 25 h of culture in medium 199 (to 1.5 x fed control), but was maintained by glucagon plus octanoate. Dibutyryl or 8-bromo cyclic AMP increased PDH kinase activity 2-2.2-fold in hepatocytes from fed rats, but phenylephrine and isoproterenol (isoprenaline) were without effect. Insulin blocked the action of glucagon to increase PDH kinase activity and decreased the effect of octanoate and octanoate plus glucagon. It is suggested that the effects of starvation to increase activities of PDH kinase and of KAP in liver are mediated by alterations in circulating concentrations of glucagon, fatty acids and insulin and in hepatic cyclic AMP.
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Project description:Most cancer cells primarily produce their energy through a high rate of glycolysis followed by lactic acid fermentation even in the presence of abundant oxygen. Pyruvate dehydrogenase kinase (PDK) 1, an enzyme responsible for aerobic glycolysis via phosphorylating and inactivating pyruvate dehydrogenase (PDH) complex, is commonly overexpressed in tumors and recognized as a therapeutic target in colorectal cancer. Hemistepsin A (HsA) is a sesquiterpene lactone isolated from Hemistepta lyrata Bunge (Compositae). Here, we report that HsA is a PDK1 inhibitor can reduce the growth of colorectal cancer and consequent activation of mitochondrial ROS-dependent apoptotic pathway both in vivo and in vitro. Computational simulation and biochemical assays showed that HsA directly binds to the lipoamide-binding site of PDK1, and subsequently inhibits the interaction of PDK1 with the E2 subunit of PDH complex. As a result of PDK1 inhibition, lactate production was decreased, but oxygen consumption was increased. Mitochondrial ROS levels and mitochondrial damage were also increased. Consistent with these observations, the apoptosis of colorectal cancer cells was promoted by HsA with enhanced activation of caspase-3 and -9. These results suggested that HsA might be a potential candidate for developing a novel anti-cancer drug through suppressing cancer metabolism.
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Project description:Mitochondrial pyruvate dehydrogenase kinase 2 (PDHK2) phosphorylates the pyruvate dehydrogenase multienzyme complex (PDC) and thereby controls the rate of oxidative decarboxylation of pyruvate. The activity of PDHK2 is regulated by a variety of metabolites such as pyruvate, NAD (+), NADH, CoA, and acetyl-CoA. The inhibitory effect of pyruvate occurs through the unique binding site, which is specific for pyruvate and its synthetic analogue dichloroacetate (DCA). The effects of NAD (+), NADH, CoA, and acetyl-CoA are mediated by the binding site that recognizes the inner lipoyl-bearing domain (L2) of the dihydrolipoyl transacetylase (E2). Both allosteric sites are separated from the active site of PDHK2 by more than 20 A. Here we show that mutations of three amino acid residues located in the vicinity of the active site of PDHK2 (R250, T302, and Y320) make the kinase resistant to the inhibitory effect of DCA, thereby uncoupling the active site from the allosteric site. In addition, we provide evidence that substitutions of R250 and T302 can partially or completely uncouple the L2-binding site. Based on the available structural data, R250, T302, and Y320 stabilize the "open" and "closed" conformations of the built-in lid that controls the access of a nucleotide into the nucleotide-binding cavity. This strongly suggests that the mobility of ATP lid is central to the allosteric regulation of PDHK2 activity serving as a conformational switch required for communication between the active site and allosteric sites in the kinase molecule.
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Project description:The pyruvate dehydrogenase complex (PDC) is a multienzyme assembly that converts pyruvate to acetyl-CoA. As pyruvate and acetyl-CoA play central roles in cellular metabolism, understanding PDC regulation is pivotal to understanding the larger metabolic network. The activity of mammalian PDC is regulated through reversible phosphorylation governed by at least four isozymes of pyruvate dehydrogenase kinase (PDK). Deciphering which kinase regulates PDC in organisms at specific times or places has been challenging. In this study, we analyzed mouse strains carrying targeted mutations of individual isozymes to explore their role in regulating PDC activity. Analysis of protein content of PDK isozymes in major metabolic tissues revealed that PDK1 and PDK2 were ubiquitously expressed, whereas PDK3 and PDK4 displayed a rather limited tissue distribution. Measurement of kinase activity showed that PDK1 is the principal isozyme regulating hepatic PDC. PDK2 was largely responsible for inactivation of PDC in tissues of muscle origin and brown adipose tissue (BAT). PDK3 was the principal kinase regulating pyruvate dehydrogenase activity in kidney and brain. In a well-fed state, the tissue levels of PDK4 protein were fairly low. In most tissues tested, PDK4 ablation had little effect on the overall rates of inactivation of PDC in kinase reaction. Taken together, these data strongly suggest that the activity of PDC is regulated by different isozymes in different tissues. Furthermore, it appears that the overall flux through PDC in a given tissue largely reflects the properties of the PDK isozyme that is principally responsible for the regulation of PDC activity in that tissue.
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Project description:Hepatocytes were isolated by collagenase perfusion of livers from rats that had been allowed access to a carbohydrate-rich diet or laboratory chow or had been deprived of food 48h before use. By incubation with l-[4,5-(3)H]leucine and precipitation with anti-(L-type pyruvate kinase) sera the rates of synthesis and degradation of L-type pyruvate kinase were measured in freshly prepared cells and hepatocytes maintained in monolayer culture for up to 5 days. Hepatocytes from carbohydrate-rich-diet-fed rats synthesized more L-type pyruvate kinase than did cells from chow-fed animals, which in turn synthesized more than cells from 48h-starved rats. Hepatocytes maintained in culture for up to 5 days synthesized L-type pyruvate kinase at similar rates to freshly prepared cells. The degradation of [(3)H]leucine-labelled L-type pyruvate kinase was shown to be biphasic. A phase with t((1/2)) (half-time) 4.9h and a duration of 8-10h was followed by a phase with t((1/2)) 79.2h. Cells from chow-fed and carbohydrate-rich-diet-fed rats showed similar patterns of degradation of L-type pyruvate kinase. The addition of 2mm-fructose and 0.1mum-insulin to the culture medium increased the t((1/2)) of the rapid phase to 12h in cells isolated from carbohydrate-rich-diet-fed rats, but not in cells from chow-fed rats. The secondary, slower, phase of degradation remained unaffected. The degradation of fructose 1,6-bisphosphatase and total cell protein followed first-order kinetics. The half-life of fructose 1,6-bisphosphatase was 41.0h in cells from chow-fed animals and 48.5h in cells from carbohydrate-rich-diet-fed donors. Fructose and insulin did not affect the rate of enzyme degradation. We propose that there is a role for protein catabolism in the short-term and long-term control of L-type pyruvate kinase concentration.
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