Aim
The aim of this study was to investigate the effects of 4 consecutive simulated night shifts on glucose homeostasis, mitochondrial function and central and peripheral rhythmicities compared with ...a simulated day shift schedule.
Methods
Seventeen healthy adults (8M:9F) matched for sleep, physical activity and dietary/fat intake participated in this study (night shift work n = 9; day shift work n = 8). Glucose tolerance and insulin sensitivity before and after 4 nights of shift work were measured by an intravenous glucose tolerance test and a hyperinsulinaemic euglycaemic clamp respectively. Muscles biopsies were obtained to determine insulin signalling and mitochondrial function. Central and peripheral rhythmicities were assessed by measuring salivary melatonin and expression of circadian genes from hair samples respectively.
Results
Fasting plasma glucose increased (4.4 ± 0.1 vs. 4.6 ± 0.1 mmol L−1; P = .001) and insulin sensitivity decreased (25 ± 7%, P < .05) following the night shift, with no changes following the day shift. Night shift work had no effect on skeletal muscle protein expression (PGC1α, UCP3, TFAM and mitochondria Complex II‐V) or insulin‐stimulated pAkt Ser473, pTBC1D4Ser318 and pTBC1D4Thr642. Importantly, the metabolic changes after simulated night shifts occurred despite no changes in the timing of melatonin rhythmicity or hair follicle cell clock gene expression across the wake period (Per3, Per1, Nr1d1 and Nr1d2).
Conclusion
Only 4 days of simulated night shift work in healthy adults is sufficient to reduce insulin sensitivity which would be expected to increase the risk of T2D.
5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and caffeine, which activate AMP-activated protein kinase (AMPK) and cause sarcoplasmic reticulum calcium release, respectively, have been shown ...to increase mitochondrial biogenesis in L6 myotubes. Nitric oxide (NO) donors also increase mitochondrial biogenesis. Since neuronal and endothelial NO synthase (NOS) are calcium dependent and are also phosphorylated by AMPK, we hypothesized that NOS inhibition would attenuate the activation of mitochondrial biogenesis in response to AICAR and caffeine. L6 myotubes either were not treated (control) or were exposed acutely or for 5 h/day over 5 days to 100 microM of N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), 100 microM S-nitroso-N-acetyl-penicillamine (SNAP) (NO donor) +/- 100 microM L-NAME, 2 mM AICAR +/- 100 microM L-NAME, or 5 mM caffeine +/- 100 microM L-NAME (n = 12/treatment). Acute AICAR administration increased (P < 0.05) phospho- (P-)AMPK, but also increased P-CaMK, with resultant chronic increases in peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), cytochrome-c oxidase (COX)-1, and COX-4 protein expression compared with control cells. NOS inhibition, which had no effect on AICAR-stimulated P-AMPK, surprisingly increased P-CaMK and attenuated the AICAR-induced increases in COX-1 and COX-4 protein. Caffeine administration, which increased P-CaMK without affecting P-AMPK, increased COX-1, COX-4, PGC-1 alpha, and citrate synthase activity. NOS inhibition, surprisingly, greatly attenuated the effect of caffeine on P-CaMK and attenuated the increases in COX-1 and COX-4 protein. SNAP increased all markers of mitochondrial biogenesis, and it also increased P-AMPK and P-CaMK. In conclusion, AICAR and caffeine increase mitochondrial biogenesis in L6 myotubes, at least in part, via interactions with NOS.
The aim of this research was to examine the impact of the xanthine oxidase (XO) inhibitor allopurinol on the skeletal muscle activation of cell signaling kinases' and adaptations to mitochondrial ...proteins and antioxidant enzymes following acute endurance exercise and endurance training. Male Sprague-Dawley rats performed either acute exercise (60 min of treadmill running, 27 m/min, 5% incline) or 6 wk of endurance training (5 days/wk) while receiving allopurinol or vehicle. Allopurinol treatment reduced XO activity to 5% of the basal levels (P < 0.05), with skeletal muscle uric acid levels being almost undetectable. Following acute exercise, skeletal muscle oxidized glutathione (GSSG) significantly increased in allopurinol- and vehicle-treated groups despite XO activity and uric acid levels being unaltered by acute exercise (P < 0.05). This suggests that the source of ROS was not from XO. Surprisingly, muscle GSSG levels were significantly increased following allopurinol treatment. Following acute exercise, allopurinol treatment prevented the increase in p38 MAPK and ERK phosphorylation and attenuated the increase in mitochondrial transcription factor A (mtTFA) mRNA (P < 0.05) but had no effect on the increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor-2, GLUT4, or superoxide dismutase mRNA. Allopurinol also had no impact on the endurance training-induced increases in PGC-1α, mtTFA, and mitochondrial proteins including cytochrome c, citrate synthase, and β-hydroxyacyl-CoA dehydrogenase. In conclusion, although allopurinol inhibits cell signaling pathways in response to acute exercise, the inhibitory effects of allopurinol appear unrelated to exercise-induced ROS production by XO. Allopurinol also has little effect on increases in mitochondrial proteins following endurance training.
Departments of Physiology, 1 The University of Melbourne, Parkville, Victoria; 2 Monash University, Clayton, Victoria; 3 Naresuan University, Phitsanulok, Thailand; and 4 St. Vincent's Institute and ...Commonwealth Scientific and Industrial Research Organisation Molecular and Health Sciences, Fitzroy, Victoria, Australia
Submitted 23 September 2005
; accepted in final form 26 October 2005
We compared in human skeletal muscle the effect of absolute vs. relative exercise intensity on AMP-activated protein kinase (AMPK) signaling and substrate metabolism under normoxic and hypoxic conditions. Eight untrained males cycled for 30 min under hypoxic conditions (11.5% O 2 , 111 ± 12 W, 72 ± 3% hypoxia O 2 peak ; 72% Hypoxia) or under normoxic conditions (20.9% O 2 ) matched to the same absolute (111 ± 12 W, 51 ± 1% normoxia O 2 peak ; 51% Normoxia) or relative (to O 2 peak ) intensity (171 ± 18 W, 73 ± 1% normoxia O 2 peak ; 73% Normoxia). Increases ( P < 0.05) in AMPK activity, AMPK Thr 172 phosphorylation, ACC Ser 221 phosphorylation, free AMP content, and glucose clearance were more influenced by the absolute than by the relative exercise intensity, being greatest in 73% Normoxia with no difference between 51% Normoxia and 72% Hypoxia. In contrast to this, increases in muscle glycogen use, muscle lactate content, and plasma catecholamine concentration were more influenced by the relative than by the absolute exercise intensity, being similar in 72% Hypoxia and 73% Normoxia, with both trials higher than in 51% Normoxia. In conclusion, increases in muscle AMPK signaling, free AMP content, and glucose disposal during exercise are largely determined by the absolute exercise intensity, whereas increases in plasma catecholamine levels, muscle glycogen use, and muscle lactate levels are more closely associated with the relative exercise intensity.
metabolic regulation; glucose kinetics; contraction
Address for reprint requests and other correspondence: G. McConell, Dept. of Physiology, Univ. of Melbourne, Parkville, 3010 Australia (e-mail: mcconell{at}unimelb.edu.au )
Reduced activation of exercise responsive signalling pathways have been reported in response to acute exercise after training; however little is known about the adaptive responses of the ...mitochondria. Accordingly, we investigated changes in mitochondrial gene expression and protein abundance in response to the same acute exercise before and after 10-d of intensive cycle training. Nine untrained, healthy participants (mean±SD; VO(2peak) 44.1±17.6 ml/kg/min) performed a 60 min bout of cycling exercise at 164±18 W (72% of pre-training VO(2peak)). Muscle biopsies were obtained from the vastus lateralis muscle at rest, immediately and 3 h after exercise. The participants then underwent 10-d of cycle training which included four high-intensity interval training sessions (6×5 min; 90-100% VO(2peak)) and six prolonged moderate-intensity sessions (45-90 min; 75% VO(2peak)). Participants repeated the pre-training exercise trial at the same absolute work load (64% of pre-training VO(2peak)). Muscle PGC1-α mRNA expression was attenuated as it increased by 11- and 4- fold (P<0.001) after exercise pre- and post-training, respectively. PGC1-α protein expression increased 1.5 fold (P<0.05) in response to exercise pre-training with no further increases after the post-training exercise bout. RIP140 protein abundance was responsive to acute exercise only (P<0.01). COXIV mRNA (1.6 fold; P<0.01) and COXIV protein expression (1.5 fold; P<0.05) were increased by training but COXIV protein expression was decreased (20%; P<0.01) by acute exercise pre- and post-training. These findings demonstrate that short-term intensified training promotes increased mitochondrial gene expression and protein abundance. Furthermore, acute indicators of exercise-induced mitochondrial adaptation appear to be blunted in response to exercise at the same absolute intensity following short-term training.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Key points
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Reactive oxygen‐based molecules generated within muscle fibres in both exercise and pathological conditions can greatly affect muscle function. These and consequent reactions can lead ...to either decreased or increased force response by the contractile proteins, but the mechanisms are unknown.
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This study demonstrates that the increase in force response appears to be due to a specific chemical process, known as S‐glutathionylation, of a particular cysteine residue present on the troponin I molecule in fast‐twitch muscle fibres, which is involved in sensing and responding to changes in intracellular calcium levels.
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S‐Glutathionylation can occur when glutathione, the primary cellular anti‐oxidant, reacts with oxidized cysteine residues.
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S‐Glutathionylation of troponin I not only helps protect the molecule from oxidative stress, but evidently also makes the contractile apparatus much more sensitive to calcium ions.
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This process seemingly occurs in exercising humans and is likely to be an important mechanism helping delay onset of muscle fatigue.
Oxidation can decrease or increase the Ca2+ sensitivity of the contractile apparatus in rodent fast‐twitch (type II) skeletal muscle fibres, but the reactions and molecular targets involved are unknown. This study examined whether increased Ca2+ sensitivity is due to S‐glutathionylation of particular cysteine residues. Skinned muscle fibres were directly activated in heavily buffered Ca2+ solutions to assess contractile apparatus Ca2+ sensitivity. Rat type II fibres were subjected to S‐glutathionylation by successive treatments with 2,2′‐dithiodipyridine (DTDP) and glutathione (GSH), and displayed a maximal increase in pCa50 (−log10Ca2+ at half‐maximal force) of ∼0.24 pCa units, with little or no effect on maximum force or Hill coefficient. Partial similar effect was produced by exposure to oxidized gluthathione (GSSG, 10 mm) for 10 min at pH 7.1, and near‐maximal effect by GSSG treatment at pH 8.5. None of these treatments significantly altered Ca2+ sensitivity in rat type I fibres. Western blotting showed that both the DTDP–GSH and GSSG–pH 8.5 treatments caused marked S‐glutathionylation of the fast troponin I isoform (TnIf) present in type II fibres, but not of troponin C (TnC) or myosin light chain 2. Both the increased Ca2+ sensitivity and glutathionylation of TnIf were blocked by N‐ethylmaleimide (NEM). S‐Nitrosoglutathione (GSNO) also increased Ca2+ sensitivity, but only in conditions where it caused S‐glutathionylation of TnIf. In human type II fibres from vastus lateralis muscle, DTDP–GSH treatment also caused similar increased Ca2+ sensitivity and S‐glutathionylation of TnIf. When the slow isoform of TnI in type I fibres of rat was partially substituted (∼30%) with TnIf, DTDP–GSH treatment caused a significant increase in Ca2+ sensitivity (∼0.08 pCa units). TnIf in type II fibres from toad and chicken muscle lack Cys133 present in mammalian TnIf, and such fibres showed no change in Ca2+ sensitivity with DTDP–GSH nor any S‐glutathionylation of TnIf (latter examined only in toad). Following 40 min of cycling exercise in human subjects (at ∼60% peak oxygen consumption), TnIf in vastus lateralis muscle displayed a marked increase in S‐glutathionylation (∼4‐fold). These findings show that S‐glutathionylation of TnIf, most probably at Cys133, increases the Ca2+ sensitivity of the contractile apparatus, and that this occurs in exercising humans, with likely beneficial effects on performance.
Department of Physiology, The University of Melbourne, Parkville, Victoria, Australia
Submitted 15 May 2006
; accepted in final form 2 August 2006
The purpose of this study was to determine whether ...nitric oxide synthase (NOS) inhibition decreased basal and exercise-induced skeletal muscle mitochondrial biogenesis. Male Sprague-Dawley rats were assigned to one of four treatment groups: NOS inhibitor N G -nitro- L -arginine methyl ester ( L -NAME, ingested for 2 days in drinking water, 1 mg/ml) followed by acute exercise, no L -NAME ingestion and acute exercise, rest plus L -NAME, and rest without L -NAME. The exercised rats ran on a treadmill for 53 ± 2 min and were then killed 4 h later. NOS inhibition significantly ( P < 0.05; main effect) decreased basal peroxisome proliferator-activated receptor- coactivator 1 (PGC-1 ) mRNA levels and tended ( P = 0.08) to decrease mtTFA mRNA levels in the soleus, but not the extensor digitorum longus (EDL) muscle. This coincided with significantly reduced basal levels of cytochrome c oxidase (COX) I and COX IV mRNA, COX IV protein and COX enzyme activity following NOS inhibition in the soleus, but not the EDL muscle. NOS inhibition had no effect on citrate synthase or -hydroxyacyl CoA dehydrogenase activity, or cytochrome c protein abundance in the soleus or EDL. NOS inhibition did not reduce the exercise-induced increase in peroxisome proliferator-activated receptor- coactivator 1 (PGC-1 ) mRNA in the soleus or EDL. In conclusion, inhibition of NOS appears to decrease some aspects of the mitochondrial respiratory chain in the soleus under basal conditions, but does not attenuate exercise-induced mitochondrial biogenesis in the soleus or in the EDL.
contraction; metabolic regulation; peroxisome proliferator-activated receptor- coactivator 1
Address for reprint requests and other correspondence: G. Wadley, Dept. of Physiology, The Univ. of Melbourne, Parkville 3010, Australia (e-mail: gdwadley{at}unimelb.edu.au )
Nitric oxide (NO) appears to play a role in contraction-stimulated glucose uptake in isolated rodent skeletal muscle; however, no studies have examined this question in humans. Seven healthy men ...completed two 30-min bouts of supine cycling exercise at 60 +/- 2% peak pulmonary oxygen uptake (VO2 peak), separated by 90 min of rest. The NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA; total dose 5 mg/kg body weight) or saline (control) were administered via the femoral artery for the final 20 min of exercise in a randomized blinded crossover design. L-Arginine (5 mg/kg body weight) was co-infused during the final 5 min of each exercise bout. Leg blood flow (LBF) was measured by thermodilution in the femoral vein, and leg glucose uptake was calculated as the product of LBF and femoral arteriovenous (AV) glucose difference. L-NMMA infusion significantly (P < 0.05) reduced leg glucose uptake compared with control (48 +/- 12% lower at 15 min, mean +/- SE). The reduction in glucose uptake was due solely to a decrease in AV glucose difference, as there was no effect of L-NMMA infusion on LBF during exercise. Co-infusion of L-arginine restored glucose uptake during L-NMMA infusion to levels similar to control. These results indicate that NO production contributes substantially to exercise-mediated skeletal muscle glucose uptake in humans independent of skeletal muscle blood flow.
Nitric oxide is a potential regulator of mitochondrial biogenesis. Therefore, we investigated if mice deficient in endothelial
nitric oxide synthase (eNOS â/â ) or neuronal NOS (nNOS â/â ) ...have attenuated activation of skeletal muscle mitochondrial biogenesis in response to exercise. eNOS â/â , nNOS â/â and C57Bl/6 (CON) mice (16.3 ± 0.2 weeks old) either remained in their cages (basal) or ran on a treadmill (16 m min â1 , 5% grade) for 60 min ( n = 8 per group) and were killed 6 h after exercise. Other eNOS â/â , nNOS â/â and CON mice exercise trained for 9 days (60 min per day) and were killed 24 h after the last bout of exercise training.
eNOS â/â mice had significantly higher nNOS protein and nNOS â/â mice had significantly higher eNOS protein in the EDL, but not the soleus. The basal mitochondrial biogenesis markers NRF1,
NRF2α and mtTFA mRNA were significantly ( P < 0.05) higher in the soleus and EDL of nNOS â/â mice whilst basal citrate synthase activity was higher in the soleus and basal PGC-1α mRNA higher in the EDL. Also, eNOS â/â mice had significantly higher basal citrate synthase activity in the soleus but not the EDL. Acute exercise increased ( P < 0.05) PGC-1α mRNA in soleus and EDL and NRF2α mRNA in the EDL to a similar extent in all genotypes. In addition, short-term
exercise training significantly increased cytochrome c protein in all genotypes ( P < 0.05) in the EDL. In conclusion, eNOS and nNOS are differentially involved in the basal regulation of mitochondrial biogenesis
in skeletal muscle but are not critical for exercise-induced increases in mitochondrial biogenesis in skeletal muscle.
High doses of the antioxidant vitamin C prevent the increases in skeletal muscle mitochondrial biogenesis after exercise training. Since exercise training effects rely on the acute stimulus of each ...exercise bout, we examined whether vitamin C supplementation also attenuates the increases in skeletal muscle metabolic signaling and mitochondrial biogenesis in response to an acute exercise bout. Male Sprague-Dawley rats performed 60 min of treadmill running (27 m/min, 5% grade) or remained sedentary. For 7 days before this, one-half of the rats received water containing 500 mg/kg body wt vitamin C. Acute exercise significantly (P<0.05) increased the phosphorylation of p38 MAPK, AMP-activated kinase-alpha, and activating transcription factor (ATF)-2 and the ratio of oxidized to total glutathione (GSSG/TGSH) in the gastrocnemius. However, vitamin C had no effect on these increases. Similarly, vitamin C did not prevent the exercise-induced increases in peroxisome proliferator-activated receptor-gamma coactivator-1alpha, nuclear respiratory factor (NRF)-1, NRF-2, mitochondrial transcription factor A, glutathione peroxidase-1, MnSOD, extracellular SOD, or glucose transporter 4 (P<0.05) mRNA after exercise. Surprisingly, vitamin C supplementation significantly increased the basal levels of GSSG/TGSH, NRF-1, and NRF-2 mRNA and basal ATF-2 phosphorylation. In summary, despite other studies in rats showing that vitamin C supplementation prevents increases in skeletal muscle mitochondrial biogenesis and antioxidant enzymes with exercise training, vitamin C had no affect on the acute exercise-induced increases of these markers.
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