Andrea J. Morash

Acclimatization of skeletal muscle mitochondria to high-altitude hypoxia during an ascent of Everest

Ascent to high altitude is associated with a fall in the partial pressure of inspired oxygen (hypobaric hypoxia). For oxidative tissues such as skeletal muscle, resultant cellular hypoxia necessitates acclimatization to optimize energy metabolism and restrict oxidative stress, with changes in gene and protein expression that alter mitochondrial function. It is known that lowlanders returning from high altitude have decreased muscle mitochondrial densities, yet the underlying transcriptional mechanisms and time course are poorly understood. To explore these, we measured gene and protein expression plus ultrastructure in muscle biopsies of lowlanders at sea level and following exposure to hypobaric hypoxia. Subacute exposure (19 d after initiating ascent to Everest base camp, 5300 m) was not associated with mitochondrial loss. After 66 d at altitude and ascent beyond 6400 m, mitochondrial densities fell by 21%, with loss of 73% of subsarcolemmal mitochondria. Correspondingly, levels of the transcriptional coactivator PGC‐1α fell by 35%, suggesting down‐regulation of mitochondrial biogenesis. Sustained hypoxia also decreased expression of electron transport chain complexes I and IV and UCP3 levels. We suggest that during subacute hypoxia, mitochondria might be protected from oxidative stress. However, following sustained exposure, mitochondrial biogenesis is deactivated and uncoupling down‐regulated, perhaps to improve the efficiency of ATP production.—Levett, D. Z., Radford, E. J., Menassa, D. A., Graber, E. F., Morash, A. J., Hoppeler, H., Clarke, K., Martin, D. C., Ferguson‐Smith, A. C., Montgomery, H. E., Grocott, M. P. W., Murray, A. J., Caudwell Xtreme Everest Research Group. Acclimatization of skeletal muscle mitochondria to high‐altitude hypoxia during an ascent of Everest. FASEB J. 26, 1431‐1441 (2012). www.fasebj.org

Intertissue regulation of carnitine palmitoyltransferase I (CPTI): Mitochondrial membrane properties and gene expression in rainbow trout (Oncorhynchus mykiss)

Carnitine palmitoyltransferase (CPT) I is regulated by several genetic and non-genetic factors including allosteric inhibition, mitochondrial membrane composition and/or fluidity and transcriptional regulation of enzyme content. To determine the intrinsic differences in these regulating factors that may result in differences between tissues in fatty acid oxidation ability, mitochondria were isolated from red, white and heart muscles and liver tissue from rainbow trout. Maximal activity (V(max)) for beta-oxidation enzymes and citrate synthase per mg tissue protein as well as CPT I in isolated mitochondria followed a pattern across tissues of red muscle>heart>white muscle>liver suggesting both quantitative and qualitative differences in mitochondria. CPT I inhibition showed a similar pattern with the highest malonyl-CoA concentration to inhibit activity by 50% (IC(50)) found in red muscle while liver had the lowest. Tissue malonyl-CoA content was highest in white muscle with no differences between the other tissues. Interestingly, the gene expression profiles did not follow the same pattern as the tissue enzyme activity. CPT I mRNA expression was greatest in heart>red muscle>white muscle>liver. In contrast, PPARalpha mRNA was greatest in the liver>red muscle>heart>white muscle. There were no significant differences in the mRNA expression of PPARbeta between tissues. As well, no significant differences were found in the mitochondrial membrane composition between tissues, however, there was a tendency for red muscle to exhibit higher proportions of PUFAs as well as a decreased PC:PE ratio, both of which would indicate increased membrane fluidity. In fact, there were significant correlations between IC(50) of CPT I for malonyl-CoA and indicators of membrane fluidity across tissues. This supports the notion that sensitivity of CPT I to its allosteric regulator could be modulated by changes in mitochondrial membrane composition and/or fluidity.

Effects of dietary fatty acid composition on the regulation of carnitine palmitoyltransferase (CPT) I in rainbow trout (Oncorhynchus mykiss)

Dietary fatty acid composition, particularly polyunsaturated fatty acids, can affect both genetic and non-genetic regulatory mechanisms of carnitine palmitoyltransferase (CPT) I, the main regulatory enzyme of mitochondrial fatty acid oxidation. We aimed to determine how these regulatory mechanisms were affected by changes in the fatty acid composition of the diet in fish. Specifically, we fed rainbow trout (Oncorhynchus mykiss) either a high polyunsaturated fatty acid (PUFA) diet, a high saturated fatty acid (SFA) diet or a mixed fatty acid control (CTL) diet for 8 weeks to determine if modifications of the dietary fatty acids would affect 1) the genetic expression of CPT I and its transcription factor peroxisome proliferator activated receptor (PPAR), 2) the mitochondrial membrane composition and if these modifications would affect CPT I sensitivity to malonyl-CoA, and 3) levels of malonyl-CoA in the tissues. We found that fish fed the high PUFA diet significantly increased CPT I mRNA expression in red muscle, liver and adipose tissue, while PPAR alpha and beta expressions were variable across tissues. Few significant changes were observed in the mitochondrial membrane composition with the exception of DHA in the red muscle. There were no significant differences in CPT I sensitivity to malonyl-CoA or the malonyl-CoA content of the tissues with either experimental diet. Our present data suggest that changes in gene expression of CPT I and PPARs is the main regulatory mechanism controlling CPT I function in fish using our experimental diet.

Genome duplication events have led to a diversification in the CPT I gene family in fish

The enzyme carnitine palmitoyltransferase (CPT) I is a major regulator of mitochondrial fatty acid oxidation in vertebrates. Numerous genome duplication events throughout evolution have given rise to three (in mammals) or multiple (in fish) genetically and functionally different isoforms of this enzyme. In particular, these isoforms represent a diversification of kinetic and regulatory properties stemming from mutations at the genomic and proteomic levels. Phylogenetic reconstructions reveal a comprehensive view of the CPT I family in vertebrates and genomic modifications leading to structural changes in proteins and functional differences between tissues and taxa. In a model fish species (rainbow trout), the presence of five CPT I isoforms suggests repeated duplication events in bony fishes and salmonids. Subsequently, an array of nucleotide and amino acid substitutions in the isoforms may contribute to a tissue-specific and a previously observed species-specific difference in the IC(50) for malonyl-CoA. Moreover, all five isoforms are expressed in trout at the mRNA level in skeletal muscle, heart, liver, kidney, and intestine. In general, transcript levels of the beta-isoforms were higher in muscle tissues, while levels of the alpha-isoforms were higher in other tissues. Rainbow trout also exhibit developmental plasticity in relative mRNA expression of CPT I isoforms from fry to juvenile to adult stage. Thus the evolution of CPT I has resulted in a very diverse family of isoforms. These differences represent a degree of specificity in the ability of species to regulate function at the protein and tissue levels, which, in turn, may allow for precise control of lipid oxidation in individual tissues during physiological perturbations.

A product of its environment: the epaulette shark (Hemiscyllium ocellatum) exhibits physiological tolerance to elevated environmental CO2

Ocean acidification is predicted to affect the performance of marine species, but little is known about the effects on sharks. We found that long-term exposure to elevated CO2 did not affect the epaulette shark, possibly because it experiences fluctuating environmental conditions in its shallow coral reef habitat.

Changes in HIF-1α protein, pyruvate dehydrogenase phosphorylation, and activity with exercise in acute and chronic hypoxia

Exercise under acute hypoxia elicits a large increase in blood lactate concentration ([La](b)) compared with normoxic exercise. However, several studies in humans show that with the transition to chronic hypoxia, exercise [La](b) returns to normoxic levels. Although extensively examined over the last decades, the muscle-specific mechanisms responsible for this phenomenon remain unknown. To assess the changes in skeletal muscle associated with a transition from acute to chronic hypoxia, CD-1 mice were exposed for 24 h (24H), 1 wk (1WH), or 4 wk (4WH) to hypobaric hypoxia (equivalent to 4,300 m), exercised under 12% O(2), and compared with normoxic mice (N) at 21% O(2). Since the enzyme pyruvate dehydrogenase (PDH) plays a major role in the metabolic fate of pyruvate (oxidation vs. lactate production), we assessed the changes in its activity and regulation. Here we report that when run under hypoxia, 24H mice exhibited the highest blood and intramuscular lactate of all groups, while the 1WH group approached N group values. Concomitantly, the 24H group exhibited the lowest PDH activity, associated with a higher phosphorylation (inactive) state of the Ser(232) residue of PDH, a site specific to PDH kinase-1 (PDK1). Furthermore, protein levels of PDK1 and its regulator, the hypoxia inducible factor-1α (HIF-1α), were both elevated in the 24H group compared with N and 1WH groups. Overall, our results point to a novel mechanism in muscle where the HIF-1α pathway is desensitized in the transition from acute to chronic hypoxia, leading to a reestablishment of PDH activity and a reduction in lactate production by the exercising muscles.

Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism

BackgroundInsulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of β-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms.ResultsHerein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARβ/δ- and PPARα-dependent mechanism. Enhanced PPARβ/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα−/− mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation.ConclusionsNitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle.

Tissue-specific changes in fatty acid oxidation in hypoxic heart and skeletal muscle

Exposure to hypobaric hypoxia is sufficient to decrease cardiac PCr/ATP and alters skeletal muscle energetics in humans. Cellular mechanisms underlying the different metabolic responses of these tissues and the time-dependent nature of these changes are currently unknown, but altered substrate utilization and mitochondrial function may be a contributory factor. We therefore sought to investigate the effects of acute (1 day) and more sustained (7 days) hypoxia (13% O₂) on the transcription factor peroxisome proliferator-activated receptor α (PPARα) and its targets in mouse cardiac and skeletal muscle. In the heart, PPARα expression was 40% higher than in normoxia after 1 and 7 days of hypoxia. Activities of carnitine palmitoyltransferase (CPT) I and β-hydroxyacyl-CoA dehydrogenase (HOAD) were 75% and 35% lower, respectively, after 1 day of hypoxia, returning to normoxic levels after 7 days. Oxidative phosphorylation respiration rates using palmitoyl-carnitine followed a similar pattern, while respiration using pyruvate decreased. In skeletal muscle, PPARα expression and CPT I activity were 20% and 65% lower, respectively, after 1 day of hypoxia, remaining at this level after 7 days with no change in HOAD activity. Oxidative phosphorylation respiration rates using palmitoyl-carnitine were lower in skeletal muscle throughout hypoxia, while respiration using pyruvate remained unchanged. The rate of CO₂ production from palmitate oxidation was significantly lower in both tissues throughout hypoxia. Thus cardiac muscle may remain reliant on fatty acids during sustained hypoxia, while skeletal muscle decreases fatty acid oxidation and maintains pyruvate oxidation.

Regulation of Carnitine Palmitoyltransferase (CPT) I during Fasting in Rainbow Trout (Oncorhynchus mykiss) Promotes Increased Mitochondrial Fatty Acid Oxidation

Periods of fasting, in most animals, are fueled principally by fatty acids, and changes in the regulation of fatty acid oxidation must exist to meet this change in metabolic substrate use. We examined the regulation of carnitine palmitoyltransferase (CPT) I, to help explain changes in mitochondrial fatty acid oxidation with fasting. After fasting rainbow trout (Oncorhynchus mykiss) for 5 wk, the mitochondria were isolated from red muscle and liver to determine (1) mitochondrial fatty acid oxidation rate, (2) CPT I activity and the concentration of malonyl-CoA needed to inhibit this activity by 50% (IC50), (3) mitochondrial membrane fluidity, and (4) CPT I (all five known isoforms) and peroxisome proliferator-activated receptor (PPARα and PPARβ) mRNA levels. Fatty acid oxidation in isolated mitochondria increased during fasting by 2.5- and 1.75-fold in liver and red muscle, respectively. Fasting also decreased sensitivity of CPT I to malonyl-CoA (increased IC50), by two and eight times in red muscle and liver, respectively, suggesting it facilitates the rate of fatty acid oxidation. In the liver, there was also a significant increase CPT I activity per milligram mitochondrial protein and in whole-tissue PPARα and PPARβ mRNA levels. However, there were no changes in mitochondrial membrane fluidity in either tissue, indicating that the decrease in CPT I sensitivity to malonyl-CoA is not due to bulk fluidity changes in the membrane. However, there were significant differences in CPT I mRNA levels during fasting. Overall, these data indicate some important changes in the regulation of CPT I that promote the increased mitochondrial fatty acid oxidation that occurs during fasting in trout.

Cryopreservation of placental biopsies for mitochondrial respiratory analysis

Mitochondrial function is required to support energetically-demanding processes in the placenta. As such, a compromise in mitochondrial function could severely impact fetal growth and development. Respirometry is a highly useful method for studying mitochondrial function, but is not possible in freeze-thawed mitochondria, which become uncoupled. We have developed a novel method that permits respiratory analysis of cryopreserved placental tissue. We studied mitochondrial function in 7 normal human placentas, analysing both fresh and cryopreserved samples. We found no impairments in respiration following cryopreservation in the delivery suite, with enhanced coupling, as indicated by higher respiratory control ratios, than in fresh placental samples transported to the laboratory on ice.