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Hexokinase-mitochondrial interactions regulate glucose metabolism differentially in adult and neonatal cardiac myocytes.

ABSTRACT: In mammalian tumor cell lines, localization of hexokinase (HK) isoforms to the cytoplasm or mitochondria has been shown to control their anabolic (glycogen synthesis) and catabolic (glycolysis) activities. In this study, we examined whether HK isoform differences could explain the markedly different metabolic profiles between normal adult and neonatal cardiac tissue. We used a set of novel genetically encoded optical imaging tools to track, in real-time in isolated adult (ARVM) and neonatal (NRVM) rat ventricular myocytes, the subcellular distributions of HKI and HKII, and the functional consequences on glucose utilization. We show that HKII, the predominant isoform in ARVM, dynamically translocates from mitochondria and cytoplasm in response to removal of extracellular glucose or addition of iodoacetate (IAA). In contrast, HKI, the predominant isoform in NRVM, is only bound to mitochondria and is not displaced by the above interventions. In ARVM, overexpression of HKI, but not HKII, increased glycolytic activity. In neonatal rat ventricular myocytes (NVRM), knockdown of HKI, but not HKII, decreased glycolytic activity. In conclusion, differential interactions of HKI and HKII with mitochondria underlie the different metabolic profiles of ARVM and NRVM, accounting for the markedly increased glycolytic activity of NRVM.

SUBMITTER: Calmettes G

PROVIDER: S-EPMC3787771 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Hexokinase-mitochondrial interactions regulate glucose metabolism differentially in adult and neonatal cardiac myocytes.

Calmettes Guillaume G John Scott A SA Weiss James N JN Ribalet Bernard B

The Journal of general physiology 20131001 4

In mammalian tumor cell lines, localization of hexokinase (HK) isoforms to the cytoplasm or mitochondria has been shown to control their anabolic (glycogen synthesis) and catabolic (glycolysis) activities. In this study, we examined whether HK isoform differences could explain the markedly different metabolic profiles between normal adult and neonatal cardiac tissue. We used a set of novel genetically encoded optical imaging tools to track, in real-time in isolated adult (ARVM) and neonatal (NRV ...[more]

PMID: 24081983

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Project description:Choline and methionine serve essential roles in the liver that may interact with glucose metabolism. Our objectives were to quantify glucose export, cellular glycogen, and expression of genes controlling oxidation and gluconeogenesis in primary bovine neonatal hepatocytes exposed to increasing concentrations of choline chloride (CC) and D,L-methionine (DLM) with or without fatty acids (FA). Primary hepatocytes isolated from 3 Holstein calves were maintained as monolayer cultures for 24 h before treatment with CC (61, 128, 2028, 4528 μmol/L) and DLM (16, 30, 100, 300 μmol/L) with or without a 1 mmol/L FA cocktail in a factorial design. After 24 h, media was harvested to quantify glucose, β-hydroxybutyrate (BHB), and cells harvested to quantify glycogen, DNA, and gene expression. No interactions between CC and DLM were detected. The potential two-way interaction between CC or DLM and FA was partitioned into three contrasts when P ≤ 0.20: linear without FA, linear with FA, difference of slope. Fatty acids did not affect glucose or cellular glycogen but increased pyruvate carboxylase (PC), cytosolic and mitochondrial phosphoenolpyruvate carboxykinase (PEPCKc, PEPCKm), and glucose-6-phosphatase (G6PC) expression. Increasing CC decreased glucose but increased cellular glycogen. Expression of PC and PEPCKc was increased by CC during FA treatment. Increasing DLM did not affect metabolites or PC expression, although PEPCKc was marginally decreased. Methionine did not affect G6PC, while CC had a marginal quadratic effect on G6PC. Oxidative and gluconeogenic enzymes appear to respond to FA in primary bovine neonatal hepatocytes. Increased PC and PEPCKc by CC during FA treatment suggest increased gluconeogenic capacity. Changes in G6PC may have shifted glucose-6-phosphate towards cellular glycogen; however, subsequent examination of G6PC protein is needed. Unaltered PC and marginally decreased PEPCKc suggest increased oxidative capacity with DLM, although BHB export was unaltered. The differential regulation supports unique effects of CC and DLM within bovine hepatocytes.

Project description:BackgroundAdult mammalian cardiac myocytes are generally assumed to be terminally differentiated; nonetheless, a small fraction of cardiac myocytes have been shown to replicate during ventricular remodeling. However, the expression of Replication Factor C (RFC; RFC140/40/38/37/36) and DNA polymerase δ (Pol δ) proteins, which are required for DNA synthesis and cell proliferation, in the adult normal and hypertrophied hearts has been rarely studied.MethodsWe performed qRT-PCR and Western blot analysis to determine the levels of RFC and Pol δ message and proteins in the adult normal cardiac myocytes and cardiac fibroblasts, as well as in adult normal and pulmonary arterial hypertension induced right ventricular hypertrophied hearts. Immunohistochemical analyses were performed to determine the localization of the re-expressed DNA replication and cell cycle proteins in adult normal (control) and hypertrophied right ventricle. We determined right ventricular cardiac myocyte polyploidy and chromosomal missegregation/aneuploidy using Fluorescent in situ hybridization (FISH) for rat chromosome 12.ResultsRFC40-mRNA and protein was undetectable, whereas Pol δ message was detectable in the cardiac myocytes isolated from control adult hearts. Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts. Immunohistochemical analyses revealed that in addition to RFC40, proliferative and mitotic markers such as cyclin A, phospho-Aurora A/B/C kinase and phospho-histone 3 were also re-expressed/up-regulated simultaneously in the cardiac myocytes. Interestingly, FISH analyses demonstrated cardiac myocytes polyploidy and chromosomal missegregation/aneuploidy in these hearts. Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers.ConclusionOur novel findings suggest that transcription of RFC40 is suppressed in the normal adult cardiac myocytes and its insufficient re-expression may be responsible for causing chromosomal missegregation/aneuploidy and in cardiac myocytes during right ventricular hypertrophy.

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Hexokinase-mitochondrial interactions regulate glucose metabolism differentially in adult and neonatal cardiac myocytes.

Publications

Hexokinase-mitochondrial interactions regulate glucose metabolism differentially in adult and neonatal cardiac myocytes.

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