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ERK1/2 inhibition prevents contraction-induced increase in plasma membrane FAT/CD36 content and FA uptake in rodent muscle
Abstract
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.
... 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). Multiple intracellular signaling cascades are activated by muscle contraction, and the signals induced by the activation of these molecules regulate the changes in substrate metabolism with muscle contraction (Rasmussen and Winder 1997;Widegren et al. 1998;Wojtaszewski et al. 1999Wojtaszewski et al. , 2000Wojtaszewski et al. , 2003Ihlemann et al. 2000;Ryder et al. 2000;Kaushik et al. 2001;Chen et al. 2003;Richter et al. 2004;Raney et al. 2005;Turcotte et al. 2005;Wright et al. 2005;Turcotte 2006, 2008;Rose et al. 2006;Jensen et al. 2007b;Witczak et al. 2007Witczak et al. , 2010Abbott et al. 2009). The most important signaling pathways implicated in the regulation of fuel utilization during muscle contraction include the cascades associated with AMP-activated protein kinase (AMPK), calcium (Ca 2ϩ )/calmodulin-dependent protein kinases (CaMK), and extracellular signal-regulated kinase 1/2 (ERK1/2). ...
... It has further been shown that acute regulation of FA transport capacity, during acute muscle contraction or exercise, is mediated in large part by the translocation of one or more of these FA transport proteins from intracellular pools to the plasma membrane. This process may occur via the action of several putative adaptor proteins and vesiclemembrane (v-SNAREs) and target-membrane (t-SNAREs) associated soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins Chabowski et al. 2005;Turcotte et al. 2005;Han et al. 2007;Schwenk et al. 2007Schwenk et al. , 2010Habets et al. 2009;Jeppesen et al. 2009Jeppesen et al. , 2011. In the context of contractioninduced signaling, data on FA uptake capacity and FA transporter proteins suggest the involvement of cellular signaling cascades in the regulation of FA uptake during acute muscle contraction or exercise. ...
... As mentioned above, the evidence shows that intracellular signals other than low [AMP] or high [Ca 2ϩ ] are induced by acute muscle contraction, and that these signals may be key for appropriate regulation of substrate use in contracting skeletal muscle (Widegren et al. 2001;Donsmark et al. 2003;Raney and Turcotte 2008;Abbott et al. 2011). More specifically, data suggest that the ERK1/2 signaling pathway of the mitogenactivated protein (MAP) kinase family may be involved in the regulation of various aspects of contraction-induced FA metabolism including FA uptake and oxidation (Richter et al. 2004;Roepstorff et al. 2004a;Turcotte et al. 2005;Turcotte 2006, 2007;Watt et al. 2006). ERK1/2 proteins are phosphorylated and activated by the upstream kinase MAP/ ERK kinase 1/2 (MEK1/2) (Seger and Krebs 1995). ...
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.
... 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). ...
... 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.
... Strikingly, this was completely (basal) or partially (AICAR stimulated) recovered by increased mitochondrial uncoupling in DTG mice, despite a highly suppressed AMPKa2 activity and AMPKa1 protein expression as well as no compensatory increase in AMPKa1 activity. Other kinases have been suggested to play a role in skeletal muscle fatty acid uptake, such as the extra cellular signaling receptor kinase (ERK1/2) [68]. Expression and translocation of CD36 in rodent SM seems to require activation of ERK1/2 signaling [68]. ...
... Other kinases have been suggested to play a role in skeletal muscle fatty acid uptake, such as the extra cellular signaling receptor kinase (ERK1/2) [68]. Expression and translocation of CD36 in rodent SM seems to require activation of ERK1/2 signaling [68]. We recently showed that ERK2 is activated in SM of UCP1-TG mice [5]. ...
Transgenic (UCP1-TG) mice with ectopic expression of UCP1 in skeletal muscle (SM) show a phenotype of increased energy expenditure, improved glucose tolerance and increase substrate metabolism in SM. To investigate the potential role of skeletal muscle AMPKα2 activation in the metabolic phenotype of UCP1-TG mice we generated double transgenic (DTG) mice, by crossing of UCP1-TG mice with DN-AMPKα2 mice overexpressing a dominant negative α2 subunit of AMPK in SM which resulted in an impaired AMPKα2 activity by 90±9% in SM of DTG mice. Biometric analysis of young male mice showed decreased body weight, lean and fat mass for both UCP1-TG and DTG compared to WT and DN-AMPKα2 mice. Energy intake and weight-specific total energy expenditure were increased, both in UCP1-TG and DTG mice. Moreover, glucose tolerance, insulin sensitivity and fatty acid oxidation were not altered in DTG compared to UCP1-TG. Also uncoupling induced induction and secretion of fibroblast growth factor 21 (FGF21) from SM was preserved in DTG mice. However, voluntary physical cage activity as well as ad libitum running wheel access during night uncovered a severe activity intolerance of DTG mice. Histological analysis showed a progressive degenerative morphology in SM of DTG mice which was not observed in SM of UCP1-TG mice. Moreover, ATP-depletion related cellular stress response via heat shock protein 70 was highly induced, whereas capillarization regulator VEGF was suppressed in DTG muscle. In addition, AMPKα2-mediated induction of mitophagy regulator ULK1 was suppressed in DTG mice, as well as mitochondrial respiratory capacity and content. In conclusion, we demonstrate that AMPKα2 is dispensable for SM mitochondrial uncoupling induced metabolic effects on whole body energy balance, glucose homeostasis and insulin sensitivity. But strikingly, activation of AMPKα2 seems crucial for maintaining SM function, integrity and the ability to compensate chronic metabolic stress induced by SM mitochondrial uncoupling.
... Parallel to that GLUT4 translocation to the muscle membrane is accelerated by muscle contraction (Etgen et al. 1996) leading to increased GU (Ploug et al. 1984;Wasserman et al. 1991) and glucose oxidation (Wasserman et al. 2011). Similarly, FFAU, which is mediated by different transport proteins (Holloway et al. 2008), increases but is likely to be less dependent on substrate delivery (Turcotte et al. 2005). Although the muscle BF and the release of FFA from adipose tissue increases during submaximal exercise (i.e. ...
... However, our findings do not support this notion, as we observed a decrease in FFA extraction from rest (*34 %) to exercise (*15 %). This, together with an almost 13-fold increase in FFA delivery from resting values, likely suggests that FFAU is less dependent on FFA delivery during low-intensity exercise as earlier proposed (Turcotte et al. 2005). ...
The purpose of the present study was to investigate the regional differences in glucose and fatty acid uptake within skeletal muscle during exercise. Blood flow (BF), glucose uptake (GU) and free fatty acid uptake (FFAU) were measured in four different regions (vastus lateralis, VL; rectus femoris, RF; vastus intermedius, VI; and vastus medialis, VM) of the quadriceps femoris (QF) muscle during low-intensity, knee-extension exercise using positron emission tomography. BF was higher in VI than in VL, RF and VM (P < 0.05). FFAU was higher in VI (P < 0.001) but also in VM (P < 0.05) compared with VL and RF. In contrast, GU was higher in RF compared with VL (P < 0.05) but was not significantly different to VM or VI (both P = NS). FFAU within these four muscle regions correlated significantly with BF (r = 0.951, P < 0.05), whereas no significant relationship was observed between GU and BF (r = 0.352, P = NS). Therefore, skeletal muscle FFAU, but not GU, appears to be associated with BF during low-intensity exercise. The present results also indicate considerable regional differences in substrate use within working QF muscle. As such, an important methodological outcome from these results is that one sample from a specific part of the QF muscle does not represent the response in the entire QF muscle group.
... Taken together, these data suggest a functional role of AMPK in the dynamic regulation of FAT/CD36 traffi cking and, ultimately, FA uptake. However, Turcotte, Raney, and Todd have shown that contraction-induced FAT/CD36 translocation and increases in FA uptake are prevented when the rat hindquarter is perfused with an inhibitor of the extracellular signal-regulated kinase (ERK) 1/2 and can occur independently of AMPK activation ( 20 ). The equivocal fi ndings suggest the need for further evidence in order to elucidate the regulatory mechanism behind FAT/CD36 traffi cking and the effect on FA uptake in skeletal muscle. ...
... 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 ). However, in the present study, FAT/CD36 translocation occurred rapidly, before changes in ERK1/2 and AMPK activation, suggesting that neither ERK1/2 nor AMPK are essential for FAT/ CD36 translocation or FA uptake during contraction in rat hindlimb muscle ( Figs. 6, 7 ). ...
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.
... We previously found that NTS enhanced, while AICAR inhibited, FA uptake and that AICAR-inhibited FA uptake was abrogated in the presence of NTS 7 . ERK1/2 inhibition prevents FA uptake in rat cardiac myocytes, indicating that ERK1/2 signaling plays a positive role in FA uptake 49 . Ca 2+ mediates the activation of NTS-NTSR1-ERK1/2 signaling; thus, these findings are consistent with our previous 7 and current findings that NTS-NTSR1-ERK1/2 signaling promotes FA uptake in small intestinal epithelial cells. ...
We previously demonstrated that neurotensin, a 13-amino-acid gut hormone peptide, enhances small intestinal epithelial cell fatty acid uptake through inhibition of AMPK. Here, utilizing Drosophila and mouse models in vivo, as well as mouse and human small intestinal epithelial organoids or monolayers ex vivo, we determine the targets of neurotensin and AMPK associated with obesity and aging. High-fat diet and aging decreased AMPK and insulin signaling, which was prevented by neurotensin deficiency. High-fat diet feeding increased FABP1 protein expression in wild-type mice; this effect was attenuated in neurotensin-deficient mice. AICAR and metformin increased AMPK phosphorylation in young but not in aged small intestinal epithelial cells. By contrast, AICAR and metformin inhibited FABP1 mRNA and protein expression. Moreover, cytosolic colocalization of AMPKα1 and FABP1 was noted in IEC-6 cells. AMPK phosphorylation and FABP1 expression was decreased in aged wild-type small intestinal epithelial cells; however, this effect was reversed in neurotensin-deficient cells. Results from human duodenal organoids confirm the effects of neurotensin, palmitic acid and metformin on AMPK phosphorylation and FABP1. Finally, overexpressing neurotensin in enteroendocrine cells reduced the lifespan of Drosophila ; neurotensin deficiency extended the lifespan of mice fed a high-fat diet. Our findings indicate that neurotensin inhibits AMPK and increases FABP1 in small intestinal epithelial cells under conditions of obesity. Neurotensin deficiency preserves AMPK and FABP1 levels, thus attenuating some of the negative effects of obesity and aging.
... enrichment of long-chain fatty acid transporters (fatty acid translocase (FAT, also known as CD36), plasma membrane-associated fatty acid binding protein (FABPpm) and fatty acid transport protein 1 (FATP1) and FATP4) 132 , which have varying capacities for increasing NEFA uptake and oxidation in muscle 133 . Upon exercise, the exocytosis of CD36 (and possibly other transporters) seems independent of AMPK 134 but could involve calcium/CaMK kinase (CaMKK) 135 and MAPK/ERK kinase 1 (MEK1) and MEK2 signalling 136 . Furthermore, although FABPpm is structurally identical to mitochondrial aspartate aminotransferase (mAspAT), these proteins serve distinct functions within their respective subcellular compartments (that is, FABPpm-mediated transport of long-chain NEFAs across the sarcolemma versus mAspAT-based delivery of reducing equivalents into mitochondria) 137 . ...
Viewing metabolism through the lens of exercise biology has proven an accessible and practical strategy to gain new insights into local and systemic metabolic regulation. Recent methodological developments have advanced understanding of the central role of skeletal muscle in many exercise-associated health benefits and have uncovered the molecular underpinnings driving adaptive responses to training regimens. In this Review, we provide a contemporary view of the metabolic flexibility and functional plasticity of skeletal muscle in response to exercise. First, we provide background on the macrostructure and ultrastructure of skeletal muscle fibres, highlighting the current understanding of sarcomeric networks and mitochondrial subpopulations. Next, we discuss acute exercise skeletal muscle metabolism and the signalling, transcriptional and epigenetic regulation of adaptations to exercise training. We address knowledge gaps throughout and propose future directions for the field. This Review contextualizes recent research of skeletal muscle exercise metabolism, framing further advances and translation into practice.
... 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.
... There is good evidence that the expression of CD36 caused by HFD is secondary to the activation of the ERK1/2 (MAPK) pathway: Activation of ERK1/2 drives both CD36 expression and lipid accumulation (56,57), while inhibition of ERK1/2 activity reduces CD36 expression in hepatocytes (55). LL-37 inhibits ERK phosphorylation in macrophages (58) and the activation of ERK with EGF reverses both the inhibition of CD36 expression and the lipid accumulation caused by LL-37 in adipocytes and hepatocytes (55). ...
Metabolic endotoxemia has been suggested to play a role in the pathophysiology of metaflammation, insulin-resistance and ultimately type-2 diabetes mellitus (T2DM). The role of endogenous antimicrobial peptides (AMPs), such as the cathelicidin LL-37, in T2DM is unknown. We report here for the first time that patients with T2DM compared to healthy volunteers have elevated plasma levels of LL-37. In a reverse-translational approach, we have investigated the effects of the AMP, peptide 19-2.5, in a murine model of high-fat diet (HFD)-induced insulin-resistance, steatohepatitis and T2DM. HFD-fed mice for 12 weeks caused obesity, an impairment in glycemic regulations, hypercholesterolemia, microalbuminuria and steatohepatitis, all of which were attenuated by Peptide 19-2.5. The liver steatosis caused by feeding mice a HFD resulted in the activation of nuclear factor kappa light chain enhancer of activated B cells (NF-ĸB) (phosphorylation of inhibitor of kappa beta kinase (IKK)α/β, IκBα, translocation of p65 to the nucleus), expression of NF-ĸB-dependent protein inducible nitric oxide synthase (iNOS) and activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome, all of which were reduced by Peptide 19-2.5. Feeding mice, a HFD also resulted in an enhanced expression of the lipid scavenger receptor cluster of differentiation 36 (CD36) secondary to activation of extracellular signal-regulated kinases (ERK)1/2, both of which were abolished by Peptide 19-2.5. Taken together, these results demonstrate that the AMP, Peptide 19-2.5 reduces insulin-resistance, steatohepatitis and proteinuria. These effects are, at least in part, due to prevention of the expression of CD36 and may provide further evidence for a role of metabolic endotoxemia in the pathogenesis of metaflammation and ultimately T2DM. The observed increase in the levels of the endogenous AMP LL-37 in patients with T2DM may serve to limit the severity of the disease.
... More importantly, activating AMPK with the drug AICAR can induce all LCFAs to transfer to the plasma membrane (107). Studies of other signaling cascade pathways have shown that inhibition of extracellular signal receptor kinase (ERK1/2) can prevent FAT/CD36 translocation (108,109), and stimulation of protein kinase C can induce the shift of FAT/CD36. The internal Ca 2+ concentration is related to signal transport, and subsequent further research proves that CaMKK located upstream of AMPK mainly affects LCFA transport (110). ...
It is widely recognized that chemical, physical, and biological factors can singly or synergistically evoke the excessive production of oxidative stress in pulmonary tissue that followed by pulmonary lesions and pneumonia. In addition, metabolic and endocrine disorder-induced diseases such as diabetes and obesity often expressed higher susceptibility to pulmonary infections, and presented severe symptoms which increasing the mortality rate. Therefore, the connection between the lesion of the lungs and the metabolic/endocrine disorders is an interesting and essential issue to be addressed. Studies have noticed a similar pathological feature in both infectious pneumonia and metabolic disease-intercurrent pulmonary lesions, that is, from the view of molecular pathology, the accumulation of excessive reactive oxygen species (ROS) in pulmonary tissue accompanying with activated pro-inflammatory signals. Meanwhile, Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) and nuclear factor erythroid-2-related factor 2 (Nrf2) signaling plays important role in metabolic/endocrine homeostasis and infection response, and it's closely associated with the anti-oxidative capacity of the body. For this reason, this review will start from the summary upon the implication of ROS accumulation, and to discuss how AMPK-Nrf2 signaling contributes to maintaining the metabolic/endocrine homeostasis and attenuates the susceptibility of pulmonary infections.
... Importantly, lipid transporters are regulated by the ERK signaling pathway. ERK phosphorylation has been found to increase CD36 expression in the membrane of muscle cells during muscle contraction [35]. However, MDA-MB-231 barely express CD36 transporters, also known as fatty acid translocase (FAT), whereas they express fatty acid binding protein 5 (FABP5) ( Figure 5A) [36]. ...
Metabolic reprogramming in tumor cells is considered one of the hallmarks of cancer. Many studies have been carried out in order to elucidate the effects of tumor cell metabolism on invasion and tumor progression. However, little is known about the immediate substrate preference in tumor cells. In this work, we wanted to study this short-time preference using the highly invasive, hormone independent breast cancer cell line MDA-MB-231. By means of Seahorse and uptake experiments, our results point to a preference for glucose. However, although both glucose and glutamine are required for tumor cell proliferation, MDA-MB-231 cells can survive two days in the absence of glucose, but not in the absence of glutamine. On the other hand, the presence of glucose increased palmitate uptake in this cell line, which accumulates in the cytosol instead of going to the plasma membrane. In order to exert this effect, glucose needs to be converted to glycerol-3 phosphate, leading to palmitate metabolism through lipid synthesis, most likely to the synthesis of triacylglycerides. The effect of glucose on the palmitate uptake was also found in other triple-negative, invasive breast cancer cell lines, but not in the non-invasive ones. The results presented in this work suggest an important and specific role of glucose in lipid biosynthesis in triple-negative breast cancer.
... In our experiment, lipolysis stimulated by acetate may be via ERK1/2 signal pathway. In rodent skeletal muscle, ERK1/2 inhibition prevents the contraction-induced increase in plasma membrane FATP content and lipid uptake [46]. We could not confirm the function of ERK1/2 in acetate-regulated lipid use in skeletal muscle because acetate decreased the p-ERK1/2 protein in skeletal muscle, which is the opposite performance with the FATP and FABP genes expression. ...
Simple Summary
Lots of short-chain fatty acids (SCFAs) are produced in the rabbit cecum after dietary fiber fermentation. In addition to supplying energy, SCFAs could regulate lipid metabolism, but the related mechanism is still unknown. In our experiment, we study the effect of acetate (major SCFAs, 70–80%) on rabbit lipid metabolism. The present study found that acetate alters the process of lipid metabolism in rabbit liver, skeletal muscle and adipose tissue, and inferred some signaling pathways related to the process. A mechanism of acetate-regulating lipid metabolism is useful to identify the function in fat metabolism of microbiological products from rabbit and rabbit processes for nutrition metabolism.
Abstract
Short-chain fatty acids (SCFAs) (a microbial fermentation production in the rabbit gut) have an important role in many physiological processes, which may be related to the reduced body fat of rabbits. In the present experiment, we study the function of acetate (a major SCFA in the rabbit gut) on fat metabolism. Ninety rabbits (40 days of age) were randomly divided into three groups: a sham control group (injection of saline for four days); a group experiencing subcutaneous injection of acetate for four days (2 g/kg BM per day, one injection each day, acetate); and a pair-fed sham treatment group. The results show that acetate-inhibited lipid accumulation by promoting lipolysis and fatty acid oxidation and inhibiting fatty acid synthesis. Activated G protein-coupled receptor 41/43, adenosine monophosphate activated protein kinase (AMPK) and extracellular-signal-regulated kinase (ERK) 1/2 signal pathways were likely to participate in the regulation in lipid accumulation of acetate. Acetate reduced hepatic triglyceride content by inhibiting fatty acid synthesis, enhancing fatty acid oxidation and lipid output. Inhibited peroxisome proliferator-activated receptor α (PPARα) and activated AMPK and ERK1/2 signal pathways were related to the process in liver. Acetate reduced intramuscular triglyceride level via increasing fatty acid uptake and fatty acid oxidation. PPARα was associated with the acetate-reduced intracellular fat content.
... Potential redundancies in the adaptation process must also be considered. For example, contracting skeletal muscle activates AMPK via its upstream kinase Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) [135], and changes in proteins regulating fat metabolism such as FAT/CD36 are mediated in part by contraction-induced signalling of the ERK1/2 pathway [136]. Thus, many of the common adaptations to endurance training may be observed in the absence of RONS signalling and be minimally influenced by antioxidant supplementation. ...
Endurance training leads to a variety of adaptations at the cellular and systemic levels that serve to minimise disruptions in whole-body homeostasis caused by exercise. These adaptations are differentially affected by training volume, training intensity, and training status, as well as by nutritional choices that can enhance or impair the response to training. A variety of supplements have been studied in the context of acute performance enhancement, but the effects of continued supplementation concurrent to endurance training programs are less well characterised. For example, supplements such as sodium bicarbonate and beta-alanine can improve endurance performance and possibly training adaptations during endurance training by affecting buffering capacity and/or allowing an increased training intensity, while antioxidants such as vitamin C and vitamin E may impair training adaptations by blunting cellular signalling but appear to have little effect on performance outcomes. Additionally, limited data suggest the potential for dietary nitrate (in the form of beetroot juice), creatine, and possibly caffeine, to further enhance endurance training adaptation. Therefore, the objective of this review is to examine the impact of dietary supplements on metabolic and physiological adaptations to endurance training.
... We hypothesize that its role in the tight regulation of ERK1/2 signaling might be related to the regulation of fatty acid metabolism. This assumption is based on studies showing that inhibition of ERK1/2 prevented contraction-induced translocation of fatty acid translocase (FAT/CD36) to the plasma membrane (Turcotte et al. 2005;Raney and Turcotte 2007). Thus, uptake of fatty acids during contraction was not increased when ERK1/2 activity was blocked. ...
Physical activity promotes specific adaptations in most tissues including skeletal muscle. Acute exercise activates numerous signaling cascades including pathways involving mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK)1/2, which returns to pre-exercise level after exercise. The expression of MAPK phosphatases (MKPs) in human skeletal muscle and their regulation by exercise have not been investigated before. In this study, we used mRNA sequencing to monitor regulation of MKPs in human skeletal muscle after acute cycling. In addition, primary human myotubes were used to gain more insights into the regulation of MKPs. The two ERK1/2-specific MKPs, dual specificity phosphatase 5 (DUSP5) and DUSP6, were the most regulated MKPs in skeletal muscle after acute exercise. DUSP5 expression was ninefold higher immediately after exercise and returned to pre-exercise level within 2 h, whereas DUSP6 expression was reduced by 43% just after exercise and remained below pre-exercise level after 2 h recovery. Cultured myotubes express both MKPs, and incubation with dexamethasone (Dex) mimicked the in vivo expression pattern of DUSP5 and DUSP6 caused by exercise. Using a MAPK kinase inhibitor, we showed that stimulation of ERK1/2 activity by Dex was required for induction of DUSP5. However, maintaining basal ERK1/2 activity was required for basal DUSP6 expression suggesting that the effect of Dex on DUSP6 might involve an ERK1/2-independent mechanism. We conclude that the altered expression of DUSP5 and DUSP6 in skeletal muscle after acute endurance exercise might affect ERK1/2 signaling of importance for adaptations in skeletal muscle during exercise.
... In nonimmune cells, the MAPK-dependent metabolic rewiring is largely linked to changes in gene and protein expression. ERK1/2 can also influence fatty acid uptake and FAO by controlling CD36 expression and mediating the inhibitory phosphorylation of lipid synthesis-related enzymes, including ACC [390]. The p38 kinases can regulate glucose uptake via poorly understood mechanisms. ...
The immune system is a central determinant of organismal health. Functional immune responses require quiescent immune cells to rapidly grow, proliferate, and acquire effector functions when they sense infectious agents or other insults. Specialized metabolic programs are critical regulators of immune responses, and alterations in immune metabolism can cause immunological disorders. There has thus been growing interest in understanding how metabolic processes control immune cell functions under normal and pathophysiological conditions. In this chapter, we summarize how metabolic programs are tuned and what the physiological consequences of metabolic reprogramming are as they relate to immune cell homeostasis, differentiation, and function.
... The working hypothesis is that Ca 2þ and AMPK play important roles in the translocation of FA transporters to the muscle membrane. Turcotte et al. (2005) showed that inhibition of extracellular regulated kinase 1/2 (ERK1/2), stimulated by Ca 2þ -dependent PKC, prevented the normal contraction-induced increase in plasma membrane FAT/CD36 content and FA uptake in rodent muscle. A study by Jeppesen et al. (2011) using AMPKa2-null mice suggested that skeletal muscle FAT/CD36 trafficking and FA uptake was independent of AMPK as all responses to contraction and exercise were normal when compared with wild-type skeletal muscles. ...
During exercise, the supply of adenosine triphosphate (ATP) is essential for the energy-dependent processes that underpin ongoing contractile activity. These pathways involve both substrate-level phosphorylation, without any need for oxygen, and oxidative phosphorylation that is critically dependent on oxygen delivery to contracting skeletal muscle by the respiratory and cardiovascular systems and on the supply of reducing equivalents from the degradation of carbohydrate, fat, and, to a limited extent, protein fuel stores. The relative contribution of these pathways is primarily determined by exercise intensity, but also modulated by training status, preceding diet, age, gender, and environmental conditions. Optimal substrate availability and utilization before, during, and after exercise is critical for maintaining exercise performance. This review provides a brief overview of exercise metabolism, with expanded discussion of the regulation of muscle glucose uptake and fatty acid uptake and oxidation.
... We hypothesize that its role in the tight regulation of ERK1/2 signaling might be related to the regulation of fatty acid metabolism. This assumption is based on studies showing that inhibition of ERK1/2 prevented contraction-induced translocation of fatty acid translocase (FAT/CD36) to the plasma membrane (Turcotte et al. 2005;Raney and Turcotte 2007). Thus, uptake of fatty acids during contraction was not increased when ERK1/2 activity was blocked. ...
Dual specificity phosphatase (DUSP) 5 and 6 specifically dephosphorylate ERK1/2; thus, they are important regulators of ERK1/2 signaling. DUSP5 is located in the nucleus, whereas DUSP6 is found in the cytoplasm. Both DUSPs bind also to unphosphorylated ERK1/2 and sequester it in nucleus and cytoplasm, respectively. Depending on the stimulus, e.g. growth factors, DUSP5 and/or DUSP6 expression is promoted or reduced. Thereby, a stimulus-dependent pattern of DUSP5 and DUSP6 determines the activity of ERK1/2 in a compartment-specific manner. This might be an important regulatory mechanism in determining physiological consequences of ERK1/2 signaling. In human skeletal muscle, ERK1/2 phosphorylation is increased by acute exercise, and upon cessation rapidly reduced. However, the regulation and importance of DUSP5 and DUSP6 by exercise has not been studied so far.
Sedentary men categorized either as control (BMI = 23.5 ± 2.0 kg/m2; normal fasting and 2 h serum glucose levels; n = 13) or dysglycemic (BMI = 28.9 ± 2.5 kg/m2; fasting glucose ≥ 5.6 mmol/L and/or 2 h serum glucose ≥ 7.8 mmol/L; n = 11) were subjected to acute cycling (45 min, 70 % VO2max). Skeletal muscle biopsies were taken before, after, and 2 h after exercise. RNA was isolated and analyzed by high throughput mRNA sequencing followed by differential gene expression analysis. Primary human myotubes were acutely exposed to stimulation with dexamethasone (dex) mimicking the increase of cortisol during exercise, and ionomycin (iono), which increases intracellular Ca2+ concentrations. ERK1/2 phosphorylation was analyzed by Western blot and expression of DUSP5 and DUSP6 by qRT-PCR.
Acute exercise promoted DUSP5 expression 9.1-fold (p<0.001) directly after exercise while DUSP6 was reduced by 43 %, with no difference between the groups. After 2 h rest DUSP5 returned to basal values whereas DUSP6 was still 30 % lower compared to baseline. Basal DUSP5 and DUSP6 levels were not different between the groups. During in vitro differentiation of human skeletal muscle cells, DUSP5 expression decreased by 50 % from day 0 to day 1 and remained stable thereafter. DUSP6 expression increased during differentiation (day 6 vs. day 0: 3.5-fold, p<0.005, n=3) in parallel with enhanced expression of myogenin (4-fold) and MHCIIa (10-fold). Incubation with dex increased ERK1/2 phosphorylation after 15 min (1.6-fold, p<0.001, n=3), which returned to basal level after 30 min. Moreover, dex stimulation promoted DUSP5 expression 2.6-fold (p<0.05, n=4-6) after 1 h, which returned to basal level after 4 h. DUSP6 expression was significantly reduced by 42 % after 2 h and 4 h, and returned to basal level after 6 h. A similar pattern but with different kinetics was observed for iono, which increased DUSP5 expression 2-fold after 6 h, whereas DUSP6 was reduced by 40 % after 3 h and returned to basal level after 6 h.
The prominent regulation of DUSP5 and DUSP6 expression by acute exercise in human skeletal muscle indicates an important role in generating a specific spatio-temporal pattern of ERK1/2 activity during exercise. Stimulation of human myotubes with dex and iono mimicked these pattern of DUSP5 and DUSP6 in vitro, and enables future studies aiming to decipher the physiological consequences of a proper regulation of DUSP5 and DUSP6.
... A previous report has shown that ERK mediates CD36 expression 20 . In our studies, exposure of differentiated human adipocytes to LL-37 (1-10 μM) reduced ERK phosphorylation, which was partially reversed by the ERK activator epidermal growth factor (EGF) ( Figure 5A). ...
Background and objectives:
Obesity is a global epidemic which increases the risk of the metabolic syndrome. Cathelicidin (LL-37 and mCRAMP) is an antimicrobial peptide with an unknown role in obesity. We hypothesize that cathelicidin expression correlates with obesity and modulates fat mass and hepatic steatosis.
Materials and methods:
Male C57BL/6J mice were fed a high-fat diet. Streptozotocin was injected into mice to induce diabetes. Experimental groups were injected with cathelicidin and CD36 overexpressing lentiviruses. Human mesenteric fat adipocytes, mouse 3T3-L1 differentiated adipocytes, and human HepG2 hepatocytes were used in the in vitro experiments. Cathelicidin levels in non-diabetic, prediabetic, and Type II diabetic patients were measured by ELISA.
Results:
Lentiviral cathelicidin overexpression reduced hepatic steatosis and decreased the fat mass of high-fat diet-treated diabetic mice. Cathelicidin overexpression reduced mesenteric fat and hepatic fatty acid translocase (CD36) expression that was reversed by lentiviral CD36 overexpression. Exposure of adipocytes and hepatocytes to cathelicidin significantly inhibited CD36 expression and reduced lipid accumulation. Serum cathelicidin protein levels were significantly increased in non-diabetic and prediabetic patients with obesity, compared to non-diabetic patients with normal body mass index (BMI) values. Prediabetic patients had lower serum cathelicidin protein levels than non-diabetic subjects.
Conclusions:
Cathelicidin inhibits the CD36 fat receptor and lipid accumulation in adipocytes and hepatocytes, leading to a reduction of fat mass and hepatic steatosis in vivo. Circulating cathelicidin levels are associated with increased BMI. Our results demonstrate that cathelicidin modulates the development of obesity.International Journal of Obesity accepted article preview online, 10 May 2016. doi:10.1038/ijo.2016.90.
... Numerous data in both animal and human models support a mechanism of protein translocation (from the cytoplasm to the plasma membrane) of CD36, FABP pm , FATP1, and FATP4 Turcotte et al., 2005;Jain et al., 2009), but not FATP6 (Jain et al., 2009). Along with muscle contraction from acute exercise, hormonal stimuli (i.e. ...
Long-chain fatty acid (LCFA) movement into skeletal muscle involves a highly mediated process in which lipid rafts are utilised in the cellular membrane, involving numerous putative plasma membrane-associated LCFA transport proteins. The process of LCFA uptake and oxidation is of particular metabolic significance both at rest and during light to moderate exercise. A comprehensive systematic search of electronic databases was undertaken to investigate whether exercise alters protein and / or gene expression of putative LCFA transport proteins. There were 31 studies meeting all eligibility criteria, of these 13 utilised an acute exercise protocol and 18 examined chronic exercise adaptations. 17 involved a study design incorporating an exercise stimulus, while the remaining 14 incorporated an exercise and diet stimulus. Divergent data relating to acute exercise, as well as prolonged exercise training (≥3 weeks), on protein content (PC) response was identified for proteins CD36, FABPpm and CAV1. Messenger ribonucleic acid (mRNA) data did not always correspond to functional PC, supporting previous suggestions of a disconnect due to potentially limiting factors post gene expression. The large array of study designs, cohorts and primary dependent variables within the studies included in the present review elucidate the complexity of the interaction between exercise and LCFA transport proteins. Summary of the results in the present review validate the need for further targeted investigation within this topic, and provide an important information base for such research. This article is protected by copyright. All rights reserved.
... ERK 1/2 is phosphorylated during moderate intensity exercise and this effect is mediated by local muscle specific factors as opposed to systemic factors [18]. Inhibition of ERK 1/2 in muscle prevents the translocation of FAT/CD36 during muscle contraction suggesting an alternative regulation of fatty acid uptake to that of AMPK [19]. 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. ...
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.
... The contraction-stimulated increase in skeletal muscle PM FAT/CD36 and FABPpm has been shown previously in skeletal muscle (23,85,212). This study is the first to report that muscle contraction also induces the translocation of FATP1 and FATP4. ...
... Skeletal muscle-specific overexpression of FAT/CD36 resulted in increased FA oxidation but only during muscle contraction and not at rest, suggesting that LCFA transport is not simply regulated by FAT/CD36 expression levels 113 . Instead it is thought that FAT/CD36 resides in internal pool that can be sequestered to the plasma membrane, as occurs with acute contraction of skeletal muscle 114 . ...
... Activation of p38 MAPK in the skeletal muscle is involved in stimulating mitochondrial biogenesis and oxidative capacity through activating the downstream target Peroxisome proliferator-activated receptor gamma coactivator 1-alpha [58] and glucose transport through the translocation of glucose transporter 4 (GLUT 4) [59]. ERK1/2 triggers cluster differentiation 36 (CD36), which stimulates fatty acid oxidation and fatty acid uptake [60] and is also thought to be partly involved in glucose uptake [61]. Together, activation of these pathways in the skeletal muscle can promote signalling that enhances nutrient utilisation [62] and might be beneficial for treatment of obesity and associated disease states. ...
... 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.
... In rats, the fatty acid transport capacity of skeletal muscle can be increased by chronic training, and decreased by denervation (100). (173). In contrast, increasing cyclic AMP (cAMP) levels in cardiac myocytes did not induce FAT/CD36 translocation (119), suggesting catecholamine elevations associated with exercise are not responsible for the increase in fatty acid transport. ...
This thesis is an investigation of the role of fatty acid translocase (FAT/CD36), plasma membrane associated fatty acid binding protein (FABPpm), and carnitine palmitoyltransferase I (CPTI) in transporting long-chain fatty acids (LCFAs) across mitochondrial membranes. Maximal CPTI activity, as well as the sensitivity of CPTI for its substrate palmitoyl-CoA (P-CoA) and its inhibitor malonyl-CoA (M-CoA), were measured in mitochondria isolated from human vastus lateralis muscles at rest and following muscle contraction. Exercise did not alter maximal CPTI activity or the sensitivity of CPTI for P-CoA. In contrast, exercise progressively attenuated the ability of M-CoA to inhibit CPTI activity. Mitochondrial FAT/CD36 protein content was also measured at rest, during, and following 2h of cycling at ~60% maximal oxygen uptake. Exercise progressively increased the content of mitochondrial FAT/CD36 (+59%), which was significantly (p< 0.05) correlated with palmitate oxidation during exercise (r = 0.52), while palmitate oxidation was inhibited ~80% by the administration of a specific FAT/CD36 inhibitor. These data suggest that alterations in CPTI M-CoA sensitivity and increases in mitochondrial FAT/CD36 coordinate exercise-induced increases in fatty acid oxidation. FABPpm, another plasma membrane transport protein, has identical amino acid sequence to mitochondrial aspartate aminotransferase (mAspAT). Since FABPpm contributes to plasma membrane fatty acid transport, the role of FABPpm with respect to mitochondrial LCFA transport was investigated. However, unlike FAT/CD36, muscle contraction did not induce an increase in mitochondrial FABPpm protein in rat or human skeletal muscle. In addition, electrotransfecting FABPpm cDNA into rat skeletal muscle upregulated this protein in mitochondriaby 80% without altering mitochondrial palmitate oxidation. In contrast, electrotransfection increased mAspAT activity by 90%, and this was correlated (r = 0.75p< 0.01) with FABPpm protein. These data suggest that FABPpm does not contribute to the regulation of mitochondrial LCFA transport. Previously, it has been suggested that mitochondria from obese individuals contain an inherent dysfunction to oxidize LCFAs. In age-matched lean (BMI = 23.3 0.7kgm²) and obese (BMI = 37.6 2.2kgm²) individuals, isolated mitochondrial palmitate oxidation was not altered. In addition, mitochondrial FAT/CD36 content was not different in lean and obese individuals. In contrast, citrate synthase and -hydroxyacyl-CoA dehydrogenase, common markers of total mitochondrial content, were decreased with obesity. Therefore, the decrease in mitochondrial content appeared to account for the observed reductions in whole-muscle LCFA oxidation.
... While the exact signalling mechanisms inducing FAT/CD36 translocation to the plasma membrane in muscle remain elusive, AICAR can induce the translocation of all LCFA transport proteins to the plasma membrane; therefore, AMPK may be involved ( Luiken et al., 2003;Chabowski et al., 2005). Other signalling cascades have also been implicated, as inhibition of extra cellular signalling receptor kinase (ERK1/2) can prevent contraction-induced FAT/CD36 translocation ( Turcotte et al., 2005) and activating protein kinase C in cardiac myocytes induces FAT/CD36 translocation, suggesting a possible role for Ca 2+ (Luiken et al., 2004). This has been further strengthened by recent work showing that fatty acid transport is increased in response to caffeine, which increases cytosolic Ca 2+ levels, a response attenuated with inhibitors for CaMKII and CaMKK signalling ( Abbott et al., 2009). ...
Skeletal muscle plays an important role in regulating whole-body energy expenditure given it is a major site for glucose and lipid oxidation. Obesity and type 2 diabetes are causally linked through their association with skeletal muscle insulin resistance, while conversely exercise is known to improve whole body glucose homeostasis simultaneously with muscle insulin sensitivity. Exercise activates skeletal muscle AMP-activated protein kinase (AMPK). AMPK plays a role in regulating exercise capacity, skeletal muscle mitochondrial content and contraction-stimulated glucose uptake. Skeletal muscle AMPK is also thought to be important for regulating fatty acid metabolism; however, direct genetic evidence in this area is currently lacking. This review will discuss the current paradigms regarding the influence of AMPK in regulating skeletal muscle fatty acid metabolism and mitochondrial biogenesis at rest and during exercise, and highlight the potential implications in the development of insulin resistance.
... Nevertheless, direct evidence of AMPK-mediated translocation in skeletal muscle is still lacking. However, it would appear that if AMPK plays a role in FAT/CD36 translocation with muscle contraction, its activation is not sufficient for translocation as ERK1/2 signaling is also required (Turcotte et al. 2005). Interestingly, recent findings show that FAT/CD36 is also present in the mitochondrial membrane and may be involved along with CPT-1 in the transportation of acyl-CoAs into the mitochondria, and that its concentration there increases with chronic muscle contraction (Holloway et al. 2008). ...
AMP-activated protein kinase (AMPK) has emerged as a key regulator of skeletal muscle fat metabolism. Because abnormalities in skeletal muscle metabolism contribute to a variety of clinical diseases and disorders, understanding AMPK’s role in the muscle is important. It was originally shown to stimulate fatty acid (FA) oxidation decades ago, and since then much research has been accomplished describing this role. In this brief review, we summarize much of these data, particularly in relation to changes in FA oxidation that occur during skeletal muscle exercise. Potential roles for AMPK exist in regulating FA transport into the mitochondria via interactions with acetyl-CoA carboxylase, malonyl-CoA decarboxylase, and perhaps FA transporter/CD36 (FAT/CD36). Likewise, AMPK may regulate transport of FAs into the cell through FAT/CD36. AMPK may also regulate capacity for FA oxidation by phosphorylation of transcription factors such as CREB or coactivators such as PGC-1α.
... In our cardiomyocytes, aliskiren did not decrease expression of (P)Rr or Igf2/M6Pr receptors, so the angiotensin II-independent effects of a potential renin increase at the cardiac level should perhaps be further considered in relation to clinical treatment. It has also recently been demonstrated that the ERK1/2 pathway activation is involved in regulating cardiac fatty-acid metabolism [82] and in the contraction-induced increase in plasma-membrane CD-36 and fatty-acid uptake in rodent myocytes [83]. These latter findings could constitute a nexus between our functional data showing that aliskiren stimulates fatty-acid uptake and increases ERK1/2 phosphorylation. ...
We investigated whether the direct renin inhibitor aliskiren can affect metabolism in cardiomyocytes from rat, mouse and human sources.
At 10-50 μmol/L, aliskiren significantly increased medium-chain-fatty-acid uptake in primary-cultured neonatal-rat and HL-1 adult-mouse-derived cardiomyocytes (BODIPY-induced fluorescence intensity). The fatty-acid transporter CD-36 was correspondingly translocated to, but the glucose transporter Glut-4 away from, the sarcoplasmic reticulum/plasma membrane, in primary-cultured neonatal-rat (CD-36, Glut-4) and adult-human (CD-36) cardiomyocytes (confocal immunocytochemistry). Immunoblotting showed that aliskiren induced phosphorylation of ERK1/2 in cardiomyocytes from all three sources; responses were dose- and time-dependent, unaffected by renin treatment, and did not cause alterations in expression of (P)R or Igf2/M6P receptors. Microarray analysis of the complete genome of aliskiren-treated neonatal-rat cardiomyocytes, with RT-qPCR and immunoblot confirmation assays in rat and human primary cardiomyocytes, showed that aliskiren up-regulated mRNA and increased protein expression of several enzymes important in lipid and glucose metabolism and in cholesterol biosynthesis. Cardiomyocyte cell-cycle and viability were unaffected by aliskiren.
Aliskiren can induce changes in fatty-acid and glucose uptake and expression of key enzymes of lipid and cholesterol metabolism, which are not associated with increased expression of (P)R or Igf2/M6P receptors, in cultured cardiomyocytes.
... Low AMPK␣ 2 activity did not affect the phosphorylation state of other signaling intermediates known to be implicated in the regulation of substrate use in skeletal muscle. Consistent with our previous results and those of others (26,35,47), ERK1/2 phosphorylation was increased during muscle contraction but was not affected by the DN transgene. Overall, our results suggest that AMPK␣ 2 activation is not necessary to observe a rise in CaMKI and ERK1/2 phosphorylation during moderate-intensity muscle contraction. ...
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.
... FAT/CD36 (fatty acid translocase/Cluster of Differentiation 36) is a class B scavenger receptor with strong homology with human CD36 (glycoprotein IV) and has been shown to function as a plasma membrane LCFA (long-chain fatty acid) transporter in various tissues, including skeletal muscle, heart, liver, adipose tissue and the small intestine [1][2][3][4][5][6][7]. Additionally, in heart and skeletal muscle, intracellular pools of FAT/CD36 exist which can be induced to translocate to the plasma membrane to regulate LCFA uptake through the activation of several signalling cascades, including AMPK (AMP-activated protein kinase) [8], CaMKK (Ca 2 + /calmodulin-dependent protein kinase kinase) [9], ERK1/2 (extracellular-signal-regulated kinase 1/2) [10] and insulin [11]. ...
FAT/CD36 (fatty acid translocase/Cluster of Differentiation 36), a plasma membrane fatty-acid transport protein, has been found on mitochondrial membranes; however, it remains unclear where FAT/CD36 resides on this organelle or its functional role within mitochondria. In the present study, we demonstrate, using several different approaches, that in skeletal muscle FAT/CD36 resides on the OMM (outer mitochondrial membrane). To determine the functional role of mitochondrial FAT/CD36 in this tissue, we determined oxygen consumption rates in permeabilized muscle fibres in WT (wild-type) and FAT/CD36-KO (knockout) mice using a variety of substrates. Despite comparable muscle mitochondrial content, as assessed by unaltered mtDNA (mitochondrial DNA), citrate synthase, β-hydroxyacyl-CoA dehydrogenase, cytochrome c oxidase complex IV and respiratory capacities [maximal OXPHOS (oxidative phosphorylation) respiration] in WT and KO mice, palmitate-supported respiration was 34% lower in KO animals. In contrast, palmitoyl-CoA-supported respiration was unchanged. These results indicate that FAT/CD36 is key for palmitate-supported respiration. Therefore we propose a working model of mitochondrial fatty-acid transport, in which FAT/CD36 is positioned on the OMM, upstream of long-chain acyl-CoA synthetase, thereby contributing to the regulation of mitochondrial fatty-acid transport. We further support this model by providing evidence that FAT/CD36 is not located in mitochondrial contact sites, and therefore does not directly interact with carnitine palmitoyltransferase-I as original proposed.
... To strengthen our metabolic data, we measured the effects of metformin on ACC phosphorylation and PM CD36 content. Multiple studies have indicated that CD36 is an important PM protein that carries FA from plasma to the cytosol (5,23,29) and that PM CD36 content and FA uptake often change in a parallel manner (1,22,67). In line with this, our results show that, in parallel with our FA uptake data, PM (but not total) CD36 protein content was reduced with metformin treatment. ...
Metformin is known to improve insulin sensitivity in part via a rise in AMP-activated protein kinase (AMPK) activity and alterations in muscle metabolism. However, a full understanding of how metformin alters AMPK-α(1) vs. AMPK-α(2) activation remains unknown. To study this question, L6 skeletal muscle cells were treated with or without RNAi oligonucleotide sequences to downregulate AMPK-α(1) or AMPK-α(2) protein expression and incubated with or without 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or metformin and/or insulin. In contrast to AICAR, which preferentially activated AMPK-α(2), metformin preferentially activated AMPK-α(1) in a dose- and time-dependent manner. Metformin increased (P < 0.05) glucose uptake and plasma membrane (PM) Glut4 in a dose- and time-dependent manner. Metformin significantly reduced palmitate uptake (P < 0.05) and oxidation (P < 0.05), and this was accompanied by a similar decrease (P < 0.05) in PM CD36 content but with no change in acetyl-CoA carboxylase (ACC) phosphorylation (P > 0.05). AICAR and metformin similarly increased (P < 0.05) nuclear silent mating-type information regulator 2 homolog 1 (SIRT1) activity. Downregulation of AMPK-α(1) completely prevented the metformin-induced reduction in palmitate uptake and oxidation but only partially reduced the metformin-induced increase in glucose uptake. Downregulation of AMPK-α(2) had no effect on metformin-induced glucose uptake, palmitate uptake, and oxidation. The increase in SIRT1 activity induced by metformin was not affected by downregulation of either AMPK-α(1) or AMPK-α(2). Our data indicate that, in muscle cells, the inhibitory effects of metformin on fatty acid metabolism occur via preferential phosphorylation of AMPK-α(1), and the data indicate that cross talk between AMPK and SIRT1 does not favor either AMPK isozyme.
... Beyond that, the data suggest that an adequate stress response to physical activity is important to initiate essential adaptations to exercise, not only to prevent tissue damage but also to improve exercise performance and to achieve health benefits. The muscular stress response has been implicated in the upregulation of enzymes and co-activators important for lipid and glucose metabolism in rodents (1,98,118). Antioxidant treatment to reduce the exercisedependent oxidative stress depresses muscle force production (25,99), prevents training-induced adaptations in endurance performance and mitochondrial biogenesis (38) and abrogates improvements of insulin sensitivity (100) and cardiovascular parameters (129). ...
Recent research on the effectiveness of training interventions indicates major alterations of hepatic lipid metabolism and suggests a substantial and beneficial adaptation of the liver to regular physical activity in humans. However, while various' data demonstrate the response of the working skeletal muscle to acute exercise and training, considerably less is known about the molecular events in the liver during and after increased physical activity. Here we discuss recent studies performed in rodents, that elucidate the acute hepatic response to one single bout of exercise with particular emphasis on stress response-related pathways. The acute transcriptional response to one exercise bout comprises three-times more hepatic transcripts than those expressed in soleus muscle, with a significantly more pronounced up- or downregulation of hepatic genes. Evaluation of the affected pathways shows that the liver responds to acute exercise with a rapid activation of the mitogen-activated protein kinase (MAPK) signalling pathway, of the p53 protein, and of interleukin (IL)-6-type cytokine signalling pathways, resulting in a marked transcriptional upregulation of stress response genes (e.g., transcription factors of the Fos/Jun-family, growth arrest and DNA damage (GADD)45gamma, and p53-target genes) and genes typically induced by energy depletion, e.g., insulin-like growth factor binding protein (IGFBP)-1, peroxisome proliferator-activated receptor coactivator (PGC)1alpha. One explanation for the marked differential expression of hepatic genes immediately after exercise is the induction of energetic stress. After non-exhaustive exercise energy depletion predominantly occurs in the liver not as much in the working muscle, and during exercise, the liver is exposed to altered concentrations of insulin and glucagon in the portal vein. Furthermore, lower plasma glucose levels post-exercise are related to increased expression levels of stress response genes. It appears that the unique function of the liver to supply glucose for the working muscle renders this organ especially susceptible for exercise-induced cellular stress that leads to the marked induction of defense adaptations. These results give rise to the question whether these molecular events are linked not only to stress defense but to the metabolic adaptations of the liver to exercise.
Physical activity plays a fundamental role in human health and disease. Exercise has been shown to improve a wide variety of disease states, and the scientific community is committed to understanding the precise molecular mechanisms that underlie the exquisite benefits. This review provides an overview of molecular responses to acute exercise and chronic training, particularly energy mobilization and generation, structural adaptation, inflammation, and immune regulation. Further it offers a detailed discussion on known molecular signals and systemic regulators activated during various forms of exercise and their role in orchestrating health benefits. Critically, the increasing use of multi-omic technologies is explored with an emphasis on how multi-omic and multi-tissue studies contribute to a more profound understanding of exercise biology. These data inform anticipated future advancement in the field and highlight the prospect of integrating exercise with pharmacology for personalized disease prevention and treatment.
The bone-muscle unit refers to the reciprocal regulation between bone and muscle by mechanical interaction and tissue communication via soluble factors. The receptor activator of NF-κB ligand (RANKL) stimulation induces mitochondrial biogenesis and increases the oxidative capacity in osteoclasts and adipocytes. RANKL may bind to the membrane bound receptor activator of NF-κB (RANK) or to osteoprotegerin (OPG), a decoy receptor that inhibits RANK-RANKL activation. RANK is highly expressed in skeletal muscle, but the contribution of RANKL to healthy skeletal muscle fiber remains elusive. Here we show that RANKL stimulation in C2C12-derived myotubes induced activation of mitochondrial biogenesis pathways as detected by RNA-seq and western blot. RANKL expanded the mitochondrial reticulum, as shown by mitochondrial DNA quantification and MitoTracker staining, and boosted the spare respiratory capacity. Using MEK and MAPK inhibitors, we found that RANKL signals via ERK and p38 to induce mitochondrial biogenesis. The soleus from OPG-/- and OPG+/- mice showed higher respiratory rates compared to C57BL6/J wild-type (WT) mice, which correlates with high serum RANKL levels. RANKL infusion using a mini-osmotic pump in WT mice increased the number of mitochondria, boosted the respiratory rate, increased succinate dehydrogenase (SDH) activity in skeletal muscle, and improved the fatigue resistance of gastrocnemius. Therefore, our findings reveal a new role of RANKL as an osteokine-like protein that impacts muscle fiber metabolism.
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.
CD36 is a multifunctional glycoprotein, expressed in different types of cells and known to play a significant role in the pathophysiology of the host. The structural studies revealed that the scavenger receptor consists of short cytosolic domains, two transmembrane domains and a large ectodomain. The ectodomain serves as a receptor for a diverse number of endogenous and exogenous ligands. The CD36-specific ligands are involved in regulating the immune response during infectious and non-infectious diseases in the host. The role of CD36 in regulating the innate immune response during Pneumonia, Tuberculosis, Malaria, Leishmaniasis, HIV and Sepsis in a ligand-mediated fashion. Apart from infectious diseases, it is also considered to be involved in metabolic disorders such as Atherosclerosis, Alzheimer’s, cancer and Diabetes. The ligand binding to scavenger receptor modulates the CD36 down-stream innate immune response, and it can be exploited to design suitable immuno-modulators. Hence, the current review focused on the role of the CD36 in innate immune response and therapeutic potentials of novel heterocyclic compounds as CD36 ligands during infectious and non-infectious diseases.
Fatty acid transport proteins rapidly translocate to the plasma membrane in response to various stimuli, including insulin, influencing lipid uptake into muscle. However, our understanding of the mechanisms regulating post-prandial fatty acid transporter subcellular location remains limited. We demonstrate that the response of fatty acid transporters to insulin stimulation is extremely brief and not temporally matched in the post-prandial state. We further show that high-fat diet-induced accumulation of fatty acid transporters on the plasma membrane can occur in the absence of insulin. Altogether, these data suggest that insulin is not the primary signal regulating fatty acid transporter relocation in vivo. This article is protected by copyright. All rights reserved.
Carbohydrates and long-chain fatty acids are the predominant substrates for cardiac energy production. While the mechanism and regulation of myocardial carbohydrate (glucose, lactate) uptake have been unraveled in detail in the 1990s, insight into fatty acid uptake originates from more recent studies. Fatty acid movement across the sarcolemma is facilitated by membrane-associated proteins, specifically CD36, membrane-associated fatty acid-binding protein (FABPpm) and selected fatty acid transport protein (FATP) isoforms, and is up- or downregulated through changes in sarcolemmal content of (primarily) CD36. The recruitment of CD36 from an endosomal storage pool to the sarcolemma, which is under the control of various physiological stimuli (including insulin and contraction), represents a pivotal step in the overall regulation of myocardial fatty acid uptake and utilization. Dysregulation of the intracellular cycling of CD36 underlies various cardiac metabolic diseases. As a result, the mechanism and regulation of myocardial glucose uptake by GLUT4 cycling and of fatty acid uptake by CD36 cycling are very similar. Likely, manipulation of the presence and/or activity of substrate transporters for glucose and fatty acids in the sarcolemma holds promise as therapeutic approach to alter cardiac substrate preference in disease so as to regain metabolic homeostasis and rectify cardiac functioning.
Exercise has been shown to induce the translocation of fatty acid translocase (FAT/CD36), a fatty acid transport protein, to both plasma and mitochondrial membranes. While previous studies have examined signals involved in the induction of FAT/CD36 translocation to sarcolemmal membranes, to date the signaling events responsible for FAT/CD36 accumulation on mitochondrial membranes have not been investigated. In the current study muscle contraction rapidly increased FAT/CD36 on plasma membranes (7.5 minutes), while in contrast, FAT/CD36 only increased on mitochondrial membranes after 22.5 minutes of muscle contraction, a response that was exercise-intensity dependent. Considering that previous research has shown that AMP activated protein kinase (AMPK) α2 is not required for FAT/ CD36 translocation to the plasma membrane, we investigated whether AMPK α2 signaling is necessary for mitochondrial FAT/CD36 accumulation. Administration of 5-Aminoimidazole- 4-carboxamide ribonucleotide (AICAR) induced AMPK phosphorylation, and resulted in FAT/CD36 accumulation on SS mitochondria, suggesting AMPK signaling may mediate this response. However, SS mitochondrial FAT/CD36 increased following acute treadmill running in both wild-type (WT) and AMPKα 2 kinase dead (KD) mice. These data suggest that AMPK signaling is not required for SS mitochondrial FAT/CD36 accumulation. The current data also implicates alternative signaling pathways that are exercise-intensity dependent, as IMF mitochondrial FAT/CD36 content only occurred at a higher power output. Taken altogether the current data suggests that activation of AMPK signaling is sufficient but not required for exercise-induced accumulation in mitochondrial FAT/CD36. © 2015 Monaco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Long-chain fatty acid uptake has now been shown to occur via a highly regulated, protein-mediated mechanism involving plasma membrane fatty acid transporters. This process is especially important in skeletal muscle, a tissue with a highly variable metabolic rate that constitutes approximately 40% of body mass. We review the evidence that skeletal muscle fatty acid transport is acutely and chronically regulated by muscle contraction and insulin, largely by the fatty acid transporter CD36. We also examine recent data suggesting that CD36 may contribute to regulating fatty acid oxidation by mitochondria. In addition, we review evidence showing that skeletal muscle insulin resistance is associated with the dysregulation of CD36-mediated fatty acid transport, and that the insulin-sensitizing effects of proliferator-activated receptor- coactivator-1α may depend on limiting CD36 upregulation. Taken altogether, it is apparent that skeletal muscle fatty acid transport is central to the regulation of whole-body lipid metabolism in health and disease.
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.
During fasting, rapid metabolic adaptations are required to maintain energy homeostasis. This occurs by a coordinated regulation of energy/nutrient-sensing pathways leading to transcriptional activation and repression of specific sets of genes. The aim of the study was to investigate how short-term fasting affects whole-body energy homeostasis and skeletal muscle energy/nutrient-sensing pathways and transcriptome in humans. For this purpose, twelve young healthy men were studied during a 24-hour fast. Whole-body glucose/lipid oxidation rates were determined by indirect calorimetry and blood and skeletal muscle biopsies were collected and analyzed at baseline and after 10 and 24h of fasting. As expected, fasting induced a time-dependent decrease in plasma insulin and leptin levels, whereas levels of ketone bodies and free fatty acids increased. This was associated with a metabolic shift from glucose towards lipid oxidation. At the molecular level, activation of the protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR) pathways was time-dependently reduced in skeletal muscle during fasting, whereas the AMP-activated protein kinase (AMPK) activity remained unaffected. Furthermore, we report some changes in the phosphorylation and/or content of forkhead protein 1 (FoxO1), sirtuins 1 (SIRT1) and class IIa histone deacetylase 4 (HDAC4), suggesting that these pathways might be involved in the transcriptional adaptation to fasting. Finally, transcriptome profiling identified genes that were significantly regulated by fasting in skeletal muscle at both early and late time-points. Collectively, our study provides a comprehensive map of the main energy/nutrient-sensing pathways and transcriptomic changes during short-term adaptation to fasting in human skeletal muscle.
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.
G protein-coupled receptor 55 (GPR55) is a putative cannabinoid receptor that is expressed in several tissues involved in regulating energy homeostasis, including the hypothalamus, gastrointestinal tract, pancreas, liver, white adipose and skeletal muscle. GPR55 has been shown to have a role in cancer and gastrointestinal inflammation, as well as in obesity and type 2 diabetes mellitus (T2DM). Despite this, the (patho)physiological role of GPR55 in cell dysfunction is still poorly understood, largely because of the limited identification of downstream signalling targets. Nonetheless, research has suggested that GPR55 modulation would be a useful pharmacological target in metabolically active tissues to improve treatment of diseases such as obesity and T2DM. Further research is essential to gain a better understanding of the role that this receptor might have in these and other pathophysiological conditions.
GPR43 is a receptor for short-chain fatty acids. Preliminary data suggest a putative role for GPR43 in regulating systemic health via processes including inflammation, carcinogenesis, gastrointestinal function, and adipogenesis. GPR43 is involved in secretion of gastrointestinal peptides, which regulate appetite and gastrointestinal motility. This suggests GPR43 may have a role in weight control. Moreover, GPR43 regulates plasma lipid profile and inflammatory processes, which further indicates that GPR43 could have the ability to modulate the etiology and pathogenesis of metabolic diseases such as obesity, type 2 diabetes mellitus, and cardiovascular disease. This review summarizes the current evidence regarding the ability of GPR43 to mediate both systemic and tissue specific functions and how GPR43 may be modulated in the treatment of metabolic disease.
The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, also known as the MEK/ERK1/2 kinase cascade, has recently been implicated in the regulation of lipid metabolism and fatty liver disease. However, its functional effect on cellular fatty acid composition is unknown. Herein, we examined the effect of a pharmacological inhibitor of MEK, the upstream kinase activator of ERK1/2, on fatty acid composition of hepatocellular carcinoma cell line HepG2.
HepG2 cells cultured in RPMI-1640 were exposed to the commonly used ERK1/2 pathway inhibitor PD98059 and were investigated with respect to fatty acid composition by gas-liquid chromatography.
Exposure of cells to the ERK1/2 pathway inhibitor induced an increase in monounsaturated fatty acids and the fatty acid desaturation index and a decrease in polyunsaturated fatty acid content. Specifically, we showed a significant increase of oleic acid (18:1n-9; +29%, P=0.003) and arachidonic acid (20:4n-6)/linoleic acid (18:2n-6) ratio (3.5-fold; P<0.001) in HepG2 cells.
Cellular fatty acid composition of HepG2 cells appeared to be differentially regulated by ERK1/2 pathway, thus suggesting related metabolic pathways as potential mediators of the effects of ERK1/2 signaling on hepatic fatty acid composition.
In all eukaryotes, AMP-activated protein kinase (AMPK) integrates metabolic signals to influence energy balance through the regulation of multiple biochemical pathways. The ability of AMPK to maintain adequate ATP supply at the cellular level plays an important role in the cardiomyocyte since maintaining adequate ATP supply is essential for proper contractile function. In addition, AMPK controls a variety of essential biological processes that also impacts cardiomyocyte function and survival. In this chapter we will discuss the metabolic role of AMPK in modulating glucose and fatty acid transport, oxidation, storage, and the mechanisms by which AMPK influences contractile function and structural remodeling in healthy and diseased hearts. Specifically, we discuss the role that AMPK plays in a variety of conditions such as ischemia/reperfusion injury, cardiac hypertrophy, glycogen storage cardiomyopathy, and Wolff–Parkinson–White syndrome, as well as cardiac dysfunction associated with obesity, insulin resistance, and diabetes. Overall, this chapter summarizes the existing data about the role of AMPK in the heart as well as the role that AMPK plays in the metabolic remodeling that occurs in cardiac disease. Moreover, we pose some pertinent questions, the answers of which may help provide additional insight into the metabolic role of AMPK in diseased hearts. This information may eventually help in the development of therapeutic approaches that target AMPK or AMPK-regulated pathways to improve function of metabolically remodeled diseased hearts.
SMITH, B. K., A. BONEN, and G. P. HOLLOWAY. A dual mechanism of action for skeletal muscle FAT/CD36 during exercise. Exerc. Sport Sci. Rev., Vol. 40, No. 4, pp. 211-217, 2012. Carnitine palmitoyltransferase I has been viewed historically as the sole regulator of fatty acid oxidation. However, we have identified fatty acid translocase/CD36 as an additional control point. Specifically, fatty acid translocase/CD36 seems to have a novel dual mechanism of action with regard to fatty acid oxidation during exercise, influencing transport of lipids across the sarcolemmal membrane and into the mitochondria.
Fatty acid transport proteins are present on the plasma membrane and are involved in the uptake of long-chain fatty acids into skeletal muscle. The present study determined whether acute endurance exercise increased the plasma membrane content of fatty acid transport proteins in rat and human skeletal muscle and whether the increase was accompanied by an increase in long-chain fatty acid transport in rat skeletal muscle. Sixteen subjects cycled for 120 min at ∼60 ± 2% Vo(2) peak. Two skeletal muscle biopsies were taken at rest and again following cycling. In a parallel study, eight Sprague-Dawley rats ran for 120 min at 20 m/min, whereas eight rats acted as nonrunning controls. Giant sarcolemmal vesicles were prepared, and protein content of FAT/CD36 and FABPpm was measured in human and rat vesicles and whole muscle homogenate. Palmitate uptake was measured in the rat vesicles. In human muscle, plasma membrane FAT/CD36 and FABPpm protein contents increased 75 and 20%, respectively, following 120 min of exercise. In rat muscle, plasma membrane FAT/CD36 and FABPpm increased 20 and 30%, respectively, and correlated with a 30% increase in palmitate transport following 120 min of running. These data suggest that the translocation of FAT/CD36 and FABPpm to the plasma membrane in rat skeletal muscle is related to the increase in fatty acid transport and oxidation that occurs with endurance running. This study is also the first to demonstrate that endurance cycling induces an increase in plasma membrane FAT/CD36 and FABPpm content in human skeletal muscle, which is predicted to increase fatty acid transport.
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.
Hormonally stimulated lipolysis occurs by activation of cyclic AMP-dependent protein kinase (PKA) which phosphorylates hormone-sensitive
lipase (HSL) and increases adipocyte lipolysis. Evidence suggests that catecholamines not only can activate PKA, but also
the mitogen-activated protein kinase pathway and extracellular signal-regulated kinase (ERK). We now demonstrate that two
different inhibitors of MEK, the upstream activator of ERK, block catecholamine- and β3-stimulated lipolysis by ∼30%. Furthermore, treatment of adipocytes with dioctanoylglycerol, which activates ERK, increases
lipolysis, although MEK inhibitors decrease dioctanoylglycerol-stimulated activation of lipolysis. Using a tamoxifen regulatable
Raf system expressed in 3T3-L1 preadipocytes, exposure to tamoxifen causes a 14-fold activation of ERK within 15–30 min and
results in ∼2-fold increase in HSL activity. In addition, when differentiated 3T3-L1 cells expressing the regulatable Raf
were exposed to tamoxifen, a 2-fold increase in lipolysis is observed. HSL is a substrate of activated ERK and site-directed
mutagenesis of putative ERK consensus phosphorylation sites in HSL identified Ser600 as the site phosphorylated by active ERK. When S600A HSL was expressed in 3T3-L1 cells expressing the regulatable Raf, tamoxifen
treatment fails to increase its activity. Thus, activation of the ERK pathway appears to be able to regulate adipocyte lipolysis
by phosphorylating HSL on Ser600 and increasing the activity of HSL.
The molecular signaling mechanisms by which muscle contractions lead to changes in glucose metabolism and gene expression remain largely undefined. We assessed whether exercise activates MAP kinase proteins (ERK1/2, SEK1, and p38 MAP kinase) as well as Akt and PYK2 in skeletal muscle from healthy volunteers obtained during and after one-leg cycle ergometry at approximately 70% VO2max. Exercise led to a marked increase in ERK1/2 phosphorylation, which rapidly decreased to resting levels upon recovery. Exercise increased phosphorylation of SEK1 and p38 MAP kinase to a lesser extent than ERK1/2. In contrast to ERK1/2, p38 MAP kinase phosphorylation was increased in nonexercised muscle upon cessation of exercise. Phosphorylation of the transcription factor CREB was increased in nonexercised muscle upon cessation of exercise. Exercise did not activate Akt or increase tyrosine phosphorylation of PYK2. Thus, exercise has divergent effects on parallel MAP kinase pathways, of which only p38 demonstrated a systemic response. However, our data do not support a role of Akt or PYK2 in exercise/contraction-induced signaling in human skeletal. Activation of the different MAP kinase pathways by physical exercise appears to be important in the regulation of transcriptional events in skeletal muscle.
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.
Giant sarcolemmal vesicles were isolated from rat heart and hindlimb muscles for a) characterization of long-chain fatty acid transport in the absence of metabolism and b) comparison of fatty acid transport protein expression with fatty acid transport. Giant vesicles contained cytosolic fatty acid binding protein. Palmitate uptake was completely divorced from its metabolism. All palmitate taken up was recovered in the intravesicular cytosol as unesterified FA. Palmitate uptake by heart vesicles exhibited a K m of 9.7 nm, similar to that of muscle (K m = 9.7 nm). Vmax (2.7 pmol/mg protein/s) in heart was 8-fold higher than in muscle (0.34 pmol/mg protein/s). Palmitate uptake was inhibited in heart (55-80%) and muscle (31-50%) by trypsin, phloretin, sulfo-N-succinimidyloleate (SSO), or a polyclonal antiserum against the 40 kDa plasma membrane fatty acid binding protein (FABPpm). Palmitate uptake by heart and by red and white muscle vesicles correlated well with the expression of fatty acid translocase (FAT/CD36) and fatty acid binding protein FABPpm, which may act in concert. The expression of fatty acid transport protein (FATP), was 10-fold lower in heart vesicles than in white muscle vesicles. It is concluded that long-chain fatty acid uptake by heart and muscle vesicles is largely protein-mediated, involving FAT/CD36 and FABPpm. The role of FATP in muscle and heart remains uncertain.
Increasing evidence has implicated the membrane protein CD36 (FAT) in binding and transport of long chain fatty acids (FA). To determine the physiological role of CD36, we examined effects of its overexpression in muscle, a tissue that depends on FA for its energy needs and is responsible for clearing a major fraction of circulating FA. Mice with CD36 overexpression in muscle were generated using the promoter of the muscle creatine kinase gene (MCK). Transgenic (MCK-CD36) mice had a slightly lower body weight than control litter mates. This reflected a leaner body mass with less overall adipose tissue, as evidenced by magnetic resonance spectroscopy. Soleus muscles from transgenic animals exhibited a greatly enhanced ability to oxidize fatty acids in response to stimulation/contraction. This increased oxidative ability was not associated with significant alterations in histological appearance of muscle fibers. Transgenic mice had lower blood levels of triglycerides and fatty acids and a reduced triglyceride content of very low density lipoproteins. Blood cholesterol levels were slightly lower, but no significant decrease in the cholesterol content of major lipoprotein fractions was measured. Blood glucose was significantly increased, while insulin levels were similar in the fed state and higher in the fasted state. However, glucose tolerance curves, determined at 20 weeks of age, were similar in control and transgenic mice. In summary, the study documented, in vivo, the role of CD36 to facilitate cellular FA uptake. It also illustrated importance of the uptake process in muscle to overall FA metabolism and glucose utilization.
The transmembrane protein CD36 has been identified in isolated cell studies as a putative transporter of long chain fatty acids. In humans, an association between CD36 deficiency and defective myocardial uptake of the fatty acid analog 15-(p-iodophenyl)-3-(R, S)-methyl pentadecanoic acid (BMIPP) has been reported. To determine whether this association represents a causal link and to assess the physiological role of CD36, we compared tissue uptake and metabolism of two iodinated fatty acid analogs BMIPP and 15-(p-iodophenyl) pentadecanoic acid (IPPA) in CD36 null and wild type mice. We also investigated the uptake and lipid incorporation of palmitate by adipocytes isolated from both groups. Compared with wild type, uptake of BMIPP and IPPA was reduced in heart (50-80%), skeletal muscle (40-75%), and adipose tissues (60-70%) of null mice. The reduction was associated with a 50-68% decrease in label incorporation into triglycerides and in 2-3-fold accumulation of label in diglycerides. Identical results were obtained from studies of [(3)H]palmitate uptake in isolated adipocytes. The block in diglyceride to triglyceride conversion could not be explained by changes in specific activities of the key enzymes long chain acyl-CoA synthetase and diacylglycerol acyltransferase, which were similar in tissues from wild type and null mice. It is concluded that CD36 facilitates a large fraction of fatty acid uptake by heart, skeletal muscle, and adipose tissues and that CD36 deficiency in humans is the cause of the reported defect in myocardial BMIPP uptake. In CD36-expressing tissues, uptake regulates fatty acid esterification at the level of diacylglycerol acyltransferase by determining fatty acyl-CoA supply. The membrane transport step may represent an important control site for fatty acid metabolism in vivo.
Skeletal muscle GLUT-4 transcription in response to treatment with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), a known activator of AMP-activated protein kinase (AMPK), was studied in rats and mice. The increase in GLUT-4 mRNA levels in response to a single subcutaneous injection of AICAR, peaked at 13 h in white and red quadriceps muscles but not in the soleus muscle. The mRNA level of chloramphenicol acyltransferase reporter gene which is driven by 1,154 or 895 bp of the human GLUT-4 proximal promoter was increased in AICAR-treated transgenic mice, demonstrating the transcriptional upregulation of the GLUT-4 gene by AICAR. However, this induction of transcription was not apparent with 730 bp of the promoter. In addition, nuclear extracts from AICAR-treated mice bound to the consensus sequence of myocyte enhancer factor-2 (from -473 to -464) to a greater extent than from saline-injected mice. Thus AMP-activated protein kinase activation by AICAR increases GLUT-4 transcription by a mechanism that requires response elements within 895 bp of human GLUT-4 proximal promoter and that may be cooperatively mediated by myocyte enhancer factor-2.
It is well known that muscle contraction and insulin can independently translocate GLUT-4 from an intracellular depot to the plasma membrane. Recently, we have shown that the fatty acid transporter FAT/CD36 is translocated from an intracellular depot to the plasma membrane by muscle contraction (<30 min) (Bonen et al. J Biol Chem 275: 14501-14508, 2000). In the present study, we examined whether insulin also induced the translocation of FAT/CD36 in rat skeletal muscle. In studies in perfused rat hindlimb muscles, we observed that insulin increased fatty acid uptake by +51%. Insulin increased the rate of palmitate incorporation into triacylglycerols, diacylglycerols, and phospholipids (P < 0.05) while reducing muscle palmitate oxidation (P < 0.05). Perfusing rat hindlimb muscles with insulin increased plasma membrane FAT/CD36 by +48% (P < 0.05), whereas concomitantly the intracellular FAT/CD36 depot was reduced by 68% (P < 0.05). These insulin-induced effects on FAT/CD36 translocation were inhibited by the phosphatidylinositol 3-kinase inhibitor LY-294002. Thus these studies have shown for the first time that insulin can induce the translocation of FAT/CD36 from an intracellular depot to the plasma membrane. This reveals a previously unknown level of regulation of fatty acid transport by insulin and may well have important consequences in furthering our understanding of the relation between fatty acid metabolism and insulin resistance.
Exercise increases glucose transport in muscle by activating 5'-AMP-activated protein kinase (AMPK), but subsequent events are unclear. Presently, we examined the possibility that AMPK increases glucose transport through atypical protein kinase Cs (aPKCs) by activating proline-rich tyrosine kinase-2 (PYK2), ERK pathway components, and phospholipase D (PLD). In mice, treadmill exercise rapidly activated ERK and aPKCs in mouse vastus lateralis muscles. In rat extensor digitorum longus (EDL) muscles, (a) AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-d-riboside (AICAR), activated PYK2, ERK and aPKCs; (b) effects of AICAR on ERK and aPKCs were blocked by tyrosine kinase inhibitor, genistein, and MEK1 inhibitor, PD98059; and (c) effects of AICAR on aPKCs and 2-deoxyglucose (2-DOG) uptake were inhibited by genistein, PD98059, and PLD-inhibitor, 1-butanol. Similarly, in L6 myotubes, (a) AICAR activated PYK2, ERK, PLD, and aPKCs; (b) effects of AICAR on ERK were inhibited by genistein, PD98059, and expression of dominant-negative PYK2; (c) effects of AICAR on PLD were inhibited by MEK1 inhibitor UO126; (d) effects of AICAR on aPKCs were inhibited by genistein, PD98059, 1-butanol, and expression of dominant-negative forms of PYK2, GRB2, SOS, RAS, RAF, and ERK; and (e) effects of AICAR on 2DOG uptake/GLUT4 translocation were inhibited by genistein, PD98059, UO126, 1-butanol, cell-permeable myristoylated PKC-zeta pseudosubstrate, and expression of kinase-inactive RAF, ERK, and PKC-zeta. AMPK activator dinitrophenol had effects on ERK, aPKCs, and 2-DOG uptake similar to those of AICAR. Our findings suggest that effects of exercise on glucose transport that are dependent on AMPK are mediated via PYK2, the ERK pathway, PLD, and aPKCs.
Long-chain fatty acids are an important constituent of the diet and they contribute to a multitude of cellular pathways and functions. Uptake of long-chain fatty acids across plasma membranes is the first step in fatty acid utilization, and recent evidence supports an important regulatory role for this process. Although uptake of fatty acids involves two components, passive diffusion through the lipid bilayer and protein-facilitated transfer, the latter component appears to play the major role in mediating uptake by key tissues. Identification of several proteins as fatty acid transporters, and emerging evidence from genetically altered animal models for some of these proteins, has contributed significant insight towards understanding the limiting role of transport in the regulation of fatty acid utilization. We are also beginning to better appreciate how disturbances in fatty acid utilization influence general metabolism and contribute to metabolic pathology.
Physical exercise is a significant stimulus for the regulation of multiple metabolic and transcriptional processes in skeletal muscle. For example, exercise increases skeletal muscle glucose uptake, and, after exercise, there are increases in the rates of both glucose uptake and glycogen synthesis. A single bout of exercise can also induce transient changes in skeletal muscle gene transcription and can alter rates of protein metabolism, both of which may be mechanisms for chronic adaptations to repeated bouts of exercise. A central issue in exercise biology is to elucidate the underlying molecular signaling mechanisms that regulate these important metabolic and transcriptional events in skeletal muscle. In this review, we summarize research from the past several years that has demonstrated that physical exercise can regulate multiple intracellular signaling cascades in skeletal muscle. It is now well established that physical exercise or muscle contractile activity can activate three of the mitogen-activated protein kinase signaling pathways, including the extracellular signal-regulated kinase 1 and 2, the c-Jun NH(2)-terminal kinase, and the p38. Exercise can also robustly increase activity of the AMP-activated protein kinase, as well as several additional molecules, including glycogen synthase kinase 3, Akt, and the p70 S6 kinase. A fundamental goal of signaling research is to determine the biological consequences of exercise-induced signaling through these molecules, and this review also provides an update of progress in this area.
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 AMPK (5'AMP-activated protein kinase) is becoming recognized as a critical regulator of energy metabolism. However, many of these effects in muscle metabolism have been ascribed to AMPK based on the use of the unspecific activator AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside). Using mouse models in which AMPK activity has been specifically blocked (kinase dead) or knocked out we and others have been able to conduct studies gaining more conclusive data on the role of AMPK in muscle metabolism. In this mini-review focus is on AMPK and its regulatory role for glucose transport and GS (glycogen synthase) activity in skeletal muscle, indicating that AMPK is a GS kinase in vivo which might influence GS activity during exercise and that AMPK is involved in AICAR/hypoxia-induced glucose transport but not or only partially in contraction-stimulated glucose transport.
We investigated the importance of the two catalytic alpha-isoforms of the 5'-AMP-activated protein kinase (AMPK) in 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) and contraction-induced glucose uptake in skeletal muscle. Incubated soleus and EDL muscle from whole-body alpha2- or alpha1-AMPK knockout (KO) and wild type (WT) mice were incubated with 2.0 mm AICAR or electrically stimulated to contraction. Both AICAR and contraction increased 2DG uptake in WT muscles. KO of alpha2, but not alpha1, abolished AICAR-induced glucose uptake, whereas neither KO affected contraction-induced glucose uptake. AICAR and contraction increased alpha2- and alpha1-AMPK activity in wild type (WT) muscles. During AICAR stimulation, the remaining AMPK activity in KO muscles increased to the same level as in WT. During contraction, the remaining AMPK activity in alpha2-KO muscles was elevated by 100% probably explained by a 2-3-fold increase in alpha1-protein. In alpha1-KO muscles, alpha2-AMPK activity increased to similar levels as in WT. Both interventions increased total AMPK activity, as expressed by AMPK-P and ACCbeta-P, in WT muscles. During AICAR stimulation, this was dramatically reduced in alpha2-KO but not in alpha1-KO, whereas during contraction, both measurements were essentially similar to WT in both KO-muscles. The results show that alpha2-AMPK is the main donor of basal and AICAR-stimulated AMPK activity and is responsible for AICAR-induced glucose uptake. In contrast, during contraction, the two alpha-isoforms seem to substitute for each other in terms of activity, which may explain the normal glucose uptake despite the lack of either alpha2- or alpha1-AMPK. Alternatively, neither alpha-isoform of AMPK is involved in contraction-induced muscle glucose uptake.
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.
Sulfo-N-succinimidyl derivatives of the long-chain fatty acids, oleic and myristic, were synthesized and covalently reacted with isolated rat adipocytes. The plasma membrane proteins labeled by these compounds and the effect of labeling on the transport of long-chain fatty acids were investigated. Sulfo-N-succinimidyl oleate (SSO) and myristate (SSM) inhibited the transport of fatty acids (by about 70%). Inhibition of fatty acid transport was not a result of alterations in cell integrity, as intracellular water volume was not changed. It did not reflect effects on fatty acid metabolism, since it was observed under conditions where greater than 90% of the fatty acid taken up was recovered in the free form. The inhibitory effect was specific to the fatty acid transport system, as the transport of glucose and the permeation of retinoic acid, a substance with structural similarities to long-chain fatty acids, were unaffected. Sulfosuccinimidyl oleate reacted exclusively with a plasma membrane protein with an apparent size of 85 kDa while sulfosuccinimidyl myristate also labeled a 75-kDa protein. These proteins were among the ones labeled by diisothiocyanodisulfonic acid (DIDS) which also inhibits fatty acid transport irreversibly. The data suggest that the 85-kDa protein, which is the only one labeled by all three inhibitors is involved in facilitating membrane permeation of long-chain fatty acids.
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.
Muscle malonyl-CoA decreases during exercise or electrical stimulation, the exercise-induced decline being accompanied by changes in the kinetic properties [maximal velocity (Vmax), activation constant (Ka), and citrate concentration required to produce 50% Vmax (K0.5)] of acetyl-CoAcarboxylase (ACC) and by an increase in the AMP-activated protein kinase activity (AMPK). This study was designed to ascertain whether the exercise-induced changes are contraction mediated and, if so, to follow the time course of these changes. The left sciatic nerve of rats was stimulated at 1 Hz for 0, 2, 5, 10, 20, or 30 min, and the gastrocnemius-plantaris muscle group was then excised, frozen in liquid nitrogen, and later analyzed for malonyl-CoA and other metabolites. ACC and AMPK activities were quantitated in ammonium sulfate precipitates from homogenates prepared from the frozen muscles. The Vmax and Ka of ACC for citrate decreased and increased, respectively, over the first 10 min of stimulation, but significantly increased AMPK activity was not observed until 10 to 20 min of stimulation (P < 0.05). Stimulation increased estimated free AMP (P < 0.05). Thus exercise-induced changes in functional properties of ACC appear to be contraction mediated and are accompanied by increased AMPK activity and an increase in the estimated free AMP.
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.
As muscle goes from a resting state to exercise, the following sequence of events occurs (Figure 5.5): (1) The rise in AMP accompanying contraction allosterically activates AMPK and an AMPK kinase; (2) The activated AMPK kinase phosphorylates and further activates AMPK; (3) The activated AMPK phosphorylates and inactivates ACC; and (4) The consequent decline in malonyl-CoA (product of ACC reaction) relieves inhibition of CPT-1 and allows an increased rate of fatty acid oxidation when fatty acids become available.
The intracellular signaling proteins that lead to exercise-stimulated glucose transport in skeletal muscle have not been identified, although it is clear that there are separate signaling mechanisms for exercise- and insulin-stimulated glucose transport. We have hypothesized that the 5'AMP-activated protein kinase (AMPK) functions as a signaling intermediary in exercise-stimulated glucose uptake. This hypothesis was based on recent studies showing the following: 1) muscle contraction increases AMPK activity and 2) perfusion of rat hindlimb skeletal muscles with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a compound that results in increased AMPK activity, increased insulin-stimulated glucose uptake. In the current study, isolated rat epitrochlearis muscles were treated to contract in vitro (via electrical stimulation for 10 min) and/or incubated in the absence or presence of AICAR (2 mmol/l), insulin (1 micromol/l), or wortmannin (100 nmol/l). Both contraction and AICAR significantly increased AMPK activity, while the enzyme was not activated by insulin. AICAR, contraction, and insulin all increased 3-O-methylglucose (3MG) transport by threefold to fivefold above basal. The phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin completely blocked insulin-stimulated transport, but did not inhibit AICAR- or contraction-stimulated transport. The increase in glucose transport with the combination of maximal AICAR plus maximal insulin treatments was partially additive, suggesting that these stimuli increase glucose transport by different mechanisms. In contrast, there was no additive effect on glucose transport with the combination of AICAR plus contraction. These data suggest that AICAR and contraction stimulate glucose transport by a similar insulin-independent signaling mechanism and are consistent with the hypothesis that AMPK is involved in exercise-stimulated glucose uptake.
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.
We have investigated the activation of the extracellular signal-regulated kinases (ERK1 and ERK2) by muscle contraction and insulin in perfused rat skeletal muscle. Both stimuli activated ERK1 and ERK2 by an upstream kinase MAP/ERK kinase (MEK)-dependent mechanism, as the MEK inhibitor PD-98059 inhibited ERK phosphorylation. The presence of the phosphatidylinositol (PI) 3-kinase inhibitors LY-294002 and wortmannin totally eradicated ERK1 and ERK2 phosphorylation in response to insulin but not contraction. Insulin and muscle contraction activated muscle glucose transport, glycogen synthase, and amino acid transport independently of ERK signaling, whereas the PI 3-kinase inhibitors abolished the stimulatory effects of insulin but not those of contraction on these three cellular processes. We conclude that 1) insulin and contraction activate ERK signaling in skeletal muscle; 2) ERK signaling is not necessary for activation of glucose and amino acid transport or glycogen synthase activity by contraction and insulin in skeletal muscle; and 3) insulin-induced activation of MEK, the upstream activator of ERK, is dependent on PI 3-kinase, whereas contraction utilizes a different mechanism.
Physical exercise is a potent stimulator of mitogen-activated protein (MAP) kinase signaling. To determine if this activation is secondary to systemic responses to exercise or due to muscle contractile activity per se, an isolated muscle preparation was developed. Contractile activity in vitro significantly increased p44(MAPK) and p42(MAPK) phosphorylation by 2.9- and 2.4-fold, respectively. Contraction-stimulated MAP kinase phosphorylation was not decreased in the presence of D-tubocurarine or calphostin C, suggesting that neither neurotransmitter release nor diacylglycerol-sensitive protein kinase C mediates the contraction-induced activation of this signaling cascade. However, PD-98059, an inhibitor of MAP kinase kinase (MEK), inhibited the contraction-induced increases in MAP kinase phosphorylation. PD-98059 did not alter contraction-induced increases in glucose uptake or glycogen synthase activity, demonstrating that MAP kinase signaling is not necessary for these important metabolic effects of contractile activity in skeletal muscle. These data suggest that contractile activity of the skeletal muscle fibers per se, and not responses to neurotransmitter release, hormones, or other systemic factors, is responsible for the stimulation of MAP kinase signaling with physical exercise.
We used muscle contraction, which increases fatty acid oxidation, as a model to determine whether fatty acid transport is
acutely regulated by fatty acid translocase (FAT/CD36). Palmitate uptake by giant vesicles, obtained from skeletal muscle,
was increased by muscle contraction. Kinetic studies indicated that muscle contraction increased V
max, butKm
remained unaltered. Sulfo-N-succinimidyl oleate, a specific inhibitor of FAT/CD36, fully blocked the contraction-induced increase in palmitate uptake.
In giant vesicles from contracting muscles, plasma membrane FAT/CD36 was also increased in parallel with the increase in long
chain fatty acid uptake. Further studies showed that like GLUT-4, FAT/CD36 is located in both the plasma membrane and intracellularly
(endosomally). With muscle contraction, FAT/CD36 at the surface of the muscle was increased, while concomitantly, FAT/CD36
in the intracellular pool was reduced. Similar responses were observed for GLUT-4. We conclude that fatty acid uptake is subject
to short term regulation by muscle contraction and involves the translocation of FAT/CD36 from intracellular stores to the
sarcolemma, analogous to the regulation of glucose uptake by GLUT-4.
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.
Altered muscle fatty acid (FA) metabolism may contribute to the presence of muscle insulin resistance in the genetically obese Zucker rat. To determine whether FA uptake and disposal are altered in insulin-resistant muscle, we measured palmitate uptake, oxidation, and incorporation into di- and triglycerides in isolated rat hindquarters, as well as muscle plasma membrane fatty acid-binding protein (FABP(PM)) content of lean (n = 16, fa/+) and obese (n = 15, fa/fa) Zucker rats (12 weeks of age). Hindquarters were perfused with 7 mmol/l glucose, 1,000 micromol/l albumin-bound palmitate, and albumin-bound [1-(14)C]palmitate at rest (no insulin). Glucose uptake was 42% lower in the obese than in the lean rats and indicated the presence of muscle insulin resistance. Fractional and total rates of palmitate uptake were 42 and 74% higher in the obese than in the lean rats and were associated with higher muscle FABP(PM) content (r(2) = 0.69, P < 0.05). The percentage of palmitate oxidized was not significantly different between groups. FA disposal to storage was altered according to fiber type. When compared with lean rats, the rate of triglyceride synthesis in red muscle was 158% higher in obese rats, and the rate of palmitate incorporation into diglycerides in white muscle was 93% higher in obese rats. Pre- and postperfusion muscle triglyceride levels were higher in both red and white muscles of the obese rats. These results show that increased FA uptake and altered FA disposal to storage may contribute to the development of muscle insulin resistance in obese Zucker rats.
Eukaryotic cells possess systems for sensing nutritional stress and inducing compensatory mechanisms that minimize the consumption of ATP while utilizing alternative energy sources. Such stress can also be imposed by increased energy needs, such as in skeletal muscle of exercising animals. In these studies, we consider the role of the metabolic sensor, AMP-activated protein kinase (AMPK), in the regulation of glucose transport in skeletal muscle. Expression in mouse muscle of a dominant inhibitory mutant of AMPK completely blocked the ability of hypoxia or AICAR to activate hexose uptake, while only partially reducing contraction-stimulated hexose uptake. These data indicate that AMPK transmits a portion of the signal by which muscle contraction increases glucose uptake, but other AMPK-independent pathways also contribute to the response.
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.
Exercise has numerous growth and metabolic effects in skeletal muscle, including changes in glycogen metabolism, glucose and amino acid uptake, protein synthesis and gene transcription. However, the mechanism(s) by which exercise regulates intracellular signal transduction to the transcriptional machinery in the nucleus, thus modulating gene expression, is largely unknown. This review will provide insight on potential intracellular signalling mechanisms by which muscle contraction/exercise leads to changes in gene expression. Mitogen-activated protein kinase (MAPK) cascades are associated with increased transcriptional activity. The MAPK family members can be separated into distinct parallel pathways including the extracellular signal-regulated kinase (ERK) 1/2, the stress-activated protein kinase cascades (SAPK1/JNK and SAPK2/p38) and the extracellular signal-regulated kinase 5 (ERK5). Acute exercise elicits signal transduction via MAPK cascades in direct response to muscle contraction. Thus, MAPK pathways appear to be potential physiological mechanisms involved in the exercise-induced regulation of gene expression in skeletal muscle.
To determine the effects of brief food restriction on fatty acid (FA) metabolism, hindlimbs of F344/BN rats fed either ad libitum (AL) or food restricted (FR) to 60% of baseline food intake for 28 days were perfused under hyperglycemic-hyperinsulinemic conditions (20 mM glucose, 1 mM palmitate, 1,000 microU/ml insulin, [3-(3)H]glucose, and [1-(14)C]palmitate). Basal glucose and insulin levels were significantly lower (P < 0.05) in FR vs. AL rats. Palmitate uptake (34.3 +/- 2.7 vs. 24.5 +/- 3.1 nmol/g/min) and oxidation (3.8 +/- 0.2 vs. 2.7 +/- 0.3 nmol.g(-1).min(-1)) were significantly higher (P < 0.05) in FR vs. AL rats, respectively. Glucose uptake was increased in FR rats and was accompanied by significant increases in red and white gastrocnemius glycogen synthesis, indicating an improvement in insulin sensitivity. Although muscle triglyceride (TG) levels were not significantly different between groups, glucose uptake and total preperfusion TG concentration were negatively correlated (r(2) = 0.27, P < 0.05). In conclusion, our results show that under hyperglycemic-hyperinsulinemic conditions, brief FR resulted in an increase in FA oxidative disposal that may contribute to the improvement in insulin sensitivity.
The purpose of this investigation was to examine the activation (phosphorylation) and total protein content of MAPK signalling cascade proteins (ERK 1/2, p90RSK, Mnk 1, eIF4E, p38 MAPK, JNK/SAPK, MKP 1) at rest and following exercise, in sedentary young and old men. Eight young (22 +/- 1 years; YM) and eight old (79 +/- 3 years; OM) men underwent a resting muscle biopsy of the vastus lateralis; they then performed a knee extensor resistance exercise session (29 contractions at approximately 70 % of max), followed by a post-exercise biopsy. Western immunoblot analysis demonstrated that the OM had higher resting phosphorylation of ERK 1/2, p90RSK, Mnk 1, p38 MAPK and JNK/SAPK proteins versus YM (P < 0.05). The resistance exercise bout caused an increase in phosphorylation of the ERK 1/2, p90RSK and Mnk 1 proteins (P < 0.05) in the YM. Conversely, the OM had a decrease in ERK 1/2, p90RSK, Mnk 1, p38 MAPK and JNK/SAPK phosphorylation (P < 0.05) after the exercise bout. Neither group showed a change in eIF4E phosphorylation. The total amount of protein expression of the MAPK signalling proteins was not different between the YM and OM, except that there was a higher (P < 0.05) MKP 1 protein content in the OM. This investigation is the first to provide evidence that MAPK proteins are differentially activated at rest and in response to a bout of resistance exercise in skeletal muscle of young and old men. These findings may have implications for other processes (e.g. transcription and translation) involved in skeletal muscle type and growth, when examining the changes occurring with ageing muscle before and after resistance exercise/training.
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.
It has been assumed that the uptake of long chain fatty acids (LCFAs) into skeletal muscle and the heart muscle, as well as other tissues, occurred via passive diffusion. In recent years our work has shown that the LCFA uptake into skeletal muscle is a highly regulated process. The use of giant sarcolemmal vesicles obtained from skeletal muscle and heart has been used to demonstrate that LCFA uptake into these tissues occurs via a protein-mediated mechanism involving the 40 kDa plasma membrane associated fatty acid binding protein (FABPpm) and the 88 kDa fatty acid translocase, the homologue of human CD36 (FAT/CD36). Both are ubiquitously expressed proteins and correlate with LCFA uptake into heart and muscle, consistent with the known differences in LCFA metabolism in these tissues. It has recently been found that FAT/CD36 is present in an intracellular (endosomal) compartment from which it can be translocated to the plasma membrane within minutes by muscle contraction and by insulin, to stimulate LCFA uptake. In rodent models of obesity and type 1 diabetes LCFA uptake into heart and muscle is also increased, either by permanently relocating FAT/CD36 to the plasma membrane without altering its expression (obesity) or by increasing the expression of both FAT/CD36 and FABPpm (type 1 diabetes). Chronic leptin treatment decreases LCFA transporters and transport in muscle. Clearly, recent evidence has established that LCFA uptake into heart and muscle is regulated acutely and chronically.
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.
Increased palmitate transport and oxidation in skeletal muscles of transgenic mice that overexpress fatty acid translocase (FAT/CD36)
- Bonen A.