James Lally

Repeated transient mRNA bursts precede increases in transcriptional and mitochondrial proteins during training in human skeletal muscle

Exercise training induces mitochondrial biogenesis, but the time course of molecular sequelae that accompany repetitive training stimuli remains to be determined in human skeletal muscle. Therefore, throughout a seven‐session, high‐intensity interval training period that increased (12%), we examined the time course of responses of (a) mitochondrial biogenesis and fusion and fission proteins, and (b) selected transcriptional and mitochondrial mRNAs and proteins in human muscle. Muscle biopsies were obtained 4 and 24 h after the 1st, 3rd, 5th and 7th training session. PGC‐1α mRNA was increased >10‐fold 4 h after the 1st session and returned to control within 24 h. This ‘saw‐tooth’ pattern continued until the 7th bout, with smaller increases after each bout. In contrast, PGC‐1α protein was increased 24 h after the 1st bout (23%) and plateaued at +30–40% between the 3rd and 7th bout. Increases in PGC‐1β mRNA and protein were more delayed and smaller, and did not persist. Distinct patterns of increases were observed in peroxisome proliferator‐activated receptor (PPAR) α and γ protein (1 session), PPAR β/δ mRNA and protein (5 sessions) and nuclear respiratory factor‐2 protein (3 sessions) while no changes occurred in mitochondrial transcription factor A protein. Citrate synthase (CS) and β‐HAD mRNA were rapidly increased (1 session), followed 2 sessions later (session 3) by increases in CS and β‐HAD activities, and mitochondrial DNA. Changes in COX‐IV mRNA (session 3) and protein (session 5) were more delayed. Training also increased mitochondrial fission proteins (fission protein‐1, >2‐fold; dynamin‐related protein‐1, 47%) and the fusion protein mitofusin‐1 (35%) but not mitofusin‐2. This study has provided the following novel information: (a) the training‐induced increases in transcriptional and mitochondrial proteins appear to result from the cumulative effects of transient bursts in their mRNAs, (b) training‐induced mitochondrial biogenesis appears to involve re‐modelling in addition to increased mitochondrial content, and (c) the ‘transcriptional capacity’ of human muscle is extremely sensitive, being activated by one training bout.

Modest PGC-1α Overexpression in Muscle in Vivo Is Sufficient to Increase Insulin Sensitivity and Palmitate Oxidation in Subsarcolemmal, Not Intermyofibrillar, Mitochondria

PGC-1α overexpression in skeletal muscle, in vivo, has yielded disappointing and unexpected effects, including disrupted cellular integrity and insulin resistance. These unanticipated results may stem from an excessive PGC-1α overexpression in transgenic animals. Therefore, we examined the effects of a modest PGC-1α overexpression in a single rat muscle, in vivo, on fuel-handling proteins and insulin sensitivity. We also examined whether modest PGC-1α overexpression selectively targeted subsarcolemmal (SS) mitochondrial proteins and fatty acid oxidation, because SS mitochondria are metabolically more plastic than intermyofibrillar (IMF) mitochondria. Among metabolically heterogeneous rat hindlimb muscles, PGC-1α was highly correlated with their oxidative fiber content and with substrate transport proteins (GLUT4, FABPpm, and FAT/CD36) and mitochondrial proteins (COXIV and mTFA) but not with insulin-signaling proteins (phosphatidylinositol 3-kinase, IRS-1, and Akt2), nor with 5′-AMP-activated protein kinase, α2 subunit, and HSL. Transfection of PGC-1α into the red (RTA) and white tibialis anterior (WTA) compartments of the tibialis anterior muscle increased PGC-1α protein by 23-25%. This also induced the up-regulation of transport proteins (FAT/CD36, 35-195%; GLUT4, 20-32%) and 5′-AMP-activated protein kinase, α2 subunit (37-48%), but not other proteins (FABPpm, IRS-1, phosphatidylinositol 3-kinase, Akt2, and HSL). SS and IMF mitochondrial proteins were also up-regulated, including COXIV (15-75%), FAT/CD36 (17-30%), and mTFA (15-85%). PGC-1α overexpression also increased palmitate oxidation in SS (RTA, +116%; WTA, +40%) but not in IMF mitochondria, and increased insulin-stimulated phosphorylation of AKT2 (28-43%) and rates of glucose transport (RTA, +20%; WTA, +38%). Thus, in skeletal muscle in vivo, a modest PGC-1α overexpression up-regulated selected plasmalemmal and mitochondrial fuel-handling proteins, increased SS (not IMF) mitochondrial fatty acid oxidation, and improved insulin sensitivity.

Greater Transport Efficiencies of the Membrane Fatty Acid Transporters FAT/CD36 and FATP4 Compared with FABPpm and FATP1 and Differential Effects on Fatty Acid Esterification and Oxidation in Rat Skeletal Muscle

In selected mammalian tissues, long chain fatty acid transporters (FABPpm, FAT/CD36, FATP1, and FATP4) are co-expressed. There is controversy as to whether they all function as membrane-bound transporters and whether they channel fatty acids to oxidation and/or esterification. Among skeletal muscles, the protein expression of FABPpm, FAT/CD36, and FATP4, but not FATP1, correlated highly with the capacities for oxidative metabolism (r ≥ 0.94), fatty acid oxidation (r ≥ 0.88), and triacylglycerol esterification (r ≥ 0.87). We overexpressed independently FABPpm, FAT/CD36, FATP1, and FATP4, within a normal physiologic range, in rat skeletal muscle, to determine the effects on fatty acid transport and metabolism. Independent overexpression of each fatty acid transporter occurred without altering either the expression or plasmalemmal content of other fatty acid transporters. All transporters increased fatty acid transport, but FAT/CD36 and FATP4 were 2.3- and 1.7-fold more effective than FABPpm and FATP1, respectively. Fatty acid transporters failed to alter the rates of fatty acid esterification into triacylglycerols. In contrast, all transporters increased the rates of long chain fatty acid oxidation, but the effects of FABPpm and FAT/CD36 were 3-fold greater than for FATP1 and FATP4. Thus, fatty acid transporters exhibit different capacities for fatty acid transport and metabolism. In vivo, FAT/CD36 and FATP4 are the most effective fatty acid transporters, whereas FABPpm and FAT/CD36 are key for stimulating fatty acid oxidation.

In obese rat muscle transport of palmitate is increased and is channeled to triacylglycerol storage despite an increase in mitochondrial palmitate oxidation

Intramuscular triacylglycerol (IMTG) accumulation in obesity has been attributed to increased fatty acid transport and/or to alterations in mitochondrial fatty acid oxidation. Alternatively, an imbalance in these two processes may channel fatty acids into storage. Therefore, in red and white muscles of lean and obese Zucker rats, we examined whether the increase in IMTG accumulation was attributable to an increased rate of fatty acid transport rather than alterations in subsarcolemmal (SS) or intermyofibrillar (IMF) mitochondrial fatty acid oxidation. In obese animals selected parameters were upregulated, including palmitate transport (red: +100%; white: +51%), plasmalemmal FAT/CD36 (red: +116%; white: +115%; not plasmalemmal FABPpm, FATP1, or FATP4), IMTG concentrations (red: approximately 2-fold; white: approximately 4-fold), and mitochondrial content (red +30%). Selected mitochondrial parameters were also greater in obese animals, namely, palmitate oxidation (SS red: +91%; SS white: +26%; not IMF mitochondria), FAT/CD36 (SS: +65%; IMF: +65%), citrate synthase (SS: +19%), and beta-hydroxyacyl-CoA dehydrogenase activities (SS: +20%); carnitine palmitoyltransferase-I activity did not differ. A comparison of lean and obese rat muscles revealed that the rate of change in IMTG concentration was eightfold greater than that of fatty acid oxidation (SS mitochondria), when both parameters were expressed relative to fatty transport. Thus fatty acid transport, esterification, and oxidation (SS mitochondria) are upregulated in muscles of obese Zucker rats, with these effects being most pronounced in red muscle. The additional fatty acid taken up is channeled primarily to esterification, suggesting that upregulation in fatty acid transport as opposed to altered fatty acid oxidation is the major determinant of intramuscular lipid accumulation.

The deacetylase enzyme SIRT1 is not associated with oxidative capacity in rat heart and skeletal muscle and its overexpression reduces mitochondrial biogenesis

Deacetylation of PGC‐1α by SIRT1 is thought to be an important step in increasing PGC‐1α transcriptional activity, since in muscle cell lines SIRT1 induces PGC‐1α protein expression and mitochondrial biogenesis. We examined the relationship between SIRT1 protein and activity, PGC‐1α and markers of mitochondrial density, (a) across a range of metabolically heterogeneous skeletal muscles and the heart, and when mitochondrial biogenesis was stimulated by (b) chronic muscle stimulation (7 days) and (c) AICAR administration (5 days), and finally, (d) we also examined the effects of SIRT1 overexpression on mitochondrial biogenesis and PGC‐1α. SIRT1 protein and activity were correlated (r= 0.97). There were negative correlations between SIRT1 protein and PGC‐1α (r=−0.95), COX IV (r=−0.94) and citrate synthase (r=−0.97). Chronic muscle stimulation and AICAR upregulated PGC‐1α protein (22–159%) and oxidative capacity (COX IV, 20–69%); in each instance SIRT1 protein was downregulated by 20–40%, while SIRT1 intrinsic activity was increased. SIRT1 overexpression in rodent muscle increased SIRT1 protein (+240%) and doubled SIRT1 activity, but PGC‐1α (−25%), mtTFA (−14%) and COX IV (−10%) proteins were downregulated. Taken altogether these experiments are not consistent with the notion that SIRT1 protein plays an obligatory regulatory role in the process of PGC‐1α‐mediated mitochondrial biogenesis in mammalian muscle.

PGC-1α increases skeletal muscle lactate uptake by increasing the expression of MCT1 but not MCT2 or MCT4

We examined the relationship between PGC-1alpha protein; the monocarboxylate transporters MCT1, 2, and 4; and CD147 1) among six metabolically heterogeneous rat muscles, 2) in chronically stimulated red (RTA) and white tibialis (WTA) muscles (7 days), and 3) in RTA and WTA muscles transfected with PGC-1alpha-pcDNA plasmid in vivo. Among rat hindlimb muscles, there was a strong positive association between PGC-1alpha and MCT1 and CD147, and between MCT1 and CD147. A negative association was found between PGC-1alpha and MCT4, and CD147 and MCT4, while there was no relationship between PGC-1alpha or CD147 and MCT2. Transfecting PGC-1alpha-pcDNA plasmid into muscle increased PGC-1alpha protein (RTA +23%; WTA +25%) and induced the expression of MCT1 (RTA +16%; WTA +28%), but not MCT2 and MCT4. As a result of the PGC-1alpha-induced upregulation of MCT1 and its chaperone CD147 (+29%), there was a concomitant increase in the rate of lactate uptake (+20%). In chronically stimulated muscles, the following proteins were upregulated, PGC-1alpha in RTA (+26%) and WTA (+86%), MCT1 in RTA (+61%) and WTA (+180%), and CD147 in WTA (+106%). In contrast, MCT4 protein expression was not altered in either RTA or WTA muscles, while MCT2 protein expression was reduced in both RTA (-14%) and WTA (-10%). In these studies, whether comparing oxidative capacities among muscles or increasing their oxidative capacities by PGC-1alpha transfection and chronic muscle stimulation, there was a strong relationship between the expression of PGC-1alpha and MCT1, and PGC-1alpha and CD147 proteins. Thus, MCT1 and CD147 belong to the family of metabolic genes whose expression is regulated by PGC-1alpha in skeletal muscle.

In Vivo, Fatty Acid Translocase (CD36) Critically Regulates Skeletal Muscle Fuel Selection, Exercise Performance, and Training-induced Adaptation of Fatty Acid Oxidation

Background: CD36-mediated lipid transport may regulate muscle fuel selection and adaptation. Results: CD36 ablation impaired fatty acid oxidation and prevented its exercise training-induced up-regulation. Without altering mitochondrial content, CD36 overexpression mimicked exercise training effects on fatty acid oxidation. Conclusion: CD36 contributes to regulating fatty acid oxidation and adaptation in a mitochondrion-independent manner. Significance: This work identified another mechanism regulating muscle fatty acid oxidation. For ∼40 years it has been widely accepted that (i) the exercise-induced increase in muscle fatty acid oxidation (FAO) is dependent on the increased delivery of circulating fatty acids, and (ii) exercise training-induced FAO up-regulation is largely attributable to muscle mitochondrial biogenesis. These long standing concepts were developed prior to the recent recognition that fatty acid entry into muscle occurs via a regulatable sarcolemmal CD36-mediated mechanism. We examined the role of CD36 in muscle fuel selection under basal conditions, during a metabolic challenge (exercise), and after exercise training. We also investigated whether CD36 overexpression, independent of mitochondrial changes, mimicked exercise training-induced FAO up-regulation. Under basal conditions CD36-KO versus WT mice displayed reduced fatty acid transport (−21%) and oxidation (−25%), intramuscular lipids (less than or equal to −31%), and hepatic glycogen (−20%); but muscle glycogen, VO2max, and mitochondrial content and enzymes did not differ. In acutely exercised (78% VO2max) CD36-KO mice, fatty acid transport (−41%), oxidation (−37%), and exercise duration (−44%) were reduced, whereas muscle and hepatic glycogen depletions were accelerated by 27–55%, revealing 2-fold greater carbohydrate use. Exercise training increased mtDNA and β-hydroxyacyl-CoA dehydrogenase similarly in WT and CD36-KO muscles, but FAO was increased only in WT muscle (+90%). Comparable CD36 increases, induced by exercise training (+44%) or by CD36 overexpression (+41%), increased FAO similarly (84–90%), either when mitochondrial biogenesis and FAO enzymes were up-regulated (exercise training) or when these were unaltered (CD36 overexpression). Thus, sarcolemmal CD36 has a key role in muscle fuel selection, exercise performance, and training-induced muscle FAO adaptation, challenging long held views of mechanisms involved in acute and adaptive regulation of muscle FAO.

Testosterone increases lactate transport, monocarboxylate transporter (MCT) 1 and MCT4 in rat skeletal muscle

We have examined the effects of administration of testosterone for 7 days on monocarboxylate transporter (MCT) 1 and MCT4 mRNAs and proteins in seven metabolically heterogeneous rat hindlimb muscles and in the heart. In addition, we also examined the effects of testosterone treatment on plasmalemmal MCT1 and MCT4, and lactate transport into giant sarcolemmal vesicles prepared from red and white hindlimb muscles and the heart. Testosterone did not alter MCT1 or MCT4 mRNA, except in the plantaris muscle. Testosterone increased MCT1 (20%–77%, P < 0.05) and MCT4 protein (29%–110%, P< 0.05) in five out of seven muscles examined. In contrast, in the heart MCT1 protein was not increased (P> 0.05), and MCT 4 mRNA and protein were not detected. There was no correlation between the testosterone‐induced increments in MCT1 and MCT4 proteins. Muscle fibre composition was not associated with testosterone‐induced increments in MCT1 protein. In contrast, there was a strong positive relationship between the testosterone‐induced increments in MCT4 protein and the fast‐twitch fibre composition of rat muscles. Lactate transport into giant sarcolemmal vesicles was increased in red (23%, P< 0.05) and white muscles (21%, P< 0.05), and in the heart (58%, P< 0.05) of testosterone‐treated animals (P< 0.05). However, plasmalemmal MCT1 protein (red, +40%, P< 0.05; white, +39%, P< 0.05) and plasmalemmal MCT4 protein (red, +25%, P< 0.05; white, +48%, P< 0.05) were increased only in skeletal muscle. In the heart, plasmalemmal MCT1 protein was reduced (−20%, P< 0.05). In conclusion, these studies have shown that testosterone induces an increase in both MCT1 and MCT4 proteins and their plasmalemmal content in skeletal muscle. However, the testosterone‐induced effect was tissue‐specific, as MCT1 protein expression was not altered in the heart. In the heart, the testosterone‐induced increase in lactate transport cannot be explained by changes in plasmalemmal MCT1 content, but in skeletal muscle the increase in the rate of lactate transport was associated with increases in plasmalemmal MCT1 and MCT4.

Treatment of nonalcoholic fatty liver disease: role of AMPK

Nonalcoholic fatty liver disease (NAFLD) is a growing worldwide epidemic and an important risk factor for the development of insulin resistance, type 2 diabetes, nonalcoholic steatohepatitis (NASH), and hepatic cellular carcinoma (HCC). Despite the prevalence of NAFLD, lifestyle interventions involving exercise and weight loss are the only accepted treatments for this disease. Over the last decade, numerous experimental compounds have been shown to improve NAFLD in preclinical animal models, and many of these therapeutics have been shown to increase the activity of the cellular energy sensor AMP-activated protein kinase (AMPK). Because AMPK activity is reduced by inflammation, obesity, and diabetes, increasing AMPK activity has been viewed as a viable therapeutic strategy to improve NAFLD. In this review, we propose three primary mechanisms by which AMPK activation may improve NAFLD. In addition, we examine the mechanisms by which AMPK is activated. Finally, we identify 27 studies that have used AMPK activators to reduce NAFLD. Future considerations for studies examining the relationship between AMPK and NAFLD are highlighted.

AMP‐activated protein kinase is required for exercise‐induced peroxisome proliferator‐activated receptor γ co‐activator 1α translocation to subsarcolemmal mitochondria in skeletal muscle

The transcriptional co‐activator peroxisome proliferator‐activated receptor γ co‐activator 1α (PGC‐1α), in concert with mitochondrial transcription factor A (Tfam), has been implicated in the direct regulation of the mitochondrial genome. In humans, rats and mice, acute exercise was found to promote PGC‐1α translocation to subsarcolemmal (SS) mitochondria. In rats, treatment with 5‐aminoimidazole‐4‐carboxamide‐1‐β‐ribofuranoside induced both PGC‐1α and Tfam translocation and in addition PGC‐1α and Tfam were found to co‐locolize with α‐tubulin. In mice, rendering AMP‐activated protein kinase (AMPK) inactive prevented PGC‐1α translocation. These data suggest that exercise causes translocation of PGC‐1α preferentially to SS mitochondria in an AMPK‐dependent manner.