Mechanism responsible for inactivation of skeletal muscle pyruvate dehydrogenase complex in starvation and diabetes.
(17/1585)
Regulation of the activity of the pyruvate dehydrogenase complex in skeletal muscle plays an important role in fuel selection and glucose homeostasis. Activation of the complex promotes disposal of glucose, whereas inactivation conserves substrates for hepatic glucose production. Starvation and diabetes induce a stable increase in pyruvate dehydrogenase kinase activity in skeletal muscle mitochondria that promotes phosphorylation and inactivation of the complex. The present study shows that these metabolic conditions induce a large increase in the expression of PDK4, one of four pyruvate dehydrogenase kinase isoenzymes expressed in mammalian tissues, in the mitochondria of gastrocnemius muscle. Refeeding starved rats and insulin treatment of diabetic rats decreased pyruvate dehydrogenase kinase activity and also reversed the increase in PDK4 protein in gastrocnemius muscle mitochondria. Starvation and diabetes also increased the abundance of PDK4 mRNA in gastrocnemius muscle, and refeeding and insulin treatment again reversed the effects of starvation and diabetes. These findings suggest that an increase in amount of this enzyme contributes to hyperphosphorylation and inactivation of the pyruvate dehydrogenase complex in these metabolic conditions. It was further found that feeding rats WY-14,643, a selective agonist for the peroxisome proliferator-activated receptor-alpha (PPAR-alpha), also induced large increases in pyruvate dehydrogenase kinase activity, PDK4 protein, and PDK4 mRNA in gastrocnemius muscle. Since long-chain fatty acids activate PPAR-alpha endogenously, increased levels of these compounds in starvation and diabetes may signal increased expression of PDK4 in skeletal muscle. (+info)
Coordinate induction of energy gene expression in tissues of mitochondrial disease patients.
(18/1585)
We have examined the transcript levels of a variety of oxidative phosphorylation (OXPHOS) and associated bioenergetic genes in tissues of a patient carrying the myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) A3243G mitochondrial DNA (mtDNA) mutation and the skeletal muscles of 14 patients harboring other pathogenic mtDNA mutations. The patients' tissues, which harbored 88% or more mutant mtDNA, had increased levels of mtDNA transcripts, increased nuclear OXPHOS gene transcripts including the ATP synthase beta subunit and the heart-muscle isoform of the adenine nucleotide translocator, and increased ancillary gene transcripts including muscle mitochondrial creatine phosphokinase, muscle glycogen phosphorylase, hexokinase I, muscle phosphofructokinase, the E1alpha subunit of pyruvate dehydrogenase, and the ubiquinone oxidoreductase. A similar coordinate induction of bioenergetic genes was observed in the muscle biopsies of severe pathologic mtDNA mutations. The more significant coordinated expression was found in muscle from patients with the MELAS, myoclonic epilepsy with ragged red fibers, and chronic progressive external ophthalmoplegia deletion syndromes, with ragged red muscle fibers and mitochondrial paracrystalline inclusions. High levels of mutant mtDNAs were linked to a high induction of the mtDNA and nuclear OXPHOS genes and of several associated bioenergetic genes. These observations suggest that human tissues attempt to compensate for OXPHOS defects associated with mtDNA mutations by stimulating mitochondrial biogenesis, possibly mediated through redox-sensitive transcription factors. (+info)
A stop-codon mutation in the human mtDNA cytochrome c oxidase I gene disrupts the functional structure of complex IV.
(19/1585)
We have identified a novel stop-codon mutation in the mtDNA of a young woman with a multisystem mitochondrial disorder. Histochemical analysis of a muscle-biopsy sample showed virtually absent cytochrome c oxidase (COX) stain, and biochemical studies confirmed an isolated reduction of COX activity. Sequence analysis of the mitochondrial-encoded COX-subunit genes identified a heteroplasmic G-->A transition at nucleotide position 6930 in the gene for subunit I (COX I). The mutation changes a glycine codon to a stop codon, resulting in a predicted loss of the last 170 amino acids (33%) of the polypeptide. The mutation was present in the patient's muscle, myoblasts, and blood and was not detected in normal or disease controls. It was not detected in mtDNA from leukocytes of the patient's mother, sister, and four maternal aunts. We studied the genetic, biochemical, and morphological characteristics of transmitochondrial cybrid cell lines, obtained by fusing of platelets from the patient with human cells lacking endogenous mtDNA (rho0 cells). There was a direct relationship between the proportion of mutant mtDNA and the biochemical defect. We also observed that the threshold for the phenotypic expression of this mutation was lower than that reported in mutations involving tRNA genes. We suggest that the G6930A mutation causes a disruption in the assembly of the respiratory-chain complex IV. (+info)
T(3) stimulates resting metabolism and UCP-2 and UCP-3 mRNA but not nonphosphorylating mitochondrial respiration in mice.
(20/1585)
The molecular basis for variations in resting metabolic rate (RMR) within a species is unknown. One possibility is that variations in RMR occur because of variations in uncoupling protein 2 (UCP-2) and uncoupling protein 3 (UCP-3) expression, resulting in mitochondrial proton leak differences. We tested the hypothesis that UCP-2 and -3 mRNAs positively correlate with RMR and proton leak. We treated thyroidectomized and sham-operated mice with triiodothyronine (T(3)) or vehicle and measured RMR, liver, and skeletal muscle mitochondrial nonphosphorylating respiration and UCP-2 and -3 mRNAs. T(3) stimulated RMR and liver UCP-2 and gastrocnemius UCP-2 and -3 expression. Mitochondrial respiration was not affected by T(3) and did not correlate with UCP-2 and -3 mRNAs. Gastrocnemius UCP-2 and -3 expression did correlate with RMR. We conclude 1) T(3) did not influence intrinsic mitochondrial properties such as membrane structure and composition, and 2) variations in UCP-2 and -3 expression may partly explain variations in RMR. One possible explanation for these data is that T(3) stimulates the leak in vivo but not in vitro because a posttranslational regulator of UCP-2 and -3 is not retained in the mitochondrial fraction. (+info)
Temperature-dependent expression of cytochrome-c oxidase in Antarctic and temperate fish.
(21/1585)
Seasonal acclimation versus permanent adaptation to low temperatures leads to a differential response in the expression of cytochrome-c oxidase (CCO) in temperate and Antarctic eelpouts. Although eurythermal eelpout from the North Sea (Zoarces viviparus) displayed a cold-induced rise of CCO activity in white muscle, enzyme activity in the cold stenothermal Antarctic eelpout Pachycara brachycephalum failed to reflect such a compensatory increase. In Antarctic eelpout, CCO activity correlates with transcript levels of mitochondrial encoded subunits of CCO (CCO I and CCO II), whereas cold-acclimated eelpout from the North Sea show lower enzyme activities than expected on the basis of mitochondrial mRNA levels. In these animals, CCO expression at low temperatures may be limited either by nuclear CCO transcripts or by posttranscriptional processes. These may comprise translation of the subunits or assembly of the CCO holoenzyme. mRNA levels of CCO IV, one of the nuclear encoded subunits, increased strongly during cold acclimation, indicating that the expression of CCO is likely not message limited in cold-acclimated Z. viviparus. Our data suggest that seasonal cold acclimation of Z. viviparus results in a modification of the relationship between transcription and translation or posttranslational processes. In permanently cold-adapted P. brachycephalum, on the other hand, CCO expression shows similar characteristics as in the warm-acclimated confamilial species, e.g., low levels of enzyme activity correlated with low levels of mitochondrial message. (+info)
Mitochondrial clearance of cytosolic Ca(2+) in stimulated lizard motor nerve terminals proceeds without progressive elevation of mitochondrial matrix [Ca(2+)].
(22/1585)
This study used fluorescent indicator dyes to measure changes in cytosolic and mitochondrial [Ca(2+)] produced by physiological stimulation of lizard motor nerve terminals. During repetitive action potential discharge at 10-50 Hz, the increase in average cytosolic [Ca(2+)] reached plateau at levels that increased with increasing stimulus frequency. This stabilization of cytosolic [Ca(2+)] was caused mainly by mitochondrial Ca(2+) uptake, because drugs that depolarize mitochondria greatly increased the stimulation-induced elevation of cytosolic [Ca(2+)], whereas blockers of other Ca(2+) clearance routes had little effect. Surprisingly, during this sustained Ca(2+) uptake the free [Ca(2+)] in the mitochondrial matrix never exceeded a plateau level of approximately 1 microM, regardless of stimulation frequency or pattern. When stimulation ceased, matrix [Ca(2+)] decreased over a slow ( approximately 10 min) time course consisting of an initial plateau followed by a return to baseline. These measurements demonstrate that sustained mitochondrial Ca(2+) uptake is not invariably accompanied by progressive elevation of matrix free [Ca(2+)]. Both the plateau of matrix free [Ca(2+)] during stimulation and its complex decay after stimulation could be accounted for by a model incorporating reversible formation of an insoluble Ca salt. This mechanism allows mitochondria to sequester large amounts of Ca(2+) while maintaining matrix free [Ca(2+)] at levels sufficient to activate Ca(2+)-dependent mitochondrial dehydrogenases, but below levels that activate the permeability transition pore. (+info)
Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia.
(23/1585)
It has been shown that nitric oxide (NO), synthesized by the inducible NO synthase (iNOS) expressed in the diaphragm during endotoxemia, participates in the development of muscular contractile failure. The aim of the present study was to investigate whether this deleterious action of NO was related to its effects on cellular oxidative pathways. Rats were inoculated with E. coli lipopolysaccharide (LPS) or sterile saline solution (controls) and studied at 3 and 6 h after inoculation. iNOS protein and activity could be detected in the rat diaphragm as early as 3 h after LPS, with a sustained steady-state concentration of 0.5 microM NO in the muscle associated with increased detection of hydrogen peroxide (H(2)O(2)). In vitro, the same NO concentration produced a marked increase in H(2)O(2) production by isolated control diaphragm mitochondria, thus reflecting a higher intramitochondrial concentration of nondiffusible superoxide anion (O(2)(-.)). In a similar way, whole diaphragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H(2)O(2) production associated with uncoupling and decreased phosphorylating capacity. Simultaneous with the maximal impairment in respiration (6 h after LPS), nitration of mitochondrial proteins (a peroxynitrite footprint) was detected and diaphragmatic force was reduced. Functional mitochondrial abnormalities, nitration of mitochondrial proteins, and the decrease in force were significantly attenuated by administration of the NOS inhibitor L-NMMA. These results show that increased and sustained NO levels lead to a consecutive formation of O(2)(-.) that reacts with NO to form peroxynitrite, which in turn impairs mitochondrial function, which probably contributes to the impairment of muscle contractility. during endotoxemia. (+info)
Gene expression profile of aging and its retardation by caloric restriction.
(24/1585)
The gene expression profile of the aging process was analyzed in skeletal muscle of mice. Use of high-density oligonucleotide arrays representing 6347 genes revealed that aging resulted in a differential gene expression pattern indicative of a marked stress response and lower expression of metabolic and biosynthetic genes. Most alterations were either completely or partially prevented by caloric restriction, the only intervention known to retard aging in mammals. Transcriptional patterns of calorie-restricted animals suggest that caloric restriction retards the aging process by causing a metabolic shift toward increased protein turnover and decreased macromolecular damage. (+info)