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Regulation of contraction-induced FA uptake and oxidation by AMPK and ERK1/2 is intensity dependent in rodent muscle
Abstract
Muscle contraction activates AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK1/2), two signaling molecules involved in the regulation of muscle metabolism. The purpose of this study was to determine whether activation of AMPK and/or ERK1/2 contributes to the regulation of muscle fatty acid (FA) uptake and oxidation in contracting muscle. Rat hindquarters were perfused during rest (R) or electrical stimulation (E) of increasing intensity by manipulating train duration (E1 = 25 ms, E2 = 50 ms, E3 = 100 ms, E4 = 200 ms). For matched FA delivery, FA uptake was significantly greater than R during E1, E2, and E3 (7.8 +/- 0.7 vs. 14.4 +/- 0.3, 16.9 +/- 0.8, 15.2 +/- 0.5 nmol.min(-1).g(-1), respectively, P < 0.05), but not during E4 (8.3 +/- 0.3 nmol.min(-1).g(-1), P > 0.05). FA oxidation was significantly greater than R during E1 and E2 (1.5 +/- 0.1 vs. 2.3 +/- 0.2, 2.5 +/- 0.2 nmol.min(-1).g(-1), P < 0.05) before returning to resting levels for E3 and E4 (1.8 +/- 0.1 and 1.5 +/- 0.2 nmol.min(-1).g(-1), P > 0.05). A positive correlation was found between FA uptake and ERK1/2 phosphorylation from R to E3 (R(2) = 0.55, P < 0.05) and between FA oxidation and ERK1/2 phosphorylation from R to E2 (R(2) = 0.76, P < 0.05), correlations that were not maintained when the data for E4 and E3 and E4, respectively, were included in the analysis (R(2) = 0.04 and R(2) = 0.03, P > 0.05). A positive correlation was also found between FA uptake and FA oxidation and AMPK activity for all exercise intensities (R(2) = 0.57, R(2) = 0.65 respectively, P < 0.05). These results, in combination with previous data from our laboratory, suggest that ERK1/2 and AMPK are the predominant signaling molecules regulating FA uptake and oxidation during low- to moderate-intensity muscle contraction and during moderate- to high-intensity muscle contraction, respectively.
... The utilization of fatty acids (FAs) as fuel sources increases significantly during muscle contraction or exercise. Multiple studies have shown that the rates of FA uptake and oxidation during exercise and muscle contraction can increase up to 3 or 4 times the basal rates (Romijn et al. 1993;Dyck and Bonen 1998;Turcotte et al. 1998;Bergman et al. 1999;Bonen et al. 2000;van Loon et al. 2001;Chen et al. 2003;Raney and Turcotte 2006;Helge et al. 2007). Studies have also shown that intramuscular triacylglycerol (IMTG) contributes to total energy expenditure during acute muscle contraction or exercise (Romijn et al. 1993;Donsmark et al. 2004;Roepstorff et al. 2004aRoepstorff et al. , 2005bWatt et al. 2004Watt et al. , 2006Prats et al. 2006). ...
... However, the relationship between contraction intensity and FA uptake and oxidation during muscle contraction or exercise is not linear in nature. Multiple studies in humans performing cycling or knee extension exercises, and in isolated rodent muscle preparations, have determined that FA uptake and oxidation are generally higher during low (25%-40% V O 2 peak) to moderate (40%-65% V O 2 peak) intensity muscle contraction than at rest or during muscle contraction of higher intensity (75%-85% V O 2 peak) (Turcotte et al. 1992;Romijn et al. 1993;van Loon et al. 2001;Chen et al. 2003;Raney and Turcotte 2006;Helge et al. 2007). For example, in trained and untrained subjects performing bicycling exercise, the contribution of FA to total energy expenditure calculated from tracer and indirect calorimetry data decreases from low-intensity (25%-40% V O 2 peak) and moderate-intensity (55%-65% V O 2 peak) exercise, to highintensity (75%-85% V O 2 peak) exercise (Romijn et al. 1993;van Loon et al. 2001;Chen et al. 2003). ...
... Conversely, in subjects performing graded knee extension exercise in which thigh FA uptake does not increase from low (25% maximal power), to moderate (65% maximal power), or high (85% maximal power) intensity exercise, thigh plasma FA oxidation increases with an increase in exercise intensity (Helge et al. 2007). In rat hindquarters perfused with matched FA delivery during rest or electrical stimulation of increasing intensity, both FA uptake and oxidation increase from rest to muscle contraction of low and moderate intensity, and decrease during muscle contraction of the highest intensity (Raney and Turcotte 2006). Taken together, these data suggest that molecular signaling events must be present within the muscle cells to regulate the magnitude and direction of the independent shifts in FA uptake and oxidation that occur during exercise or acute muscle contraction. ...
The regulation of fatty acid utilization during muscle contraction and exercise remains to be fully elucidated. Evidence suggests that the metabolic responses of skeletal muscle induced by the contraction-induced changes in energy demand are mediated by the activation of a multitude of intracellular signaling cascades. This review addresses the roles played by 3 intracellular signaling cascades of interest in the regulation of fatty acid uptake and oxidation in contracting skeletal muscle; namely, the AMP-activated protein kinase (AMPK), calcium/calmodulin-dependent protein kinases (CaMKs), and the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling cascades. Data delineating the potential role of AMPK in cross-talk with CaMKII, CaMK kinase (CaMKK), and ERK1/2 are presented. Collectively, data show that in perfused rodent muscle, regulation of fatty acid uptake and oxidation occurs via (i) CaMKII signaling via both AMPK-dependent and -independent cascades, (ii) CaMKK signaling via both AMPK-dependent and -independent cascades, (iii) AMPK signaling in a time- and intensity-dependent manner, and (iv) ERK1/2 signaling in an intensity-dependent manner.
... EVIDENCE SUGGESTS THAT MULTIPLE cellular signals regulate the changes in substrate metabolism with muscle contraction, including the AMP-activated protein kinase (AMPK) signaling cascade (2,34,35,52). During states of low energy, such as muscle contraction or exercise, it is widely accepted that liver kinase B1 (LKB1) acts as an upstream AMPK kinase that phosphorylates and activates AMPK and initiates a multitude of signaling events to maintain energy homeostasis (39 -41). ...
... The length of the GSP muscle group was adjusted at the initiation of electrical stimulation to obtain maximal active tension, and the GSP muscle group was connected to a Grass stimulator (model S48, Grass Telefactor, West Warwick, RI) to induce isometric muscle contractions via delivery of 15-V trains of 100 Hz lasting 50 ms with impulse duration of 1 ms with 30 trains/min. This moderate-intensity protocol has been shown to maximize FA metabolism (34). ...
... Muscle sample preparation. For Western blot analysis, frozen GSP muscle samples (40 mg) were powdered under liquid N2 and homogenized in 500 l of ice-cold RIPA buffer, as previously described (34,36). The total cell homogenate was then transferred to a microcentrifuge tube and vortexed frequently for 1 h, whereupon the samples were centrifuged at 4,500 g at 4°C for 1 h. ...
AMP-activated protein kinase (AMPK) is a fuel sensor in skeletal muscle with multiple downstream signaling targets that may be triggered by increases in intracellular Ca(2+) concentration ([Ca(2+)]). The purpose of this study was to determine whether increases in intracellular [Ca(2+)] induced by caffeine act solely via AMPKα(2) and whether AMPKα(2) is essential to increase glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle. Hindlimbs from wild-type (WT) or AMPKα(2) dominant-negative (DN) transgene mice were perfused during rest (n = 11), treatment with 3 mM caffeine (n = 10), or muscle contraction (n = 11). Time-dependent effects on glucose and FA uptake were uncovered throughout the 20-min muscle contraction perfusion period (P < 0.05). Glucose uptake rates did not increase in DN mice during muscle contraction until the last 5 min of the protocol (P < 0.05). FA uptake rates were elevated at the onset of muscle contraction and diminished by the end of the protocol in DN mice (P < 0.05). FA oxidation rates were abolished in the DN mice during muscle contraction (P < 0.05). The DN transgene had no effect on caffeine-induced FA uptake and oxidation (P > 0.05). Glucose uptake rates were blunted in caffeine-treated DN mice (P < 0.05). The DN transgene resulted in a greater use of intramuscular triglycerides as a fuel source during muscle contraction. The DN transgene did not alter caffeine- or contraction-mediated changes in the phosphorylation of Ca(2+)/calmodulin-dependent protein kinase I or ERK1/2 (P > 0.05). These data suggest that AMPKα(2) is involved in the regulation of substrate uptake in a time-dependent manner in contracting muscle but is not necessary for regulation of FA uptake and oxidation during caffeine treatment.
... source for skeletal muscle at rest and during muscle contraction, when FA uptake and oxidation rates can increase up to three to four times the basal rates (43,47). Evidence suggests that multiple cellular signals regulate this increase in FA metabolism with muscle contraction, including the AMP-activated protein kinase (AMPK) signaling cascade (6,36,37,53). Although strong evidence indicates that AMPK is a key signaling intermediate that regulates changes in substrate use in contracting skeletal muscle (13,26,55,59), data also show that AMPK may not be the sole signal mediating contractioninduced changes in substrate uptake and FA oxidation (37,38). ...
... Standard rat chow and water were provided ad libitum to the rats. Animals were randomly divided into four experimental groups: rest (R, n ϭ 16), caffeine (C, n ϭ 14), AICAR (A, n ϭ 16), and muscle contraction (MC, n ϭ 14) of moderate intensity (23,36). Each group of animals was further divided into treatment with dimethyl sulfoxide (DMSO) as control vehicle or with STO-609 (Calbiochem, Gibbstown, NJ), an inhibitor of CaMKK (41), dissolved in DMSO. ...
... A S48 Grass stimulator (Grass Telefactor, West Warwick, RI) was used to induce isometric muscle contractions via delivery of 15-V trains of 100 Hz lasting 50 ms with impulse duration of 1 ms. This moderate-intensity protocol has been shown to maximize FA metabolism (36). A modular chart recorder (Cole Parmer, Vernon Hills, IL) was used to measure the tension developed by the gastrocnemius-soleus-plantaris muscle group during the 20-min muscle stimulation protocol. ...
Multiple signals have been shown to be involved in regulation of fatty acid (FA) and glucose metabolism in contracting skeletal muscle. This study aimed to determine whether a Ca(2+)-stimulated kinase, CaMKK, is involved in regulation of contraction-induced substrate metabolism and whether it does so in an AMP-activated protein kinase (AMPK)-dependent manner. Rat hindlimbs were perfused at rest (n = 16), with 3 mM caffeine (n = 15), with 2 mM 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR; n = 16), or during moderate-intensity muscle contraction (MC; n = 14) and with or without 5 microM STO-609, a CaMKK inhibitor. FA uptake and oxidation increased (P < 0.05) 64% and 71% by caffeine, 42% and 93% by AICAR, and 65% and 143% by MC. STO-609 abolished (P < 0.05) caffeine- and MC-induced FA uptake and oxidation but had no effect with AICAR treatment. Glucose uptake increased (P < 0.05) 104% by caffeine, 85% by AICAR, and 130% by MC, and STO-609 prevented the increase in glucose uptake in caffeine and muscle contraction groups. CaMKKbeta activity increased (P < 0.05) 113% by caffeine treatment and 145% by MC but was not affected by AICAR treatment. STO-609 prevented the caffeine- and MC-induced increase in CaMKKbeta activity. Caffeine, AICAR, and MC increased (P < 0.05) AMPKalpha2 activity by 295%, 11-fold, and 7-fold but did not affect AMPKalpha1 activity. STO-609 decreased (P < 0.05) AMPKalpha2 activity induced by caffeine treatment and MC by 60% and 61% but did not affect AICAR-induced activity. Plasma membrane transport protein content of CD36 and glucose transporter 4 (GLUT4) increased (P < 0.05) with caffeine, AICAR, and MC, and STO-609 prevented caffeine- and MC-induced increases in protein content. These results show the importance of Ca(2+)-dependent signaling via CaMKK activation in the regulation of substrate uptake and FA oxidation in contracting rat skeletal muscle and agree with the notion that CaMKK is an upstream kinase of AMPK in the regulation of substrate metabolism in skeletal muscle.
... However, it wasn't until just recently that potential signaling mechanisms involved in the regulation of contraction-induced FA uptake and oxidation were identified. Our lab and others have provided evidence suggesting that AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK1/2) may be involved in the regulation of contractioninduced FA uptake and oxidation in skeletal muscle (19,20,30,35). Despite these conclusions, the regulation of contraction-induced FA uptake and oxidation is still not completely understood. ...
... Isometric muscle contractions were induced by stimulating the sciatic nerve electrically with supramaximal 15-V trains of 100 Hz with impulse duration of 1 ms, delivered every 2 s and lasting 50 ms, which is considered moderate-intensity muscle contraction (12). We chose this train duration because a previous study in our lab demonstrated that FA metabolism is maximized during this moderate-intensity stimulation protocol (19). During the 20-min muscle stimulation, the tension developed by the gastrocnemius-soleus-plantaris muscle group was recorded with a modular chart recorder (Cole Parmer, Vernon Hills, IL). ...
... The supernatant was used to determine total AMPK activity, while isoform-specific AMPK and CaMKII activity was determined in immunoprecipitates from 200 -400 g of supernatant protein after overnight incubation at 4°C with ϳ1.5 g of affinity-purified isoform-specific goat IgG against ␣2-AMPK and CaMKII, respectively, in 20 -40 l of protein A/G-agarose beads (Santa Cruz Biotechnology). 32 P-ATP incorporation into SAMS peptide was used to measure total AMPK and ␣2-AMPK activity from the respective preparations (19,35). We have shown previously that the moderate-intensity muscle contraction protocol used in the current study does not significantly increase ␣1-AMPK activity (19), and therefore ␣1-AMPK activity was not determined. ...
Calcium-calmodulin/dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK1/2) have each been implicated in the regulation of substrate metabolism during exercise. The purpose of this study was to determine whether CaMKII is involved in the regulation of FA uptake and oxidation and, if it is involved, whether it does so independently of AMPK and ERK1/2. Rat hindquarters were perfused at rest with (n = 16) or without (n = 10) 3 mM caffeine, or during electrical stimulation (n = 14). For each condition, rats were subdivided and treated with 10 muM of either KN92 or KN93, inactive and active CaMKII inhibitors, respectively. Both caffeine treatment and electrical stimulation significantly increased FA uptake and oxidation. KN93 abolished caffeine-induced FA uptake, decreased contraction-induced FA uptake by 33%, and abolished both caffeine- and contraction-induced FA oxidation (P < 0.05). Caffeine had no effect on ERK1/2 phosphorylation (P > 0.05) and increased alpha(2)-AMPK activity by 68% (P < 0.05). Electrical stimulation increased ERK1/2 phosphorylation and alpha(2)-AMPK activity by 51% and 3.4-fold, respectively (P < 0.05). KN93 had no effect on caffeine-induced alpha(2)-AMPK activity, ERK1/2 phosphorylation, or contraction-induced ERK1/2 phosphorylation (P > 0.05). Alternatively, it decreased contraction-induced alpha(2)-AMPK activity by 51% (P < 0.05), suggesting that CaMKII lies upstream of AMPK. These results demonstrate that regulation of contraction-induced FA uptake and oxidation occurs in part via Ca(2+)-independent activation of ERK1/2 as well as Ca(2+)-dependent activation of CaMKII and AMPK.
... Comme indiqué plus haut, une accumulation d'AG intramyocellulaire a été observée dans le muscle vastus lateralis humain lors de l'augmentation de l'intensité de l'exercice de 65% à 90% VO 2 max malgré une diminution de [AG] P (Kiens et al., 1999), ce qui suggère que le transport à travers le sarcolemme ne limiterait pas l'oxydation des AG à haute intensité d'exercice. Des résultats obtenus sur le modèle du muscle isolé perfusé de rat (Raney & Turcotte, 2006) ont révélé une relation entre la captation des AG et leur oxydation, mais seulement à des intensités de contraction faibles ou modérées. Lorsque l'intensité s'accroît encore, la captation des AG reste supérieure à celle des niveaux de base alors que l'OxAG diminue jusqu'au niveau du repos (Raney & Turcotte, 2006), ce qui confirme que le transport des AG à travers la membrane n'est pas un facteur limitant pour l'OxAG lors du passage à l'exercice intense. ...
... Des résultats obtenus sur le modèle du muscle isolé perfusé de rat (Raney & Turcotte, 2006) ont révélé une relation entre la captation des AG et leur oxydation, mais seulement à des intensités de contraction faibles ou modérées. Lorsque l'intensité s'accroît encore, la captation des AG reste supérieure à celle des niveaux de base alors que l'OxAG diminue jusqu'au niveau du repos (Raney & Turcotte, 2006), ce qui confirme que le transport des AG à travers la membrane n'est pas un facteur limitant pour l'OxAG lors du passage à l'exercice intense. ...
Lipid metabolism is involved during muscle exercise. Energetic contribution of lipids increases during long lasting endurance exercise of moderate intensity (40% à 60% of VO2max). As well as circulating free fatty acids, intramyocellular lipid storages (IMCL) are postulated to be used during performances longer than 4 hours. Due the the lack experimental evidences untill today, a first study was undertaken on 10 athletes (40 ± 6 yrs) during a 24h running. Results obtained on vastus lateralis muscle showed a significant 56% and 45% decrease of IMCL in type I and IIA fibres respectively while glycogen decreased only in type I fibres. These data indicate a more efficient catabolism of IMCL than those of glycogen in fast twitch fibres during ultra endurance exercise, of which mechanism remains to be explored. IMCL accumulates during ageing or overweighting and may constitute a risk of insulin resistance (IR). A combined 14 weeks endurance (ET) and resistance (RT) training was followed by older (73 ± 6 yrs) and overweighted (58 ± 5 yrs) subjects. In the two groups IMCL increased (p<0.05) in vastus lateralis muscle (after ET) but remained stable in deltoidus muscle (after RT) and was linked to an increase (p<0.05) of β-oxydation enzymatic capacity after ET. Muscle ceramides, a category of lipids implicated in IR, decreased (p=0.052) after ET and not after RT. These results confirm that increase in IMCL is not a metabolic risk factor and that ET induces a decrease of both ceramides and IR
... However, it is possible that AMPK is located upstream of ERK 1/2 and therefore would not be affected by the inhibition of ERK 1/2. Nonetheless, in subsequent studies Raney and colleagues provided evidence that both ERK1/2 and AMPK play a significant role in fatty acid uptake and oxidation during muscle contraction [20,21]. It seems that in low to moderate exercise intensities, ERK1/2 plays a more critical role than AMPK in fatty acid transport and oxidation where as in moderate to high exercise intensities, AMPK signaling may become more important than ERK1/2 [21]. ...
... Nonetheless, in subsequent studies Raney and colleagues provided evidence that both ERK1/2 and AMPK play a significant role in fatty acid uptake and oxidation during muscle contraction [20,21]. It seems that in low to moderate exercise intensities, ERK1/2 plays a more critical role than AMPK in fatty acid transport and oxidation where as in moderate to high exercise intensities, AMPK signaling may become more important than ERK1/2 [21]. ...
A single bout of contractile activity produces a multitude of time- and intensity-dependent hormonal and cellular perturbations within skeletal muscle. With the onset of contractile activity, cytosolic and mitochondrial [Ca2+] levels are rapidly increased and, depending on the relative intensity of the exercise, metabolite concentrations change. These contraction-induced metabolic disturbances activate several key kinases and phosphatases involved in signal transduction. Chief among these are the calcium-dependent signalling pathways that respond to elevated Ca2+ concentrations (including Ca2+/calmodulin-dependent kinase [CaMK], Ca2+-dependent protein kinase C [PKC] and the Ca2+/calmodulin-dependent phosphatase calcineurin); the 5'-adenosine monophosphate-activated protein kinase (AMPK), several of the mitogen-activated protein kinases (MAPK), and protein kinase B/Akt. In addition, there are exercise-induced central nervous system (CNS) stimulatory effects on various hormones and endocrine organs that promote the oxidation of carbohydrate-based fuels. With repeated bouts of contractile activity (i.e. exercise training) there are numerous and coordinated biochemical adaptations in skeletal muscle that decrease the reliance on carbohydrate-based fuels and enhance the oxidation of lipid-based fuels via reduced sympathetic nervous system responses to any given submaximal exercise intensity. These adaptations serve to minimize cellular disturbances during subsequent exercise bouts. Accordingly, chronic adaptations in skeletal muscle are likely to be the result of the cumulative effects of repeated bouts of exercise, with the initial signaling responses leading to such adaptations occurring after each (acute) training session. This chapter will summarize our current understanding of the hormonal and cellular control of bioenergetics in human skeletal muscle.
... Despite increased epinephrine and norepinephrine concentrations with increasing exercise intensities, TAG oxidation remains stimulated in muscle and adipose tissue (Hagström-Toft et al, 1998;Quish et al, 2005), but relative carbohydrate contribution to total energy provision is greater than that of fatty acids. AMPK activity is increased with more frequent muscle contraction (from working at higher exercise intensities where ATP utilization and Ca 2+ concentration are increased) (Raney and Turcotte, 2006). Activation of the AMPK pathway increases the amount of GLUT4 translocated to the plasma membrane (Schwenk et al, 2010), which facilitates glucose uptake and ultimately carbohydrate oxidation. ...
... NEFA uptake would likely be enhanced with AMPK-induced translocation of CD36 to the plasma membrane. AMPK activation occurs during moderate intensity exercise when signals such as plasma insulin concentration are reduced and intracellular Ca 2+ concentrations are elevated compared to rest or during low intensity exercise (Raney and Turcotte, 2006). AMPK inhibits ACC activity leading to a reduction in malonyl-CoA production and removal of the inhibition on CPT1 (Brusq et al, 2006). ...
... More recently it has also been shown that, except for FATP6, muscle fatty acid transporters can potentially be induced to translocate to the plasma membrane during electrically stimulated muscle contraction or exercise (Turcotte et al. 2005;Jain et al. 2009;Jeppesen et al. 2011;Bradley et al. 2012;McFarlan et al. 2012), as well as to the t-tubules during muscle contraction (Stefanyk et al. 2012). It appears that ERK1/2 and AMPK are the predominant signalling molecules regulating fatty acid uptake during low-to moderate-intensity muscle contraction and during moderate-to high-intensity muscle contraction, respectively (Turcotte et al. 2005;Raney & Turcotte, 2006). These signals induce the translocation of CD36 to the J Physiol 591.18 sarcolemma, but possibly not FABPpm (Turcotte et al. 2005;Raney & Turcotte, 2006;Jeppesen et al. 2009). ...
... It appears that ERK1/2 and AMPK are the predominant signalling molecules regulating fatty acid uptake during low-to moderate-intensity muscle contraction and during moderate-to high-intensity muscle contraction, respectively (Turcotte et al. 2005;Raney & Turcotte, 2006). These signals induce the translocation of CD36 to the J Physiol 591.18 sarcolemma, but possibly not FABPpm (Turcotte et al. 2005;Raney & Turcotte, 2006;Jeppesen et al. 2009). This latter group (Jeppesen et al. 2011) has, however, also reported that AMPK is not necessarily essential in the regulation of CD36 translocation and FA uptake in skeletal muscle during muscle contraction (Jeppesen et al. 2011). ...
Regulation of skeletal muscle fatty acid oxidation (FAO) and adaptation to exercise-training have long been thought to depend on fatty acid (FA) delivery to muscle, their diffusion into muscle, and muscle mitochondrial content and biochemical machinery. However, FA entry into muscle occurs via a regulatable, protein-mediated mechanism, involving several transport proteins. Among these CD36 is key. Muscle contraction and pharmacological agents induce CD36 to translocate to the cell surface, a response that is regulates FA transport, and hence FAO. In exercising CD36 KO mice, exercise duration (-44% check), and FA transport (-41%) and oxidation (-37%) are comparably impaired, while carbohydrate metabolism is augmented. In trained CD36 KO mice, training-induced upregulation of FAO is not observed, despite normal training-induced increases in mitochondrial density and enzymes. Transfecting CD36 into sedentary WT muscle (+41%), comparable to training-induced CD36 increases (+44%) in WT muscle, markedly upregulates FAO to rates observed in trained WT mice, but without any changes in mitochondrial density and enzymes. Evidently, in vivo, CD36-mediated FA transport is key for muscle fuel selection and training-induced FAO upregulation, independent of mitochondrial adaptations. This CD36 molecular mechanism challenges the view that skeletal muscle FAO is solely regulated by muscle mitochondrial content and machinery.
... As mentioned above, an accumulation of intramyocellular FAs was observed in human vastus lateralis muscle when exercise intensity was increased from 65% ˙ V O 2 ,peak to 90% ˙ V O 2 ,peak despite a decrease in plasma FA concentration (Kiens et al. 1999), suggesting that the transport across the sarcolemma was not limiting FA oxidation at high exercise intensities. Data from studies in the perfused rat hindlimb model (Raney & Turcotte, 2006) have revealed a relation between FA uptake and oxidation, but only at low to moderate contraction intensities. When increasing to higher intensities, FA uptake was still elevated compared to basal levels, despite FA oxidation being decreased to resting values (Raney & Turcotte, 2006), supporting the ...
... Data from studies in the perfused rat hindlimb model (Raney & Turcotte, 2006) have revealed a relation between FA uptake and oxidation, but only at low to moderate contraction intensities. When increasing to higher intensities, FA uptake was still elevated compared to basal levels, despite FA oxidation being decreased to resting values (Raney & Turcotte, 2006), supporting the ...
Fatty acids (FAs) as fuel for energy utilization during exercise originate from different sources: FAs transported in the circulation either bound to albumin or as triacylglycerol (TG) carried by very low density lipoproteins and FAs from lipolysis of muscle TG stores. Despite a high rate of energy expenditure during high intensity exercise the total FA oxidation is suppressed to below that observed during moderate intensity exercise. Although this has been known for many years, the mechanisms behind this phenomenon are still not fully elucidated. A failure of adipose tissue to deliver sufficient FAs to exercising muscle has been proposed, but evidence is emerging that factors within the muscle might be of more importance. The high rate of glycolysis during high intensity exercise might be the 'driving force' via the increased production of acetyl-CoA, which in turn is trapped by carnitine. This will lead to decreased availability of free carnitine for long chain FA transport into mitochondria. This review summarizes our present view on how FA metabolism is regulated during exercise with a special focus on the limitations in FA oxidation in the transition from moderate to high intensity exercise in humans.
... This was consistent with a previous study, revealing the effects of exercise on MAPKs depended on training types, in which lowintensity or accustomed exercise collectively utilized ERK1/2 (Raney & Turcotte, 2006). Activated ERK1/2 was reported to inhibit myoblast differentiation (Knight & Kothary, 2011), explaining LMHFV could only suppress ERK1/2 phosphorylation and promote AChRs clustering when applied from D1 to D6 of myoblast differentiation. ...
Neuromuscular junction (NMJ) degeneration is one of pathological factors of sarcopenia. Low‐magnitude high‐frequency vibration (LMHFV) was reported effective in alleviating the sarcopenia progress. However, no previous study has investigated treatment effects of LMHFV targeting NMJ degeneration in sarcopenia. We first compared morphological differences of NMJ between sarcopenic and non‐sarcopenic subjects, as well as young and old C57BL/6 mice. We then systematically characterized the age‐related degeneration of NMJ in SAMP8 against its control strain, SAMR1 mice, from 3 to 12 months old. We also investigated effects of LMHFV in SAMP8 on the maintenance of NMJ during the onset of sarcopenia with respect to the Agrin‐LRP4‐MuSK‐Dok7 pathway and investigated the mechanism related to ERK1/2 signaling. We observed sarcopenic/old NMJ presented increased acetylcholine receptors (AChRs) cluster fragmentation and discontinuity than non‐sarcopenic/young NMJ. In SAMP8, NMJ degeneration (morphologically at 6 months and functionally at 8 months) was observed associated with the sarcopenia onset (10 months). SAMR1 showed improved NMJ morphology and function compared with SAMP8 at 10 months. Skeletal muscle performance was improved at Month 4 post‐LMHFV treatment. Vibration group presented improved NMJ function at Months 2 and 6 posttreatment, accompanied with alleviated morphological degeneration at Month 4 posttreatment. LMHFV increased Dok7 expression at Month 4 posttreatment. In vitro, LMHFV could promote AChRs clustering in myotubes by increasing Dok7 expression through suppressing ERK1/2 phosphorylation. In conclusion, NMJ degeneration was observed associated with the sarcopenia onset in SAMP8. LMHFV may attenuate NMJ degeneration and sarcopenia progression by increasing Dok7 expression through suppressing ERK1/2 phosphorylation.
... Furthermore, gene set enrichment analysis identified MAPK signaling as a pathway that was strongly influenced by exercise but attenuated in people with obesity. The MAPK pathway is a key component in exercise-induced metabolic adaptations (Raney & Turcotte, 2006;Widegren et al., 1998;Yu et al., 2003) and has been shown to be upregulated in aerobic exercise in humans (Boppart et al., 2000;Widegren et al., 1998;Yu et al., 2001). Given F I G U R E 4 Exercise-induced transcriptional patterns in skeletal muscle in obese and non-obese adults. ...
Obesity is associated with several skeletal muscle impairments which can be improved through an aerobic exercise prescription. The possibility that exercise responsiveness is diminished in people with obesity has been suggested but not well‐studied. The purpose of this study was to investigate how obesity influences acute exercise responsiveness in skeletal muscle and circulating amino metabolites. Non‐obese (NO; n = 19; 10F/9M; BMI = 25.1 ± 2.8 kg/m2) and Obese (O; n = 21; 14F/7M; BMI = 37.3 ± 4.6 kg/m2) adults performed 30 min of single‐leg cycling at 70% of VO2peak. 13C6‐Phenylalanine was administered intravenously for muscle protein synthesis measurements. Serial muscle biopsies (vastus lateralis) were collected before exercise and 3.5‐ and 6.5‐h post‐exercise to measure protein synthesis and gene expression. Targeted plasma metabolomics was used to quantitate amino metabolites before and 30 and 90 min after exercise. The exercise‐induced fold change in mixed muscle protein synthesis trended (p = 0.058) higher in NO (1.28 ± 0.54‐fold) compared to O (0.95 ± 0.42‐fold) and was inversely related to BMI (R2 = 0.140, p = 0.027). RNA sequencing revealed 331 and 280 genes that were differentially expressed after exercise in NO and O, respectively. Gene set enrichment analysis showed O had six blunted pathways related to metabolism, cell to cell communication, and protein turnover after exercise. The circulating amine response further highlighted dysregulations related to protein synthesis and metabolism in adults with obesity at the basal state and in response to the exercise bout. Collectively, these data highlight several unique pathways in individuals with obesity that resulted in a modestly blunted exercise response.
... Activation of MAPKs is associated with the sensing of extracellular changes such as growth factors and cytokines but via the sensing of cellular stress from diverse stimuli such as osmotic, oxidative, and mechanical stress (679). In the context of acute exercise, ERK1/2 has been demonstrated to have a mechanistic role in the regulation of fatty acid oxidation in skeletal muscle at low-to-moderate intensities of exercise via the regulation of fatty acid uptake through CD36 translocation to the plasma membrane, in addition to an effect on fatty oxidation independent of effects on fatty acid uptake (680)(681)(682). In fact, this series of experiments from a perfused rat hindlimb model implicate convergence between CaMKII and CaMKK signaling via both AMPK-dependent and -independent regulation and ERK1/2 signaling in both a time-and intensity-dependent manner (683). ...
Repeated, episodic bouts of skeletal muscle contraction undertaken frequently as structured exercise training is a potent stimulus for physiological adaptation in many organs. Specifically in skeletal muscle, remarkable plasticity is demonstrated by the remodeling of muscle structure and function in terms of muscular size, force, endurance, and contractile velocity as a result of the functional demands induced by various types of exercise training. This plasticity, and the mechanistic basis for adaptations to skeletal muscle in response to exercise training, is underpinned by activation and/or repression of molecular pathways and processes induced in response to each individual acute exercise session. These pathways include the transduction of signals arising from neuronal, mechanical, metabolic, and hormonal stimuli through complex signal transduction networks, which are linked to a myriad of effector proteins involved in the regulation of pre- and post-transcriptional processes, and protein translation and degradation processes. This review therefore describes acute exercise-induced signal transduction and the molecular responses to acute exercise in skeletal muscle including emerging concepts such as epigenetic pre- and post-transcriptional regulation, and the regulation of protein translation and degradation. A critical appraisal of methodological approaches and the current state of knowledge informs a series of recommendations offered as future directions in the field.
... During muscle contraction and exercise, signaling pathways such as calcium/calmodulin-dependent protein kinase kinase (CaMKK) [156], extracellular regulated kinases 1/2 (ERK1/2) [140] and p38 mitogen-activated protein kinase [157] are activated, some of which have been linked to translocation of FAT/CD36 to the plasma membrane. ...
Since the lipid profile is altered by physical activity, the study of lipid metabolism is a remarkable element in understanding if and how physical activity affects the health of both professional athletes and sedentary subjects. Although not fully defined, it has become clear that resistance exercise uses fat as an energy source. The fatty acid oxidation rate is the result of the following processes: (a) triglycerides lipolysis, most abundant in fat adipocytes and intramuscular triacylglycerol (IMTG) stores, (b) fatty acid transport from blood plasma to muscle sarcoplasm, (c) availability and hydrolysis rate of intramuscular triglycerides, and (d) transport of fatty acids through the mitochondrial membrane. In this review, we report some studies concerning the relationship between exercise and the aforementioned processes also in light of hormonal controls and molecular regulations within fat and skeletal muscle cells.
... Intra-workout CHO ingestion may also blunt the interleukin-6 response to exercise (Akerstrom, Krogh-Madsen, Petersen, & Pedersen, 2009). Future studies are needed to determine the impact of these changes, acute exercise responses are not predictive of longer-term adaptations (Cochran et al., 2014), which may be influenced to the contraction-induced signaling in mitochondrial adaptations and redundancies in the adaptive process (Raney & Turcotte, 2006). ...
Endurance performance is the result of optimal training targeting
cardiovascular, metabolic, and peripheral muscular adaptations
and is coupled to effective nutrition strategies via the use of
macronutrient manipulations surrounding training and potential
supplementation with ergogenic aids. It is important to note that training and nutrition may differ according to the individual needs of the athlete and can markedly impact the physiological response to training. Herein, we discuss various aspects of endurance training adaptations, nutritional strategies and their contributions to towards performance.
... Phosphorylation of ACC2 Ser212 does not appear to play a role in regulating fatty acid oxidation during ex vivo contraction and exercise (218), providing evidence to support that AMPK-ACC2-independent pathways regulate fatty acid oxidation during muscle contractile activity. In addition, AMPK does not seem essential for increasing FAT/CD36 translocation and fatty acid uptake during ex vivo contraction (219), which may limit fatty acid oxidation during low to moderate contraction intensities (220). Considering these findings, the importance of AMPK in regulating muscle fatty acid oxidation during exercise appears sparse given the genetic evidence obtained from various transgenic mouse models. ...
Skeletal muscle possesses a remarkable ability to adapt to various physiologic conditions. AMPK is a sensor of intracellular energy status that maintains energy stores by fine-tuning anabolic and catabolic pathways. AMPK's role as an energy sensor is particularly critical in tissues displaying highly changeable energy turnover. Due to the drastic changes in energy demand that occur between the resting and exercising state, skeletal muscle is one such tissue. Here, we review the complex regulation of AMPK in skeletal muscle and its consequences on metabolism (e.g., substrate uptake, oxidation, and storage as well as mitochondrial function of skeletal muscle fibers). We focus on the role of AMPK in skeletal muscle during exercise and in exercise recovery. We also address adaptations to exercise training, including skeletal muscle plasticity, highlighting novel concepts and future perspectives that need to be investigated. Furthermore, we discuss the possible role of AMPK as a therapeutic target as well as different AMPK activators and their potential for future drug development.-Kjøbsted, R., Hingst, J. R., Fentz, J., Foretz, M, Sanz, M.-N., Pehmøller, C., Shum, M., Marette, A., Mounier, R., Treebak, J. T., Wojtaszewski, J. F. P., Viollet, B., Lantier, L. AMPK in skeletal muscle function and metabolism.
... Other kinases, such as extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein (MAP) kinases, have been suggested as contraction-induced regulators of CD36 translocation [21] but appear not to be essential [15]. During muscle contractions, cytosolic Ca 2+ levels are immediately increased. ...
This review summarizes how fatty acid (FA) oxidation is regulated in skeletal muscle during exercise. From the available evidence it seems that acetyl-CoA availability in the mitochondrial matrix adjusts FA oxidation to exercise intensity and duration. This is executed at the step of mitochondrial fatty acyl import, as the extent of acetyl group sequestration by carnitine determines the availability of carnitine for the carnitine palmitoyltransferase 1 (CPT1) reaction. The rate of glycolysis seems therefore to be central to the amount of β-oxidation-derived acetyl-CoA that is oxidized in the tricarboxylic acid (TCA) cycle. FA oxidation during exercise is also determined by FA availability to mitochondria, dependent on trans-sarcolemmal FA uptake via cluster of differentiation 36/SR-B2 (CD36) and FAs mobilized from myocellular lipid droplets.
... Perhaps the most robust evidence to corroborate the isoform-specific modification of resting AMPK activity and muscle lipid status comes from the paired data from each subject's resting biopsy samples: compared to a high carbohydrate diet, fat adaptation significantly increased muscle triglyceride concentration (Figure 3.3), with such changes being strongly correlated to difference in basal AMPK2 activity (r = 0.82, p<0.05). Raney and Turcotte (2006) have also reported a positive association between fatty acid uptake and oxidation and AMPK2 activity in the perfused rat hindlimb model. However, it should be noted that correlational data cannot determine causality and it is possible that factors other than muscle lipid status may play a role in modifying AMPKα2 activity. ...
Nutrient availability and exercise training are two potent modulators of skeletal muscle adaptation. While the effects of nutrient provision or training alone on muscle adaptation have been well researched, the interactive effects of these two stressors on skeletal muscle adaptation are less well understood, especially in well-trained athletes. Furthermore, until recently, the mechanisms that underlie the changes in muscle metabolism that result from this nutrient-training interaction are largely unknown. Accordingly, the primary aim of the studies undertaken in this book was to enhance our understanding of how nutrient availability in skeletal muscle interacts with endurance exercise to modify skeletal muscle adaptation in already well-trained athletes. In this regard, nutrient levels were manipulated by two different strategies: first by prescribing a high fat, low-CHO diet, and secondly by manipulating the athletes training schedule. The subsequent training adaptation was studied by examining changes in whole-body metabolism and enzymatic activities as well as changes to selected cell signaling pathways that are related to both nutrient availability and training stimuli.
... Perhaps the most robust evidence to corroborate the isoform-specific modification of resting AMPK activity and muscle lipid status comes from the paired data from each subject's resting biopsy samples: compared to a high carbohydrate diet, fat adaptation significantly increased muscle triglyceride concentration (Figure 3.3), with such changes being strongly correlated to difference in basal AMPK2 activity (r = 0.82, p<0.05). Raney and Turcotte (2006) have also reported a positive association between fatty acid uptake and oxidation and AMPK2 activity in the perfused rat hindlimb model. However, it should be noted that correlational data cannot determine causality and it is possible that factors other than muscle lipid status may play a role in modifying AMPKα2 activity. ...
... For example, in LKB1-deficient mice, although ACC phosphorylation in both resting and contracted conditions is reduced, the level of malonyl-CoA remain unaltered and the influence of muscle contraction on this coenzyme is somewhat weak (132). Furthermore, studies also suggest that contraction-induced increases in fatty acid oxidation are independent of AMPK activity (133). Muscle blood flow is also an important compensatory mechanism wherein increased metabolic demands of active muscle groups is readily met by increases in local blood flow which serves to enhance muscle perfusion and performance. ...
AMPK is a serine/threonine kinase that is found in all eukaryotes and is ubiquitously expressed in all organ systems. Once activated, AMPK stimulates hepatic fatty acid oxidation and ketogenesis, inhibits cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibits adipocyte lipolysis and lipogenesis, stimulates skeletal muscle fatty acid oxidation and muscle glucose uptake, and modulates insulin secretion by the pancreas. Thus its importance in many critical cellular processes is well established. For cells it is critical that energy supply and demand are closely matched. AMPK is recognized as a critical integrator of this balance. It is known to be allosterically activated by an increased AMP:ATP ratio. Activation of the kinase switches on catabolic pathways while switching off anabolic ones. It also acts as a redox sensor in endothelial cells where oxidative stress can disturb NO signaling. Abnormal NO signaling leads to disturbed vasodilatory responses. By inhibiting the formation of reactive oxygen species in the endothelium, AMPK can optimize the redox balance in the vasculature. Here, we review the role of AMPK in the cell.
... The activity of ERK1/2 is associated with various aspects of lipid metabolism. It seems to be involved in the phosphorylation of ACC and hormone-sensitive lipase (12) as well as fatty acid uptake during muscle activity (45,59). Therefore, the increased ERK2 in UCP1 Tg mice could explain their increased SM fatty acid metabolism. ...
Ectopic expression of uncoupling protein 1 (UCP1) in skeletal muscle (SM) mitochondria considerably increases lifespan in high fat diet fed UCP1 TG mice in comparison to wildtype (WT). In order to clarify the underlying mechanisms we investigated substrate metabolism as well as oxidative stress damage and antioxidant defense in SM of low fat and high fat fed mice. TG mice showed an increased protein expression of phosphorylated AMP activated protein kinase, markers of lipid turn over (pACC, FAT/CD36), and an increased SM ex-vivo fatty acid oxidation. Surprisingly, UCP1 TG mice showed elevated lipid peroxidative protein modifications with no changes in glycoxidation or direct protein oxidation. This was paralleled by an induction of catalase and superoxide dismutase activity, an increased redox signaling (MAPK signaling pathway), and increased expression of stress protective heat shock protein 25. We conclude that increased skeletal muscle mitochondrial uncoupling in vivo does not reduce the oxidative stress status in the muscle cell. Moreover it increases lipid metabolism and reactive lipid-derived carbonyls. This stress induction in turn increases the endogenous antioxidant defense system and redox signaling. All together our data argue for an adaptive role of reactive species as essential signaling molecules for health and longevity.
... The right and left hindlimbs or adipose tissue of nonperfused CD-and HFD-fed WT and DN mice were used for the following preparations. For Western blot analysis, frozen muscle and adipose tissue samples (40 mg) were powdered under liquid N 2 and homogenized in 500 μl of ice-cold RIPA buffer as previously described (Raney et al. 2005;Raney & Turcotte, 2006). The total cell homogenate was then transferred to a microcentrifuge tube and vortexed frequently for 1 h, whereupon the samples were centrifuged at 4500g at 4 • C for 1 h. ...
Owing to its critical role in the regulation of skeletal muscle metabolism, AMP-activated protein kinase (AMPK) remains a central focus of research for the treatment of insulin resistance. The purpose of the present study was to determine the role of AMPKα2 activity in the regulation of glucose uptake and fatty acid (FA) metabolism in insulin-resistant skeletal muscle. Male C57BL/6 mice were divided into groups fed a control diet (CD) or high-fat (60%) diet (HFD) for 6 weeks and were either wild-type (WT) or possessed an AMPKα2 dominant negative transgene (DN). After 6 weeks, hindlimbs of CD (n = 10) and HFD mice (n = 10) were perfused with or without 450 μU ml(-1) insulin. Muscles of CD (n = 8) and HFD mice (n = 8) were used for measurement of basal protein expression. In CD mice, low AMPKα2 activity did not affect basal FA uptake (FAU), but it increased basal FA oxidation (FAO) by 28% and prevented the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased basal FAU by 147% (P < 0.05). In both WT and DN mice, HFD abolished the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased SIRT1 activity and decreased Protein Tyrosine Phosphatase 1B (PTP1B) expression and Akt(Thr308) phosphorylation (P < 0.05). Adipose tissue protein expression of interleukin-6 and tumour necrosis factor α was increased by HFD in WT mice but not in DN mice (P < 0.05). Skeletal muscle interleukin-15 expression was decreased in both feeding conditions in the DN mice (P < 0.05). The data from this study suggest that in insulin-resistant conditions low AMPKα2 activity impacts the regulation of skeletal muscle FA metabolism via changes in SIRT1 activity, PTP1B expression and Akt phosphorylation and the expression of adipose tissue pro-inflammatory markers.
... Therefore, because we examined FAT/ CD36 translocation during treadmill exercise, it seems unlikely that upregulation of alternative signaling pathways could have contributed to the maintenance of FA uptake in the AMPK KD mice. Downloaded from support of these fi ndings, Raney and Turcotte ( 42 ) and Raney et al. ( 43 ) have shown that increased FA uptake and FA oxidation during low-intensity muscle contractions occur independently of AMPK activation. Furthermore, they suggested that ERK1/2 may be a primary regulator of FAT/ CD36 translocation and FA uptake ( 20 ). ...
The aim of this study was to investigate the molecular mechanisms regulating FA translocase CD36 (FAT/CD36) translocation and FA uptake in skeletal muscle during contractions. In one model, wild-type (WT) and AMP-dependent protein kinase kinase dead (AMPK KD) mice were exercised or extensor digitorum longus (EDL) and soleus (SOL) muscles were contracted, ex vivo. In separate studies, FAT/CD36 translocation and FA uptake in response to muscle contractions were investigated in the perfused rat hindlimb. Exercise induced a similar increase in skeletal muscle cell surface membrane FAT/CD36 content in WT (+34%) and AMPK KD (+37%) mice. In contrast, 5-aminoimidazole-4-carboxamide ribonucleoside only induced an increase in cell surface FAT/CD36 content in WT (+29%) mice. Furthermore, in the perfused rat hindlimb, muscle contraction induced a rapid (1 min, +15%) and sustained (10 min, +24%) FAT/CD36 relocation to cell surface membranes. The increase in cell surface FAT/CD36 protein content with muscle contractions was associated with increased FA uptake, both in EDL and SOL muscle from WT and AMPK KD mice and in the perfused rat hindlimb. This suggests that AMPK is not essential in regulation of FAT/CD36 translocation and FA uptake in skeletal muscle during contractions. However, AMPK could be important in regulation of FAT/CD36 distribution in other physiological situations.
... It will be of great interest to study whether the hepatic activation of the pathways found in the wholegenome-expression analysis is involved in the metabolic adaptation of the liver to exercise and beyond that in the beneficial effects of physical activity for the maintenance of insulin sensitivity. The activation of MAPK signalling in the skeletal muscle has been implicated in increased fatty acid uptake and oxidation [48]. Future studies will show how hepatic MAPK signalling is linked to the regulation of hepatic glucose and lipid metabolism during exercise or to the beneficial effects of regular physical activity on hepatic fat content as demonstrated in human studies [49, 50]. ...
We aimed to identify, in the liver of mice, signal transduction pathways that show a pronounced regulation by acute exercise. We also aimed to elucidate the role of metabolic stress in this response.
C57Bl6 mice performed a 60 min run on a treadmill under non-exhaustive conditions. Hepatic RNA and protein lysates were prepared immediately after running and used for whole-genome-expression analysis, quantitative real-time PCR and immunoblotting. A subset of mice recovered for 3 h after the treadmill run. A further group of mice performed the treadmill run after having received a vitamin C- and vitamin E-enriched diet over 4 weeks.
The highest number of genes differentially regulated by exercise in the liver was found in the mitogen-activated protein kinase (MAPK) signalling pathway, with a pronounced and transient upregulation of the transcription factors encoded by c-Fos (also known as Fos), c-Jun (also known as Jun), FosB (also known as Fosb) and JunB (also known as Junb) and phosphorylation of hepatic MAPK. Acute exercise also activated the p53 signalling pathway. A major role for oxidative stress is unlikely since the antioxidant-enriched diet did not prevent the activation of the MAPK pathway. In contrast, lower plasma glucose levels after running were related to enhanced levels of MAPK signalling proteins, similar to the upregulation of Igfbp1 and Pgc-1alpha (also known as Ppargc1a). In the working muscle the activation of the MAPK pathway was weak and not related to plasma glucose concentrations.
Metabolic stress evidenced as low plasma glucose levels appears to be an important determinant for the activation of the MAPK signalling pathway and the transcriptional response of the liver to acute exercise.
... We have also demonstrated that although contractionstimulated AMPK activity is reduced in AMPK DN mice, ACC phosphorylation and fatty acid oxidation are maintained (137). In addition, it has also been reported that low-intensity contraction can increase fatty acid oxidation independent of increased AMPK activity (402,403) and that AICAR and contraction may have additive effects on fatty acid oxidation (455). ...
The function and survival of all organisms is dependent on the dynamic control of energy metabolism, when energy demand is matched to energy supply. The AMP-activated protein kinase (AMPK) alphabetagamma heterotrimer has emerged as an important integrator of signals that control energy balance through the regulation of multiple biochemical pathways in all eukaryotes. In this review, we begin with the discovery of the AMPK family and discuss the recent structural studies that have revealed the molecular basis for AMP binding to the enzyme's gamma subunit. AMPK's regulation involves autoinhibitory features and phosphorylation of both the catalytic alpha subunit and the beta-targeting subunit. We review the role of AMPK at the cellular level through examination of its many substrates and discuss how it controls cellular energy balance. We look at how AMPK integrates stress responses such as exercise as well as nutrient and hormonal signals to control food intake, energy expenditure, and substrate utilization at the whole body level. Lastly, we review the possible role of AMPK in multiple common diseases and the role of the new age of drugs targeting AMPK signaling.
... One example of this dichotomy is observed in LKB1-deficient mice, which, despite no activation of AMPKa2 during muscle contraction, have only modest suppression of ACC2 phosphorylation (Koh et al. 2006;Thomson et al. 2007). Low-intensity muscle contractions can increase fatty acid oxidation independent of AMPK activity (Raney and Turcotte 2006;Raney et al. 2005), and AICAR and contraction have additive effects on fatty acid oxidation . In humans, both endurance training and exercise in women, compared with men, results in increased rates of fatty acid oxidation, despite reduced activation of AMPK signalling during exercise (McConell et al. 2005;Roepstorff et al. 2006). ...
During moderate-intensity exercise, fatty acids are the predominant substrate for working skeletal muscle. The release of fatty acids from adipose tissue stores, combined with the ability of skeletal muscle to actively fine tune the gradient between fatty acid and carbohydrate metabolism, depending on substrate availability and energetic demands, requires a coordinated system of metabolic control. Over the past decade, since the discovery that AMP-activated protein kinase (AMPK) was increased in accordance with exercise intensity, there has been significant interest in the proposed role of this ancient stress-sensing kinase as a critical integrative switch controlling metabolic responses during exercise. In this review, studies examining the role of AMPK as a regulator of fatty acid metabolism in both adipose tissue and skeletal muscle during exercise will be discussed. Exercise induces activation of AMPK in adipocytes and regulates triglyceride hydrolysis and esterfication through phosphorylation of hormone sensitive lipase (HSL) and glycerol-3-phosphate acyl-transferase, respectively. In skeletal muscle, exercise-induced activation of AMPK is associated with increases in fatty acid uptake, phosphorylation of HSL, and increased fatty acid oxidation, which is thought to occur via the acetyl-CoA carboxylase-malony-CoA-CPT-1 signalling axis. Despite the importance of AMPK in regulating fatty acid metabolism under resting conditions, recent evidence from transgenic models of AMPK deficiency suggest that alternative signalling pathways may also be important for the control of fatty acid metabolism during exercise.
... Perhaps the most robust evidence to corroborate the isoform-specific modification of resting AMPK activity and muscle lipid status comes from the paired data from each subject's resting biopsy samples: compared with a HCHO diet, fat adaptation significantly increased muscle triglyceride concentration (Fig. 3), with such changes being strongly correlated to difference in basal AMPK-␣ 2 activity (r ϭ 0.82, P ϭ 0.04). Raney and Turcotte (29) have also reported a positive association between FA uptake and oxidation and AMPK-␣ 2 activity in the perfused rat hindlimb model. However, it should be noted that correlational data cannot determine causality, and it is possible that factors other than muscle lipid status may play a role in modifying AMPK-␣ 2 activity. ...
We have previously reported that 5 days of a high-fat diet followed by 1 day of high-carbohydrate intake (Fat-adapt) increased rates of fat oxidation and decreased rates of muscle glycogenolysis during submaximal cycling compared with consumption of an isoenergetic high-carbohydrate diet (HCHO) for 6 days (Burke et al. J Appl Physiol 89: 2413-2421, 2000; Stellingwerff et al. Am J Physiol Endocrinol Metab 290: E380-E388, 2006). To determine potential mechanisms underlying shifts in substrate selection, eight trained subjects performed Fat-adapt and HCHO. On day 7, subjects performed 1-h cycling at 70% peak O2 uptake. Muscle biopsies were taken immediately before and after exercise. Resting muscle glycogen content was similar between treatments, but muscle triglyceride levels were higher after Fat-adapt (P < 0.05). Resting AMPK-α1 and -α2 activity was higher after Fat-adapt (P = 0.02 and P = 0.05, respectively), while the phosphorylation of AMPK's downstream target, acetyl-CoA carboxylase (pACC at Ser 221), tended to be elevated after Fat-adapt (P = 0.09). Both the respiratory exchange ratio (P < 0.01) and muscle glycogen utilization (P < 0.05) were lower during exercise after Fat-adapt. Exercise increased AMPK-α1 activity after HCHO (P = 0.03) but not Fat-adapt. Exercise was associated with an increase in pACC at Ser221 for both dietary treatments (P < 0.05), with postexercise pACC Ser221 higher after Fat-adapt (P = 0.02). In conclusion, compared with HCHO, Fat-adapt increased resting muscle triglyceride stores and resting AMPK- α1 and -α2 activity. Fat-adapt also resulted in higher rates of whole body fat oxidation, reduced muscle glycogenolysis, and attenuated the exercise-induced rise in AMPK-α1 and AMPK-α2 activity compared with HCHO. Our results demonstrate that AMPK-α1 and AMPK-α2 activity and fuel selection in skeletal muscle in response to exercise can be manipulated by diet and/or the interactive effects of diet and exercise training.
Muscle atrophy exacerbates disease outcomes and increases mortality, whereas the preservation of skeletal muscle mass and function play pivotal roles in ensuring long-term health and overall quality-of-life. Muscle atrophy represents a significant clinical challenge, involving the continued loss of muscle mass and strength, which frequently accompany the development of numerous types of cancer. Cancer cachexia is a highly prevalent multifactorial syndrome, and although cachexia is one of the main causes of cancer-related deaths, there are still no approved management strategies for the disease. The etiology of this condition is based on the upregulation of systemic inflammation factors and catabolic stimuli, resulting in the inhibition of protein synthesis and enhancement of protein degradation. Numerous necessary cellular processes are disrupted by cachectic pathology, which mediate intracellular signalling pathways resulting in the net loss of muscle and organelles. However, the exact underpinning molecular mechanisms of how these changes are orchestrated are incompletely understood. Much work is still required, but structured exercise has the capacity to counteract numerous detrimental effects linked to cancer cachexia. Primarily through the stimulation of muscle protein synthesis, enhancement of mitochondrial function, and the release of myokines. As a result, muscle mass and strength increase, leading to improved mobility, and quality-of-life. This review summarises existing knowledge of the complex molecular networks that regulate cancer cachexia and exercise, highlighting the molecular interplay between the two for potential therapeutic intervention. Finally, the utility of mass spectrometry-based proteomics is considered as a way of establishing early diagnostic biomarkers of cachectic patients.
This chapter summarizes how fatty acid (FA) oxidation is regulated in skeletal muscle during exercise and the role of obesity in regulation of FA oxidation in skeletal muscle. The substrates fueling increased FA oxidation in skeletal muscle during exercise are mainly circulating FAs, although hydrolysis of circulating triacylglycerol (TG) in very-low-density lipoproteins (VLDL-TG) and especially lipolysis of intramuscular TG (IMTG) also appear to contribute to some extent. Several steps are involved in FA uptake and oxidation in skeletal muscle and could all be of importance in the regulation of FA oxidation during exercise. Besides trans-sarcolemmal FA uptake via fatty acid transporters, it appears that intramyocellular carnitine content plays an important regulatory step in regulation of substrate selection during exercise. Interestingly, individuals with obesity exhibit a compromised ability to oxidize FAs and to increase FA oxidation in response to lipid exposure (reduced metabolic flexibility). Skeletal muscle mitochondrial function appears to be related to this defect. It remains controversial whether this impaired FA oxidative capacity in obesity diminishes the ability to increase and properly regulate FA oxidation during an acute, single exercise bout. However, despite these initial impairments in FA oxidation capacity in the obese situation, endurance exercise training can rescue the capacity for FA oxidation and the metabolic flexibility in the skeletal muscle of individuals with obesity at least to equivalent levels of their lean counterparts.
Neuromuscular junction (NMJ) degeneration is one of the pathological factors of sarcopenia. Low-magnitude high-frequency vibration (LMHFV) was previously reported effective in alleviating the progress of sarcopenia in SAMP8 mice. In this study, we observed more acetylcholine receptors (AChRs) branching in sarcopenic NMJ in humans. Utilizing a validated non-sarcopenic (SAMR1) versus sarcopenic (SAMP8) animal model, NMJ degeneration was observed to precede the onset of sarcopenia where the SAMR1 mice presented better NMJ function and more intact NMJ structure. After the treatment of LMHFV in SAMP8, progress of sarcopenia and NMJ degeneration were both alleviated with increased Dok7 expression, one protein important for NMJ assembly and function. In vitro, when Dok7 expression was knocked down, myotube diameter and acetylcholine receptors (AChRs) cluster formation were decreased, that could not be retarded by LMHFV. Further mechanistic investigation showed that LMHFV could suppress ERK1/2 activation and the blockade of ERK1/2 phosphorylation promoted AChRs clustering and increased Dok7 expression. In myotubes with ERK1/2 phosphorylation blocked, knocking down Dok7 could still reduce AChRs cluster formation, which could not be retarded by LMHFV. Therefore, effectiveness of LMHFV on Dok7 expression in NMJ degeneration was shown to act through suppressing ERK1/2 phosphorylation.
A single bout of exercise can potentiate the effect of insulin on skeletal muscle glucose uptake via activation of the AMPK-TBC1D4 pathway, which suggests a positive correlation between AMPK activation and insulin sensitization. Additionally, prolonged fasting in rodents is known to upregulate and thereby synergistically enhance the effect of exercise on muscle AMPK activation. Therefore, fasting may potentiate the insulin-sensitizing effect of exercise. In the present study, we mimicked exercise by in situ muscle contraction and evaluated the effect of a 36 h fast on muscle contraction-induced insulin sensitization. Male Wistar rats weighing 150-170 g were allocated to either a 36-h fasting or feeding group. The extensor digitorum longus (EDL) muscles were electrically contracted via the common peroneal nerve for 10 min followed by a 3 h recovery period. EDL muscles were dissected and incubated in the presence or absence of submaximal insulin. Our results demonstrated that acute muscle contraction and 36 h of fasting additively upregulated AMPK pathway activation. Insulin-stimulated muscle glucose uptake and site-specific TBC1D4 phosphorylation were enhanced by prior muscle contraction in 36-h fasted rats, but not in fed rats. Moreover, enhanced insulin-induced muscle glucose uptake and Akt phosphorylation due to 36 h of fasting was associated with a decrease in tribbles homolog 3 (TRB3), a negative regulator of Akt activation. In conclusion, fasting and prior muscle contraction synergistically enhance insulin-stimulated TBC1D4 phosphorylation and glucose uptake, which is associated with augmented AMPK pathway activation in rodents.
[Purpose]
The purpose of this review is to discuss current views regarding the acute effects of phytochemicals, exercise, and exercise plus phytochemicals on fatty acid oxidation.
[Methods]
Data acquired from human and animal studies were comprehensively assessed to determine the single and combined effects of phytochemicals and exercise on fatty acid oxidation. In addition, underlying mechanisms associated with those conditions that may contribute to the regulation of fat metabolism are discussed.
[Results]
Although not all phytochemicals are effective at increasing fatty acid oxidation, some significantly improve the rate of fatty acid oxidation at rest. In addition, dietary supplementation of p-synephrine, catechins, or anthocyanins in combination with moderately intense exercise has the additive effect of increasing fatty acid oxidation, but not total energy expenditure during exercise.
[Conclusion]
The data reported from current reviewed studies suggest positive outcomes regarding facilitation of fatty acid oxidation from the combined effects of certain phytochemicals with exercise. Those data provide new insight for developing a strategy to boost fat loss and control weight in obese patients.
Regular exercise contributes to maintaining the skeletal muscle mass and quality, which may prevent type II diabetes, hypertension, coronary heart disease, and/or sarcopenia. Exercise/muscle contraction induces activation or inactivation of the intracellular molecules for a short period, which results in an increased glucose uptake, fatty acid oxidation, and protein synthesis. Exercise also affects transcription factors and coactivators, which change the target gene expression and are related to muscle adaptations such as increasing glucose transport-related protein, mitochondrial biogenesis, and the muscle fiber type transition over a long period. Alterations of these molecules are mediated by changes in the intracellular Ca2+ level, energy status level, and/or the activated mitogen-activated protein kinase (MAPK) signaling pathway. In this section, the intracellular signaling pathway induced by skeletal muscle contraction is discussed.
The use of nutritional supplements is common in highly competitive sports but also popular among recreational athletes. Nutritional antioxidants (AO) as essential compounds of diet are often consumed by athletes as additional oral supplementation with the goal of reducing exercise-induced oxidative stress, enhancing performance and preventing poor antioxidant status. Exercise has been shown to induce an augmented generation of reactive oxygen species (ROS) via different mechanisms. There is further evidence that ROS formation in response to vigorous physical exertion can result in oxidative stress. Recent research has also revealed the important role of ROS as signaling molecules. In this context, ROS can modulate contractile function of skeletal muscle and induce gene expression via redox-sensitive transcription pathways which represents an important regulatory mechanism potentially involved in the process of training adaptation. Although the protective role of several dietary AO in the biological system is well established, current data failed to show that additional oral supplementation of AO increased exercise performance. Furthermore, clear indicative findings that AO can reduce exercise-induce oxidative stress or muscle damage are not available as yet. Further arguments against the long-term consumption of large doses of antioxidants involve their suppressive effects on some immune functions and signalling processes and the potential side effects as described for selected AOs. Furthermore, it is currently unclear whether regular vigorous exercise increases the need for an additional intake of AO. Prevention of poor antioxidative status is achieved best by consuming complex mixtures of AO compounds via increased consumption of fruits and vegetables. Additional intake of AO in small doses may be considered on an individual basis in athletes who exhibit an extreme energy expenditure, lacking adequate nutrition or/and attempting to limit their energy intake.
Cellular respiration depends upon a coordinated response of the cardiovasculature and metabolism to meet changing energy demands in muscle. Even though the adjustments in blood flow, O2 gradient, and myoglobin (Mb) saturation will enhance O2 flux to the mitochondria at the initiation of contraction, the relative contribution of Mb vs. Hb remains an issue for contentious debate. Some researchers have ascribed no significant role for Mb in supplying O2 during muscle contraction. Since Mb has an extremely high affinity for O2, it cannot readily release its O2 store. Hb must then supply all the O2 from the onset of contraction. This viewpoint underpins many interpretations of the noninvasive near-infrared spectroscopy (NIRS) data. Although NIRS cannot discriminate between the Hb and Mb signals, many researchers have assumed that NIRS monitors only Hb oxygen saturation and desaturation kinetics. The present chapter introduces an experiment system that allows for the observations of Mb saturation during muscle contraction. The results then provide insights into the relative Hb vs. Mb contribution in the NIRS signal and into the mechanisms of oxygen delivery and consumption regulation.
New Findings
What is the topic of this review?
This report addresses novel mechanisms regulating the utilization of long‐chain fatty acids, with emphasis on FAT/CD36 and lipolysis of intramuscular triacylglycerol in skeletal muscle during exercise and contractions.
What advances does it highlight?
Recent findings show that adipose triglyceride lipase (ATGL) and hormone‐sensitive lipase (HSL) collectively account for at least 98% of total triacylglycerol lipase activity in skeletal muscle during muscle contractions. The relative importance of HSL and ATGL for breakdown of intramuscular triacylglycerol during muscle contractions is discussed. Collectively, these findings contribute to the understanding of skeletal muscle lipid metabolism during exercise and muscle contractions.
Exercise increases the utilization of lipids in muscle. The sources of lipids are long‐chain fatty acids taken up from the plasma and fatty acids released from stores of intramuscular triacylglycerol by the action of intramuscular lipases. In the present review, we focus on the role of fatty acid binding proteins, particularly fatty acid translocase/cluster of differentiation 36 (FAT/CD36), in the exercise‐ and contraction‐induced increase in uptake of long‐chain fatty acids in muscle. The FAT/CD36 translocates from intracellular depots to the surface membrane upon initiation of exercise/muscle contractions. This occurs independently of AMP‐activated protein kinase, and data suggest that Ca ²⁺ ‐related signalling is responsible. The FAT/CD36 has an important role; long‐chain fatty acid uptake is markedly decreased in FAT/CD36 knockout mice during contractions/exercise compared with wild‐type control mice. In skeletal muscle, 98% of the lipase activity is accounted for by adipose triglyceride lipase and hormone‐sensitive lipase. Give that inhibition or knockout of hormone‐sensitive lipase does not impair lipolysis in muscle during contraction, the data point to an important role of adipose triglyceride lipase in regulation of muscle lipolysis. Although the molecular regulation of the lipases in muscle is not understood, it is speculated that intramuscular lipolysis may be regulated in part by the availability of the plasma concentration of long‐chain fatty acids.
AMP-activated protein kinase (AMPK) has been studied extensively and postulated to be a target for the treatment and/or prevention of metabolic disorders such as insulin resistance. Exercise training has been deemed a beneficial treatment for obesity and insulin resistance. Further, exercise is a feasible method to combat high fat diet-induced alterations in insulin sensitivity. The purpose of this study was to determine if AMPKα2 activity is required to gain beneficial effects of exercise training with high fat-feeding. Wild type (WT) and AMPKα2 dominant negative (DN) male mice were fed standard diet (SD), underwent voluntary wheel running (TR), fed high fat diet (HFD), or trained with HFD (TR + HFD). By week 6, TR, irrespective of genotype, decreased blood glucose and increased citrate synthase activity in both diet groups and decreased insulin levels in HFD groups. Hindlimb perfusions were performed and in WT mice with SD, TR increased insulin-mediated palmitate uptake (76.7%) and oxidation (>2 fold). These training-induced changes were not observed in the DN mice. With HFD, TR decreased palmitate oxidation (61-64%) in both WT and DN and increased palmitate uptake (112%) in the WT with no effects on palmitate uptake in the DN. With SD, TR increased ERK1/2 and JNK1/2 phosphorylation regardless of genotype. With HFD, TR reduced JNK1/2 phosphorylation regardless of genotype, CPT1 expression in WT and CD36 expression in both DN and WT. These data suggest that low AMPKα2 signaling disrupts, in part, the exercise training-induced adaptations in insulin-stimulated metabolism in skeletal muscle following high fat diet.
There is an inverse relationship between cancer incidence and cardiorespiratory fitness in large population studies. Mechanistic insights into these observations may strengthen the rationale for encouraging exercise fitness in the clinics for cancer prevention and may promote the development of new preventive strategies.
Studying the multifaceted activities of p53, a critical tumor suppressor gene, has revealed various cellular pathways necessary for adapting to environmental stresses. Genetic connections are being made between p53 and an increasing number of metabolic activities such as oxidative phosphorylation, glycolysis and fatty acid oxidation. In-vivo mouse models show that p53 plays an important role in determining both basal aerobic exercise capacity and its improvement by training.
The genetic pathways by which p53 regulates metabolism and exercise may help explain significant epidemiologic observations connecting cardiorespiratory fitness and cancer. Further understanding of these molecular pathways through human translational studies may promote the development of new cancer preventive strategies.
The performance of prolonged (>90min), continuous, endurance exercise is limited by endogenous carbohydrate (CHO) stores. Accordingly, for many decades, sports nutritionists and exercise physiologists have proposed a number of diet-training strategies that have the potential to increase fatty acid availability and rates of lipid oxidation and thereby attenuate the rate of glycogen utilization during exercise. Because the acute ingestion of exogenous substrates (primarily CHO) during exercise has little effect on the rates of muscle glycogenolysis, recent studies have focused on short-term (<12weeks) diet-training interventions that increase endogenous substrate stores (i.e., muscle glycogen and lipids) and alter patterns of substrate utilization during exercise. One such strategy is fat adaptation, an intervention in which well-trained endurance athletes consume a high-fat, low-CHO diet for up to 2weeks while undertaking their normal training and then immediately follow this by CHO restoration (consuming a high-CHO diet and tapering for 13days before a major endurance event). Compared with an isoenergetic CHO diet for the same intervention period, this dietary periodization protocol increases the rate of whole-body and muscle fat oxidation while attenuating the rate of muscle glycogenolysis during submaximal exercise. Of note is that these metabolic perturbations favouring the oxidation of fat persist even in the face of restored endogenous CHO stores and increased exogenous CHO availability. Here we review the current knowledge of some of the potential mechanisms by which skeletal muscle sustains high rates of fat oxidation in the face of high exogenous and endogenous CHO availability.
Long-chain fatty acids and lipids serve a wide variety of functions in mammalian homeostasis, particularly in the formation and dynamic properties of biological membranes and as fuels for energy production in tissues such as heart and skeletal muscle. On the other hand, long-chain fatty acid metabolites may exert toxic effects on cellular functions and cause cell injury. Therefore, fatty acid uptake into the cell and intracellular handling need to be carefully controlled. In the last few years, our knowledge of the regulation of cellular fatty acid uptake has dramatically increased. Notably, fatty acid uptake was found to occur by a mechanism that resembles that of cellular glucose uptake. Thus, following an acute stimulus, particularly insulin or muscle contraction, specific fatty acid transporters translocate from intracellular stores to the plasma membrane to facilitate fatty acid uptake, just as these same stimuli recruit glucose transporters to increase glucose uptake. This regulatory mechanism is important to clear lipids from the circulation postprandially and to rapidly facilitate substrate provision when the metabolic demands of heart and muscle are increased by contractile activity. Studies in both humans and animal models have implicated fatty acid transporters in the pathogenesis of diseases such as the progression of obesity to insulin resistance and type 2 diabetes. As a result, membrane fatty acid transporters are now being regarded as a promising therapeutic target to redirect lipid fluxes in the body in an organ-specific fashion.
AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, has been proposed to function as a fuel gauge to monitor cellular energy status in response to nutritional environmental variations. AMPK system is a regulator of energy balance that, once activated by low energy status, switches on ATP-producing catabolic pathways (such as fatty acid oxidation and glycolysis), and switches off ATP-consuming anabolic pathways (such as lipogenesis), both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression. Numerous observations obtained with pharmacological activators and agents that deplete intracellular ATP have been supportive of AMPK playing a role in the control of energy metabolism but none of these studies have provided conclusive evidence. Relatively recent developments in our understanding of precisely how AMPK complexes might operate to control energy metabolism is due in part to the development of transgenic and knockout mouse models. Although there are inevitable caveats with genetic models, some important findings have emerged. In the present review, we discuss recent findings obtained from animal models with inhibition or activation of AMPK signaling pathway.
Mitogen-activated protein kinases (MAPKs) and NF-kappaB are two major regulators of gene transcription and metabolism in response to oxidative, energetic, and mechanical stress in skeletal muscle. Chronic activation of these signaling pathways has been implicated in the development and perpetuation of various pathologies, such as diabetes and cachexia. However, both MAPK and NF-kappaB are also stimulated by exercise, which promotes improvements in fuel homeostasis and can prevent skeletal muscle atrophy. This review will first discuss the major MAPK signaling modules in skeletal muscle, their differential activation by exercise, and speculated functions on acute substrate metabolism and exercise-induced gene expression. Focus will then shift to examination of the NF-kappaB pathway, including its mechanism of activation by cellular stress and its putative mediation of exercise-stimulated adaptations in antioxidant status, tissue regeneration, and metabolism. Although limited, there is additional evidence to suggest cross talk between MAPK and NF-kappaB signals with exercise. The objectives herein are twofold: 1) to determine how and why exercise activates MAPK and NF-kappaB; and 2) to resolve their paradoxical activation during diseased and healthy conditions.
In the last 30 years, the role of reactive oxygen species (ROS) in exercise physiology has received considerable attention. Acute physical exertion has been shown to induce an augmented generation of ROS in skeletal muscle via different mechanisms. There is evidence that ROS formation in response to vigorous physical exertion can result in oxidative stress. More recent research has revealed the important role of ROS as signaling molecules. ROS modulate contractile function in unfatigued and fatigued skeletal muscle. Furthermore, involvement of ROS in the modulation of gene expression via redox-sensitive transcription pathways represents an important regulatory mechanism, which has been suggested to be involved in the process of training adaptation. In this context, the adaptation of endogenous antioxidant systems in response to regular training reflects a potential mechanism responsible for augmented tolerance of skeletal muscle to exercise-induced stress. The present review outlines current knowledge and more recent findings in this area by focussing on major sources of ROS production, oxidative stress, tissue damage, contractile force, and redox-regulated gene expression in exercising skeletal muscle.
Data show that extracellular signal-regulated kinase 1/2 (ERK1/2) may be involved in the regulation of fatty acid (FA) uptake during muscle contraction via stimulation of CD36 translocation to the plasma membrane. The perfused hind limb model was used to determine (1) the importance of ERK1/2 signaling on contraction-induced FA uptake and (2) the effect of ERK1/2-mediated FA uptake on contraction-induced FA oxidation. We perfused rat hind limbs with 8 mmol/L glucose, 550 micromol/L palmitate, and no insulin at rest in the absence of inhibitor and during moderate-intensity electrical stimulation and dose-dependent pharmacologic inhibition of ERK1/2 using increasing concentrations of PD98059 (P1 = none, P2 = 10 micromol/L, P3 = 20 micromol/L, P4 = 50 micromol/L). Increasing PD98059 concentration resulted in a gradual decrease in contraction-induced ERK1/2 phosphorylation, and this was accompanied by a decrease in contraction-induced FA uptake (concentration required for 50% inhibition [IC(50)] = 15.8 +/- 1.6 mumol/L) and in plasma membrane CD36 content (IC(50) = 8.7 +/- 0.3 micromol/L) (P < .05). Percent FA oxidation was significantly lower in P3 and P4 compared with P1 and P2. Based on IC(50) values, FA oxidation demonstrated a greater sensitivity than FA uptake to changes in ERK1/2 phosphorylation (IC(50) = 5.4 +/- 0.3 micromol/L) (P < .05). A positive correlation was found between FA uptake and plasma membrane CD36 content (R(2) = 0.85, P < .05). Plasma membrane CD36 content, FA uptake, and FA oxidation each shared a positive correlation with ERK1/2 phosphorylation (R(2) = 0.64, 0.66, and 0.71, respectively; P < .05). These results suggest that during moderate-intensity muscle contraction, ERK1/2 phosphorylation is required for translocation of CD36 to the plasma membrane and the subsequent increase in FA uptake. In addition, these data suggest that ERK1/2 signaling may be involved in the regulation of FA oxidation independently of its effects on FA uptake.
Growing evidence suggests that activation of mitogen-activated protein kinase (MAPK) signal transduction mediates changes
in muscle gene expression in response to exercise. Nevertheless, little is known about upstream or downstream regulation of
MAPK in response to muscle contraction. Here we show that ex vivo muscle contraction stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38MAPK phosphorylation. Phosphorylation of ERK1/2 or p38MAPK was unaffected by protein kinase C inhibition (GF109203X), suggesting that protein kinase C is not involved in mediating
contraction-induced MAPK signaling. Contraction-stimulated phosphorylation of ERK1/2 and p38MAPK was completely inhibited by pretreatment with PD98059 (MAPK kinase inhibitor) and SB203580 (p38MAPKinhibitor), respectively. Muscle contraction also activated MAPK downstream targets p90 ribosomal S6 kinase (p90Rsk), MAPK-activated protein kinase 2 (MAPKAP-K2), and mitogen- and stress-activated protein kinase 1 (MSK1). Use of PD98059
or SB203580 revealed that stimulation of p90Rsk and MAPKAP-K2 most closely reflects ERK and p38MAPK stimulation, respectively. Stimulation of MSK1 in contracting skeletal muscle required the activation of both ERK and p38MAPK. These data demonstrate that muscle contraction, separate from systemic influence, activates MAPK signaling. Furthermore,
we are the first to show that contractile activity stimulates MAPKAP-K2 and MSK1.
• Contemporary stable isotope methodology was applied in combination with muscle biopsy sampling to accurately quantify substrate utilisation and study the regulation of muscle fuel selection during exercise. • Eight cyclists were studied at rest and during three consecutive 30 min stages of exercise at intensities of 40, 55 and 75 % maximal workload (Wmax). A continuous infusion of [U-13C]palmitate and [6,6-2H2]glucose was administered to determine plasma free fatty acid (FFA) oxidation and estimate plasma glucose oxidation, respectively. Biopsy samples were collected before and after each exercise stage. • Muscle glycogen and plasma glucose oxidation rates increased with every increment in exercise intensity. Whole-body fat oxidation increased to 32 ± 2 kJ min−1 at 55 % Wmax, but declined at 75 % Wmax (19 ± 2 kJ min−1). This decline involved a decrease in the oxidation rate of both plasma FFA and triacylglycerol fat sources (sum of intramuscular plus lipoprotein-derived triacylglycerol), and was accompanied by increases in muscle pyruvate dehydrogenase complex activation and acetylation of the carnitine pool, resulting in a decline in muscle free carnitine concentration. • We conclude that the most likely mechanism for the reduction in fat oxidation during high-intensity exercise is a downregulation of carnitine palmitoyltransferase I, either by this marked decline in free carnitine availability or by a decrease in intracellular pH.
The effects of exogenous oleate on glucose uptake, lactate production and glycogen concentration in resting and contracting skeletal muscle were studied in the perfused rat hindquarter. In preliminary studies with aged erythrocytes at a haemoglobin concentration of 8g/100ml in the perfusion medium, 1.8mm-oleate had no effect on glucose uptake or lactate production. During these studies it became evident that O(2) delivery was inadequate with aged erythrocytes. Perfusion with rejuvenated human erythrocytes at a haemoglobin concentration of 12g/100ml resulted in a 2-fold higher O(2) uptake at rest and a 4-fold higher O(2) uptake during muscle contraction than was obtained with aged erythrocytes. Rejuvenated erythrocytes were therefore used in subsequent experiments. Glucose uptake and lactate production by the well-oxygenated hindquarter were inhibited by one-third, both at rest and during muscle contraction, when 1.8mm-oleate was added to the perfusion medium. Addition of oleate also significantly protected against glycogen depletion in the fast-twitch red and slow-twitch red types of muscle, but not in white muscle, during sciatic-nerve stimulation. In the absence of added oleate, glucose was confined to the extracellular space in resting muscle. Addition of oleate resulted in intracellular glucose accumulation in red muscle. Contractile activity resulted in accumulation of intracellular glucose in all three muscle types, and this effect was significantly augmented in the red types of muscle by perfusion with oleate. The concentrations of citrate and glucose 6-phosphate were also increased in red muscle perfused with oleate. We conclude that, as in the heart, availability of fatty acids has an inhibitory effect on glucose uptake and glycogen utilization in well-oxygenated red skeletal muscle.
Seven men were studied during 30 min of treadmill exercise (approximately 70% VO2 max) to determine the effects of increased availability of plasma free fatty acids (FFA) and elevated plasma insulin on the utilization of muscle glycogen. This elevation of plasma FFA (1.01 mmol/1) with heparin (2,000 units) decreased the rate of muscle glycogen depletion by 40% as compared to the control experiment (FFA = 0.21 mmol/1). The ingestion of 75 g of glucose 45 min before exercise produced a 3.3-fold increase in plasma insulin and a 38% rise in plasma glucose at 0 min of exercise. Subsequent exercise increased muscle glycogen utilization and total carbohydrate (CHO) oxidation 17 and 13%, respectively, when compared to the control trial. This elevation of plasma insulin produced hypoglycemia (less than 3.5 mmol/1) in most subjects throughout the exercise. These data illustrate the regulatory influence of both plasma insulin and FFA on the rate of CHO usage during prolonged severe muscular activity.
We have investigated the role of triglyceride-fatty acid cycling in amplifying control of the net flux of fatty acids in response to exercise and in recovery from exercise. Five normal volunteers were infused with [1-13C]palmitate and D-5-glycerol throughout rest, 4 h of treadmill exercise at 40% maximum O2 consumption, and 2 h of recovery. Total fat oxidation was quantified by indirect calorimetry. Lipolysis (rate of appearance of glycerol) increased from 2.1 +/- 0.3 to 6.0 +/- 1.2 mumol.kg-1.min-1 after 30 min of exercise and progressively increased thereafter to a value of 10.5 +/- 0.8 mumol.kg-1.min-1 after 4 h. Lipolysis decreased rapidly during the first 20 min of recovery, but it was still significantly elevated after 2 h of recovery. The rate of appearance of free fatty acids followed the same pattern of response. Seventy percent of released fatty acids were reesterified at rest, and this value decreased to 25% within the first 30 min of exercise. Reesterification remained less than 35% of lipolysis until the start of recovery, at which time the value rose to 90%. In exercise, more than one-half the increase in fat oxidation could be attributed to the reduction in the percent reesterification. Most of the change in percent reesterification during exercise and recovery was caused by changes in extracellular cycling of fatty acids released into plasma. We conclude that triglyceride-fatty acid cycling plays an important role in enabling a rapid response of fatty acid metabolism to major changes in energy metabolism.
The effects of sympathoadrenal manipulations on the exercise-induced alterations in blood glucose, plasma free fatty acids (FFA), and insulin were investigated in intact and adrenodemedullated rats. Exercise consisted of strenuous swimming against a countercurrent for 15 min. Before, during, and after swimming, blood samples were taken through a permanent heart catheter. Adrenodemedullation (Adm) markedly reduced the exercise-induced increase in both glucose and FFA. This effect was counteracted by intravenous infusion of epinephrine (E, 20 ng/min). Intravenous infusion of 50 ng E/min into Adm rats caused an exaggerated increase in glucose. In two additional experiments 1) specific adrenoceptor agonists and antagonists were administered to exercising intact and Adm rats, and 2) E or norepinephrine (NE; 20 ng/min) was infused into intact resting rats. The results suggest that E from the adrenal medulla directly affects glucose and insulin but not FFA concentrations in the blood. NE released from peripheral sympathetic nerve endings probably acts in two different ways: as neurotransmitter on liver and pancreas and as a hormone on adipose tissue.
AMP-activated protein kinase (AMPK) and Ca2+/calmodulin (CaM)-dependent protein kinase I (CaMKI) are protein kinases that are regulated both by allosteric activation
(AMP and Ca2+/CaM, respectively) and by phosphorylation by upstream protein kinases (AMPK kinase (AMPKK) and CaMKI kinase (CaMKIK), respectively).
We now report that AMPKK can activate CaMKI and that, conversely, CaMKIK can activate AMPK. CaMKIK is 68-fold more effective
at activating CaMKI than AMPK, while AMPKK is 17-fold more effective at activating AMPK than CaMKI. Our results suggest that
CaMKIK and AMPKK are distinct enzymes dedicated to their respective kinase targets but with some overlap in their substrate
specificities. The availability of alternative substrates for AMPKK and CaMKIK allowed the unequivocal demonstration that
AMP and Ca2+/calmodulin promote the activation of AMPK and CaMKI, respectively, via three independent mechanisms: 1) direct activation
of AMPK and CaMKI, 2) activation of AMPKK and CaMKIK, and 3) by binding to AMPK and CaMKI, inducing exposure of their phosphorylation
sites. Since AMP and Ca2+/calmodulin each has a triple effect in its respective system, in vivo, the two systems would be expected to be exquisitely sensitive to changes in concentration of their respective activating
ligands.
Stable isotope tracers and indirect calorimetry were used to evaluate the regulation of endogenous fat and glucose metabolism in relation to exercise intensity and duration. Five trained subjects were studied during exercise intensities of 25, 65, and 85% of maximal oxygen consumption (VO2max). Plasma glucose tissue uptake and muscle glycogen oxidation increased in relation to exercise intensity. In contrast, peripheral lipolysis was stimulated maximally at the lowest exercise intensity, and fatty acid release into plasma decreased with increasing exercise intensity. Muscle triglyceride lipolysis was stimulated only at higher intensities. During 2 h of exercise at 65% VO2max plasma-derived substrate oxidation progressively increased over time, whereas muscle glycogen and triglyceride oxidation decreased. In recovery from high-intensity exercise, although the rate of lipolysis immediately decreased, the rate of release of fatty acids into plasma increased, indicating release of fatty acids from previously hydrolyzed triglycerides. We conclude that, whereas carbohydrate availability is regulated directly in relation to exercise intensity, the regulation of lipid metabolism seems to be more complex.
In the present study we examined the hypothesis that fatty acid oxidation is less during high-intensity exercise than during moderate-intensity exercise because of inhibition of long-chain fatty acid entry into the mitochondria. Six volunteers exercised at 40% peak oxygen consumption (VO2peak) for 60 min and at 80% VO2peak for 30 min on two different occasions. [1-13C]oleate, a long-chain fatty acid, and [1-14C]octanoate, a medium-chain fatty acid, were infused for the duration of the studies. Lipids and heparin were infused during exercise at 80% VO2peak to prevent the expected decrease in plasma free fatty acid (FFA) concentration. Plasma oleate and total FFA availability were similar in the two experiments. Oleate oxidation decreased from 2.8 +/- 0.6 (40% VO2peak) to 1.8 +/- 0.2 mumol.kg-1.min-1 (80% VO2peak, P < 0.05), whereas octanoate oxidation increased from 1.0e-05 +/- 1.0e-06 (40% VO2peak) to 1.3e-05 +/- 5.1e-06 mumol.kg-1.min-1 (80% VO2peak, P < 0.05). Furthermore, the percentage of oleate uptake oxidized decreased from 67.7 +/- 2.8% (40% VO2peak) to 51.8 +/- 4.6% (80% VO2peak, P < 0.05), whereas the percentage of octanoate oxidized was similar during exercise at 40 and 80% VO2peak (84.8 +/- 2.7 vs. 89.3 +/- 2.7%, respectively). Our data suggest that, in addition to suboptimal FFA availability, fatty acid oxidation is likely limited during high-intensity exercise because of direct inhibition of long-chain fatty acid entry into mitochondria.
We have examined whether 1) fatty acid (FA) uptake, 2) FA transporter expression, and 3) FA metabolism are increased when the oxidative capacity of skeletal muscle is increased. The oxidative capacities of red and white tibialis anterior and extensor digitorum longus muscles were increased via chronic stimulation (10 Hz, 24 h/day for 7 days). The contralateral muscles served as controls. After 7 days of increased muscle activity 1) palmitate uptake by giant sarcolemmal vesicles was increased twofold (P < 0.05), 2) the expression of FA translocase (FAT)/CD36 was increased at both the mRNA (3.2- to 10-fold) and protein (3.4-fold) levels, and 3) palmitate oxidation and esterification into triacylglycerols and phospholipids were increased 1.5-, 2.7-, and 1.7-fold, respectively (P < 0.05). These data show that when the oxidative capacity of muscle is increased, there is a parallel increase in the rate of FA transport and FA transporters at the sarcolemmal membrane, which is associated with the enhanced expression of the membrane transporter FAT/CD36.
This study investigated intramuscular triacylglycerol (IMTG) and glycogen utilisation, pyruvate dehydrogenase activation (PDHa) and acetyl group accumulation during prolonged moderate intensity exercise. Seven endurance-trained men cycled for 240 min at 57 % maximal oxygen consumption (V(O2,max)) and duplicate muscle samples were obtained at rest and at 10, 120 and 240 min of exercise. We hypothesised that IMTG utilisation would be augmented during 2-4 h of exercise, while PDHa would be decreased secondary to reduced glycogen metabolism. IMTG was measured on both muscle samples at each time point and the coefficient of variation was 12.3 +/- 9.4 %. Whole body respiratory exchange ratio (RER) decreased from 0.89 +/- 0.01 at 30 min to 0.83 +/- 0.01 at 150 min and remained low throughout exercise. Plasma glycerol and free fatty acids (FFAs) had increased compared with rest at 90 min and progressively increased until exercise cessation. Although plasma glucose tended to decrease with exercise, this was not significant. IMTG was reduced at 120 min compared with rest (0 min, 15.6 +/- 0.8 mmol kg(-1) d.m.; 120 min, 12.8 +/- 0.7 mmol kg(-1) d.m.) but no further reduction in IMTG was observed at 240 min. Muscle glycogen was 468 +/- 49 mmol kg(-1) d.m. at rest and decreased at 120 min and again at 240 min (217 +/- 48 and 144 + 47 mmol kg(-1) d.m.). PDHa increased above rest at 10 and 120 min, but decreased at 240 min, which coincided with reduced whole body carbohydrate oxidation. Muscle pyruvate and ATP were unchanged with exercise. Acetyl CoA increased at 10 min and remained elevated throughout exercise. Acetylcarnitine increased at exercise onset but returned to resting values by 240 min. Contrary to our first hypothesis, significant utilisation of IMTG occurred during the first 2 h of moderate exercise but not during hours 2-4. The reduced utilisation of intramuscular fuels during the last 120 min was offset by greater FFA delivery and oxidation. Consistent with the second hypothesis, PDHa decreased late in moderate exercise and closely matched the estimates of lower carbohydrate flux. Although the factor underlying the PDHa decrease was not apparent, reduced pyruvate provision secondary to diminished glycolytic flux is the most likely mechanism.
gACRP30, the globular subunit of adipocyte complement-related protein of 30 kDa (ACRP30), improves insulin sensitivity and increases fatty acid oxidation. The mechanism by which gACRP30 exerts these effects is unknown. Here, we examined if gACRP30 activates AMP-activated protein kinase (AMPK), an enzyme that has been shown to increase muscle fatty acid oxidation and insulin sensitivity. Incubation of rat extensor digitorum longus (EDL), a predominantly fast twitch muscle, with gACRP30 (2.5 micro g/ml) for 30 min led to 2-fold increases in AMPK activity and phosphorylation of both AMPK on Thr-172 and acetyl CoA carboxylase (ACC) on Ser-79. Accordingly, concentration of malonyl CoA was diminished by 30%. In addition, gACRP30 caused a 1.5-fold increase in 2-deoxyglucose uptake. Similar changes in malonyl CoA and ACC were observed in soleus muscle incubated with gACRP30 (2.5 micro g/ml), although no significant changes in AMPK activity or 2-deoxyglucose uptake were detected. When EDL was incubated with full-length hexameric ACRP30 (10 micro g/ml), AMPK activity and ACC phosphorylation were not altered. Administration of gACRP30 (75 micro g) to C57 BL6J mice in vivo led to increased AMPK activity and ACC phosphorylation and decreased malonyl CoA concentration in gastrocnemius muscle within 15-30 min. Both in vivo and in vitro, activation of AMPK was the first effect of gACRP30 and was transient, whereas alterations in malonyl CoA and ACC occurred later and were more sustained. Thus, gACRP30 most likely exerts its actions on muscle fatty acid oxidation by inactivating ACC via activation of AMPK and perhaps other signal transduction proteins.
The metabolic role of 5'AMP-activated protein kinase (AMPK) in regulation of skeletal muscle metabolism in humans is unresolved. We measured isoform-specific AMPK activity and beta-acetyl-CoA carboxylase (ACCbeta) Ser(221) phosphorylation and substrate balance in skeletal muscle of eight athletes at rest, during cycling exercise for 1 h at 70% peak oxygen consumption, and 1 h into recovery. The experiment was performed twice, once in a glycogen-loaded (glycogen concentration approximately 900 mmol/kg dry wt) and once in a glycogen-depleted (glycogen concentration approximately 160 mmol/kg dry wt) state. At rest, plasma long-chain fatty acids (FA) were twofold higher in the glycogen-depleted than in the loaded state, and muscle alpha1 AMPK (160%) and alpha2 AMPK (145%) activities and ACCbeta Ser(221) phosphorylation (137%) were also significantly higher in the glycogen-depleted state. During exercise, alpha2 AMPK activity, ACCbeta Ser(221) phosphorylation, plasma catecholamines, and leg glucose and net FA uptake were significantly higher in the glycogen-depleted than in the glycogen-loaded state without apparent differences in muscle high-energy phosphates. Thus exercise in the glycogen-depleted state elicits an enhanced uptake of circulating fuels that might be associated with elevated muscle AMPK activation. It is concluded that muscle AMPK activity and ACCbeta Ser(221) phosphorylation at rest and during exercise are sensitive to the fuel status of the muscle. During exercise, this dependence may in part be mediated by humoral factors.
Contraction of rat cardiac myocytes induces translocation of fatty acid translocase (FAT)/CD36 and GLUT4 from intracellular stores to the sarcolemma, leading to enhanced rates of long-chain fatty acid (FA) and glucose uptake, respectively. Because intracellular AMP/ATP is elevated in contracting cardiac myocytes, we investigated whether activation of AMP-activated protein kinase (AMP kinase) is involved in contraction-inducible FAT/CD36 translocation. The cell-permeable adenosine analog 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and the mitochondrial inhibitor oligomycin, similar to 4-Hz electrostimulation, evoked a more than threefold activation of cardiomyocytic AMP kinase. Both AICAR and oligomycin stimulated FA uptake into noncontracting myocytes by 1.4- and 2.0-fold, respectively, but were ineffective in 4 Hz-contracting myocytes. These findings indicate that both agents stimulate FA uptake by a similar mechanism as electrostimulation, involving activation of AMP kinase, as evidenced from phosphorylation of acetyl-CoA carboxylase. Furthermore, the stimulating effects of both AICAR and oligomycin were antagonized by blocking FAT/CD36 with sulfo-N-succinimidylpalmitate, but not by inhibiting phosphatidylinositol 3-kinase with wortmannin, indicating the involvement of FAT/CD36, but excluding a role for insulin signaling. Subcellular fractionation showed that oligomycin was able to mobilize intracellularly stored FAT/CD36 to the sarcolemma. We conclude that AMP kinase regulates cardiac FA use through mobilization of FAT/CD36 from a contraction-inducible intracellular storage compartment.
The effect of exercise intensity on skeletal muscle AMP-activated protein kinase (AMPK) signaling and substrate metabolism was examined in eight men cycling for 20 min at each of three sequential intensities: low (40 +/- 2% VO(2) peak), medium (59 +/- 1% VO(2) peak), and high (79 +/- 1% VO(2) peak). Muscle free AMP/ATP ratio only increased at the two higher exercise intensities (P < 0.05). AMPK alpha 1 (1.5-fold) and AMPK alpha 2 (5-fold) activities increased from low to medium intensity, with AMPK alpha 2 activity increasing further from medium to high intensity. The upstream AMPK kinase activity was substantial at rest and only increased 50% with exercise, indicating that, initially, signaling through AMPK did not require AMPK kinase posttranslational modification. Acetyl-CoA carboxylase (ACC)-beta phosphorylation was sensitive to exercise, increasing threefold from rest to low intensity, whereas neuronal NO synthase (nNOS) micro phosphorylation was only observed at the higher exercise intensities. Glucose disappearance (tracer) did not increase from rest to low intensity, but increased sequentially from low to medium to high intensity. Calculated fat oxidation increased from rest to low intensity in parallel with ACC beta phosphorylation, then declined during high intensity. These results indicate that ACC beta phosphorylation is especially sensitive to exercise and tightly coupled to AMPK signaling and that AMPK activation does not depend on AMPK kinase activation during exercise.
Endurance training induces a partial fast-to-slow muscle phenotype transformation and mitochondrial biogenesis but no growth. In contrast, resistance training mainly stimulates muscle protein synthesis resulting in hypertrophy. The aim of this study was to identify signaling events that may mediate the specific adaptations to these types of exercise. Isolated rat muscles were electrically stimulated with either high frequency (HFS; 6x10 repetitions of 3 s-bursts at 100 Hz to mimic resistance training) or low frequency (LFS; 3 h at 10 Hz to mimic endurance training). HFS significantly increased myofibrillar and sarcoplasmic protein synthesis 3 h after stimulation 5.3- and 2.7-fold, respectively. LFS had no significant effect on protein synthesis 3 h after stimulation but increased UCP3 mRNA 11.7-fold, whereas HFS had no significant effect on UCP3 mRNA. Only LFS increased AMPK phosphorylation significantly at Thr172 by approximately 2-fold and increased PGC-1alpha protein to 1.3 times of control. LFS had no effect on PKB phosphorylation but reduced TSC2 phosphorylation at Thr1462 and deactivated translational regulators. In contrast, HFS acutely increased phosphorylation of PKB at Ser473 5.3-fold and the phosphorylation of TSC2, mTOR, GSK-3beta at PKB-sensitive sites. HFS also caused a prolonged activation of the translational regulators p70 S6k, 4E-BP1, eIF-2B, and eEF2. These data suggest that a specific signaling response to LFS is a specific activation of the AMPK-PGC-1alpha signaling pathway which may explain some endurance training adaptations. HFS selectively activates the PKB-TSC2-mTOR cascade causing a prolonged activation of translational regulators, which is consistent with increased protein synthesis and muscle growth. We term this behavior the "AMPK-PKB switch." We hypothesize that the AMPK-PKB switch is a mechanism that partially mediates specific adaptations to endurance and resistance training, respectively.
The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKalphaThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs. We have employed three cell lines deficient in LKB1 to study AMPK regulation and phosphorylation, HeLa, A549, and murine embryo fibroblasts derived from LKB(-/-) mice. In HeLa and A549 cells, mannitol, 2-deoxyglucose, and ionomycin, but not 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), treatment activates AMPK by alphaThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase, are largely inhibited by the Ca(2+)/ calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609. AMPKK activity in HeLa cell lysates measured in vitro is totally inhibited by STO-609 with an IC50 comparable with that of the known CaMKK isoforms, CaMKKalpha and CaMKKbeta. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, alphaThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKalpha and CaMKKbeta. Lastly, the activation of AMPK in response to ionomycin and 2-deoxyglucose is not impaired in LKB1(-/-) murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo.
This chapter focuses on some modern methods of regression. Smoothers attempt to estimate a regression line without forcing to have a particular shape, such as a straight line. Cleveland's smoother: a technique derived by Cleveland is known as a locally weighted running-line smoother. The idea is that, although a regression line might not be linear over the entire range of X values, it will be approximately linear over small intervals of X. S-PLUS function lowess: the built-in S-PLUS function computes Cleveland's smoother. The value for p, the span, defaults to 2/3. Running-interval smoother: To estimate some measure of location for Y, given some value for X, a running interval smoother searches for all points close to the value of X that are of interest and then simply computes the measure of location based on the corresponding Y values. In contrast, Cleveland's method uses the k nearest points, with k fixed and chosen in advance. Later, predictor is discussed. S-PLUS functions rungen and runmean: The S-PLUS function rungen computes a running interval smooth assuming that there is only one predictor. The S-PLUS function runmean is a bit more convenient to use. These regression methods have been useful in most of its applications.
During the last half century hundreds of papers published in statistical journals have documented general conditions where reliance on least squares regression and Pearson's correlation can result in missing even strong associations between variables. Moreover, highly misleading conclusions can be made, even when the sample size is large. There are, in fact, several fundamental concerns related to non-normality, outliers, heteroscedasticity, and curvature that can result in missing a strong association. Simultaneously, a vast array of new methods have been derived for effectively dealing with these concerns. The paper (1) reviews why least squares regression and classic inferential methods can fail, (2) provides an overview of the many modern strategies for dealing with known problems, including some recent advances, and (3) illustrates that modern robust methods can make a practical difference in our understanding of data. Included are some general recommendations regarding how modern methods might be used.
We determined whether the cell permeable molecule AICAR, whose metabolite activates AMP-activated protein kinase (AMPK) in cells, affected glycogen metabolism in rat seleus muscle preparations in vitro. The basal and insulin-stimulated rates of radiochemical lactate formation, net lactate release and glycogen synthesis were determined. AICAR stimulated net lactate release (but not radiochemical lactate formation) only at a basal concentration of insulin. An increased rate of glycogenolysis was the likely cause of increased net lactate release as glycogen phosphorylase activity was significantly increased by AICAR. AICAR-stimulated net lactate release and phosphorylase activity were potently inhibited by insulin.
To evaluate the effects of contractions on the kinetics of uptake and oxidation of palmitate in a physiological muscle preparation, rat hindquarters were perfused with glucose (6 mmol/l), albumin-bound [1-14C]palmitate, and varying amounts of albumin-bound palmitate (200-2,200 micro mol/l) at rest and during muscle contractions. When plotted against the unbound palmitate concentration, palmitate uptake and oxidation displayed simple Michaelis-Menten kinetics with estimated maximal velocity (Vmax) and Michaelis-Menten constant (Km) values of 42.8 +/- 3.8 (SE) nmol . min-1 . g-1 and 13.4 +/- 3.4 nmol/l for palmitate uptake and 3.8 +/- 0.4 nmol . min-1 . g-1 and 8.1 +/- 2.9 nmol/l for palmitate oxidation, respectively, at rest. Whereas muscle contractions increased the Vmax for both palmitate uptake and oxidation to 91.6 +/- 10.1 and 16.5 +/- 2.3 nmol . min-1 . g-1, respectively, the Km remained unchanged. Vmax and Km estimates obtained from Hanes-Woolf plots (substrate concentration/velocity vs. substrate concentration) were not significantly different. In the resting perfused hindquarter, an increase in palmitate delivery from 31.9 +/- 0.9 to 48.7 +/- 1.2 micro mol . g-1 . h-1 by increasing perfusate flow was associated with a decrease in the fractional uptake of palmitate so that the rates of uptake and oxidation of palmitate remained unchanged. It is concluded that the rates of uptake and oxidation of long-chain fatty acids (LCFA) saturate with an increase in the concentration of unbound LCFA in perfused skeletal muscle and that muscle contractions, but not an increase in plasma flow, increase the Vmax for LCFA uptake and oxidation. The data are consistent with the notion that uptake of LCFA in muscle may be mediated in part by a transport system.
We studied the effect of local muscle adaptations on free fatty acid (FFA) metabolism during prolonged exercise in trained and untrained subjects. Six trained (T) and six untrained (UT) young human males exercised for 3 h at 60% of their individual maximal dynamic knee extension capacity. The contribution of blood and plasma metabolites as well as intramuscular substrates to oxidative metabolism in the thigh was calculated from arteriovenous differences and femoral-venous blood flow as well as from muscle biopsies in subjects that were continuously infused with [1-14C]palmitate. Arterial plasma FFA concentration increased over time in both T and UT. Fractional uptake of FFA across the thigh remained unchanged over time in T (15%) but decreased in UT (from 15 to 7%), especially during the last hour of exercise. Thus FFA uptake increased linearly over time in T (96 +/- 20 to 213 +/- 20 mumol.min-1.kg-1), whereas it leveled off after 2 h in UT (74 +/- 16 to 133 +/- 46) even though FFA delivery increased similarly in T and UT. Percentage oxidation was similar in T and UT; thus total FFA oxidation was higher in T. Glucose uptake increased in both groups over time and was significantly higher in UT during the last hour of exercise. In conclusion, during prolonged knee extension exercise, FFA uptake increases linearly with FFA delivery in the trained thigh, whereas in the untrained thigh uptake becomes saturated with time. This difference partly explains the increased lipid oxidation in T vs. UT and suggests, furthermore, that local muscle adaptations to training are important for the utilization of FFA during prolonged exercise.
Little is known about the contribution of plasma free fatty acid (FFA) and intramuscular triacylglycerol (TG) as substrates for energy production during prolonged electrical stimulation of skeletal muscle. The purpose of this study was to investigate the effects of continuous and intermittent electrical stimulation protocols of different intensities on exogenous FFA oxidation, exogenous FFA incorporation into intracellular TG, and intracellular TG content in the isolated in vitro rat flexor digitorum brevis muscle preparation. Muscles were electrically stimulated for 0.5 h continuously at 0.2 Hz or intermittently (30 s on, 60 s off) at 0.2, 0.4, 0.8, and 5.0 Hz while incubated at 37 degrees C in 0.5 mM palmitate-3% bovine serum albumin medium (pH 7.4) in the presence of insulin (100 microU/ml) and glucose (11 mM). Control muscles were frozen immediately after excision or incubated for 0.5 h. At similar frequencies, less exogenous FFA esterification and more exogenous FFA oxidation occurred during continuous than during intermittent stimulation. As the frequency of intermittent stimulation increased, the amount of exogenous FFA esterified decreased and the amount of exogenous FFA oxidized increased. The data also indicate that at least a portion of TG was constantly being hydrolyzed during electrical stimulation. Under stimulation conditions in which exogenous FFA esterification was below the control (resting muscle) level, intramuscular TG content was significantly decreased compared with control TG content values. Thus both plasma FFA and intramuscular TG are substrates for energy production during electrical stimulation. However, the stimulation parameters employed affect the quantities utilized.
Malonyl-CoA, an inhibitor of fatty acid oxidation in skeletal muscle mitochondria, decreases in rat skeletal muscle during exercise or in response to electrical stimulation. Regulation of rat skeletal muscle acetyl-CoA carboxylase (ACC), the enzyme that synthesizes malonyl-CoA, was studied in vitro and in vivo. Avidin-Sepharose affinity-purified ACC from hindlimb skeletal muscle was phosphorylated by purified liver AMP-activated protein kinase with a concurrent decrease in ACC activity. AMP-activated protein kinase was quantitated in resuspended ammonium sulfate precipitates of the fast-twitch red (type IIa fibers) region of the quadriceps muscle. Rats running on a treadmill at 21 m/min up a 15% grade show a 2.4-fold activation of AMP-activated protein kinase concurrently with a marked decrease in ACC activity in the resuspended ammonium sulfate precipitates at all citrate concentrations ranging from 0 to 20 mM. Malonyl-CoA decreased from a resting value of 1.85 +/- 0.29 to 0.50 +/- 0.09 nmol/g in red quadriceps muscle after 30 min of treadmill running. The activation of the AMP-activated protein kinase with consequent phosphorylation and inactivation of ACC may be one of the primary events in the control of malonyl-CoA and hence fatty acid oxidation during exercise.
Malonyl-CoA is synthesized by acetyl-CoA carboxylase (ACC) and is an inhibitor of fatty acid oxidation. Exercise induces a decline in skeletal muscle malonyl-CoA, which is accompanied by inactivation of ACC and increased activity of AMP-activated protein kinase (AMPK). This study was designed to determine the effect of exercise intensity on the enzyme kinetics of ACC, malonyl-CoA levels, and AMPK activity in skeletal muscle. Male Sprague-Dawley rats were killed (pentobarbital sodium anesthesia) at rest or after 5 min of exercise (10, 20, 30, or 40 m/min at 5% grade). The fast-twitch red and white regions of the quadriceps muscle were excised and frozen in liquid nitrogen. A progressive decrease in red quadriceps ACC maximal velocity (from 28.6 +/- 1.5 to 14.3 +/- 0.7 nmol . g-1 . min-1, P < 0.05), an increase in activation constant for citrate, and a decrease in malonyl-CoA (from 1.9 +/- 0.2 to 0.9 +/- 0.1 nmol/g, P < 0.05) were seen with the increase in exercise intensity from rest to 40 m/min. AMPK activity increased more than twofold. White quadriceps ACC activity decreased only during intense exercise. We conclude that the extent of ACC inactivation during short-term exercise is dependent on exercise intensity.
5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) has previously been reported to be taken up into cells and phosphorylated to form ZMP, an analog of 5'-AMP. This study was designed to determine whether AICAR can activate AMP-activated protein kinase (AMPK) in skeletal muscle with consequent phosphorylation of acetyl-CoA carboxylase (ACC), decrease in malonyl-CoA, and increase in fatty acid oxidation. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine red blood cells, 200 microU/ml insulin, and 10 mM glucose with or without AICAR (0.5-2.0 mM). Perfusion with medium containing AICAR was found to activate AMPK in skeletal muscle, inactivate ACC, and decrease malonyl-CoA. Hindlimbs perfused with 2 mM AICAR for 45 min exhibited a 2.8-fold increase in fatty acid oxidation and a significant increase in glucose uptake. No difference was observed in oxygen uptake in AICAR vs. control hindlimb. These results provide evidence that decreases in muscle content of malonyl-CoA can increase the rate of fatty acid oxidation.
We examined the oxidation and esterification of palmitate and the hydrolysis and oxidation of intramuscular lipids in isolated soleus muscles at rest and during tetanic contractions (2-40 tetani/min). Muscles were pulsed with [14C]palmitate to prelabel all intramuscular lipid pools. Muscles remained at rest or were then stimulated to contract at 2, 8, 20, or 40 tetani/min (30 min) in the presence of [3H]palmitate. Palmitate oxidation was increased 412% at 2 tetani/min (P < 0.05) and 880% at 8 tetani/min (P < 0.05). During contraction there was an absolute increase in esterification of palmitate to triacylglycerol in proportion with the increasing rate of palmitate oxidation. Intramuscular lipid oxidation provided approximately 77% of the total muscle energy compared with approximately 3% provided by exogenous palmitate under all conditions, with carbohydrate sources (glycogen and glucose) providing approximately 20% of the total energy. Thus, during muscle contraction, the oxidation rates of both exogenous and intramuscular lipids are increased in proportion to each other, while concomitantly palmitate is esterified in proportion to its oxidation.
To evaluate the effects of endurance training in rats on fatty acid metabolism, we measured the uptake and oxidation of palmitate in isolated rat hindquarters as well as the content of fatty acid-binding proteins in the plasma membranes (FABP(PM)) of red and white muscles from 16 trained (T) and 18 untrained (UT) rats. Hindquarters were perfused with 6 mM glucose, 1,800 microM palmitate, and [1-(14)C]palmitate at rest and during electrical stimulation (ES) for 25 min. FABP(PM) content was 43-226% higher in red than in white muscles and was increased by 55% in red muscles after training. A positive correlation was found to exist between succinate dehydrogenase activity and FABP(PM) content in muscle. Palmitate uptake increased by 64-73% from rest to ES in both T and UT and was 48-57% higher in T than UT both at rest (39.8 +/- 3.5 vs. 26.9 +/- 4. 4 nmol. min(-1). g(-1), T and UT, respectively) and during ES (69.0 +/- 6.1 vs. 43.9 +/- 4.4 nmol. min(-1). g(-1), T and UT, respectively). While the rats were resting, palmitate oxidation was not affected by training; palmitate oxidation during ES was higher in T than UT rats (14.8 +/- 1.3 vs. 9.3 +/- 1.9 nmol. min(-1). g(-1), T and UT, respectively). In conclusion, endurance training increases 1) plasma free fatty acid (FFA) uptake in resting and contracting perfused muscle, 2) plasma FFA oxidation in contracting perfused muscle, and 3) FABP(PM) content in red muscles. These results suggest that an increased number of these putative plasma membrane fatty acid transporters may be available in the trained muscle and may be implicated in the regulation of plasma FFA metabolism in skeletal muscle.
5'AMP-activated protein kinase (AMPK) has been suggested to be a key regulatory protein in exercise signaling of muscle glucose transport. To test this hypothesis, we investigated whether muscle glycogen levels affect AMPK activation and glucose transport stimulation similarly during contractions. Rats were preconditioned by a combination of swimming exercise and diet to obtain a glycogen-supercompensated group (high muscle glycogen content [HG]) with approximately 3-fold higher muscle glycogen levels than a glycogen-depleted group (low muscle glycogen content [LG]). In perfused fast-twitch muscles, contractions induced significant increases in AMPK activity and glucose transport and decreases in acetyl-CoA carboxylase (ACC) activity in both HG and LG groups. Contraction-induced glucose transport was nearly 2-fold (P < 0.05) and AMPK activation was 3-fold (P < 0.05) higher in the LG group compared with the HG group, whereas ACC deactivation was not different between groups. Thus, there was a significant positive correlation between AMPK activity and glucose transport in contracting fast-twitch muscles (r = 0.80, P < 0.01). However, in slow-twitch muscles with HG, glucose transport was increased 6-fold (P < 0.05) during contractions, whereas AMPK activity did not increase. In contracting slow-twitch muscles with LG, the increase in AMPK activity (315%) and the decrease in ACC activity (54 vs. 34% at 0.2 mmol/l citrate, LG vs. HG) was higher (P < 0.05) compared with HG muscles, whereas the increase in glucose transport was identical in HG and LG. In conclusion, in slow-twitch muscles, high glycogen levels inhibit contraction-induced AMPK activation without affecting glucose transport. This observation suggests that AMPK activation is not an essential signaling step in glucose transport stimulation in skeletal muscle.
Previous studies have indicated that frequency of stimulation is a major determinant of glucose transport in contracting muscle. We have now studied whether this is so also when total force development or metabolic rate is kept constant. Incubated soleus muscles were electrically stimulated to perform repeated tetanic contractions at four different frequencies (0.25, 0.5, 1, and 2 Hz) for 10 min. Resting length was adjusted to achieve identical total force development or metabolic rate (glycogen depletion and lactate accumulation). Overall, at constant total force development, glucose transport (2-deoxyglucose uptake) increased with stimulation frequency (P < 0.05; basal: 25 +/- 2, 0.25 Hz: 50 +/- 4, 0.5 Hz: 50 +/- 3, 1 Hz: 81 +/- 5, 2 Hz: 79 +/- 3 nmol. g(-1). 5 min(-1)). However, glucose transport was identical (P > 0.05) at the two lower (0.25 and 0.5 Hz) as well as at the two higher (1 and 2 Hz) frequencies. Glycogen decreased (P < 0.05; basal: 19 +/- 1, 0.25 Hz: 13 +/- 1, 0.5 Hz: 12 +/- 2, 1 Hz: 7 +/- 1, 2 Hz: 7 +/- 1 mmol/kg) and 5'-AMP-activated protein kinase (AMPK) activity increased (P < 0. 05; basal: 1.7 +/- 0.4, 0.25 Hz: 32.4 +/- 7.0, 0.5 Hz: 36.5 +/- 2.1, 1 Hz: 63.4 +/- 8.0, 2 Hz: 67.0 +/- 13.4 pmol. mg(-1). min(-1)) when glucose transport increased. Experiments with constant metabolic rate were carried out in soleus, flexor digitorum brevis, and epitrochlearis muscles. In all muscles, glucose transport was identical at 0.5 and 2 Hz (P > 0.05); also, AMPK activity did not increase with stimulation frequency. In conclusion, muscle glucose transport increases with stimulation frequency but only in the face of energy depletion and increase in AMPK activity. This indicates that contraction-induced glucose transport is elicited by metabolic demands rather than by events occurring early during the excitation-contraction coupling.
1. 5'-AMP-activated protein kinase (AMPK) has been suggested to play a key role in the regulation of metabolism in skeletal muscle. AMPK is activated in treadmill-exercised and electrically stimulated rodent muscles. Whether AMPK is activated during exercise in humans is unknown. 2. We investigated the degree of activation and deactivation of alpha-isoforms of AMPK during and after exercise. Healthy human subjects performed bicycle exercise on two separate occasions at either a low ( approximately 50% maximum rate of O2 uptake (VO2,max) for 90 min) or a high ( approximately 75% VO2,max for 60 min) intensity. Biopsies from the vastus lateralis muscle were obtained before and immediately after exercise, and after 3 h of recovery. 3. We observed a 3- to 4-fold activation of the alpha2-AMPK isoform immediately after high intensity exercise, whereas no activation was observed after low intensity exercise. The activation of alpha2-AMPK was totally reversed 3 h after exercise. In contrast, alpha1-AMPK was not activated during either of the two exercise trials. 4. The in vitro AMP dependency of alpha2-AMPK was significantly greater than that of alpha1-AMPK ( approximately 3- vs. approximately 2-fold). 5. We conclude that in humans activation of alpha2-AMPK during exercise is dependent upon exercise intensity. The stable activation of alpha2-AMPK, presumably due to the activation of an upstream AMPK kinase, is compatible with a role for this kinase complex in the regulation of skeletal muscle metabolism during exercise, whereas the lack of stable alpha1-AMPK activation makes this kinase complex a less likely candidate.
The mitogen-activated protein (MAP) kinase pathways have been highlighted as a possible link between exercise and adaptive changes in skeletal muscle. In this study, the effect of exercise intensity on the activation of the ERK/MAP kinase pathway was investigated in human skeletal muscle. One-leg exercise at low (40% maximal oxygen consumption, VO2max for 30 min) and high (75% VO2max for 30 min) intensity resulted in 11.5+8. I-fold and 39.7+/-6.3-fold (mean +/-SEM) increases in ERK1/2 phosphorylation (P<0.001), respectively. The phosphorylation of MEK1/2, the upstream kinase of ERK1/2, increased with exercise intensity (P<0.05) to 2.5+/-0.9 and 4.8+/-1.1 times the basal level at the low and high intensity, respectively. The statistical analysis revealed a systematic difference between basal, low and high intensity exercise levels for both kinases. There was no change in the phosphorylation of either kinase in the non-exercised leg. The phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB), a possible downstream target of the ERK/MAP kinase signalling pathway, was unaffected by exercise. The phosphorylation of ERK1/2 was significantly higher in purified freeze-dried compared to crude wet muscle after exercise, whereas the opposite pattern was observed for CREB. In conclusion, phosphorylation of ERK1/2 and MEK1/2 increases in an exercise intensity-dependent manner in human skeletal muscle and this seems to originate in the muscle fibres themselves.
The mechanism by which mechanical forces acting through skeletal muscle cells generate intracellular signaling, known as mechanotransduction, and the details of how gene expression and cell size are regulated by this signaling are poorly understood. Mitogen-activated protein kinases (MAPKs) are known to be involved in mechanically induced signaling in various cell types, including skeletal muscle where MAPK activation has been reported in response to contraction and passive stretch. Therefore, the investigation of MAPK activation in response to mechanical stress in skeletal muscle may yield important information about the mechanotransduction process. With the use of a rat plantaris in situ preparation, a wide range of peak tensions was generated through passive stretch and concentric, isometric, and eccentric contractile protocols, and the resulting phosphorylation of c-Jun NH(2)-terminal kinase (JNK), extracellular regulated kinase (ERK), and p38 MAPKs was assessed. Isoforms of JNK and ERK MAPKs were found to be phosphorylated in a tension-dependent manner, such that eccentric > isometric > concentric > passive stretch. Peak tension was found to be a better predictor of MAPK phosphorylation than time-tension integral or rate of tension development. Differences in maximal response amplitude and sensitivity between JNK and ERK MAPKs suggest different roles for these two kinase families in mechanically induced signaling. A strong linear relationship between p54 JNK phosphorylation and peak tension over a 15-fold range in tension (r(2) = 0.89, n = 32) was observed, supporting the fact that contraction-type differences can be explained in terms of tension and demonstrating that MAPK activation is a quantitative reflection of the magnitude of mechanical stress applied to muscle. Thus the measurement of MAPK activation, as an assay of skeletal muscle mechanotransduction, may help elucidate mechanically induced hypertrophy.
AMP-activated protein kinase (AMPK) is emerging as an important energy-sensing/signaling system in skeletal muscle. This kinase is activated allosterically by 5'-AMP and inhibited allosterically by creatine phosphate. Phosphorylation of AMPK by an upstream kinase, AMPK kinase (also activated allosterically by 5'-AMP), results in activation. It is activated in both rat and human muscle in response to muscle contraction, the extent of activation depending on work rate and muscle glycogen concentration. AMPK can also be activated chemically in resting muscle with 5-aminoimidazole-4-carboxamide-riboside, which enters the muscle and is phosphorylated to form ZMP, a nucleotide that mimics the effect of 5'-AMP. Once activated, AMPK is hypothesized to phosphorylate proteins involved in triggering fatty acid oxidation and glucose uptake. Evidence is also accumulating for a role of AMPK in inducing some of the adaptations to endurance training, including the increase in muscle GLUT-4, hexokinase, uncoupling protein 3, and some of the mitochondrial oxidative enzymes. It thus appears that AMPK has the capability of monitoring intramuscular energy charge and then acutely stimulating fat oxidation and glucose uptake to counteract the increased rates of ATP utilization during muscle contraction. In addition, this system may have the capability of enhancing capacity for ATP production when the muscle is exposed to endurance training.
To determine whether changes in long-chain fatty acid (LCFA) oxidative metabolism induced by elevated intracellular carbohydrate availability are due to changes in LCFA uptake or in mitochondrial transport capacity, rat hindquarters were perfused with 500 microM palmitate and [1-14C]palmitate or [1-14C]octanoate as well as with either low (LG) or high (HG) carbohydrate availability. Glucose uptake was higher in the HG vs. LG group (23.6 +/- 1.5 vs 4.7 +/- 0.9 micromol x g(-1) x h(-1), P < 0.05). Palmitate delivery was not significantly different between groups and averaged 97.1 +/- 4.6 nmol x min(-1) x g(-1) (P > 0.05). Fractional and total palmitate uptake values were 60% higher (P < 0.05) in the HG (0.125 +/- 0.012 and 7.4 +/- 1.2 nmol x min(-1) x g(-1)) vs. LG (0.079 +/- 0.009 and 11.8 +/- 1.5 nmol x min(-1) x g(-1)) group. Values of percent and total palmitate oxidized were significantly lower (P < 0.05) in the HG (9.1 +/- 1.1% and 1.31 +/- 0.16 nmol x min(-1) x g(-1)) vs. LG (23.4 +/- 5.2% and 0.76 +/- 0.08 nmol x min(-1) x g(-1)) group. Conversely, values of fractional uptake and percent oxidation of octanoate were not significantly different between groups (P > 0.05). Malonyl-CoA levels were inversely correlated with LCFA oxidation (P < 0.05). These results demonstrate that high carbohydrate availability in muscle is associated with a decrease in LCFA oxidation that is not due to a parallel decrease in LCFA uptake; rather, the decrease in LCFA oxidation could be due to malonyl-CoA inhibition of mitochondrial LCFA transport.
AMP-activated protein kinase (AMPK) is activated during muscle contraction in response to the increase in AMP and decrease in phosphocreatine (PCr). Once activated, AMPK has been proposed to phosphorylate a number of targets, resulting in increases in glucose transport, fatty acid oxidation, and gene transcription. Although it has been possible to directly observe phosphorylation of one of these targets, acetyl-CoA carboxylase (ACC) in vitro, it has been more difficult to obtain direct evidence of ACC phosphorylation in contracting skeletal muscle. In these experiments using a phosphoserine antibody to ACC and a phosphothreonine antibody to AMPK, evidence was obtained for phosphorylation and activation of ACC in vitro, in gastrocnemius muscle electrically stimulated at different frequencies, and in muscle from rats running on the treadmill. Significant negative linear correlations between phospho-ACC and ACC activity were observed in all models (P < 0.01). The decline in ACC activity was related to the decrease in PCr and the rise in AMP. A relationship between phospho-AMPK (threonine 172) and activity of AMPK immunoprecipitated with anti-alpha(2) subunit antibody preparation was also observed. These data provide the first evidence of a direct link between extent of phosphorylation of these proteins at sites recognized by the antibodies and activity of the enzymes in electrically stimulated muscle and in muscle of rats running on the treadmill.
Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased HSL activity in supernatant from basal but not from electrically stimulated muscle. In conclusion, in muscle, PKC can stimulate HSL through ERK. Contractions and adrenaline enhance muscle HSL activity by different signalling mechanisms. The effect of contractions is mediated by PKC, at least partly via the ERK pathway.
Exercise enhances insulin-stimulated glucose transport (GT) in skeletal muscle. Evidence suggests that 5' AMP-activated protein kinase (AMPK) and glycogen may be important for enhanced insulin sensitivity. Our goals were to investigate the effect of various in situ muscle contraction protocols on insulin-stimulated GT and assess the relationship of contraction-induced changes in AMPK and glycogen with postcontraction improvement in insulin-stimulated GT. Rats were anesthetized, both ulnar nerves were exposed, and one nerve was electrically stimulated to contract forelimb muscles. We performed a series of five experiments, sequentially varying only one contraction parameter (train duration, train rate, pulse frequency, number of 5-min bouts, or pulse duration) while holding the others constant. Both epitrochlearis muscles were dissected out and incubated for 3.5 h before measurement of GT. For each contraction parameter studied, we identified an apparent threshold value that did not induce a significant increase in insulin-stimulated GT and an apparent peak value, above which there was a plateau or decline in insulin-stimulated GT. Using other rats, we evaluated muscle AMPK phosphorylation and glycogen concentration immediately postcontraction. AMPK phosphorylation and reduction in glycogen were increased compared with resting controls in each protocol, which had previously been shown to increase insulin-stimulated GT, as well as in several protocols that did not significantly increase insulin-stimulated GT. These data suggest that contraction-induced AMPK phosphorylation and decrease in glycogen may be necessary but are not sufficient for the postcontraction increase in insulin-stimulated GT in rat skeletal muscle.
Atypical protein kinase C (aPKC) and extracellular signal-regulated kinase (ERK) are emerging as important signalling molecules in the regulation of metabolism and gene expression in skeletal muscle. Exercise is known to increase activity of aPKC and ERK in skeletal muscle but the effect of exercise intensity hereon has not been studied. Furthermore, the relationship between activity and phosphorylation of the two enzymes during exercise is unknown. Nine healthy young men exercised for 30 min on a bicycle ergometer on two occasions. One occasion consisted of three consecutive 10 min bouts of 35, 60 and 85% of peak pulmonary oxygen uptake (V̇ O2peak) and the second of one 30 min bout at 35% of V̇ O2peak. Both trials also included 30 min recovery. Muscle biopsies were obtained from the vastus lateralis muscle before and after each exercise bout. Exercise increased muscle aPKC activity at 35% V̇ O2peak, whereupon no further increase was observed at higher exercise intensities. Activation of aPKC was not accompanied by increased phosphorylation of aPKC Thr 410/403. ERK1/2 activity increased in a similar pattern to aPKC, reaching maximal activity at 35% V̇ O2peak, whereas ERK1 Thr 202/Tyr 204 and ERK2 Thr 183/Tyr 185 phosphorylation increased with increasing exercise intensity. Thus, aPKC and ERK1/2 activity in muscle during exercise did not correspond to phosphorylation of sites on aPKC or ERK1/2, respectively, which are considered important for their activation. It is concluded that assessment of aPKC and ERK1/2 activity in muscle using phosphospecific antibodies did not reflect direct activity measurements on immunoprecipitated enzyme in vitro. Thus, estimation of enzyme activity during exercise by use of phosphospecific antibodies should not be performed uncritically. In addition, increase in muscle activity of aPKC or ERK1/2 during exercise is not closely related to energy demands of the muscle but may serve other regulatory or permissive functions in muscle.
To determine the role of AMP-activated protein kinase (AMPK) activation on the regulation of fatty acid (FA) uptake and oxidation, we perfused rat hindquarters with 6 mM glucose, 10 microU/ml insulin, 550 microM palmitate, and [14C]palmitate during rest (R) or electrical stimulation (ES), inducing low-intensity (0.1 Hz) muscle contraction either with or without 2 mM 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). AICAR treatment significantly increased glucose and FA uptake during R (P < 0.05) but had no effect on either variable during ES (P > 0.05). AICAR treatment significantly increased total FA oxidation (P < 0.05) during both R (0.38 +/- 0.11 vs. 0.89 +/- 0.1 nmol x min(-1) x g(-1)) and ES (0.73 +/- 0.11 vs. 2.01 +/- 0.1 nmol x min(-1) x g(-1)), which was paralleled in both conditions by a significant increase and significant decrease in AMPK and acetyl-CoA carboxylase (ACC) activity, respectively (P < 0.05). Low-intensity muscle contraction increased glucose uptake, FA uptake, and total FA oxidation (P < 0.05) despite no change in AMPK (950.5 +/- 35.9 vs. 1,067.7 +/- 58.8 nmol x min(-1) x g(-1)) or ACC (51.2 +/- 6.7 vs. 55.7 +/- 2.0 nmol x min(-1) x g(-1)) activity from R to ES (P > 0.05). When contraction and AICAR treatment were combined, the AICAR-induced increase in AMPK activity (34%) did not account for the synergistic increase in FA oxidation (175%) observed under similar conditions. These results suggest that while AMPK-dependent mechanisms may regulate FA uptake and FA oxidation at rest, AMPK-independent mechanisms predominate during low-intensity muscle contraction.
Activation of the AMP-activated protein kinase (AMPK) results in acute changes in cellular metabolism and transcriptional events that make the cell more robust when encountering an energy challenge. AMPK is thought to be inhibited by glycogen, the major storage form of intracellular carbohydrate. We hypothesized that long-chain acyl-CoA esters (LCACEs) might also inhibit AMPK signaling. Cytosolic LCACEs are available for immediate transport and oxidation within the mitochondria and accordingly may be representative of the lipid energy charge of the cell. We found that LCACEs inhibited phosphorylation of AMPK by the recombinant AMPK kinase (AMPKK) LKB1/STRAD/MO25 in a concentration-dependent manner. Palmitoyl-CoA (PCoA) did not affect the activity of phosphothreonine-172 AMPK. PCoA potently inhibited AMPKK purified from liver. Conversely, PCoA stimulated the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. Octanoyl-CoA, palmitate, and palmitoylcarnitine did not inhibit AMPKK activity. Removal of AMP from the reaction mixture resulted in reduced AMPKK activity in the presence of PCoA. In conclusion, these results demonstrate that the AMPKK activity of LKB1/STRAD/MO25 is substrate specific and distinct from the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. They also demonstrate that LCACEs inhibit the AMPKK activity of LKB1/STRAD/MO25 in a specific manner with a dependence on both a long fatty chain and a CoA moiety. These results suggest that the AMPK signaling cascade may directly sense and respond to the lipid energy charge of the cell.
Increases in contraction-stimulated glucose transport in fast-twitch rat epitrochlearis muscle are mediated by AMPK- and Ca2+/calmodulin-dependent protein kinase (CAMK)-dependent signaling pathways. However, recent studies provide evidence suggesting that contraction-stimulated glucose transport in slow-twitch skeletal muscle is mediated through an AMPK-independent pathway. The purpose of the present study was to test the hypothesis that contraction-stimulated glucose transport in rat slow-twitch soleus muscle is mediated by an AMPK-independent/Ca2+-dependent pathway. Caffeine, a sarcoplasmic reticulum (SR) Ca2+-releasing agent, at a concentration that does not cause muscle contractions or decreases in high-energy phosphates, led to an approximately 2-fold increase in 2-deoxyglucose (2-DG) uptake in isolated split soleus muscles. This increase in glucose transport was prevented by the SR calcium channel blocker dantrolene and the CAMK inhibitor KN93. Conversely, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an AMPK activator, had no effect on 2-DG uptake in isolated split soleus muscles yet resulted in an approximately 2-fold increase in the phosphorylation of AMPK and its downstream substrate acetyl-CoA carboxylase. The hypoxia-induced increase in 2-DG uptake was prevented by dantrolene and KN93, whereas hypoxia-stimulated phosphorylation of AMPK was unaltered by these agents. Tetanic muscle contractions resulted in an approximately 3.5-fold increase in 2-DG uptake that was prevented by KN93, which did not prevent AMPK phosphorylation. Taken in concert, our results provide evidence that hypoxia- and contraction-stimulated glucose transport is mediated entirely through a Ca2+-dependent mechanism in rat slow-twitch muscle.
The purpose of this experiment was to investigate the role of extracellular signal-regulated kinase 1/2 (ERK1/2) signalling in the contraction-induced increase in muscle FA uptake.
Male Wistar rats (n = 41) were randomly assigned to either a resting or stimulated group. Within each group, animals were randomly assigned to receive PD-98059, an inhibitor of MAP/ERK kinase 1/2 (MEK1/2), a kinase upstream of ERK1/2 and perfused with 550 microM palmitate, [(14)C]palmitate, 7 mM glucose, and no insulin. In the stimulated group, electrical stimulation (ES) of supramaximal trains of 100 ms was delivered every 2 s for 20 min.
ERK1/2 phosphorylation was increased by 50% (P < 0.05) during ES but the contraction-induced increase was prevented by the addition of PD-98059. Glucose uptake increased by 3.6-fold (P < 0.05) from rest to ES in muscle perfused without PD-98059 and was not affected by the addition of PD-98059 either at rest (P > 0.05) or during ES (P > 0.05). For a matched palmitate delivery, ES increased palmitate uptake by 35% (P < 0.05). PD-98059 had no effect on palmitate uptake at rest but completely abolished the increase in palmitate uptake during ES. Plasma membrane FAT/CD36 protein content was increased by 38% during ES (P < 0.05) but the contraction-induced increase was prevented by the addition of PD-98059. AMPK activity was increased by ES (P < 0.05) but was unaffected by PD-98059.
These results show for the first time that the increase in FA uptake and in plasma membrane FAT/CD36 protein content is mediated, at least in part, by the ERK1/2 signalling pathway during muscle contraction.