Changes in mitochondrial perilipin 3 and perilipin 5 protein content in rat skeletal muscle following endurance training and acute stimulated contraction.

NEW FINDINGS: What is the central question of this study? The aim was to determine whether mitochondrial protein content of perilipin 3 (PLIN3) and perilipin 5 (PLIN5) is increased following endurance training and whether mitochondrial PLIN5 protein is increased to a greater extent in endurance-trained rats when compared with sedentary rats following acute contraction. What is the main finding and its importance? Mitochondrial PLIN3 but not PLIN5 protein was increased in endurance-trained compared with sedentary rats, suggesting a mitochondrial role for PLIN3 due to chronic exercise. Contrary to our hypothesis, acute mitochondrial PLIN5 protein was similar in both sedentary and endurance-trained rats. Endurance training results in an increased association between skeletal muscle lipid droplets and mitochondria. This association is likely to be important for the expected increase in intramuscular fatty acid oxidation that occurs with endurance training. The perilipin family of lipid droplet proteins, PLIN(2-5), are thought to play a role in skeletal muscle lipolysis. Recently, results from our laboratory demonstrated that skeletal muscle mitochondria contain PLIN3 and PLIN5 protein. Furthermore, 30 min of stimulated contraction induces an increased mitochondrial PLIN5 content. To determine whether mitochondrial content of PLIN3 and PLIN5 is altered with endurance training, Sprague-Dawley rats were randomized into sedentary or endurance-trained groups for 8 weeks of treadmill running followed by an acute (30 min) sciatic nerve stimulation to induce lipolysis. Mitochondrial PLIN3 protein was ∼1.5-fold higher in red gastrocnemius of endurance-trained rats compared with sedentary animals, with no change in mitochondrial PLIN5 protein. In addition, there was an increase in plantaris intramuscular lipid storage. Acute electrically stimulated contraction in red gastrocnemius from sedentary and endurance-trained rats resulted in a similar increase of mitochondrial PLIN5 between these two groups, with no net change in PLIN3 in either group. Plantaris intramuscular lipid content decreased to a similar extent in sedentary and endurance-trained rats. These results suggest that while total mitochondrial PLIN5 content is not altered by endurance training, PLIN5 does have an acute role in the mitochondrial fraction during muscle contraction. Conversely, mitochondrial PLIN3 does not change acutely with muscle contraction, but PLIN3 content was increased following endurance training, indicating a role in chronic adaptations of skeletal muscle.

Examination of Akt and GSK3ß in BDNF-mediated reductions in BACE1 activity in neuronal cells.

Brain-derived neurotrophic factor (BDNF) content and signaling has been identified as one potential regulator of amyloid precursor protein (APP) processing. Recently published work has demonstrated that BDNF reduces BACE1 activity while also elevating the inhibition of GSK3ß in the prefrontal cortex of male C57BL/6J mice. These results provide evidence that BDNF alters APP processing by reducing BACE1 activity, which may act through GSK3ß inhibition. The purpose of this study was to further explore the role of GSK3ß in BDNF-induced regulation on BACE1 activity. We utilized a cell culture and an in vitro activity assay model to pharmacologically target BDNF and GSK3ß signaling to confirm its involvement in the BDNF response. Treatment of differentiated SH-SY5Y neuronal cells with 75 ng/mL BDNF resulted in elevated pTrkB content, pAkt content, pGSK3ß content, and reduced BACE1 activity. An in vitro BACE1 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF reduced BACE1 activity; however, in the presence of TrkB or Akt inhibition, this effect was abolished. An in vitro ADAM10 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF did not alter ADAM10 activity. However, inhibiting BDNF signaling reduced ADAM10 activity. Collectively these studies suggest that GSK3ß inhibition may be necessary for BDNF-induced reductions in BACE1 activity. These findings will allow for the optimization of future therapeutic strategies by selectively targeting TrkB activation and GSK3ß inhibition.

Creatine and low-dose lithium supplementation separately alter energy expenditure, body mass, and adipose metabolism for the promotion of thermogenesis.

Nutraceutical approaches to promote adipose tissue thermogenesis may help to prevent obesity onset. Creatine is a critical regulator of adipose metabolic function and low-dose lithium supplementation has been shown to promote adipose thermogenesis. In the present study, we sought to directly compare the two supplements for their effects on adipose metabolism and thermogenesis. We show that both supplements increase daily energy expenditure (EE) and reduce body mass in male Sprague-Dawley rats. Lithium increased brown adipose tissue (BAT) mitochondrial and lipolytic proteins that are associated with thermogenesis, while creatine increased BAT UCP1 and mitochondrial respiration. The BAT thermogenic findings were not observed in females. White adipose tissue and skeletal muscle markers of thermogenesis were unaltered with the supplements. Together, the data show that low-dose lithium and creatine have diverging effects on markers of BAT thermogenesis and that each increase daily EE and lower body mass in a sex-dependent manner.

Lactation during hibernation in wild black bears: effects on plasma amino acids and nitrogen metabolites.

This study examined the seasonal and reproductive influences on individual plasma amino acid concentrations and nitrogen metabolites in a black bear population (Ontario, Canada). During hibernation, 11 of 23 plasma amino acids were significantly higher (13%-108%) in lactating than in nonlactating females, without an alteration in plasma total protein or total essential or nonessential amino acid levels. The greatest changes were observed in glutamine, arginine, and glycine levels. Plasma urea, urea/creatinine, and ammonia levels were significantly lower in hibernating compared with active female bears, but lactation had no effect on these parameters. Taken together these results show that lactation during hibernation is an additional metabolic challenge that results in increased mobilization of individual plasma amino acids and no accumulation of nitrogen end products, underlining the remarkable efficiency of amino acid and urea recycling in denning female black bears.

O2 uptake kinetics, pyruvate dehydrogenase activity, and muscle deoxygenation in young and older adults during the transition to moderate-intensity exercise.

The adaptation of pulmonary O(2) uptake (Vo(2)(p)) kinetics is slowed in older compared with young adults during the transition to moderate-intensity exercise. In this study, we examined the relationship between Vo(2)(p) kinetics and mitochondrial pyruvate dehydrogenase (PDH) activity in young (n = 7) and older (n = 6) adults. Subjects performed cycle exercise to a work rate corresponding to approximately 90% of estimated lactate threshold. Phase 2 Vo(2)(p) kinetics were slower (P < 0.05) in older (tau = 40 +/- 17 s) compared with young (tau = 21 +/- 6 s) adults. Relative phosphocreatine (PCr) breakdown was greater (P < 0.05) at 30 s in older compared with young adults. Absolute PCr breakdown at 6 min was greater (P < 0.05) in older compared with young adults. In young adults, PDH activity increased (P < 0.05) from baseline to 30 s, with no further change observed at 6 min. In older adults, PDH activity during baseline exercise was similar to that seen in young adults. During the exercise transition, PDH activity did not increase (P > 0.05) at 30 s of exercise but was elevated (P < 0.05) after 6 min. The change in deoxyhemoglobin (HHb) was greater for a given Vo(2)(p) in older adults, and there was a similar time course of HHb accompanying the slower Vo(2)(p) kinetics in the older adults, suggesting a slower adaptation of bulk O(2) delivery in older adults. In conclusion, the slower adaptation of Vo(2)(p) in older adults is likely a result of both an increased metabolic inertia and lower O(2) availability.

Prior heavy exercise elevates pyruvate dehydrogenase activity and speeds O2 uptake kinetics during subsequent moderate-intensity exercise in healthy young adults.

The adaptation of pulmonary oxygen uptake (.VO2) during the transition to moderate-intensity exercise (Mod) is faster following a prior bout of heavy-intensity exercise. In the present study we examined the activation of pyruvate dehydrogenase (PDHa) during Mod both with and without prior heavy-intensity exercise. Subjects (n = 9) performed a Mod(1)-heavy-intensity-Mod(2) exercise protocol preceded by 20 W baseline. Breath-by-breath .VO2 kinetics and near-infrared spectroscopy-derived muscle oxygenation were measured continuously, and muscle biopsy samples were taken at specific times during the transition to Mod. In Mod(1), PDHa increased from baseline (1.08 +/- 0.2 mmol min(-1) (kg wet wt)(-1)) to 30 s (2.05 +/- 0.2 mmol min(-1) (kg wet wt)(-1)), with no additional change at 6 min exercise (2.07 +/- 0.3 mmol min(-1) (kg wet wt)(-1)). In Mod(2), PDHa was already elevated at baseline (1.88 +/- 0.3 mmol min(-1) (kg wet wt)(-1)) and was greater than in Mod(1), and did not change at 30 s (1.96 +/- 0.2 mmol min(-1) (kg wet wt)(-1)) but increased at 6 min exercise (2.70 +/- 0.3 mmol min(-1) (kg wet wt)(-1)). The time constant of .VO2 was lower in Mod(2) (19 +/- 2 s) than Mod(1) (24 +/- 3 s). Phosphocreatine (PCr) breakdown from baseline to 30 s was greater (P < 0.05) in Mod(1) (13.6 +/- 6.7 mmol (kg dry wt)(-1)) than Mod(2) (6.5 +/- 6.2 mmol (kg dry wt)(-1)) but total PCr breakdown was similar between conditions (Mod(1), 14.8 +/- 7.4 mmol (kg dry wt)(-1); Mod(2), 20.1 +/- 8.0 mmol (kg dry wt)(-1)). Both oxyhaemoglobin and total haemoglobin were elevated prior to and throughout Mod(2) compared with Mod(1). In conclusion, the greater PDHa at baseline prior to Mod(2) compared with Mod(1) may have contributed in part to the faster .VO2 kinetics in Mod(2). That oxyhaemoglobin and total haemoglobin were elevated prior to Mod(2) suggests that greater muscle perfusion may also have contributed to the observed faster .VO2 kinetics. These findings are consistent with metabolic inertia, via delayed activation of PDH, in part limiting the adaptation of pulmonary .VO2 and muscle O2 consumption during the normal transition to exercise.

Novel aspects of the activities and subcellular distribution of enzymes of ketone body metabolism in the liver and kidney of the goldfish, Carassius auratus.

The metabolic organization of ketone body metabolism of liver and kidney of the goldfish Carassius auratus was assessed by measuring maximal activities, subcellular distribution, and stereoisomer preference of ketone body enzymes. These determinations indicate that the organization of ketone body metabolism in liver and kidney of goldfish differs from that of mammals in some respects. All the enzymes of ketone body metabolism were present in liver and kidney of goldfish, with the exception of hydroxymethylglutaryl-CoA (HMG-CoA) synthetase, which was not detected in liver. Two forms of beta-hydroxybutyrate dehydrogenase (betaHBDH) with different stereospecificity for beta-hydroxybutyrate (D- and L-beta-hydroxybutyrate) were detectable in liver and kidney. All of the ketone body enzymes measured in liver were mainly in the mitochondrial fraction, with the exception of D- and L-betaHBDH, which were cytosolic. In kidney, HMG-CoA synthase, together with HMG-CoA lyase and acetoacetyl CoA thiolase (AcoAT), were found mainly in the mitochondrial fraction. L-betaHBDH was mainly cytosolic in kidney, but by contrast with liver, D-betaHBDH was mainly found in the mitochondria, and SKT was distributed in both the mitochondrial and cytosolic compartments. J. Exp. Zool. 286:434-439, 2000.

Discrimination between viable and dead Encephalitozoon cuniculi (Microsporidian) spores by dual staining with sytox green and calcofluor white M2R.

Microsporidia are obligate intracellular parasites, recognized as causing chronic diarrhea and systemic disease in AIDS patients, organ transplant recipients, travelers, and malnourished children. Species of microsporidia that infect humans have been detected in drinking-water sources, and methods are needed to ascertain if these microsporidia are viable and capable of causing infections. In this study, Calcofluor White M2R and Sytox Green stains were used in combination to differentiate between live (freshly harvested) and dead (boiled) Encephalitozoon cuniculi spores. Calcofluor White M2R binds to chitin in the microsporidian spore wall. Dual-stained live spores appeared as turquoise-blue ovals, while dead spores appeared as white-yellow ovals at an excitation wavelength of 395 to 415 nm used for viewing the Calcofluor stain. Sytox Green, a nuclear stain, is excluded by live spores but penetrates compromised spore membranes. Dual-stained dead spores fluoresced bright yellow-green when viewed at an excitation wavelength of 470 to 490 nm, whereas live spores failed to stain with Sytox Green. After live and dead spores were mixed at various ratios, the number of viably stained spores detected in the dual-staining procedure correlated (P = 0.0025) with the expected numbers of viable spores. Spore mixtures were also assayed for infectivity in a focus-forming assay, and a correlation (P = 0.0002) was measured between the percentage of focus-forming microsporidia and the percentage of expected infectious spores in each mixture. By analysis of variance, no statistically significant differences were measured between the percentage of viably stained microsporidia and the percentage of infectious microsporidia (P = 0.964) in each mixture. These results suggest that Calcofluor White M2R and Sytox Green stains, when used together, may facilitate studies to identify viable microsporidia.

Effects of respiratory alkalosis on human skeletal muscle metabolism at the onset of submaximal exercise.

The purpose of this study was to examine the effects of respiratory alkalosis on human skeletal muscle metabolism at rest and during submaximal exercise. Subjects exercised on two occasions for 15 min at 55 % of their maximal oxygen uptake while either hyperventilating (R-Alk) or breathing normally (Con). Muscle biopsies were taken at rest and after 1 and 15 min of exercise. At rest, no effects on muscle metabolism were observed in response to R-Alk. In the first minute of exercise, there was a delayed activation of pyruvate dehydrogenase (PDH) in R-Alk compared with Con, resulting in a reduced rate of pyruvate oxidation. Also, glycogenolysis was higher in R-Alk compared with Con, which was attributed to a higher availability of the monoprotonated form of inorganic phosphate (P(i)), resulting in an elevated rate of pyruvate production. The mismatch between pyruvate production and its oxidation resulted in net lactate accumulation. These effects were not seen after 15 min of exercise, with no further differences in muscle metabolism between conditions. The results from the present study suggest that respiratory alkalosis may play an important role in lactate accumulation during the transition from rest to exercise in acute hypoxic conditions, but that other factors mediate lactate accumulation during steady-state exercise.

Correlations of plasma lipid metabolites with hibernation and lactation in wild black bears Ursus americanus.

During the denning period, black bears (Ursus americanus) are capable of enduring several months without food. At the same time, female bears that are pregnant or lactating have an added metabolic stress. Based on laboratory studies, much of the energy required to support metabolism and lactation during denning in black bears comes from lipid reserves. These lipid reserves are mobilized and the most metabolically active lipid fraction in the blood are nonesterified fatty acids (NEFA). Therefore, we hypothesized that plasma NEFAs would be higher in denning relative to active bears and in lactating relative to non-lactating female bears. We further hypothesized that in bears with elevated plasma NEFA levels, other lipid-related parameters (e.g., ketone bodies, albumin, cholesterol, lipase) would also be elevated in the plasma. Denning bears had significantly increased NEFA levels in all classes (saturates, monoenes, and polyenes). A doubling of plasma NEFA levels and a 33% increase in albumin, the plasma fatty acid binding protein, in denning bears, resulted in NEFA/albumin ratios that were higher in denning bears (4:1) compared to those of active bears (3:1). Bears became relatively ketonemic with a 17-fold increase in D-beta-hydroxybutyrate levels during the denning period. Plasma cholesterol approximately doubled and lipase was ten-fold lower in denning relative to active bears. These findings indicate a strong correlation between plasma lipid metabolites and the denning period in a wild population of black bears.