Regulation of Torpor in the Gray Mouse Lemur:Transcriptional and Translational Controls and Role of AMPK Signaling

The gray mouse lemur (Microcebus murinus) is one of few primate species that is able to enter daily torpor or prolonged hibernation in response to environmental stresses. With an emerg-ing significance...     

Primate Torpor:Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur,Microcebus murinus

Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primate...     

Induction of Antioxidant and Heat Shock Protein Responses During Torpor in the Gray Mouse Lemur,Microcebus murinus

A natural tolerance of various environmental stresses is typically supported by various cytoprotective mechanisms that protect macromolecules and promote extended viability. Among these are antioxidant defenses that help to limit damage from reactive oxygen species and chaperones that help to minimize protein misfolding or unfolding under stress conditions. To understand the molecular mechanisms that act to protect cells during primate torpor, the present study characterizes antioxidant and heat shock protein(HSP) responses in various organs of control(aroused)and torpid gray mouse lemurs, Microcebus murinus. Protein expression of HSP70 and HSP90 a was elevated to 1.26 and 1.49 fold, respectively, in brown adipose tissue during torpor as compared with control animals, whereas HSP60 in liver of torpid animals was 1.15 fold of that in control(P 0.05). Among antioxidant enzymes, protein levels of thioredoxin 1 were elevated to 2.19 fold in white adipose tissue during torpor, whereas Cu–Zn superoxide dismutase 1 levels rose to 1.1 fold in skeletal muscle(P 0.05). Additionally, total antioxidant capacity was increased to 1.6 fold in liver during torpor(P 0.05), while remaining unchanged in the five other tissues. Overall, our data suggest that antioxidant and HSP responses are modified in a tissue-specific manner during daily torpor in gray mouse lemurs. Furthermore, our data also show that cytoprotective strategies employed during primate torpor are distinct from the strategies in rodent hibernation as reported in previous studies.     

Modulation of Gene Expression in Key Survival Pathways During Daily Torpor in the Gray Mouse Lemur,Microcebus murinus

A variety of mammals employ torpor as an energy-saving strategy in environments of marginal or severe stress either on a daily basis during their inactive period or on a seasonal basis during prolonged...     

Regulation of pyruvate dehydrogenase in the common killifish, Fundulus heteroclitus, during hypoxia exposure

We examined the metabolic responses of the hypoxia-tolerant killifish (Fundulus heteroclitus) to 15 h of severe hypoxia and recovery with emphasis on muscle substrate usage and the regulation of the mitochondrial protein pyruvate dehydrogenase (PDH), which controls carbohydrate oxidation. Hypoxia survival involved a transient activation of substrate-level phosphorylation in muscle (decreases in [creatine phospate] and increases in [lactate]) during which time mechanisms to reduce overall ATP consumption were initiated. This metabolic transition did not affect total cellular [ATP], but had an impact on cellular energy status as indicated by large decreases in [ATP]/[ADP(free)] and [ATP]/[AMP(free)] and a significant loss of phosphorylation potential and Gibbs free energy of ATP hydrolysis (DeltafG'). The activity of PDH was rapidly (within 3 h) decreased by approximately 50% upon hypoxia exposure and remained depressed relative to normoxic samples throughout. Inactivation of PDH was primarily mediated via posttranslational modification following the accumulation of acetyl-CoA and subsequent activation of pyruvate dehydrogenase kinase (PDK). Estimated changes in cytoplasmic and mitochondrial [NAD(+)]/[NADH] did not parallel one another, suggesting the mitochondrial NADH shuttles do not function during hypoxia exposure. Large increases in the expression of PDK (PDK isoform 2) were consistent with decreased PDH activity; however, these changes in mRNA were not associated with changes in total PDK-2 protein content assessed using mammalian antibodies. No other changes in the expression of other known hypoxia-responsive genes (e.g., lactate dehydrogenase-A or -B) were observed in either muscle or liver.     

Pyruvate dehydrogenase E1α phosphorylation is induced by glucose but does not control metabolism-secretion coupling in INS-1E clonal β-cells

Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation. In the organelle, glucose-derived pyruvate is metabolised along the oxidative and anaplerotic pathway to generate downstream signals leading to insulin granule exocytosis. Entry into the oxidative pathway is catalysed by pyruvate dehydrogenase (PDH) and controlled in part by phosphorylation of the PDH E1α subunit blocking enzyme activity. We find that glucose but not other nutrient secretagogues induce PDH E1α phosphorylation in INS-1E cells and rat islets. INS-1E cells and primary β-cells express pyruvate dehydrogenase kinase (PDK) 1, 2 and 3, which mediate the observed phosphorylation. In INS-1E cells, suppression of the two main isoforms, PDK1 and PDK3, almost completely prevented PDH E1α phosphorylation. Under basal glucose conditions, phosphorylation was barely detectable and therefore the enzyme almost fully active (90% of maximal). During glucose stimulation, PDH is only partially inhibited (to 78% of maximal). Preventing PDH phosphorylation in situ after suppression of PDK1, 2 and 3 neither enhanced pyruvate oxidation nor insulin secretion. In conclusion, although glucose stimulates E1α phosphorylation and therefore inhibits PDH activity, this control mechanism by itself does not alter metabolism-secretion coupling in INS-1E clonal β-cells.     

Cytokine and Antioxidant Regulation in the Intestine of the Gray Mouse Lemur (Microcebus murinus) During Torpor

During food shortages, the gray mouse lemur (Microcebus murinus) of Madagascar expe-riences daily torpor thereby reducing energy expenditures. The present study aimed to understand the impacts of torpo...     

PDH activation during in vitro muscle contractions in PDH kinase 2 knockout mice: effect of PDH kinase 1 compensation

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate oxidation in skeletal muscle. PDH is deactivated by a set of PDH kinases (PDK1, PDK2, PDK3, PDK4), with PDK2 and PDK4 being the most predominant isoforms in skeletal muscle. Although PDK2 is the most abundant isoform, few studies have examined its physiological role. The role of PDK2 on PDH activation (PDHa) at rest and during muscle stimulation at 10 and 40 Hz (eliciting low- and moderate-intensity muscle contractions, respectively) in isolated extensor digitorum longus muscles was studied in PDK2 knockout (PDK2KO) and wild-type (WT) mice (n = 5 per group). PDHa activity was unexpectedly 35 and 77% lower in PDK2KO than WT muscle (P = 0.043), while total PDK activity was nearly fourfold lower in PDK2KO muscle (P = 0.006). During 40-Hz contractions, initial force was lower in PDK2KO than WT muscle (P < 0.001) but fatigued similarly to ～75% of initial force by 3 min. There were no differences in initial force or rate of fatigue during 10-Hz contractions. PDK1 compensated for the lack of PDK2 and was 1.8-fold higher in PDK2KO than WT muscle (P = 0.019). This likely contributed to ensuring that resting PDHa activity was similar between the groups and accounts for the lower PDH activation during muscle contraction, as PDK1 is a very potent inhibitor of the PDH complex. Increased PDK1 expression appears to be regulated by hypoxia inducible factor-1α, which was 3.5-fold higher in PDK2KO muscle. It is clear that PDK2 activity is essential, even at rest, in regulation of carbohydrate oxidation and production of reducing equivalents for the electron transport chain. In addition, these results underscore the importance of the overall kinetics of the PDK isoform population, rather than total PDK activity, in determining transformation of the PDH complex and PDHa activity during muscle contraction.     

Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise.

Prolonged moderate-intensity exercise is characterized by a progressive reduction in carbohydrate oxidation and concomitant increase in fat oxidation. Pyruvate dehydrogenase (PDH) controls the entry of pyruvate into oxidative pathways and is a rate-limiting enzyme for carbohydrate metabolism. PDH is controlled by the activities of a kinase (PDK, inhibitory) and phosphatase (stimulatory). To test the hypothesis that increased PDK activity was associated with decreased PDH activity and carbohydrate oxidation during an acute exercise bout, seven recreationally active men completed 4 h of cycle exercise at 55% peak oxygen consumption. Muscle samples were obtained before and at 10 min and 4 h of exercise for the measurement of PDH activity and the extraction of intact mitochondria for the measurements of PDK activity and PDK-2 and PDK-4 protein expression. Carbohydrate oxidation was reduced (P < 0.05) with exercise duration. Muscle glycogen content was lower (P < or = 0.05) at 4 h compared with rest and there was no change in muscle pyruvate content from 10 to 240 min during exercise (10 min: 0.28 +/- 0.05; 240 min: 0.35 +/- 0.09 mmol/kg dry muscle). PDH activity increased (P < 0.05) above resting values at 10 min (2.86 +/- 0.26 mmol.min(-1).kg wet muscle(-1)), but was lower than 10 min after 4 h (2.23 +/- 0.24 mmol.min(-1).kg wet muscle(-1)) of exercise. PDK-2 and PDK-4 protein expression was not different from rest at 10 min and 4 h of exercise. PDK activity at rest averaged 0.081 +/- 0.016 min(-1), was similar at 10 min, and increased (P < 0.05) to 0.189 +/- 0.013 min(-1) at 4 h. Although reduced glycolytic flux may have played a role in decreasing carbohydrate oxidation, the results suggest that increased PDK activity contributed to the reduction in PDH activity and carbohydrate oxidation late in prolonged exercise. The increased PDK activity was independent of changes in intra-mitochondrial effectors, and PDK-2 and PDK-4 protein content, suggesting that it was caused by a change in the specific activity of the existing kinases.     

Mitochondrial metabolic suppression and reactive oxygen species production in liver and skeletal muscle of hibernating thirteen-lined ground squirrels

During hibernation, animals cycle between periods of torpor, during which body temperature (T(b)) and metabolic rate (MR) are suppressed for days, and interbout euthermia (IBE), during which T(b) and MR return to resting levels for several hours. In this study, we measured respiration rates, membrane potentials, and reactive oxygen species (ROS) production of liver and skeletal muscle mitochondria isolated from ground squirrels (Ictidomys tridecemlineatus) during torpor and IBE to determine how mitochondrial metabolism is suppressed during torpor and how this suppression affects oxidative stress. In liver and skeletal muscle, state 3 respiration measured at 37°C with succinate was 70% and 30% lower, respectively, during torpor. In liver, this suppression was achieved largely via inhibition of substrate oxidation, likely at succinate dehydrogenase. In both tissues, respiration by torpid mitochondria further declined up to 88% when mitochondria were cooled to 10°C, close to torpid T(b). In liver, this passive thermal effect on respiration rate reflected reduced activity of all components of oxidative phosphorylation (substrate oxidation, phosphorylation, and proton leak). With glutamate + malate and succinate, mitochondrial free radical leak (FRL; proportion of electrons leading to ROS production) was higher in torpor than IBE, but only in liver. With succinate, higher FRL likely resulted from increased reduction state of complex III during torpor. With glutamate + malate, higher FRL resulted from active suppression of complex I ROS production during IBE, which may limit ROS production during arousal. In both tissues, ROS production and FRL declined with temperature, suggesting ROS production is also reduced during torpor by passive thermal effects.     

Enzymatic regulation of glucose disposal in human skeletal muscle after a high-fat, low-carbohydrate diet.

Whole body glucose disposal and skeletal muscle hexokinase, glycogen synthase (GS), pyruvate dehydrogenase (PDH), and PDH kinase (PDK) activities were measured in aerobically trained men after a standardized control diet (Con; 51% carbohydrate, 29% fat, and 20% protein of total energy intake) and a 56-h eucaloric, high-fat, low-carbohydrate diet (HF/LC; 5% carbohydrate, 73% fat, and 22% protein). An oral glucose tolerance test (OGTT; 1 g/kg) was administered after the Con and HF/LC diets with vastus lateralis muscle biopsies sampled pre-OGTT and 75 min after ingestion of the oral glucose load. The 90-min area under the blood glucose and plasma insulin concentration vs. time curves increased by 2-fold and 1.25-fold, respectively, after the HF/LC diet. The pre-OGTT fraction of GS in its active form and the maximal activity of hexokinase were not affected by the HF/LC diet. However, the HF/LC diet increased PDK activity (0.19 +/- 0.05 vs. 0.08 +/- 0.02 min(-1)) and decreased PDH activation (0.38 +/- 0.08 vs. 0.79 +/- 0.10 mmol acetyl-CoA.kg wet muscle(-1).min(-1)) before the OGTT vs. Con. During the OGTT, GS and PDH activation increased by the same magnitude in both diets, such that PDH activation remained lower during the HF/LC OGTT (0.60 +/- 0.11 vs. 1.04 +/- 0.09 mmol acetyl-CoA.kg(-1).min(-1)). These data demonstrate that the decreased glucose disposal during the OGTT after the 56-h HF/LC diet was in part related to decreased oxidative carbohydrate disposal in skeletal muscle and not to decreased glycogen storage. The rapid increase in PDK activity during the HF/LC diet appeared to account for the reduced potential for oxidative carbohydrate disposal.     

Regulation of pyruvate dehydrogenase (PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus)

Pyruvate dehydrogenase (PDH) is a vital regulatory enzyme that catalyzes the conversion of pyruvate into acetyl-CoA and connects anaerobic glycolysis to aerobic TCA cycle. Post-translational inhibition of PDH activity via three serine phosphorylation sites (pS232, pS293, and pS300) regulate the metabolic flux through the TCA cycle, decrease glucose utilization, and facilitate lipid metabolism during times of nutrient deprivation. As metabolic readjustment is necessary to survive hibernation, the purpose of this study was to explore the post-translational regulation of pyruvate dehydrogenase and the expression levels of four mitochondrial serine/threonine kinases (PDHKs), during torpor-arousal cycles in liver, heart, and skeletal muscle of 13-lined ground squirrels. A combination of Luminex multiplex technology and western immunoblotting were used to measure the protein expression levels of total PDH, three phosphorylation sites, S232, 293, 300, and the expression levels of the corresponding PDH kinases (PDHK1-4) during euthermic control, entrance, late torpor, and interbout arousal. Liver and heart showed strong inhibitory PDH regulation, indicating a possible decrease in glucose utilization and a possible preference for β-oxidation of fatty acids during periods of low temperature and starvation. On the contrary, skeletal muscle showed limited PDH regulation via phosphorylation, possibly due to alternate controls. Phosphorylation of PDH may play an important role in regulating aerobic and anaerobic metabolic responses during hibernation in the 13-lined ground squirrel.     

MuRF1-dependent regulation of systemic carbohydrate metabolism as revealed from transgenic mouse studies

Under various pathophysiological muscle-wasting conditions, such as diabetes and starvation, a family of ubiquitin ligases, including muscle-specific RING-finger protein 1 (MuRF1), are induced to target muscle proteins for degradation via ubiquitination. We have generated transgenic mouse lines over-expressing MuRF1 in a skeletal muscle-specific fashion (MuRF1-TG mice) in an attempt to identify the in vivo targets of MuRF1. MuRF1-TG lines were viable, had normal fertility and normal muscle weights at eight weeks of age. Comparison of quadriceps from MuRF1-TG and wild type mice did not reveal elevated multi-ubiquitination of myosin as observed in human patients with muscle wasting. Instead, MuRF1-TG mice expressed lower levels of pyruvate dehydrogenase (PDH), a mitochondrial key enzyme in charge of glycolysis, and of its regulator PDK2. Furthermore, yeast two-hybrid interaction studies demonstrated the interaction of MuRF1 with PDH, PDK2, PDK4, PKM2 (all participating in glycolysis) and with phosphorylase β (PYGM) and glycogenin (both regulating glycogen metabolism). Consistent with the idea that MuRF1 may regulate carbohydrate metabolism, MuRF1-TG mice had twofold elevated insulin blood levels and lower hepatic glycogen contents. To further examine MuRF1's role for systemic carbohydrate regulation, we performed glucose tolerance tests (GTT) in wild type and MuRF1-TG mice. During GTT, MuRF1-TG mice developed striking hyperinsulinaemia and hepatic glycogen stores, that were depleted at basal levels, became rapidly replenished. Taken together, our data demonstrate that MuRF1 expression in skeletal muscle re-directs glycogen synthesis to the liver and stimulates pancreatic insulin secretion, thereby providing a regulatory feedback loop that connects skeletal muscle metabolism with the liver and the pancreas during metabolic stress.     

The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity

Pyruvate dehydrogenase (PDH) is a mitochondrial enzyme responsible for regulating the conversion of pyruvate to acetyl-CoA for use in the tricarboxylic acid cycle. PDH is regulated through phosphorylation and inactivation by PDH kinase (PDK) and dephosphorylation and activation by PDH phosphatase (PDP). The effect of endurance training on PDK in humans has been investigated; however, to date no study has examined the effect of endurance training on PDP in humans. Therefore, the purpose of this study was to examine differences in PDP activity and PDP1 protein content in human skeletal muscle across a range of muscle aerobic capacities. This association is important as higher PDP activity and protein content will allow for increased activation of PDH, and carbohydrate oxidation. The main findings of this study were that 1) PDP activity (r(2) = 0.399, P = 0.001) and PDP1 protein expression (r(2) = 0.153, P = 0.039) were positively correlated with citrate synthase (CS) activity as a marker for muscle aerobic capacity; 2) E1α (r(2) = 0.310, P = 0.002) and PDK2 protein (r(2) = 0.229, P =0.012) are positively correlated with muscle CS activity; and 3) although it is the most abundant isoform, PDP1 protein content only explained ～ 18% of the variance in PDP activity (r(2) = 0.184, P = 0.033). In addition, PDP1 in combination with E1α explained ～ 38% of the variance in PDP activity (r(2) = 0.383, P = 0.005), suggesting that there may be alternative regulatory mechanisms of this enzyme other than protein content. These data suggest that with higher muscle aerobic capacity (CS activity) there is a greater capacity for carbohydrate oxidation (E1α), in concert with higher potential for PDH activation (PDP activity).     

Characterization of testis-specific isoenzyme of human pyruvate dehydrogenase

Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1.     

Regulation of PDK mRNA by high fatty acid and glucose in pancreatic islets

Pyruvate dehydrogenase (PDH) converts pyruvate to acetyl-CoA, links glycolysis to the Krebs cycle, and plays an important role in glucose metabolism and insulin secretion in pancreatic beta cells. In beta cells from obese and Type 2 diabetic animals, PDH activity is significantly reduced. PDH is negatively regulated by multiple pyruvate dehydrogenase kinase (PDK) isotypes (PDK subtypes 1-4). However, we do not know whether fatty acids or high glucose modulate PDKs in islets. To test this we determined PDH and PDK activities and PDK gene and protein expression in C57BL/6 mouse islets. Both high palmitate and high glucose reduced active PDH activity and increased PDK activity. The gene and protein for PDK3 were not expressed in islets. Palmitate up-regulated mRNA expression of PDK1 (2.9-fold), PDK2 (1.9-fold), and PDK4 (3.1-fold). High glucose increased PDK1 (1.8-fold) and PDK2 (2.7-fold) mRNA expression but reduced PDK4 mRNA expression by 40 percent in cultured islets. Changed PDK expression was confirmed by Western blotting. These results demonstrate that in islet cells both fat and glucose regulate PDK gene and protein expression and indicate that hyperglycemia and hyperlipidemia contribute to the decline in diabetic islet PDH activity by increasing mRNA and protein expression of PDK.     

Skeletal muscle type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression: effects of obesity and endurance training

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate metabolism in skeletal muscle. PDH is activated by PDH phosphatase (PDP) and deactivated by PDH kinase (PDK). Obesity has a large negative impact on skeletal muscle carbohydrate metabolism, whereas endurance training has been shown to improve regulatory control of skeletal muscle carbohydrate metabolism, more so when coupled with obesity. A majority of this literature has focused on PDK, with little information available on PDP. To determine the relative role of PDP in regulating skeletal muscle PDH activity with obesity and endurance training, obese and lean Zucker rats remained sedentary or were endurance trained (1 h/day, 5 days/wk) for a period of 8 wk. Soleus, red gastrocnemius, (RG), and white gastrocnemius (WG) muscles were sampled after the training period. The main findings were 1) obesity resulted in a 46% decrease in PDP activity expressed per milligram extracted mitochondrial protein only in RG, while PDP isoform content was unchanged; 2) 8 wk of endurance training led to a significant 1.4-2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; 3) 8 wk of endurance training led to a trending 1.4-2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; and 4) PDP2 protein content was not affected by obesity or training. These results suggest that decreased PDP activity in oxidative skeletal muscles may play a role in the impairment of carbohydrate metabolism in obese rats, which is reversible with endurance training.