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Evidence for the involvement of CaMKII and AMPK in Ca2+-dependent signaling pathways regulating FA uptake and oxidation in contracting rodent muscle
Abstract
Calcium-calmodulin/dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK1/2) have each been implicated in the regulation of substrate metabolism during exercise. The purpose of this study was to determine whether CaMKII is involved in the regulation of FA uptake and oxidation and, if it is involved, whether it does so independently of AMPK and ERK1/2. Rat hindquarters were perfused at rest with (n = 16) or without (n = 10) 3 mM caffeine, or during electrical stimulation (n = 14). For each condition, rats were subdivided and treated with 10 muM of either KN92 or KN93, inactive and active CaMKII inhibitors, respectively. Both caffeine treatment and electrical stimulation significantly increased FA uptake and oxidation. KN93 abolished caffeine-induced FA uptake, decreased contraction-induced FA uptake by 33%, and abolished both caffeine- and contraction-induced FA oxidation (P < 0.05). Caffeine had no effect on ERK1/2 phosphorylation (P > 0.05) and increased alpha(2)-AMPK activity by 68% (P < 0.05). Electrical stimulation increased ERK1/2 phosphorylation and alpha(2)-AMPK activity by 51% and 3.4-fold, respectively (P < 0.05). KN93 had no effect on caffeine-induced alpha(2)-AMPK activity, ERK1/2 phosphorylation, or contraction-induced ERK1/2 phosphorylation (P > 0.05). Alternatively, it decreased contraction-induced alpha(2)-AMPK activity by 51% (P < 0.05), suggesting that CaMKII lies upstream of AMPK. These results demonstrate that regulation of contraction-induced FA uptake and oxidation occurs in part via Ca(2+)-independent activation of ERK1/2 as well as Ca(2+)-dependent activation of CaMKII and AMPK.
... This study showed that the content of p-CaMKII was significantly higher in unloaded soleus muscle without metformin treatment than in all other groups. It is known that the activity of CaMKII is regulated by intracellular calcium concentration (52,53), the increase of which was previously reported upon muscle unloading (13,33). CaMKII regulates phosphorylation of numerous proteins, including AMPK (53)(54)(55) and the transcription factors (56). ...
... It is known that the activity of CaMKII is regulated by intracellular calcium concentration (52,53), the increase of which was previously reported upon muscle unloading (13,33). CaMKII regulates phosphorylation of numerous proteins, including AMPK (53)(54)(55) and the transcription factors (56). ...
... In the current study, treatment with metformin during unloading significantly decreased the content of phosphorylated CaMKII in soleus muscle when compared with the unloaded muscle without treatment. CaMK can phosphorylate AMPK, PLN, and SLP AQ: 16 (53)(54)(55). However, AMPK has allosteric regulation feature that requires binding of AMP to the second site for the increased AMPK phosphorylation (52). ...
Current study tested a hypothesis that during skeletal muscle unloading, calcium-dependent signaling pathways, markers of protein synthesis and expression of E3 ubiquitin ligases can be regulated by metformin. Thirty-two male Wistar rats were randomly assigned into one of four groups: non-treated control (3C), control rats treated with metformin (3CM), 3 days of unloading/hindlimb suspension with placebo (3HS), and 3 days of unloading treated with metformin (3HSM). In soleus muscle of HS group level of phospho-AMPK (p-AMPK) was decreased by 46% while ATP content was increased by 49% when compared with the control group. There was an increase of the level of phospho-CaMK II (483%) and an up-regulation of CaN, SERCA2a, and Calpain-1 mRNA expression (87%, 41% and 62%, respectively, p<0.05) in the HS group relative to the control. HS group also had increased mRNA expression of MuRF1, MAFbx, and ubiquitin (167%, 146% and 191%, respectively, p<0.05) when compared to the control soleus muscle. Metformin treatment impeded unloading-induced changes in soleus muscle. Conclusions: metformin treatment during three days of soleus muscle unloading: 1) prevented the decrease of p-AMPK and increase of ATP content; 2) affected regulation of calcium-dependent signaling pathways via level of CaMK II phosphorylation or CaMK II, CaN, SERCA2a, and Calpain-1 mRNA expression; 3) attenuated an increase in the expression of critical markers of ubiquitin-proteasome pathways MuRF1, MAFbx, and ubiquitin while not affecting the unloading-induced increase of ULK-1 marker of autophagic/lysosomal pathway.
... Because activated AMPK restricts HBV replication via autophagic degradation [13], and CaMKII is an upstream regulator of AMPK [48,49], the level of phosphorylated AMPK was also determined ( Figure 2, panel 3, lanes 3, 4, 9-12, 15-20, 23 and 24). Similar to CaMKII phosphorylation, the level of active, phosphorylated AMPK at T172 was lower in seven biopsy specimens from patients Nos. 19, 10, 13, 20, 21, 26 and 34, indicating that HBV-associated HCCs tend to inhibit the activation of CaMKII and AMPK. ...
... Since AMPK seems to act downstream of CaMKII ( Figure 4C,D) [48,49], the effect of AMPK overexpression was analyzed in HBV replicating cells. Similar to overexpression of CaMKII α in HBV replicating cells (Figure 4), AMPK α1 overexpression reduced HBV DNA synthesis in transiently co-transfected HepG2 and Huh7 cells ( Figure 5A Figure S3C). ...
... 2mM metformin was not cytotoxic to Huh7 cell ( Figure S4C, middle panel). Although CaMKII acts upstream of AMPK ( Figure 4C,D) [48,49], CaMKII phosphorylation was also increased by metformin ( Figure 7A, top panel, lane 2 vs. 3). Because AMPK activation induces CaMKII activation ( Figure 7A), we hypothesized that CaMKII and AMKP may form a feedback loop, thereby affecting the AKT/mTOR signaling pathway during HBV replication. ...
Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), which is involved in the calcium signaling pathway, is an important regulator of cancer cell proliferation, motility, growth, and metastasis. The effects of CaMKII on hepatitis B virus (HBV) replication have never been evaluated. Here, we found that phosphorylated, active CaMKII is reduced during HBV replication. Similar to other members of the AMPK/AKT/mTOR signaling pathway associated with HBV replication, CaMKII, which is associated with this pathway, was found to be a novel regulator of HBV replication. Overexpression of CaMKII reduced the expression of covalently closed circular DNA (cccDNA), HBV RNAs, and replicative intermediate (RI) DNAs while activating AMPK and inhibiting the AKT/mTOR signaling pathway. Findings in HBx-deficient mutant-transfected HepG2 cells showed that the CaMKII-mediated AMPK/AKT/mTOR signaling pathway was independent of HBx. Moreover, AMPK overexpression reduced HBV cccDNA, RNAs, and RI DNAs through CaMKII activation. Although AMPK acts downstream of CaMKII, AMPK overexpression altered CaMKII phosphorylation, suggesting that CaMKII and AMPK form a positive feedback loop. These results demonstrate that HBV replication suppresses CaMKII activity, and that CaMKII upregulation suppresses HBV replication from cccDNA via AMPK and the AKT/mTOR signaling pathway. Thus, activation or overexpression of CaMKII may be a new therapeutic target against HBV infection.
... The Ca 2+ released by the sarcoplasmic reticulum (SR) is a prerequisite for muscle contraction and may be a signal to the muscle to capture more blood glucose [6,7], Two parallel tracks stimulate glucose uptake, AMPK, and Ca 2+ / calmodulin-dependent protein kinase II (CaMKII). Both, AMPK and CaMKII, are necessary and sufficient to keep the amount of muscle glucose uptake to provide enough energy for muscle contraction [8][9][10]. Caffeine may stimulate Ca 2+ SR [9][10][11] release, increasing the phosphorylation of AMPK and activating CaMKII, promoting Glut4 translocation, and triggering a higher glucose uptake. ...
... Both, AMPK and CaMKII, are necessary and sufficient to keep the amount of muscle glucose uptake to provide enough energy for muscle contraction [8][9][10]. Caffeine may stimulate Ca 2+ SR [9][10][11] release, increasing the phosphorylation of AMPK and activating CaMKII, promoting Glut4 translocation, and triggering a higher glucose uptake. ...
... In addition to glucose, fatty acids are essential for cellular energy maintenance, and glycerol is related to fatty acids mobilization, from the process of lipolysis until the consequent increase of glycerol [10]. The Ca 2+ released from the SR into physical stimuli results in an increase in fatty acid uptake through increased AMPK phosphorylation, and consequently increases kinases proteins regulated by extracellular signals 1 and 2 (ERK1/2), activated by CaMKII [9][10][11]. ...
Objective
The objective of this study was to evaluate caffeine effects on cardiovascular and biochemical parameters during the recovering time of aerobic exercise in diabetic rats.
Materials and methods
24 animals of 60 days were allocated to four experimental groups: Control, Diabetic, Caffeine, and Diabetes + Caffeine. Diabetes mellitus was induced by an intraperitoneal administration of 60 mg/kg of alloxan. Once a day for 30 days, animals underwent training of forced swimming for 40 min and tied to loads of 4% of their body weight. On the 1st and 30th days of training, animals underwent a stress test, in which they performed 60 min of forced swimming with loads of 6% session of 6% of their body’s weight. Caffeine was administrated 30 min before the exercise at the test days. Cardiovascular responses such as systolic blood pressure (SBP), heart rate (HR), and double product (DP) were recorded before and after the tests. In addition, blood samples were collected for glucose, glycerol, and lactate analyses, by caudal puncture.
Results
Glucose values were lower in rats of group caffeine + diabetes when compared to animals of other groups (25%; p < 0.05). After caffeine intake, the values of HR, SBP, and DP of diabetic groups increased when compared to control groups (32%).
Conclusion
The present work demonstrated that caffeine intake associated with aerobic exercise might control glucose levels in healthy and diabetic rats. Caffeine was able to reduce glycemic values, regulate cardiovascular responses, and maintain nutrients’ availability during exercise, which may improve overall fitness and tissues’ adaptation to better function.
... During unloading, intracellular Ca 2+ concentration increases in both the myoplasm and the cell nucleus [2,33]. CaMKII activity is regulated by intracellular Ca 2+ [44,45]. In the present study, the phosphorylation of CaMKIIb was increased in the unloaded soleus muscle (3HS group) relative to the control group, but the administration of the PI3K inhibitor LY294002 prevented these changes. ...
... In the present study, the phosphorylation of CaMKIIb was increased in the unloaded soleus muscle (3HS group) relative to the control group, but the administration of the PI3K inhibitor LY294002 prevented these changes. Phosphorylation of several proteins is regulated by CaMKII, including AMPK [45,46] and several transcription factors [47]. ...
During skeletal muscle unloading, phosphoinositide 3-kinase (PI3K), and especially PI3K gamma (PI3Kγ), can be activated by changes in membrane potential. Activated IP3 can increase the ability of Ca²⁺ to enter the nucleus through IP3 receptors. This may contribute to the activation of transcription factors that initiate muscle atrophy processes. LY294002 inhibitor was used to study the role of PI3K in the ATP-dependent regulation of skeletal muscle signaling during three days of unloading. Inhibition of PI3K during soleus muscle unloading slows down the atrophic processes and prevents the accumulation of ATP and the expression of the E3 ubiquitin ligase MuRF1 and ubiquitin. It also prevents the increase in the expression of IP3 receptors and regulates the activity of Ca²⁺-dependent signaling pathways by reducing the mRNA expression of the Ca²⁺-dependent marker calcineurin (CaN) and decreasing the phosphorylation of CaMKII. It also affects the regulation of markers of anabolic signaling in unloaded muscles: IRS1 and 4E-BP. PI3K is an important mediator of skeletal muscle atrophy during unloading. Developing strategies for the localized skeletal muscle release of PI3K inhibitors might be one of the future treatments for inactivity and disease-induced muscle atrophy.
... Similar to the current study, the increased phosphorylation of CaMK IIβ in skeletal muscle after unloading was reported previously [39]. Intracellular calcium is one of the regulators of CaMKII activity [52,53]. During unloading, the concentration of intracellular calcium is increased [7,37,54]. ...
... During unloading, the concentration of intracellular calcium is increased [7,37,54]. CaMKII regulates the phosphorylation of several proteins, including AMPK [53,55,56] and some transcription factors [57]. Increased calcium concentrations induce autophosphorylation of CaMK IIβ at Thr 287 [58]. ...
Skeletal muscle abnormalities and atrophy during unloading are accompanied by the accumulation of excess calcium in the sarcoplasm. We hypothesized that calcium accumulation may occur, among other mechanisms, due to the inhibition of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity. Consequently, the use of the SERCA activator will reduce the level of calcium in the sarcoplasm and prevent the negative consequences of muscle unloading. Wistar rats were randomly assigned into one of three groups (eight rats per group): control rats with placebo (C), 7 days of unloading/hindlimb suspension with placebo (7HS), and 7 days of unloading treated with SERCA activator CDN1163 (7HSC). After seven days of unloading the soleus muscle, the 7HS group displayed increased fatigue in the ex vivo test, a significant increase in the level of calcium-dependent CaMK II phosphorylation and the level of tropomyosin oxidation, as well as a decrease in the content of mitochondrial DNA and protein, slow-type myosin mRNA, and the percentage of slow-type muscle fibers. All of these changes were prevented in the 7HSC group. Moreover, treatment with CDN1163 blocked a decrease in the phosphorylation of p70S6k, an increase in eEF2 phosphorylation, and an increase in MuRF-1 mRNA expression. Nevertheless, there were no differences in the degree of fast and slow muscle fiber atrophy between the 7HS and 7HSC groups. Conclusion: SERCA activation during 7 days of unloading prevented an increase in soleus fatigue, the decrease of slow-type myosin, mitochondrial markers, and markers of calcium homeostasis but had no effect on muscle atrophy.
... reported to regulate the activity of AMP-activated protein kinase (AMPK) (Raney & Turcotte, 2008), whereas CaMKII was suggested to regulate AMPK activity in the heart (Meng et al., 2022). In both instances, activation of the CaMKII-AMPK pathway was shown to regulate fatty acid metabolism (Meng et al., 2022;Raney & Turcotte, 2008). ...
... reported to regulate the activity of AMP-activated protein kinase (AMPK) (Raney & Turcotte, 2008), whereas CaMKII was suggested to regulate AMPK activity in the heart (Meng et al., 2022). In both instances, activation of the CaMKII-AMPK pathway was shown to regulate fatty acid metabolism (Meng et al., 2022;Raney & Turcotte, 2008). Indeed, AMPK serves as a master metabolic switch, which upon activation promotes catabolic pathways and ATP generation (Ronnett et al., 2009). ...
Ca²⁺/calmodulin‐dependent protein kinase II alpha (CaMKIIα) is a key regulator of neuronal signaling and synaptic plasticity. Synaptic activity and neurotransmitter homeostasis are closely coupled to the energy metabolism of both neurons and astrocytes. However, whether CaMKIIα function is implicated in brain energy and neurotransmitter metabolism remains unclear. Here, we explored the metabolic consequences of CaMKIIα deletion in the cerebral cortex using a genetic CaMKIIα knockout (KO) mouse. Energy and neurotransmitter metabolism was functionally investigated in acutely isolated cerebral cortical slices using stable ¹³C isotope tracing, whereas the metabolic function of synaptosomes was assessed by the rates of glycolytic activity and mitochondrial respiration. The oxidative metabolism of [U‐¹³C]glucose was extensively reduced in cerebral cortical slices of the CaMKIIα KO mice. In contrast, metabolism of [1,2‐¹³C]acetate, primarily reflecting astrocyte metabolism, was unaffected. Cellular uptake, and subsequent metabolism, of [U‐¹³C]glutamate was decreased in cerebral cortical slices of CaMKIIα KO mice, whereas uptake and metabolism of [U‐¹³C]GABA were unaffected, suggesting selective metabolic impairments of the excitatory system. Synaptic metabolic function was maintained during resting conditions in isolated synaptosomes from CaMKIIα KO mice, but both the glycolytic and mitochondrial capacities became insufficient when the synaptosomes were metabolically challenged. Collectively, this study shows that global deletion of CaMKIIα significantly impairs cellular energy and neurotransmitter metabolism, particularly of neurons, suggesting a metabolic role of CaMKIIα signaling in the brain. image
... Caffeine is a powerful metabolic stimulant in the skeletal muscle. In vitro caffeine treatment of the skeletal muscle promotes insulin-independent glucose transport [4][5][6][7][8], fatty acid oxidation [8,9], Ca 2+ release from the sarcoplasmic reticulum [10,11], and mitochondrial biogenesis [12]. We have recently demonstrated that caffeine increases the maximal capacity of contraction-stimulated AMPK activation and glucose transport in rat skeletal muscles [6]. ...
... Caffeine is a powerful metabolic stimulant in the skeletal muscle. In vitro caffeine treatment of the skeletal muscle promotes insulin-independent glucose transport [4][5][6][7][8], fatty acid oxidation [8,9], Ca 2+ release from the sarcoplasmic reticulum [10,11], and mitochondrial biogenesis [12]. We have recently demonstrated that caffeine increases the maximal capacity of contraction-stimulated AMPK activation and glucose transport in rat skeletal muscles [6]. ...
Exercise has beneficial effects on our health by stimulating metabolic activation of skeletal muscle contraction. Caffeine is a powerful metabolic stimulant in the skeletal muscle that has ergogenic effects, including enhanced muscle power output and endurance capacity. In the present study, we aim to characterize the metabolic signatures of contracting muscles with or without caffeine stimulation using liquid chromatography-mass spectrometry and capillary electrophoresis coupled to mass spectrometry. Isolated rat epitrochlearis muscle was incubated in the presence or absence or of 3 mM caffeine for 30 min. Electrical stimulation (ES) was used to induce tetanic contractions during the final 10 min of incubation. Principal component analysis and hierarchical clustering analysis detected 184 distinct metabolites across three experimental groups—basal, ES, and ES with caffeine (ES + C). Significance Analysis of Microarray identified a total of 50 metabolites with significant changes in expression, and 23 metabolites significantly changed between the ES and ES + C groups. Changes were observed in metabolite levels of various metabolic pathways, including the pentose phosphate, nucleotide synthesis, β-oxidation, tricarboxylic acid cycle, and amino acid metabolism. In particular, D-ribose 5-phosphate, IMP, O-acetylcarnitine, butyrylcarnitine, L-leucine, L-valine, and L-aspartate levels were higher in the ES + C group than in the ES group. These metabolic alterations induced by caffeine suggest that caffeine accelerates contraction-induced metabolic activations, thereby contributing to muscle endurance performance and exercise benefits to our health.
... Indeed, several reports suggest that CaMKⅡ acts upstream of AMPK (20,21) . As there have been few reports of compounds that activate AMPK via LKB1-and CaMKKβ-independent pathways, 8-PN may have a unique function in this regard. ...
8-Prenylnaringenin (8-PN) is a prenylflavonoid found in hops (Humulus lupulus L.). It has several beneficial functions, which include the inhibition of bone loss and muscle atrophy. 8-PN is a metabolite of xanthohumol, which can prevent obesity in mice; however, the effect of 8-PN on obesity is still unknown. In the present study, we found that 8-PN prevented obesity in high-fat diet-fed mice. When C57BL6/J male mice were fed 8-PN at 0.0005% or 0.005% with a high-fat diet for 8 weeks, body weight gain, fat accumulation in adipose tissue, and fatty liver induced by the high-fat diet were prevented. In mice fed a high-fat diet and 8-PN, adenosine monophosphate-activated protein kinase (AMPK) was activated in visceral adipose tissue, which was accompanied by decreased expression of a fatty acid synthesis-related factor and increased expression of a mitochondrial biosynthesis-related factor downstream of AMPK. AMPK appeared to be activated by adiponectin secretion, which was associated with increased expression of adipocyte differentiation markers in mice fed a high-fat diet and 8-PN. For the first time, this study shows that 8-PN can prevent obesity in mice and that it is effective at low concentrations that humans could consume in their daily diet.
... Gao Yongfang et al. (Gao et al., 2019) found that Ca 2+ significantly increased the activities of CaMKKβ and AMPK during beef maturation, and at the same time increased the phosphorylation level of AMPK. Raney et al. (Raney & Turcotte, 2008) demonstrated that Ca 2+ dependent activation activates AMPK through CaMKKβ, while caffeine-induced activation of AMPK is a result of elevated intracellular Ca 2+ levels. Increased Ca 2+ in cytoplasm is the primary cause of activation of CaMKKβ, which phosphorylates Thr-172 on the AMPKα subunit (Hurley et al., 2005) . ...
This study investigated the role of NO in regulating AMPK activity and beef tenderness during post-mortem maturation of bovine gluteus quadriceps. The study included treatments with L-NAME (NOS inhibitor), ODQ (sGC inhibitor), Compound C (AMPK inhibitor), and a saline control. Results showed a decline in NOS activity, NO, and cGMP levels over time (P < 0.05). The L-NAME group had lower NOS activity and NO levels compared to the control (P < 0.05). AMPK activity peaked at 6 h and then decreased, with the control group showing higher AMPK activity than the L-NAME and ODQ groups (P < 0.05). The shear force in the L-NAME group was higher than the control but lower than the Compound C group at 48 h (P < 0.05). Overall, NO was found to activate AMPK, leading to reduced shear force and improved beef tenderness, providing a basis for enhancing beef quality through endogenous factors.
... This finding is consistent with previous studies indicating that Ca2+ activation can counteract muscle atrophy [18], that Ca2+ inhibition can decrease muscle strength [19] and that lonafarnib activates the CaMKII signalling pathway in the hippocampus and induces functional recovery [20]. AMPK lies downstream of CaMKII in muscle [21], and CaMKII plays an important role in activating mitochondrial biogenesis [22]. ...
Background
Muscle atrophy, including glucocorticoid‐induced muscle wasting from treatments such as dexamethasone (DEX), results in significant reductions in muscle mass, strength and function. This study investigates the potential of lonafarnib, a farnesyltransferase inhibitor, to counteract DEX‐induced muscle atrophy by targeting key signalling pathways.
Methods
We utilized in vitro models with C2C12 myotubes treated with DEX and in vivo models with Caenorhabditis elegans and DEX‐treated Sprague–Dawley rats. Myotube morphology was assessed by measuring area, fusion index and diameter. Muscle function was evaluated by grip strength and compound muscle action potential (CMAP) in the gastrocnemius (GC) and tibialis anterior (TA) muscles. Molecular mechanisms were explored through RNA sequencing and Western blotting to assess changes in mitochondrial function and muscle signalling pathways.
Results
Lonafarnib (2 μM) significantly improved myotube area (1.49 ± 0.14 × 10⁵ μm² vs. 1.03 ± 0.49 × 10⁵ μm² in DEX, p < 0.05), fusion index (18.73 ± 1.23% vs. 13.3 ± 1.56% in DEX, p < 0.05) and myotube diameter (31.89 ± 0.89 μm vs. 21.56 ± 1.01 μm in DEX, p < 0.05) in C2C12 myotubes. In C. elegans, lonafarnib (100 μM) increased the pharyngeal pumping rate from 212 ± 7.21 contractions/min in controls to 308 ± 17.09 contractions/min at day 4 (p < 0.05), indicating enhanced neuromuscular function. In DEX‐induced atrophic rats, lonafarnib improved maximal grip strength (DEX: 13.91 ± 0.78 N vs. 1 μM lonafarnib: 16.18 ± 0.84 N and 5 μM lonafarnib: 16.71 ± 0.83 N, p < 0.05), increased muscle weight in GC, and enhanced CMAP amplitudes in both GC and TA muscles. Western blot analysis showed that lonafarnib treatment upregulated UCP3 and ANGPTL4 and increased phosphorylation of mTOR and S6 ribosomal protein (p < 0.05), indicating enhanced mitochondrial function and protein synthesis. Knockdown models further demonstrated that lonafarnib could partially rescue muscle atrophy phenotypes, indicating its action through multiple molecular pathways.
Conclusions
Lonafarnib mitigates dexamethasone‐induced muscle atrophy by enhancing mitochondrial function and activating anabolic pathways. These findings support further investigation of lonafarnib as a therapeutic agent for muscle atrophy in clinical settings.
... In the total protein fraction, we determined the level of CaMKIIb phos phorylation at Thr286. Intracellular Ca 2+ is one of the regulators of CaMKIIb activity [49,50], and the amino acid residue Thr286 of a CaMKIIb molecule is phosphorylated when Ca 2+ is elevated [46,47]. CaMKIIb phosphorylation was increased in the unloaded soleus muscle (HS group) compared to the control group (Fig. 6), but the administration of an IP3R inhibitor prevented this elevation. ...
... Contractions of skeletal muscles cause the Ca 2+ /calmodulindependent protein kinases to become active (CAMK). In particular, CAMKII, the main CAMK isoform, is phosphorylated (activated) by endurance training, while CAMKK is in control of muscle tissue contraction-induced activation of AMPK [21,22]. Because exercise regulates p38 MAPK and AMPK activation, respectively (see below), CAMKII and CAMKK may operate as upstream kinases in the control of PGC-1. ...
The purpose of this review is to describe the impact of endurance and strength physical training on the cardiovascular system by reviewing the molecular signaling pathways, which plays a key role in different muscle adaptations, and the cardiac changes in terms of metabolic and cardiac remodeling, and hemodynamics. In response to endurance-exercise, multiple signaling pathways, including Ca 2+-dependent pathways, reactive oxygen species (ROS), AMP-dependent protein kinase (AMPK), and mitogen activated protein kinases (p38 MAPK), are involved in the regulation of peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α), which controls the mitochondrial biogenesis. Strength training increases the insulin-like growth factor (IGF-1) which initiates the phosphatidylinositol 3-kinase (PI3-k)-(AKT)-(mTOR) signaling cascade, resulting in the synthesis of proteins and the muscle hypertrophy. In addition to the well-documented changes in skeletal muscle, a critical component of the response to exercise training is the dynamic cardiac remodeling, which is classified as either pathological or physiological depending on triggers.
... CaMKII can regulate the phosphorylation of a large number of different proteins, including AMPK [46][47][48]. In both P2Y1 and P2Y2 receptor inhibitor-treated groups the content of phosphorylated CaMKII was lower than in the unloaded group without treatment and was comparable to the p-CaMKII content in the control. ...
The current study aimed to investigate the hypothesis that purinergic receptors P2Y1 and P2Y2 play a regulatory role in gene expression in unloaded muscle. ATP is released from cells through pannexin channels, and it interacts with P2Y1 and P2Y2 receptors, leading to the activation of markers of protein catabolism and a reduction in protein synthesis. To test this hypothesis thirty-two rats were randomly divided into four groups (8 per group): a non-treated control group (C), a group subjected to three days of hindlimb unloading with a placebo (HS), a group subjected to three days of hindlimb unloading treated with a P2Y1 receptor inhibitor, MRS2179 (HSM), and a group subjected to three days of hindlimb unloading treated with a P2Y2 receptor inhibitor, AR-C 118925XX (HSA). This study revealed several key findings following three days of soleus muscle unloading:
1
Inhibition of P2Y1 or P2Y2 receptors prevented the accumulation of ATP, the increase in IP3 receptor content, and the decrease in the phosphorylation of GSK-3beta. This inhibition also mitigated the reduction in the rate of protein synthesis. However, it had no significant effect on the markers of mTORC1-dependent signaling.
2
Blocking P2Y1 receptors prevented the unloading-induced upregulation of phosphorylated p38MAPK and partially reduced the increase in MuRF1mRNA expression.
3
Blocking P2Y2 receptors prevented muscle atrophy during unloading, partially maintained the levels of phosphorylated ERK1/2, reduced the increase in mRNA expression of MAFbx, ubiquitin, and IL-6 receptor, prevented the decrease in phosphorylated AMPK, and attenuated the increase in phosphorylated p70S6K.
Taken together, these results suggest that the prevention of muscle atrophy during unloading, as achieved by the P2Y2 receptor inhibitor, is likely mediated through a reduction in catabolic processes and maintenance of energy homeostasis. In contrast, the P2Y1 receptor appears to play a relatively minor role in muscle atrophy during unloading.
... AMPK activation has been shown to inhibit the progression of aging via FoxO, mTOR, CREB, and sirtuin (SIRT) 1 signaling pathways [144]. AMPK activation has been correlated with increased levels of cAMP via Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) [145,146]. Because the increase in cAMP in muscles primarily depends on A2BR, an A2BR agonist could be a good therapeutic agent to counteract muscle loss in aging [115]. ...
The purinergic system has a dual role: the maintenance of energy balance and signaling within cells. Adenosine and adenosine triphosphate (ATP) are essential for maintaining these functions. Sarcopenia is characterized by alterations in the control of energy and signaling in favor of catabolic pathways. This review details the association between the purinergic system and muscle and adipose tissue homeostasis, discussing recent findings in the involvement of purinergic receptors in muscle wasting and advances in the use of the purinergic system as a novel therapeutic target in the management of sarcopenia.
... That said, the roles of CaMKs in skeletal muscle glucose transport, lipid uptake, oxidation, and plasticity, e.g., mitochondrial biogenesis, have typically been the focus of mechanistic studies using a combination of Ca 21 -releasing agents and CaMK inhibitors (533,534,536,568,662,(670)(671)(672)(673)(674)(675). The exact mechanism by which CaMKs regulate skeletal muscle gene expression EGAN AND SHARPLES 2092 ...
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.
... In recent years, many studies have found that TRPs are related to metabolism and thermogenesis in animals, and agonist or temperature activation of TRPV1, TRPV2, TRPM8 and TRPA1 can enhance metabolism, thermogenesis and browning, while the activation of TRPV4 has the opposite effect (Baboota et al., 2014;Sun et al., 2016;Ye et al., 2012;Siemens and Kamm, 2018). Silent Information Regulator T1 (SIRT1), a cellular metabolism and energy sensor, can be activated by AMP-activated protein kinase (AMPK), and calcium/ calmodulin-dependent protein kinase II (CaMKII)-AMPK signaling is thought to be involved in intracellular metabolism and fatty acid oxidation (Raney and Turcotte, 2008;Iwabu et al., 2010;Peng et al., 2010). Thus, intracellular Ca 2+ -activated CaMKII-AMPK-SIRT1 signaling pathway is essential for the study of BAT thermogenesis. ...
Transient receptor potential (TRP) channels, which can sense temperature, pressure and mechanical stimuli, were involved in many physiological and biochemical reactions. Whether thermosensitive TRP channels (Thermo-TRPs) are involved in thermoregulation in small mammals is still not clear. We measured the changes of thermo-TRPs at 4 °C, 23 °C and 30 °C in Brandt's voles (Lasiopodomys brandtii) to test the hypothesis that Thermo-TRPs are involved in cold-induced thermogenesis of brown adipose tissue (BAT) in small mammals. Results showed that air temperatures had no effect on body mass and rectal temperature, but the food intake and basal metabolic rate (BMR) in the 4 °C group were significantly higher than in the 30 °C group. Compared with 30 °C group, the protein contents of uncoupling protein 1(UCP1), TRP vanilloid 2 (TRPV2), TRP ankyrin 1 (TRPA1), TRP melastatin 2 (TRPM2), silent Information Regulator T1 (SIRT1), AMP-activated protein kinase (AMPK) and Calcium/calmodulin-dependent protein kinase II (CaMKII) in BAT increased significantly in 4 °C group, but there was no significant difference in the protein content of Thermo-TRPs in the hypothalamus among groups. Further, the expression of PRDM16 (PR domain containing 16) in inguinal white adipose tissue (iWAT) at 4 °C was significantly higher than that at 30 °C, but no difference was observed in the expression of other browning-related genes or TRPV2. In conclusion, TRP channels may participate in BAT thermoregulation through the CaMKII, AMPK, SIRT1 and UCP1 pathway in cold-acclimated Brandt's voles.
... Sarcoplasmic calcium accumulation during unloading leads to various signaling changes, involving feedback inhibition. For example, calcium-calmodulin kinase (CaMKII) is regulated by the intracellular calcium concentration [44,45]. Calcium accumulation, leading to CaMKII activation, was previously reported upon muscle unloading [8,9]. ...
A decrease in skeletal muscle contractile activity or its complete cessation (muscle unloading or disuse) leads to muscle fibers’ atrophy and to alterations in muscle performance. These changes negatively affect the quality of life of people who, for one reason or another, are forced to face a limitation of physical activity. One of the key regulatory events leading to the muscle disuse-induced changes is an impairment of calcium homeostasis, which leads to the excessive accumulation of calcium ions in the sarcoplasm. This review aimed to analyze the triggering mechanisms of calcium homeostasis impairment (including those associated with the accumulation of high-energy phosphates) under various types of muscle unloading. Here we proposed a hypothesis about the regulatory mechanisms of SERCA and IP3 receptors activity during muscle unloading, and about the contribution of these mechanisms to the excessive calcium ion myoplasmic accumulation and gene transcription regulation via excitation–transcription coupling.
... AMPK is a heterotrimeric complex composed of a, b, and g subunits. AMPK can be activated by cAMPdependent kinase, calmodulin-dependent protein kinase [12], liver kinase B1 (LKB1), and Ca 2þ /calmodulin-dependent protein kinase II [13,14]. The substrates of AMPK are involved in energy metabolism and different cellular processes [15]. ...
Background
Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of trinucleotide CAG repeat in the Huntingtin (Htt) gene. The major pathogenic pathways underlying HD involve the impairment of cellular energy homeostasis and DNA damage in the brain. The protein kinase ataxia-telangiectasia mutated (ATM) is an important regulator of the DNA damage response. ATM is involved in the phosphorylation of AMP-activated protein kinase (AMPK), suggesting that AMPK plays a critical role in response to DNA damage. Herein, we demonstrated that expression of polyQ-expanded mutant Htt (mHtt) enhanced the phosphorylation of ATM. Ginsenoside is the main and most effective component of Panax ginseng. However, the protective effect of a ginsenoside (compound K, CK) in HD remains unclear and warrants further investigation.
Methods
This study used the R6/2 transgenic mouse model of HD and performed behavioral tests, survival rate, histological analyses, and immunoblot assays.
Results
The systematic administration of CK into R6/2 mice suppressed the activation of ATM/AMPK and reduced neuronal toxicity and mHTT aggregation. Most importantly, CK increased neuronal density and lifespan and improved motor dysfunction in R6/2 mice. Conversely, CK enhanced the expression of Bcl2 protected striatal cells from the toxicity induced by the overactivation of mHtt and AMPK.
Conclusions
Thus, the oral administration of CK reduced the disease progression and markedly enhanced lifespan in the transgenic mouse model (R6/2) of HD.
... (Wang et al., 2020) found that a diet supplemented with high-level DHA promoted muscle hyperplasia by activating the AMPK/Sirt1 pathway. Ca 2+ -dependent signaling pathway maintains the normal physiological function of skeletal muscle and muscle fiber type transformation (Raney and Turcotte, 2008). Calcineurin is a Ca 2+ /calmodulin-dependent serine/ threonine-protein phosphatase, when the action potential of muscle fibers appeared, the intracellular Ca 2+ content increased, combined with CAMK and then activated CAN, which promoted the gene expression of slow muscle fibers (McCullagh et al., 2004;Swoap et al., 2000). ...
The study was carried out to explore the effect of high lipid/carbohydrate diet on muscle growth of Megalobrama amblycephala. In vivo, three experimental diets, a control diet (6% lipid and 28.13% carbohydrate, CD), a high lipid diet (11%lipid，HLD) and a high carbohydrate diet (39.6% carbohydrate, HCD), were formulated and randomly fed to Megalobrama amblycephala (average initial weight 19.80 ± 0.15 g) for 12 weeks. The results showed that there was no significant difference in specific growth rate (SGR) and weight gain rate (WGR) between the control diet group and the high energy diets group (P > 0.05). The springiness, gumminess, chewiness and muscle fiber density were significantly up-regulated in the high energy diets group than control diet group (P < 0.05). HLD, compared with HCD, significantly increased the muscle fiber cross-sectional area (CSA) and muscle fiber diameter in the white muscle (P < 0.05). High energy diets significantly elevate the transcriptional levels of Myf5, Myog and Mrf4, and reduce the transcriptional levels of MSTNa and MSTNb (P < 0.05). The transcriptional levels and protein expressions of AMPK and Sirt1 were significantly elevated in the HLD group (P < 0.05). The CAMK and CAN protein levels were significantly increased in the HCD group (P < 0.05). In vitro, the isolated primary hepatocytes from Megalobrama amblycephala were coincubated with 0.4 mM oleic acid (OA) and 40 mM d-glucose respectively. The results clearly state that 0.4 mM OA significantly increased the transcriptional levels of AMPKα1, AMPKα2 and Sirt1, the protein level of AMPK, p-AMPK, p-AMPK/AMPK and Sirt1 (P < 0.05). The protein expression of CAMK and CAN in hepatocytes treated with 40 mM d-glucose were significantly higher than that in the other two treatments (P < 0.05). In summary, high energy diets promoted the muscle fiber hyperplasia in Megalobrama amblycephala while high lipid and high carbohydrate diets may affect muscle growth through AMPK/Sirt1 pathway and Ca²⁺-dependent pathway, respectively.
... Previous articles had reported that aconitine directly interacted with L-type Ca 2+ channel on myocardiocytes membrane and increased the containing of cytosolic Ca 2+ Frontiers in Pharmacology | www.frontiersin.org June 2021 | Volume 12 | Article 646121 (Zhou et al., 2017) triggering downstream CaMKII-AMPK signaling (Raney and Turcotte, 2008). Consistent with the previous studies, our study observed the activation of CaMKII-AMPK signaling in aconitine-treated NRVMs (Supplementary Figure S1). ...
Aconitine is attracting increasing attention for its unique positive inotropic effect on the cardiovascular system, but underlying molecular mechanisms are still not fully understood. The cardiotonic effect always requires abundant energy supplement, which is mainly related to mitochondrial function. And OPA1 has been documented to play a critical role in mitochondrial morphology and energy metabolism in cardiomyocytes. Hence, this study was designed to investigate the potential role of OPA1-mediated regulation of energy metabolism in the positive inotropic effect caused by repeated aconitine treatment and the possible mechanism involved. Our results showed that repeated treatment with low-doses (0–10 μM) of aconitine for 7 days did not induce detectable cytotoxicity and enhanced myocardial contraction in Neonatal Rat Ventricular Myocytes (NRVMs). Also, we first identified that no more than 5 μM of aconitine triggered an obvious perturbation of mitochondrial homeostasis in cardiomyocytes by accelerating mitochondrial fusion, biogenesis, and Parkin-mediated mitophagy, followed by the increase in mitochondrial function and the cellular ATP content, both of which were identified to be related to the upregulation of ATP synthase α-subunit (ATP5A1). Besides, with compound C (CC), an inhibitor of AMPK, could reverse aconitine-increased the content of phosphor-AMPK, OPA1, and ATP5A1, and the following mitochondrial function. In conclusion, this study first demonstrated that repeated aconitine treatment could cause the remodeling of mitochondrial function via the AMPK–OPA1–ATP5A1 pathway and provide a possible explanation for the energy metabolism associated with cardiotonic effect induced by medicinal plants containing aconitine.
... Combining Ca 2+ with CaM activates several CaM-binding proteins including CaM kinases (CaMKI, CaMKII, and CaMKIV) (26). Of these, CaMKII is associated with the activation of CREB and AMPK, which are important factors for the induction of PGC-1α, a central regulator for mitochondrial biogenesis (27)(28)(29). We tested the relationship between PAR2 and the signaling pathways associated with calcium-related mitochondrial biogenesis in preadipocytes. ...
Protease-activated receptor 2 (PAR2) is a member of G-protein-coupled receptors and affects ligand-modulated calcium signaling. Although PAR2 signaling promotes obesity and adipose tissue inflammation in high fat- (HF-) fed conditions, its role in adipocyte differentiation under nonobesogenic conditions needs to be elucidated. Here, we used several tissues and primary-cultured adipocytes of mice lacking PAR2 to study its role in the development of adipose tissues. C57BL/6J mice with PAR2 deficiency exhibited a mild lipodystrophy-like phenotype in a chow diet-fed condition. When adipocyte differentiation was examined using primary-cultured preadipocytes, PAR2 deficiency led to a notable decrease in adipocyte differentiation and related protein expression, and PAR2 agonist treatment elevated adipocyte differentiation. Regarding the mechanism, PAR2-deficient preadipocytes exhibited impaired mitochondrial energy consumption. Further studies indicated that calcium-related signaling pathways for mitochondrial biogenesis are disrupted in the adipose tissues of PAR2-deficient mice and PAR2-deficient preadipocytes. Also, a PAR2 antagonist elevated mitochondrial reactive oxygen species and reduced the MitoTracker fluorescent signal in preadipocytes. Our studies revealed that PAR2 is important for the development of adipose tissue under basal conditions through the regulation of mitochondrial biogenesis and adipocyte differentiation.
... The overexpression of MnSOD can promote mitochondrial H 2 O 2 production through alteration of the expression and function of electron transfer chain complexes, which leads to CaMKII activation [24,25]. CaMKII is a redox-sensitive kinase upstream of AMPK [26]. The phosphorylation of CaMKII will mediate the activation of AMPK [27]. ...
Background. Manganese superoxide dismutase (MnSOD) has been reported to promote stemness of lung cancer stem-like cells (LCSLCs) which had higher glycolytic rates compared with non-CSLCs. Isovitexin exhibited an inhibitory effect on the stemness of hepatocellular carcinoma cells. However, whether isovitexin could inhibit the promotion of stemness of LCSLCs mediated by MnSOD through glycolysis remains unclear. Objective. Our study was aimed at investigating whether isovitexin inhibits lung cancer stem-like cells (LCSLCs) through MnSOD signaling blockage and glycolysis suppression. Methods. Sphere formation and soft agar assays were conducted to determine self-renewal ability. The migration and invasion of LCSLCs were determined by wound healing and transwell assay. The glycolytic activity was assessed by determination of L-lactate metabolism rate. The influences of isovitexin on MnSOD, CaMKII, and AMPK activations as well as the metabolic shift to glycolysis were determined by manipulating MnSOD expression. Results. It was found that MnSOD and glycolysis enhanced simultaneously in LCSLCs compared with parental H460 cells. Overexpression of MnSOD activated CaMKII/AMPK signaling and glycolysis in LCSLCs with increased self-renewal, migration, invasion, and expression of stemness-associated markers in vitro and elevated carcinogenicity in vivo. Knockdown of MnSOD induced an inverse effect in LCSLCs. Isovitexin blocked MnSOD/CaMKII/AMPK signaling axis and suppressed glycolysis in LCSLCs, resulting in inhibition of stemness features in LCSLCs. The knockdown of MnSOD significantly augmented isovitexin-associated inhibition of CaMKII/AMPK signaling, glycolysis, and stemness in LCSLCs. However, the overexpression of MnSOD could attenuate the inhibition of isovitexin on LCSLCs. Importantly, isovitexin notably suppressed tumor growth in nude mice bearing LCSLCs by downregulation of MnSOD expression. Conclusion. MnSOD promotion of stemness of LCSLCs derived from H460 cell line is involved in the activation of the CaMKII/AMPK pathway and induction of glycolysis. Isovitexin-associated inhibition of stemness in LCSLCs is partly dependent on blockage of the MnSOD/CaMKII/AMPK signaling axis and glycolysis suppression.
1. Introduction
Non-small-cell lung cancer (NSCLC) has a relatively poor prognosis and is a leading cause of cancer-related death worldwide. The treatment failure and low survival rates of patients with NSCLC are mainly due to drug resistance, metastasis, and recurrence of tumor [1]. Recently, a small subpopulation of lung cancer stem-like cells (LCSLCs), characterized by expression of stem cell markers, self-renewing ability, multidifferentiating potential, and high tumorigenicity in vivo, were identified and considered to be responsible for drug resistance, metastasis, and recurrence of cancers [2]. However, the molecular mechanisms of LCSLCs maintaining stemness are not fully understood.
Manganese superoxide dismutase (MnSOD), as a mitochondrial-resident enzyme, plays a vital role in cellular energy metabolism and regulation of cell proliferation and apoptosis [3]. MnSOD can protect cells against the harmful effects of reactive oxygen species (ROS), which may induce the development of numerous diseases including cancers [4]. MnSOD acts as a tumor suppressor during early stages of carcinogenesis but facilitates cancer progression at later stages of development [5]. MnSOD was reported to upregulate in malignant lung cancer tissues [6]. Hart et al. found that MnSOD could increase sustained Warburg effect in breast cancers by H2O2 production that sustained AMP-activated kinase (AMPK) activation [7]. Many studies demonstrated that CSLCs had higher glycolytic rates compared with non-CSLCs [8]. The Warburg effect is important for CSLCs keeping bioenergetic metabolism [9]. MnSOD increase may promote the stemness of LCSLCs [10] and liver cancer stem-like cells. However, whether glycolysis is involved in the process of MnSOD promoting stemness of LCSLCs remains unclear.
Isovitexin (apigenin-6-C-glucoside) is an active component of various medicinal plants and traditional Chinese medicines [11]. Isovitexin has diverse biological activities including antioxidant, anticancer, and anti-inflammatory effects [11, 12]. It has been reported that isovitexin can suppress growth of large lung carcinoma cells, amelanotic melanoma cells [13], prostate cancer cells [14], and liver cancer cells by induction of apoptosis or autophagy through the mitochondrial pathway [15]. Recently, increasing researches attempted to find a strategy to eliminate cancer stem cells using the natural products. Our recent study indicated that isovitexin could suppress self-renewal capacity of spheres from human hepatocellular carcinoma MHCC97H cells [16]. Although recent findings imply that isovitexin may be a potential candidate for the prevention of lung cancer, the effects of isovitexin on LCSLCs and its molecular mechanisms remain unclear. Therefore, the present study was aimed at clarifying whether isovitexin suppresses stemness of LCSLCs and exploring the potential molecular mechanisms.
2. Materials and Methods
2.1. Cell Culture and Sphere Formation Assay
NSCLC cell lines H460 and A549 (Chinese Academy of Sciences, China) and HBE normal human bronchial epithelial cell line (ATCC) were cultured in DMEM containing 10% FBS and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Sphere formation assay was carried out according to the methods and procedures in a previous study by our team [10]. The 2nd-generation spheroids were used as LCSLCs in this study. The single cells after treatment with the indicated concentrations of isovitexin in primary sphere culture were cultured at a cell density of 1000 cells/well in the absence of isovitexin in a 24-well plate to generate new spheroids. , in 6-day cultures.
2.2. Colony Formation Assay
Colony formation assay was performed according to the methods and procedures in the previous study by our team [16]. .
2.3. Cell Proliferation Assay
Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8; Sigma) assay. The cells were treated with isovitexin (0.0, 5.0, 10.0, 20.0, 40.0, 80.0, 160.0 μg/mL) for 48 h before adding 10 μL of CCK-8 solution to each well and incubated at 37°C for 2 h. The optical density (OD) was measured at a wavelength of 450 nm. .
2.4. Western Blot
Western blot assay was performed according to the classical experimental protocols [17]. Primary antibody information was as follows: anti-MnSOD (ab13533), anti-FoxM1 (ab175798) obtained from Abcam, anti-β-actin (A5441) obtained from Sigma-Aldrich, anti-p-AMPK (8324), anti-AMPKα (2532), anti-CaMKII (4436S), anti-p-CaMKII (12716S), anti-CD44 (3570S), anti-CD133 (5860S), anti-Bmi1 (12035S), anti-ALDH (112035S), anti-Oct4 (2788S), and anti-Nanog (5855S) obtained from Cell Signaling Technology. After incubation with secondary antibodies for 1 hour, visualization of specific bands was performed by enhanced chemiluminescence; β-actin was used as internal reference.
2.5. Glycolysis Assay
Cells grown in media in a 96-well plate were transferred to serum-free media for another 24 hours before being analyzed for glycolytic activity, by the Glycolysis Cell-Based Assay Kit (Cayman Chemical).
2.6. Wound Healing Assay
Cell migration was evaluated with a wound healing assay as provided by Saadoun et al. [18]. Briefly, cells were transfected with shRNAs or cDNA plasmid and cultured to 90% confluence. 1 mm width wounds were created and incubated in a serum-free medium for 24 hours. Then, cells were cultured with a medium including 10% fetal bovine serum. And cultures at 0 and 24 hours were fixed and photographed under a microscope.
2.7. Transwell Assay
Cells () were placed in the upper compartment of the chambers. DMEM containing 10% fetal bovine serum was added in the lower chambers. Cells were incubated for 24 hours at 37°C. And the cells on the upper face of the membrane were scraped. Then, cells on the lower face were fixed, stained, and photographed under a microscope.
2.8. Knockdown or Overexpression of MnSOD
Transduction of MnSOD-targeted shRNAs (Ad-sh MnSOD) or overexpressed plasmids (Lent-MnSOD) was performed as previously described [10]. H460 cells or LCSLCs with 40-50% confluence were incubated overnight. Then, cells were transfected with Ad-sh MnSOD plasmid or control plasmid Ad-sh GFP packaging adenoviral particles and Lent-MnSOD plasmid or control plasmid Lent-GFP packaging lentivirus particles. The infection efficiency was calculated through counting GFP-positive and living cells.
2.9. In Vivo Tumorigenicity Assay
Four-week-old Balb/c-nude mice (obtained from Animal Institute of the Chinese Academy of Medical Science) were used in this research. And in vivo experiments were carried out as described in a previous study by our team [10], according to the institutional guidelines of the Hunan Normal University (Approval No. 2015-146).
Mice were randomly divided into 3 groups () according to standard protocols for in vivo tumorigenicity assay. Each mouse was inoculated with , , and parental H460 cells in one flank subcutaneously and LCSLCs in another side, respectively. After 1 month, tumors and tumor tissue sections were prepared for histopathology analysis.
For the sake of estimating the effects of isovitexin in vivo, 100 μL suspension (/mL) was injected into each mouse subcutaneously. The mice bearing LCSLC xenograft tumor (volume about 200 mm³) were administered 200 μL of vehicle control or isovitexin (12.5, 25, and 50 mg/kg body weight, respectively) through gavage every second day for 7 times.
2.10. Immunohistochemistry Assay
5 μm sections of tissues were prepared according to standard protocols. Immunostaining was carried out using the Elivision plus kit obtained from Maixin-Bio. Primary anti-MnSOD antibodies were applied at 1 : 200 dilution. Images were shot under a microscope.
2.11. Statistical Analysis
Statistical analysis was performed by the SPSS 20.0 software and presented as . The comparisons with the control groups were performed using a two-tailed Student -test. All the pairwise comparisons between the groups were analyzed by the Tukey post hoc test using one-way ANOVA. was considered to have significant difference.
3. Results
3.1. Identification of LCSLCs Derived from NSCLC H460 Cells
The tumor sphere-forming cells (SFCs) are generally identified as cancer stem-like cells [10]. Here, human non-small-lung cancer H460 and A549 cells were cultured as suspension in stem cell-conditioned suspension medium and its stem-like features were identified. As shown in Figure 1(a), the sphere-forming ability of generations 2-4 of H460 SFCs was significantly stronger than that in the first-generation SFCs. The 2nd-generation SFCs of H460 cells were then used for establishment of a model of LCSLCs, and some important experimental results were verified in the 2nd-generation SFCs of A549 cells (LCSLCs-A549). Then, its stem-like features were further detected. The soft agar assay showed that the colony-forming rate of LCSLCs was significantly higher than that in parental H460 cells (Figure 1(b)). The results of wound healing assay and transwell assay indicated that the LCSLCs had more powerful migratory and invasive capabilities compared with parental cells (Figures 1(c) and 1(d)). Furthermore, the western blot results demonstrated that the CD133, CD44, ALDH1, Nanog, and Bmi1 protein expression levels were increased in LCSLCs compared with parental cells (Figures 1(e) and 1(f)). These results confirmed that LCSLCs derived from the 2nd-generation SFCs of the H460 cell line had cancer stem-like cell features including self-renewal ability, highly migratory and invasive potentials, and increased cancer stem cell biomarkers.
(a)
... Caffeine, a xanthine alkaloid, has been used as a ryanodine receptor agonist for inducing calcium release from intracellular calcium stores (Dettbarn et al., 1994). In skeletal muscle cells and tissue, caffeine activates calcium signaling and subsequently enhances glucose uptake, fat oxidation, and mitochondrial biogenesis (Ojuka, Jones, Han, et al., 2002;Raney & Turcotte, 2008;Wright et al., 2004Wright et al., , 2007. Additionally, caffeine inhibits phosphodiesterase activity, leading to increased cellular cyclic adenosine monophosphate (cAMP) concentration, which is followed by activation of protein kinase A (PKA) and cAMP response element-binding protein (CREB; Al-Wadei et al., 2006). ...
Abstract Myoglobin is an important regulator of muscle and whole‐body metabolism and exercise capacity. Caffeine, an activator of the calcium and cyclic AMP (cAMP)/protein kinase A (PKA) pathway, enhances glucose uptake, fat oxidation, and mitochondrial biogenesis in skeletal muscle cells. However, no study has shown that caffeine increases the endogenous expression of myoglobin in muscle cells. Further, the molecular mechanism underlying the regulation of myoglobin expression remains unclear. Therefore, our aim was to investigate whether caffeine and activators of the calcium signaling and cAMP/PKA pathway increase the expression of myoglobin in L6 myotubes and whether the pathway mediates caffeine‐induced myoglobin expression. Caffeine increased myoglobin expression and activated the cAMP/PKA pathway in L6 muscle cells. Additionally, a cAMP analog significantly increased myoglobin expression, whereas a ryanodine receptor agonist showed no significant effect. Finally, PKA inhibition significantly suppressed caffeine‐induced myoglobin expression in L6 myotubes. These results suggest that caffeine increases myoglobin expression via the cAMP/PKA pathway in skeletal muscle cells.
... Its function is to preserve ATP by inhibiting both biosynthetic and anabolic pathways while simultaneously stimulating catabolic pathways to re-establish cellular energy stores. The increased concentration of Ca 2+ during muscle contraction can also directly activate AMPK and is implicated in the regulation of numerous intracellular proteins that mediate cellular transduction, including kinase C, calcineurin, and CaMKs [81][82][83]. Both AMPK and CaMKII lead to PGC-1α activation, a member of a family of transcriptional coactivators that regulate mitochondrial biogenesis [84] (Figure 2). ...
The natural aging process is carried out by a progressive loss of homeostasis leading to a functional decline in cells and tissues. The accumulation of these changes stem from a multifactorial process on which both external (environmental and social) and internal (genetic and biological) risk factors contribute to the development of adult chronic diseases, including type 2 diabetes mellitus (T2D). Strategies that can slow cellular aging include changes in diet, lifestyle and drugs that modulate intracellular signaling. Exercise is a promising lifestyle intervention that has shown antiaging effects by extending lifespan and healthspan through decreasing the nine hallmarks of aging and age-associated inflammation. Herein, we review the effects of exercise to attenuate aging from a clinical to a cellular level, listing its effects upon various tissues and systems as well as its capacity to reverse many of the hallmarks of aging. Additionally, we suggest AMPK as a central regulator of the cellular effects of exercise due to its integrative effects in different tissues. These concepts are especially relevant in the setting of T2D, where cellular aging is accelerated and exercise can counteract these effects through the reviewed antiaging mechanisms.
... 4 Exercise activates CaMKII and results in both mitochondrial biogenesis and improved glucose transport in parallel. [5][6][7] As such, exercise can curb the accumulation of excess lipids in adipose and intramuscular tissues that may result in obesity/type 2 diabetes. This review discusses the role of exercise-induced CaMKII activation in the regulation of fatty acids and lipid metabolism. ...
Background & aims
Previous studies have reported the beneficial roles of the activation of calmodulin-dependent protein kinase (CaMK)II to many cellular functions associated with human health. This review aims at discussing its activation by exercise as well as its roles in the regulation of unsaturated, saturated, omega 3 fatty acids, and lipid metabolism.
Methods
A wide literature search was conducted using online database such as ‘PubMed’, ‘Google Scholar’, ‘Researcher’, ‘Scopus’ and the website of World Health Organization (WHO) as well as Control Disease and Prevention (CDC). The criteria for the search were mainly lipid and fatty acid metabolism, diabetes, and metabolic syndrome (MetS). A total of ninety-seven articles were included in the review.
Results
Calmodulin-dependent protein kinase activation by exercise is helpful in controlling membrane lipids related with type 2 diabetes and obesity. CaMKII regulates many health beneficial cellular functions in individuals who exercise compared with those who do not exercise. Regulation of lipid metabolism and fatty acids are crucial in the improvement of metabolic syndrome.
Conclusions
Approaches that involve CaMKII could be a new avenue for designing novel and effective therapeutic modalities in the treatment or better management of metabolic diseases such as type 2 diabetes and obesity.
... CaMKII is the dominant isoform of CaMK in human skeletal muscle (Rose et al., 2006). It is reported that exercise activates CaMKII and re-sults in both mitochondrial biogenesis and improved glucose transport in parallel (Wright et al., 2007;Raney and Turcotte, 2008;Wu et al., 1999). This parallel up-regulation has got positive implications in alleviating type 2 diabetes symptoms. ...
Individuals who exercise regularly are protected from type 2 diabetes and other metabolic syndromes, in part by enhanced gene transcription and induction of many signaling pathways crucial in correcting impaired metabolic pathways associated with a sedentary lifestyle. Exercise activates Calmodulin-dependent protein kinase (CaMK)II, resulting in increased mitochondrial oxidative capacity and glucose transport. CaMKII regulates many health beneficial cellular functions in individuals who exercise compared with those who do not exercise. The role of exercise in the regulation of carbohydrate, lipid metabolism, and insulin signaling pathways are explained at the onset. Followed by the role of exercise in the regulation of glucose transporter (GLUT)4 expression and mitochondrial biogenesis are explained. Next, the main functions of Calmodulin-dependent protein kinase and the mechanism to activate it are illustrated, finally, an overview of the role of CaMKII in regulating GLUT4 expression, mitochon-drial biogenesis, and histone modification are discussed.
... CaMKII phosphorylates transcription factors such as cAMP response element-binding protein (CREB) and myocyte enhancer factor 2 (MEF2), as well as type II histone deacetylases (HDACs) turning on transcription of genes involved in glucose metabolism and mitochondrial biogenesis. CAMK has also been shown to regulate glucose transport (128,134) as well as fatty acid uptake and oxidation (1,98) in contracting mouse and rat SkM (Figure 8.2). ...
Exercise has the ability to induce a rapid shift in metabolic demands leading to transient perturbations in whole-body homeostasis. This challenge triggers a multitude of both acute metabolic adjustments and adaptive molecular integrative responses in skeletal muscle to re-establish homeostasis. Initiating stimuli involved in inducing these responses include intracellular changes in Ca2+, cellular energy status (AMP/ATP), and reactive oxygen species (ROS) in skeletal muscle, as well as externally derived signals such as hormones and cytokines. Each of these stimuli induces intracellular signalling pathways that may mediate both exercise-induced gene regulation and exercise-induced metabolic regulation. This chapter will highlight the main intracellular signalling pathways in skeletal muscle evoked by a single bout of endurance exercise. Each section provides a detailed description of the cellular responses of the pathway and presents research findings on exercise-induced regulation of key factors in the specific signalling pathway. The chapter finishes with a perspective on basic questions within exercise-mediated intracellular signalling that remain be addressed in future studies.
... The gene Camk2b, a downstream dependent kinase of the calcium signaling pathway, promoted mitochondrial biogenesis and participated in the transformation of muscle fiber types from fast-twitch muscle to slow-twitch muscle (Al-Shanti and Stewart, 2009). In mouse muscle development, Camk2 regulated the oxidative metabolism of mouse muscle mediating by AMPK signaling (Raney and Turcotte, 2008). Therefore, we examined the expression of Camk2b, and the qPCR results demonstrated that overexpression of Mdfi significantly increased its expression. ...
Muscle development requires myoblast differentiation and muscle fiber formation. Myod family inhibitor (Mdfi) inhibits myogenic regulatory factors in NIH3T3 cells, but how Mdfi regulates myoblast myogenic development is still unclear. In the present study, we constructed an Mdfi-overexpression (Mdfi-OE) C2C12 cell line by the CRISPR/Cas9 system and performed RNA-seq on Mdfi-OE and wild-type (WT) C2C12 cells. The RNA-seq results showed that the calcium signaling pathway was the most significant. We also established the regulatory networks of Mdfi-OE on C2C12 cell differentiation and muscle fiber type transformation and identified hub genes. Further, both RNA-seq and experimental verification demonstrated that Mdfi promoted C2C12 cell differentiation by upregulating the expression of Myod, Myog, and Myosin. We also found that the positive regulation of Mdfi on fast-to-slow-twitch muscle fiber transformation is mediated by Myod, Camk2b, and its downstream genes, such as Pgc1a, Pdk4, Cs, Cox4, Acadm, Acox1, Cycs, and Atp5a1. In conclusion, our results demonstrated that Mdfi promotes C2C12 cell differentiation and positively modulates fast-to-slow-twitch muscle fiber transformation. These findings further our understanding of the regulatory mechanisms of Mdfi in myogenic development and muscle fiber type transformation. Our results suggest potential therapeutic targets for muscle- and metabolic-related diseases.
... AMPK functions as an energy sensor and regulates both cellular energy metabolism and protein synthesis (Arad et al. 2007;Dyck and Lopaschuk 2006;Frederich and Balschi 2002). It is modulated by liver kinase B1 (LKB1) (HGNC: STK11), with further modulation via changes in AMP/ADP/ATP levels, by Ca 2+ -dependent signaling, such as Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) (HGNC: CAMK2A), and Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) (HGNC: CAMKK2) as well as other direct activators including 5-aminoimidazole-4-carboxamide riboside, thienopyridone and benzimidazole derivatives, salicylate, compound-13, PT-1, and MT 63-78 (Table 1) (Kim et al. 2016;Raney and Turcotte 2008;Towler and Hardie 2007). In addition, there are indirect AMPK activators that lead to activation via AMP or Ca 2+ accumulation, such as biguanides (i.e., metformin), salicylates, thiazolidinediones, polyphenols, ginsenoside, and a-lipoic acid, among other AMPK modulators (Kim et al. 2016). ...
The number of patients diagnosed with atrial fibrillation (AF) has been rising due to increased incidence, enhanced detection methods, and greater survival rates following diagnosis. Due to this increase, AF is now the most commonly diagnosed arrhythmia in clinical practice. AF is characterized by irregular, high-frequency contractions of atrial myocytes that lead to turbulent blood flow and the potential for thrombus formation, stroke, or heart failure. These high-frequency contractions of the atrial myocytes cause an imbalance between metabolic supply and demand. Although advances have been made in understanding the pathophysiology of AF, the etiology and underlying pathogenic mechanism remain unknown. However, recent evidence suggests that cardiomyocyte metabolism involving 5′ AMP-activated protein kinase (AMPK) activation is altered in patients with AF. Here, we critically reviewed the current understanding of AMPK activation in AF and how it could affect structural, contractile, and electrophysiological cellular properties in the pathogenesis of AF.
... 4 Besides this, CaMKII has been implicated in the regulation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), 5,6 which is stimulated by high-intensity exercise 7 and muscle metabolism. [8][9][10] Nevertheless, previous evidence on the role of CaMKII in the regulation of muscle phenotype comes from extreme experimental models and information using physiological models in humans is lacking. 2 CaMKII has been implicated in muscle hypertrophy in animal models. [11][12][13] CaMKII is encoded by four genes (α, β, γ, and δ) and more than 40 splice variants have been reported 14 of which, the isoforms β M , γ, δ A , and δ D are expressed in skeletal muscle. ...
Strength training promotes a IIX-to-IIA shift in myosin heavy chain (MHC) composition, likely due to changes in sarcoplasmic [Ca2+ ] which are sensed by CaMKII. Sarcoplasmic [Ca2+ ] is in part regulated by sarcolipin (SLN) a small protein, that when overexpressed in rodents stimulates mitochondrial biogenesis and a fast-to-slow fibre type shift. The purpose of this study was to determine whether CaMKII and SLN are involved in muscle phenotype and performance changes elicited by strength training. Twenty-two men followed an 8-week velocity-based resistance training programme using the full squat exercise while monitoring repetition velocity. Subjects were randomly assigned to two resistance training programmes differing in the repetition velocity loss allowed in each set: 20% (VL20) vs 40% (VL40). Strength training caused muscle hypertrophy, improved 1RM and increased total CaMKII protein expression, particularly of the δD isoform. Phospho-Thr287 -CaMKII δD expression increased only in VL40 (+89%), which experienced greater muscle hypertrophy, and a reduction in MHC-IIX percentage. SLN expression was increased in VL20 (+33%) remaining unaltered in VL40. The changes in phospho-Thr287 -CaMKII δD were positively associated with muscle hypertrophy and the number of repetitions during training, and negatively with the changes in MHC-IIX and SLN. Most OXPHOS proteins remained unchanged, except for NDUFB8 (Complex I), which was reduced after training (-22%) in both groups. The amount of fatigue allowed in each set critically influences muscle CaMKII and SLN responses and determines muscle phenotype changes. With lower intra-set fatigue, the IIX-to-IIA MHC shift is attenuated.
... Ca 2+ -dependent protein kinase kinase β (CaMKKβ), one of three AMPK upstream kinase (AMPKK), can activate AMPK by phosphorylating Thr-172 in response to cellular Ca 2+ signaling, which was independent of the increase in AMP/ATP [11]. Meanwhile, the postmortem ATP reduction impairs the function of the ion pump, and the sarcoplasmic reticulum calcium channel loses its ability prematurely to regulate calcium ions due to oxidation, resulting in an increase in intracellular Ca 2+ level, and activation of CaMKKβ activity [12]. ...
The Musculus longissimus thoracis muscle of twenty crossbred cattle was used to investigate the activity of calcium-regulated protein kinase kinase (CaMKKβ), adenosine monophosphate-activated protein kinase (AMPK), glycolysis pathway, and apoptosis during 7 days postmortem maturation. Results showed that CaCl2 significantly increased the activity of CaMKKβ and AMPK. Meanwhile, the activation of AMPK increased the activity of rate-limiting enzymes in glycolysis (lactate dehydrogenase and pyruvate kinase), and accelerated the speed of postmortem muscle maturation. Besides, CaCl2 altered the properties of myofibrillar, affected the intramuscular environment, and induced postmortem apoptosis. Our finds indicate that CaCl2 activates AMPK, accelerating the glycolysis process and increasing mitochondrial ROS level, which was an upstream event of apoptosis. Muscle samples treated with CaCl2 had better tenderness, probably due to the degeneration of myofibrillar proteins and the occurrence of apoptosis, which may have contributed to the increased AMPK activity and accelerated glycolysis.
... In skeletal muscle, a rise in cytosolic Ca 2+ during muscle contraction leads to CaMKII activation with consequent phosphorylation of HDAC5 resulting in increased MEF2-binding to the GLUT4 gene promotor, thus leading to enhanced gene transcription [72,73]. Furthermore, there is evidence, that CaMKII is capable of activating AMP activated kinase (AMPK) [74], a metabolic stress sensor, which stimulates GLUT1 activity as well as GLUT4 expression and membrane translocation [75][76][77]. In contrast, studies in a rat skeletal muscle cell line demonstrated a dual role of CaMKII in insulin-stimulated glucose uptake: After initial stimulation of the Akt/GLUT4 pathway via CaMKIImediated Raf1 activation, a negative feedback loop with CaMKII-dependent inhibition of insulin-stimulated Akt phosphorylation, GLUT4 translocation and glucose uptake develops [78]. ...
Empagliflozin, a selective sodium-glucose co-transporter 2 (SGLT2) inhibitor, has been shown to reduce mortality and hospitalization for heart failure in diabetic patients in the EMPA-REG-OUTCOME trial (Zinman et al., 2015). Surprisingly, dapagliflozin, another SGLT2 inhibitor, exerted comparable effects on clinical endpoints even in the absence of diabetes mellitus (DAPA-HF trial) (McMurray et al., 2019). There is a myriad of suggested underlying mechanisms ranging from improved glycemic control and hemodynamic effects to altered myocardial metabolism, inflammation, neurohumoral activation and intracellular ion homeostasis.
Here, we review the effects of gliflozins on cardiac electro-mechanical coupling with an emphasis on novel CaMKII-mediated pathways and on cardiac glucose and ketone metabolism in the failing heart. We focus on empagliflozin as it is the gliflozin with the most abundant experimental evidence for direct effects on the heart. Where useful, we aim to compare empagliflozin to other gliflozins. To facilitate understanding of empagliflozin-induced alterations, we first give a short summary of the pathophysiological role of CaMKII in heart failure, as well as cardiac changes of glucose and ketone body metabolism in the failing heart.
... This process requires high levels of protein synthesis, proper mRNA translation, and energy consumption. Different signaling pathways underlie these processes, such as AMP-activated protein kinase (AMPK) signaling, which is regulated by CaMKII and liver kinase B1 (LKB1) [117,118]. In C3KO mice, during muscle regeneration after CTX injection, muscle fiber growth is arrested due to increased AMPK phosphorylation, inhibition of mTORC1, and energy shortage [71]. ...
Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a rare disease caused by mutations in the CAPN3 gene. It is characterized by progressive weakness of shoulder, pelvic, and proximal limb muscles that usually appears in children and young adults and results in loss of ambulation within 20 years after disease onset in most patients. The pathophysiological mechanisms involved in LGMDR1 remain mostly unknown, and to date, there is no effective treatment for this disease. Here, we review clinical and experimental evidence suggesting that dysregulation of Ca²⁺ homeostasis in the skeletal muscle is a significant underlying event in this muscular dystrophy. We also review and discuss specific clinical features of LGMDR1, CAPN3 functions, novel putative targets for therapeutic strategies, and current approaches aiming to treat LGMDR1. These novel approaches may be clinically relevant not only for LGMDR1 but also for other muscular dystrophies with secondary calpainopathy or with abnormal Ca²⁺ homeostasis, such as LGMD2B/LGMDR2 or sporadic inclusion body myositis.
... Additionally, SPARC increased the intracellular calcium concentration, indicating that SPARC activates AMPK via calcium-dependent signals. Calcium-dependent kinases act by regulating AMPK phosphorylation, glucose uptake, and fatty acid oxidation in response to muscle contraction and exercise (50)(51)(52). Moreover, CaMK kinases function in intact cells as AMPK kinases (53). ...
During exercise, skeletal muscles release cytokines, peptides, and metabolites that exert autocrine, paracrine, or endocrine effects on glucose homeostasis. In this study, we investigated the effects of secreted protein acidic and rich in cysteine (SPARC), an exercise‐responsive myokine, on glucose metabolism in human and mouse skeletal muscle. SPARC‐knockout mice showed impaired systemic metabolism and reduced phosphorylation of AMPK and protein kinase B in skeletal muscle. Treatment of SPARC‐knockout mice with recombinant SPARC improved glucose tolerance and concomitantly activated AMPK in skeletal muscle. These effects were dependent on AMPK‐γ3 because SPARC treatment enhanced skeletal muscle glucose uptake in wild‐type mice but not in AMPK‐γ3–knockout mice. SPARC strongly interacted with the voltage‐dependent calcium channel, and inhibition of calcium‐dependent signaling prevented SPARC‐induced AMPK phosphorylation in human and mouse myotubes. Finally, chronic SPARC treatment improved systemic glucose tolerance and AMPK signaling in skeletal muscle of high‐fat diet–induced obese mice, highlighting the efficacy of SPARC treatment in the management of metabolic diseases. Thus, our findings suggest that SPARC treatment mimics the effects of exercise on glucose tolerance by enhancing AMPK‐dependent glucose uptake in skeletal muscle.—Aoi, W., Hirano, N., Lassiter, D. G., Björnholm, M., Chibalin, A. V., Sakuma, K., Tanimura, Y., Mizushima, K., Takagi, T., Naito, Y., Zierath, J. R., Krook, A. Secreted protein acidic and rich in cysteine (SPARC) improves glucose tolerance via AMP‐activated protein kinase activation. FASEB J. 33, 10551–10562 (2019). www.fasebj.org
... This pathway has recently been connected to cellular processes such as autophagy and cell polarity [71]. AMPK signaling has been thoroughly investigated in muscle tissue during various intensity exercises and different accompanying processes: cell/tissue adaptive response [72], oxidation in muscle [73], reactive oxygen species [74], contractile activity, and energy metabolism alterations [75]. ...
Gamma-oryzanol (GO) is a popular supplement for performance horses, dogs, and humans. Previous studies indicated that GO supplementation decreases creatine kinase activity and lactate level after exercise and may affect oxidative stress in Thoroughbred horses. GO may change genes expression in equine satellite cells (ESC). The purpose of this study was to evaluate the effect of GO on miRNA, gene expression, oxidative stress, and cell damage and viability in differentiating ESC pretreated with hydrogen peroxide (H2O2). ESCs were obtained from a young horse’s skeletal muscle. ESCs were pre-incubated with GO (24 h) and then exposed to H2O2 for one hour. For the microRNA and gene expression assessment, the microarray technique was used. Identified miRNAs and genes were validated using real time-quantitative polymerase chain reaction. Several tests related to cell viability, cell damage, and oxidative stress were performed. The microarray analysis revealed differences in 17 miRNAs and 202 genes between GO-treated and control ESC. The tests related to apoptosis, cell viability, and oxidative stress showed that GO affects these processes to varying degrees. Our results suggest that GO can change miRNA and gene expression and may impact the processes involved in tissue repairing after an injury.
... AMPK is a key regulator in the maintenance of cellular FA homeostasis [29], which controls FA β-oxidation in mitochondria. As shown in Figure 6D, phosphorylation of AMPK was lower in the wt-CD36-HepG2 cells than in the NC-HepG2 cells, suggesting that CD36 overexpression inactivated the AMPK pathway. ...
Background and aims:
Fatty acid translocase CD36 (CD36) is a membrane protein with multiple immuno-metabolic functions. Palmitoylation has been suggested to regulate the distribution and functions of CD36, but little is known about its significance in NASH.
Methods:
Human liver tissue samples were obtained from patients undergoing liver biopsy for diagnostic purposes. CD36 knockout mice were injected with lentivirus vectors to express wild type CD36 and palmitoylation sites mutated CD36 in the livers. Liver histology, immunofluorescence, mRNA expression profile, subcellular distributions and functions of CD36 protein were assessed.
Results:
The localization of CD36 on the plasma membrane of hepatocytes was markedly increased in patients with NASH compared to patients with normal liver and those with simple steatosis. Increased CD36 palmitoylation and increased localization of CD36 on the plasma membrane of hepatocytes were also observed in livers of mice with NASH. Furthermore, inhibition of CD36 palmitoylation protected mice from developing NASH. The absence of palmitoylation decreased CD36 protein hydrophobicity reducing its localization on the plasma membrane as well as in lipid raft of hepatocytes. Consequently, a lack of palmitoylation decreased fatty acid uptake and CD36/Fyn/Lyn complex in HepG2 cells. Inhibition of CD36 palmitoylation not only ameliorated intracellular lipid accumulation via activating the AMPK pathway, but also inhibited inflammatory response through the inhibition of the JNK signaling pathway.
Conclusions:
Our findings demonstrate the key role of palmitoylation in regulating CD36 distributions and its functions in NASH. Inhibition of CD36 palmitoylation may represent an effective therapeutic strategy in patients with NASH.
Lay summary:
Fatty acid translocase CD36 (CD36) is a multifunctional membrane protein which contributes to the development of liver steatosis. In the present study, we demonstrated that the localization of CD36 on the plasma membrane of hepatocytes is increased in patients with NASH. Blocking the palmitoylation of CD36 reduces CD36 distribution in hepatocytes plasma membrane and protects mice from NASH. The inhibition of CD36 palmitoylation not only improved fatty acid metabolic disorders but also reduced the inflammatory response in vitro and in vivo. The present study suggests that CD36 palmitoylation is important for NASH development and inhibition of CD36 palmitoylation could be used to cure NASH.
Background. The imbalance in the ratio of protein synthesis versus protein degradation results in skeletal muscle atrophy following unloading. The onset of these processes is regulated by the sarcoplasmic concentrations of ATP and calcium (Ca ²⁺ ). We tested the hypothesis that unloading-induced inactivation of SERCA results in raised Ca ²⁺ concentrations, triggering catabolic processes. CDN1163, an activator of SERCA, was used to test this hypothesis.
Methods. Three groups of male rats were used: control rats with intraperitoneal injection of placebo (C), 3 days of unloading with placebo injection (3HS), and 3 days of unloading injected with CDN1163 (3HSC).
Results. Treatment with CDN1163 during three days of soleus muscle unloading prevented the upregulation of Ca ²⁺ and ATP, and the slow-to-fast shift in muscle fiber composition. This treatment blocked the decrease in the phosphorylation of the anabolic markers (GSK3b, eEF2, and S6(Ser240/244/ Ser235/236)), and therefore it is likely that it improved the efficiency of translation in the unloaded muscle, but it did not affect mTORC1-dependent signaling. Treatment with CDN1163 also modulated the regulation of the Ca ²⁺ -dependent signaling in muscle during unloading via SERCA1 and CSQ2, and changes in the CaMKII phosphorylation and the content of IP3R. In addition, CDN1163 prevented the upregulation of the mRNA expression of MuRF1 (but not MAFbx) and attenuated the increase of Cbl-b and ubiquitin mRNA expression during unloading.
Conclusions. Activation of SERCA with CDN1163 prevents the upregulation of Ca ²⁺ and ATP, as well as calcium-dependent and ubiquitin-proteasome pathways markers, and improves protein translation efficiency in three-day unloaded soleus muscle.
Skeletal muscle is the main site of glucose deposition in the body during meals and the major glucose utilizer during physical activity. While in both instances the supply of glucose from the circulation to the muscle is of paramount importance, in most conditions the rate limiting step in glucose uptake, storage and utilization is the transport of glucose across the muscle cell membrane. This step is dependent upon the translocation of the insulin- and contraction-responsive glucose transporter GLUT4 from intracellular storage sites to the sarcolemma and T-tubules. Here, we first analyze how glucose can traverse the capillary wall into the muscle interstitial space. We then review the molecular processes that regulate GLUT4 translocation in response to insulin and muscle contractions, and the methodologies utilized to unravel them. We further discuss how physical activity and inactivity, respectively, lead to increased and decreased insulin action in muscle and touch upon sex differences in glucose metabolism. While many key processes regulating glucose uptake in muscle are known, the advent of newer and bioinformatics tools have revealed further molecular signaling processes reaching a staggering level of complexity. Much of this molecular mapping has emerged from cellular and animal studies and more recently from application of a variety of -omics in human tissues. In the future, it will be imperative to validate the translatability of results drawn from experimental systems to human physiology.
Mammalian skeletal muscles are composed of a variety of highly specialized fibers whose selective recruitment allows muscles to fulfill their diverse functional tasks. In addition, skeletal muscle fibers can change their structural and functional properties to perform new tasks or respond to new conditions. The adaptive changes of muscle fibers can occur in response to variations in the pattern of neural stimulation, loading conditions, availability of substrates, and hormonal signals. The new conditions can be detected by multiple sensors, from membrane receptors for hormones and cytokines, to metabolic sensors, which detect high‐energy phosphate concentration, oxygen and oxygen free radicals, to calcium binding proteins, which sense variations in intracellular calcium induced by nerve activity, to load sensors located in the sarcomeric and sarcolemmal cytoskeleton. These sensors trigger cascades of signaling pathways which may ultimately lead to changes in fiber size and fiber type. Changes in fiber size reflect an imbalance in protein turnover with either protein accumulation, leading to muscle hypertrophy, or protein loss, with consequent muscle atrophy. Changes in fiber type reflect a reprogramming of gene transcription leading to a remodeling of fiber contractile properties (slow‐fast transitions) or metabolic profile (glycolytic‐oxidative transitions). While myonuclei are in postmitotic state, satellite cells represent a reserve of new nuclei and can be involved in the adaptive response. © 2013 American Physiological Society. Compr Physiol 3:1645‐1687, 2013.
IP3 receptors are found in significant quantities in muscle fibers in the sarcoplasmic reticulum, nucleus and mitochondria. We hypothesized that activation of IP3 receptors (IP3Rs) during muscle unloading may induce a weak calcium release signal, both cytosolic and nucleoplasmic, that promotes (possibly with other signaling cascades) the activation of transcription factors, leading to the expression or repression of genes involved in muscle phenotype. This hypothesis was tested by blocking IP3R during unloading of rat muscles by administering 2-APB (2-aminoethoxydiphenyl borate). Wistar rats were administered intraperitoneally at a dose of 10 mg/mg in 5 % DMSO daily. We found that the IP3R state influences the development of atrophic processes in the postural m. soleus during unloading. Administration of the IP3R blocker 2-APB to animals successfully prevented a decrease in m. soleus cross-sectional area (CSA) of both fast and slow muscle fibers. The slowdown in CSA decrease upon administration IP3R inhibitor during 7 days m. soleus unloading is associated with the prevention of a decrease in ribosomal biogenesis and an increase in the expression of autophagy markers ULK-1 and IL-6.
Redox regulation is a fundamental physiological phenomenon related to oxygen-dependent metabolism, and skeletal muscle is mainly regarded as a primary site for oxidative phosphorylation. Several studies have revealed the importance of reactive oxygen and nitrogen species (RONS) in the signaling process relating to muscle adaptation during exercise. To date, improving knowledge of redox signaling in modulating exercise adaptation has been the subject of comprehensive work and scientific inquiry. The primary aim of this review is to elucidate the molecular and biochemical pathways aligned to RONS as activators of skeletal muscle adaptation and to further identify the interconnecting mechanisms controlling redox balance. We also discuss the RONS-mediated pathways during the muscle adaptive process, including mitochondrial biogenesis, muscle remodeling, vascular angiogenesis, neuron regeneration, and the role of exogenous antioxidants.
Muscle unloading leads to signaling alterations that cause muscle atrophy and weakness. The cellular energy sensor AMPK can regulate myofiber-type shift, calcium-dependent signaling and ubiquitin-proteasome system markers. We hypothesized that the prevention of p-AMPK downregulation during the first week of muscle unloading would impede atrophy development and the slow-to-fast shift of soleus muscle fibers, and the aim of the study was to test this hypothesis. Thirty-two male Wistar rats were randomly assigned to four groups: placebo control (C), control rats treated with metformin (C + M), 7 days of hindlimb suspension (HS) + placebo (7HS), and 7 days of HS + metformin administration (7HS + M). In the soleus of the 7HS rats, we detected a slow-to-fast fiber-type shift as well as a significant downregulation of MEF-2D and p300 in the nuclei. In the 7HS group, we also found decreases in p-ACC (AMPK target) protein level and in the expression of E3 ubiquitin ligases and p-CaMK II protein level vs. the C group. The 7-day metformin treatment for soleus muscle unloading (1) prevented slow-to-fast fiber-type shift; (2) counteracted changes in the p-ACC protein level; (3) hindered changes in the nuclear protein level of the slow myosin expression activators MEF-2D and p300, but did not affect NFATc1 signaling; and (4) attenuated the unloading-induced upregulation of MuRF-1, atrogin-1, ubiquitin and myostatin mRNA expression, but did not prevent soleus muscle atrophy. Thus, metformin treatment during muscle disuse could be useful to prevent the decrease in the percentage of slow-type fatigue-resistant muscle fibers.
The study aimed to investigate the effects of four fatty acids with the same carbon-chain length and different saturations, stearic acid (SA), oleic acid (OA), linoleic acid (LA), and linolenic acid (LNA), on the growth performance and muscle quality of Megalobrama amblycephala through in vivo and in vitro experiments. A total of 320 fish with similar initial weight (34.97 ± 0.14 g) were randomly fed 3% four kinds of 18-carbon fatty acids with different carbon saturation. Growth performance was assessed at the end of the feeding trial, and samples were collected for the corresponding indicators. Results showed that the weight gain rate (WGR) and specific growth rate (SGR) of unsaturated fatty acids groups were significantly increased (P < 0.05) than the SA group. Meanwhile, unsaturated fatty acids resulted in a significant increase in white muscle texture (hardness, adhesiveness, gumminess, and chewiness) (P < 0.05). With the increase of fatty acid unsaturation, the number of muscle fibers and density were significantly increased (P < 0.05), while the sarcomere length decreased. The gene expression of sirt1 and camk were significantly up-regulated in unsaturated fatty acids groups (P < 0.05), and the genes related to muscle fiber development were also increased significantly (P < 0.05). In vitro study, the primary muscle cells of Megalobrama amblycephala were treated for 24 h according to the references. The results showed that 250 μmol of unsaturated fatty acids OA, LA, and LNA significantly up-regulated the protein levels of p-Ampk, Camk, and Pax7 associated with muscle fiber development (P < 0.05). In conclusion, unsaturated fatty acids can promote muscle fiber development and meat quality because of the different saturations, which may be realized by activating AMPK and Ca²⁺-dependent signal pathways.
An AMP-activated kinase (AMPK) signaling pathway is activated during myocardial ischemia and promotes cardiac fatty acid (FA) uptake and oxidation. Similarly, the multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is also triggered by myocardial ischemia, but its function in FA metabolism remains unclear. Here, we explored the role of CaMKII in FA metabolism during myocardial ischemia by investigating the effects of cardiac CaMKII on AMPK-acetyl-CoA carboxylase (ACC), malonyl CoA decarboxylase (MCD), and FA translocase cluster of differentiation 36 (FAT/CD36), as well as cardiac FA uptake and oxidation. Moreover, we tested whether CaMKII and AMPK are binding partners. We demonstrated that diseased hearts from patients with terminal ischemic heart disease displayed increased phosphorylation of CaMKII, AMPK, and ACC and increased expression of MCD and FAT/CD36. AC3-I mice, which have a genetic myocardial inhibition of CaMKII, had reduced gene expression of cardiac AMPK. In post-MI (myocardial infarction) AC3-I hearts, AMPK-ACC phosphorylation, MCD and FAT/CD36 levels, cardiac FA uptake, and FA oxidation were significantly decreased. Notably, we demonstrated that CaMKII interacted with AMPK α1 and α2 subunits in the heart. Additionally, AC3-I mice displayed significantly less cardiac hypertrophy and apoptosis 2 weeks post-MI. Overall, these findings reveal a unique role for CaMKII inhibition in repressing FA metabolism by interacting with AMPK signaling pathways, which may represent a novel mechanism in ischemic heart disease.
Objective:
Stromal interaction molecule 1 (STIM1) is a single-pass transmembrane endoplasmic/sarcoplasmic reticulum (E/SR) protein recognized for its role in a store operated Ca2+ entry (SOCE), an ancient and ubiquitous signaling pathway. Whereas STIM1 is known to be indispensable during development, its biological and metabolic functions in mature muscles remain unclear.
Methods:
Conditional and tamoxifen inducible muscle STIM1 knock-out mouse models were coupled with multi-omics tools and comprehensive physiology to understand the role of STIM1 in regulating SOCE, mitochondrial quality and bioenergetics, and whole-body energy homeostasis.
Results:
This study shows that STIM1 is abundant in adult skeletal muscle, upregulated by exercise, and is present at SR-mitochondria interfaces. Inducible tissue-specific deletion of STIM1 (iSTIM1 KO) in adult muscle led to diminished lean mass, reduced exercise capacity, and perturbed fuel selection in the settings of energetic stress, without affecting whole-body glucose tolerance. Proteomics and phospho-proteomics analyses of iSTIM1 KO muscles revealed molecular signatures of low-grade E/SR stress and broad activation of processes and signaling networks involved in proteostasis.
Conclusion:
These results show that STIM1 regulates cellular and mitochondrial Ca2+ dynamics, energy metabolism and proteostasis in adult skeletal muscles. Furthermore, these findings provide insight into the pathophysiology of muscle diseases linked to disturbances in STIM1-dependent Ca2+ handling.
Caffeine has been shown to stimulate multiple major regulators of cell energetics including AMP-activated protein kinase (AMPK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Additionally, caffeine induces peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and mitochondrial biogenesis. While caffeine enhances oxidative metabolism, experimental concentrations often exceed physiologically attainable concentrations through diet. This work measured the effects of low-level caffeine on cellular metabolism and gene expression in myotubes, as well as the dependence of caffeine’s effects on the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARβ/δ). C2C12 myotubes were treated with various doses of caffeine for up to 24 h. Gene and protein expression were measured via qRT-PCR and Western blot, respectively. Cellular metabolism was determined via oxygen consumption and extracellular acidification rate. Caffeine significantly induced regulators of mitochondrial biogenesis and oxidative metabolism. Mitochondrial staining was suppressed in PPARβ/δ-inhibited cells which was rescued by concurrent caffeine treatment. Caffeine-treated cells also displayed elevated peak oxidative metabolism which was partially abolished following PPARβ/δ inhibition. Similar to past observations, glucose uptake and GLUT4 content were elevated in caffeine-treated cells, however, glycolytic metabolism was unaltered following caffeine treatment. Physiological levels of caffeine appear to enhance cell metabolism through mechanisms partially dependent on PPARβ/δ.
Calcium ions that have been preloaded into isolated sarcoplasmic reticulum subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances. We report here that not only caffeine, but also Ca2+ ions, thymol, quercetin, menthol, halothane, chloroform, 1-ethyl-2-methylbenzimidazole, ryanodine, tetraphenylboron, ketoconazole, miconazole, clotrimazole, W-7, doxorubicin, 5,5'-dithiobis-(2-nitrobenzoic acid), p-chloromercuribenzoic acid, and low concentrations of Ag+ induce Ca2+ release from such triadic sarcoplasmic reticulum. All these drugs induce increased undirectional Ca2+ efflux. We believe all these drug-induced Ca2+ releases are mediated by Ca2+ efflux through the same ion channel since these releases are all greatly attenuated when light sarcoplasmic reticulum is substituted for triads and are even more pronounced when transverse tubule-free terminal cisternae are substituted for triads, and all these forms of drug-induced Ca2+ release are inhibited by submicromolar concentrations of ruthenium red, and by submillimolar concentrations of tetracaine, 9-aminoacridine, and Ba2+, yet they are not affected by nifedipine even at a concentration of 50 microM.
We have previously demonstrated that cyclic strain induces extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in endothelial cells (EC). The aim of this study was to investigate the effect of Ca2+ on the activation of ERK1/2. Bovine aortic EC were pretreated with a chelator of extracellular Ca2+, ethylaneglycol-bis(aminoethylether)-tetra-acetate (EGTA), a depleter of Ca2+ pools, 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ), or a Ca2+ channel blocker, GdCl3, and subjected to an average 10 % strain at a rate of 60 cycles/min for 10 min. BHQ and GdCl3 did not inhibit the strain-induced ERK1/2 activation. Chelation of normal extracellular Ca2+ (1.8 mM) medium with EGTA (3 mM) acutely stimulated baseline phosphorylation and activation of ERK1/2, thereby obscuring any strain-induced activation of ERK1/2. However, in EC preincubated for 24 hours in Ca2+-free medium, elevated baseline phosphorylation was minimally activated by EGTA (200 microM) such that cyclic strain stimulated ERK1/2 in the presence or absence of BHQ. These results suggest a Ca2+ independence of the ERK1/2 signaling pathway by cyclic strain.
Ca2+/calmodulin (CaM)-dependent protein kinase (CaMKII) is a ubiquitous mediator of Ca2+-linked signalling that phosphorylates a wide range of substrates to co-ordinate and regulate Ca2+-mediated alterations in cellular function. The transmission of information by the kinase from extracellular stimuli and the intracellular Ca2+ rise is not passive. Rather, its multimeric structure and autoregulation enable this enzyme to participate actively in the sensitivity, timing and location of its action. CaMKII can: (i) be activated in a Ca2+-spike frequency-dependent manner; (ii) become independent of its initial Ca2+/CaM activators; and (iii) undergo a 'molecular switch-like' behaviour, which is crucial for certain forms of learning and memory. CaMKII is derived from a family of four homologous but distinct genes, with over 30 alternatively spliced isoforms described at present. These isoforms possess diverse developmental and anatomical expression patterns, as well as subcellular localization. Six independent catalytic/autoregulatory domains are connected by a narrow stalk-like appendage to each hexameric ring within the dodecameric structure. Ca2+/CaM binding activates the enzyme by disinhibiting the autoregulatory domain; this process initiates an intra-holoenzyme autophosphorylation reaction that induces complex changes in the enzyme's sensitivity to Ca2+/CaM, including the generation of Ca2+/CaM-independent (autonomous) activity and marked increase in affinity for CaM. The role of CaMKII in Ca2+ signal transduction is shaped by its autoregulation, isoenzymic type and subcellular localization. The molecular determinants and mechanisms producing these processes are discussed as they relate to the structure-function of this multifunctional protein kinase.
Physiological hyperglycemia with hyperinsulinemia reduces fat oxidation in skeletal muscle. The mechanism responsible for this decrease in fat oxidation in human muscle is not known and may contribute to the development of insulin resistance. We hypothesized that the transfer of long-chain fatty acids (LCFAs) into the mitochondria via carnitine palmitoyltransferase-1 (CPT-1) is inhibited by increased malonyl coenzyme A (malonyl-CoA) (a known potent inhibitor of CPT-1) in human muscle during hyperglycemia with hyperinsulinemia. We studied six healthy subjects after an overnight fast and during an induced 5-hour period of hyperglycemia with hyperinsulinemia. Muscle fatty acid oxidation was calculated using stable isotope methodology combined with blood sampling from the femoral artery and vein of one leg. Muscle functional CPT-1 activity was assessed by concurrently infusing an LCFA tracer and a CPT-independent medium-chain fatty acid tracer. Muscle biopsies were obtained from the vastus lateralis after the periods of fasting and hyperglycemia with hyperinsulinemia. Hyperglycemia with hyperinsulinemia decreased LCFA oxidation, but had no effect on LCFA uptake or medium-chain fatty acid oxidation across the leg. Malonyl-CoA concentration significantly increased from 0.13 +/- 0.01 to 0.35 +/- 0.07 nmol/g during hyperglycemia with hyperinsulinemia. We conclude that hyperglycemia with hyperinsulinemia increases malonyl-CoA, inhibits functional CPT-1 activity, and shunts LCFA away from oxidation and toward storage in human muscle.
The purpose of the present investigation was to establish a method for estimating intracellular Ca(2+) concentrations ([Ca(2+)](i)) in isolated rat epitrochlearis muscles. Epitrochlearis muscles excised from 4-wk-old male Sprague-Dawley rats were loaded with a fluorescent Ca(2+) indicator, fura 2-AM, for 60-90 min at 35 degrees C in oxygenated Krebs-Henseleit buffer. After fura 2 loading and subsequent 20-min incubation, the intensities of 500-nm fluorescence, induced by 340- and 380-nm excitation lights (F(total)340 and F(total)380), were measured. The fluorescences specific to fura-2 (F(fura 2)340 and F(fura 2)380) were calculated by subtracting the non-fura 2-specific component from F(total)340 and F(total)380, respectively. The ratio of F(fura 2)340 to F(fura 2)380 was calculated as R, and the change in the ratio from the baseline value (DeltaR) was used as an index of the change in [Ca(2+)](i). In resting muscle, DeltaR was stable for 60 min. Incubation for 20 min with caffeine (3-10 mM) significantly increased DeltaR in a concentration-dependent manner. Incubation with hypoxic Krebs-Henseleit buffer for 10-60 min significantly elevated DeltaR, depending on the duration of the incubation. Incubation with 50 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide for 20 min significantly elevated DeltaR (P < 0.05). No significant increases in DeltaR were observed during incubation for 20 min with 2 mM 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside or with 2 mU/ml insulin. These results demonstrated that, by using the fura 2-AM fluorescence method, the changes in [Ca(2+)](i) can be monitored in the rat epitrochlearis muscle and suggest that the method can be utilized to observe quantitative information regarding [Ca(2+)](i) that may be involved in contraction- and hypoxia-stimulated glucose transport activity in skeletal muscle.
Cyclopiazonic acid (CPA) is a sarcoplasmic reticulum Ca2+-ATPase inhibitor that increases intracellular calcium. The role of CPA in regulating the oxidation and esterification of palmitate, the hydrolysis of intramuscular lipids, and the activation of hormone-sensitive lipase (HSL) was examined in isolated rat soleus muscles at rest. CPA (40 micro M) was added to the incubation medium to levels that resulted in subcontraction increases in muscle tension, and lipid metabolism was monitored using the previously described pulse-chase procedure. CPA did not alter the cellular energy state, as reflected by similar muscle contents of ATP, phosphocreatine, free AMP, and free ADP. CPA increased total palmitate uptake into soleus muscle (11%, P < 0.05) and was without effect on palmitate oxidation. This resulted in greater esterification of exogenous palmitate into the triacylglycerol (18%, P < 0.05) and phospholipid (89%, P < 0.05) pools. CPA decreased (P < 0.05) intramuscular lipid hydrolysis, and this occurred as a result of reduced HSL activity (20%, P < 0.05). Incubation of muscles with 3 mM caffeine, which is also known to increase Ca2+ without affecting the cellular energy state, reduced HSL activity (24%, P < 0.05). KN-93, a calcium/calmodulin-dependent kinase inhibitor (CaMKII), blocked the effects of CPA and caffeine, and HSL activity returned to preincubation values. The results of the present study demonstrate that CPA simultaneously decreases intramuscular triacylglycerol (IMTG) hydrolysis and promotes lipid storage in isolated, intact soleus muscle. The decreased IMTG hydrolysis is likely mediated by reduced HSL activity, possibly via the CaMKII pathway. These responses are not consistent with the increased hydrolysis and decreased esterification observed in contracting muscle when substrate availability and the hormonal milieu are tightly controlled. It is possible that more powerful signals or a higher [Ca2+] may override the lipid-storage effect of the CPA-mediated effects during muscular contractions.
We have previously shown that thrombin-induced endothelial cell barrier dysfunction involves cytoskeletal rearrangement and contraction, and we have elucidated the important role of endothelial cell myosin light chain kinase and the actin- and myosin-binding protein caldesmon. We evaluated the contribution of calmodulin (CaM) kinase II and extracellular signal-regulated kinase (ERK) activation in thrombin-mediated bovine pulmonary artery endothelial cell contraction and barrier dysfunction. Similar to thrombin, infection with a constitutively active adenoviral alpha-CaM kinase II construct induced significant ERK activation, indicating that CaM kinase II activation lies upstream of ERK. Thrombin-induced ERK-dependent caldesmon phosphorylation (Ser789) was inhibited by either KN-93, a specific CaM kinase II inhibitor, or U0126, an inhibitor of MEK activation. Immunofluorescence microscopy studies revealed phosphocaldesmon colocalization within thrombin-induced actin stress fibers. Pretreatment with either U0126 or KN-93 attenuated thrombin-mediated cytoskeletal rearrangement and evoked declines in transendothelial electrical resistance while reversing thrombin-induced dissociation of myosin from nondenaturing caldesmon immunoprecipitates. These results strongly suggest the involvement of CaM kinase II and ERK activities in thrombin-mediated caldesmon phosphorylation and both contractile and barrier regulation.
Neurons in the ventromedial hypothalamus mediate some counterregulatory responses to hypoglycemia and 2-deoxyglucose, but the mechanisms that mediate these responses to glucose are unclear. In the present study, ventromedial hypothalamus neurons were identified on the basis of their inhibition by the transition from 5 to 20 mmol/l glucose. Tolbutamide, which activates glucose-stimulated neurons, failed to inhibit or activate glucose-inhibited neurons. Inhibitors of glucose transport and glycolysis, in particular by the glucokinase inhibitor glucosamine, blocked the effect of glucose on glucose-inhibited neurons. Furthermore, the glucose-inhibited neurons were activated by 2-deoxyglucose, which also activates counterregulatory responses. Conversely, glucose-inhibited neurons were inhibited by glycolytic metabolites, including lactate, but not by pyruvate. These data indicate that hypoglycemia induces electrical activity in glucose-inhibited neurons by attenuating glycolysis in those neurons. Thus, counterregulatory failure could be due to relatively enhanced glycolysis in glucose-stimulated neurons during hypoglycemia and attenuation of glycolysis in glucose-inhibited neurons might reverse counterregulatory failure.
It is now generally accepted that activation of AMP-activated protein kinase (AMPK) is involved in the stimulation of glucose transport by muscle contractions. However, earlier studies provided evidence that increases in cytosolic Ca(2+) mediate the effect of muscle contractions on glucose transport. The purpose of this study was to test the hypothesis that both the increase in cytosolic Ca(2+) and the activation of AMPK are involved in the stimulation of glucose transport by muscle contractions. Caffeine causes release of Ca(2+) from the sarcoplasmic reticulum. Incubation of rat epitrochlearis muscles with a concentration of caffeine that raises cytosolic Ca(2+) to levels too low to cause contraction resulted in an approximate threefold increase in glucose transport. Caffeine treatment also resulted in increased phosphorylation of calmodulin-dependent protein kinase (CAMK)-II in epitrochlearis muscle. The stimulation of glucose transport by caffeine was blocked by the Ca(2+)-CAMK inhibitors KN62 and KN93. Activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) also resulted in an approximate threefold increase in glucose transport in the epitrochlearis. The increases in glucose transport induced by AICAR and caffeine were additive, and their combined effect was not significantly different from that induced by maximally effective contractile activity. KN62 and KN93 caused an approximately 50% inhibition of the stimulation of glucose transport by contractile activity. Our results provide evidence that both Ca(2+) and AMPK are involved in the stimulation of glucose transport by muscle contractions. They also suggest that the stimulation of glucose transport by Ca(2+) involves activation of CAMK.
Cytochrome c expression and mitochondrial biogenesis can be invoked by elevated intracellular Ca(2+) in muscle cells. To characterize the potential role of Ca(2+) as a messenger involved in mitochondrial biogenesis in muscle, we determined the effects of the Ca(2+) ionophore A-23187 on the expression of nuclear- and mitochondrially encoded genes. Treatment of myotubes with 1 microM A-23187 for 48-96 h increased nuclear-encoded beta-subunit F(1)ATPase and malate dehydrogenase (MDH) mRNA levels by 50-100% (P < 0.05) but decreased mRNA levels of glutamate dehydrogenase (GDH) by 19% (P < 0.05). mRNA levels of the cytochrome c oxidase (COX) nuclear-encoded subunits IV, Vb, and VIc were unchanged, whereas the mitochondrially encoded subunits COX II and COX III were decreased by 30 and 70%, respectively (P < 0.05). This was paralleled by a 20% decrease (P < 0.05) in COX activity. These data suggest that cytoplasmic Ca(2+) differentially regulates the mRNA level of nuclear and mitochondrial genes. The decline in COX II and III mRNA may be mediated by Tfam, because A-23187 modestly reduced Tfam levels by 48 h. A-23187 induced time-dependent increases in Egr-1 mRNA, along with the activation of ERK1/2 and AMP-activated protein kinase. MEK inhibition with PD-98059 attenuated the increase in Egr-1 mRNA. A-23187 also increased Egr-1, serum response factor, and Sp1 protein expression, transcription factors implicated in mitochondrial biogenesis. Egr-1 overexpression increased nuclear-encoded cytochrome c transcriptional activation by 1.5-fold (P < 0.05) and reduced GDH mRNA by 37% (P < 0.05) but had no effect on MDH or beta-subunit F(1)ATPase mRNA. These results indicate that changes in intracellular Ca(2+) can modify mitochondrial phenotype, in part via the involvement of Egr-1.
Intracellular mechanisms regulating fat oxidation were investigated in human skeletal muscle during exercise. Eight young, healthy, moderately trained men performed bicycle exercise (60 min, 65% peak O2 consumption) on two occasions, where they ingested either 1) a high-carbohydrate diet (H-CHO) or 2) a low-carbohydrate diet (L-CHO) before exercise to alter muscle glycogen content as well as to induce, respectively, low and high rates of fat oxidation. Leg fat oxidation was 122% higher during exercise in L-CHO than in H-CHO (P < 0.001). In keeping with this, the activity of alpha2-AMP-activated protein kinase (alpha2-AMPK) was increased twice as much in L-CHO as in H-CHO (P < 0.01) at 60 min of exercise. However, acetyl-CoA carboxylase (ACC)beta Ser221 phosphorylation was increased to the same extent (6-fold) under the two conditions. The concentration of malonyl-CoA was reduced 13% by exercise in both conditions (P < 0.05). Pyruvate dehydrogenase activity was higher during exercise in H-CHO than in L-CHO (P < 0.01). In H-CHO only, the concentrations of acetyl-CoA and acetylcarnitine were increased (P < 0.001), and the concentration of free carnitine was decreased (P < 0.01), by exercise. The data suggest that a decrease in the concentration of malonyl-CoA, secondary to alpha2-AMPK activation and ACC inhibition (by phosphorylation), contributes to the increase in fat oxidation observed at the onset of exercise regardless of muscle glycogen levels. They also suggest that, with high muscle glycogen, the availability of free carnitine may limit fat oxidation during exercise, due to its increased use for acetylcarnitine formation.
Stimulation of AMP-activated protein kinase (AMPK) in skeletal muscle and liver is seen as an exciting prospect for the treatment of type 2 diabetes. However, we have recently demonstrated that changes in AMPK activity accompany the exposure of pancreatic islet beta-cells to elevated glucose concentrations and may be involved in the activation of insulin secretion. Here, we discuss this hypothesis and explore the potential role of changes in AMPK activity in the actions of other secretagogues. Amino acids decreased AMPK activity in MIN6 beta-cells with an order of potency for inhibition: arg=leu < gln= leu + glu < glucose, which was closely correlated with the stimulation of insulin release (r2=0.76). By contrast, increases in intracellular Ca2+ concentration provoked by cell depolarization with KCl activated AMPK in the face of increased free intracellular ATP concentrations. Elevation of intracellular cAMP levels with isobutylmethylxanthine or forskolin had no effect on AMPK activity. We conclude that metabolizable amino acids regulate AMPK in the beta-cell via increases in the cytosolic ATP/AMP ratio and via phosphorylation by the upstream kinase LKB1. Intracellular Ca2+ ions may activate AMPK by calmodulin kinase 1 kinase-mediated phosphorylation. The latter may act as a novel feedback mechanism to inhibit excessive insulin secretion under some circumstances.
The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKalphaThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs. We have employed three cell lines deficient in LKB1 to study AMPK regulation and phosphorylation, HeLa, A549, and murine embryo fibroblasts derived from LKB(-/-) mice. In HeLa and A549 cells, mannitol, 2-deoxyglucose, and ionomycin, but not 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), treatment activates AMPK by alphaThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase, are largely inhibited by the Ca(2+)/ calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609. AMPKK activity in HeLa cell lysates measured in vitro is totally inhibited by STO-609 with an IC50 comparable with that of the known CaMKK isoforms, CaMKKalpha and CaMKKbeta. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, alphaThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKalpha and CaMKKbeta. Lastly, the activation of AMPK in response to ionomycin and 2-deoxyglucose is not impaired in LKB1(-/-) murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo.
AMP-activated protein kinase (AMPK) is the downstream component of a kinase cascade that plays a pivotal role in energy homeostasis. Activation of AMPK requires phosphorylation of threonine 172 (T172) within the T loop region of the catalytic alpha subunit. Recently, LKB1 was shown to activate AMPK. Here we show that AMPK is also activated by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK). Overexpression of CaMKKbeta in mammalian cells increases AMPK activity, whereas pharmacological inhibition of CaMKK, or downregulation of CaMKKbeta using RNA interference, almost completely abolishes AMPK activation. CaMKKbeta isolated from rat brain or expressed in E. coli phosphorylates and activates AMPK in vitro. In yeast, CaMKKbeta expression rescues a mutant strain lacking the three kinases upstream of Snf1, the yeast homolog of AMPK. These results demonstrate that AMPK is regulated by at least two upstream kinases and suggest that AMPK may play a role in Ca(2+)-mediated signal transduction pathways.
AMP-activated protein kinase (AMPK), which functions as a sensor of cellular energy homeostasis, was phosphorylated after norepinephrine stimulation in L6 skeletal muscle cells. This effect was mediated by alpha1-adrenoceptors, with no stimulatory effects due to interactions at alpha2- or beta-adrenoceptors. Alpha1-adrenoceptors are Gq-coupled receptors, and calcium but not phorbol esters could mimic the effect of alpha1-adrenergic stimulation; and we show that protein kinase C is not involved as an upstream signal to AMPK by alpha1-adrenergic stimulation and that the AMP-to-ATP ratio is unaltered after alpha1-adrenergic stimulation. We further show that glucose uptake mediated by alpha1- but not by beta-adrenoceptors can be inhibited by AMPK inhibition. Acetyl-CoA carboxylase (ACC) is phosphorylated at Ser218 by AMPK, and alpha1- but not beta-adrenoceptor stimulation results in phosphorylation of ACC at this residue. These results suggest a novel pathway where alpha1-adrenoceptor activation, independent of protein kinase C, leads to activation of AMPK in skeletal muscle, which contributes to alpha1-adrenoceptor-mediated increases in glucose uptake.
The Ca(2+)/calmodulin (CaM) competitive inhibitor KN-93 has previously been used to evaluate 5'-AMP-activated protein kinase (AMPK)-independent Ca(2+)-signaling to contraction-stimulated glucose uptake in muscle during intense electrical stimulation ex vivo. With the use of low-intensity tetanic contraction of mouse soleus and extensor digitorum longus (EDL) muscles ex vivo, this study demonstrates that KN-93 can potently inhibit AMPK phosphorylation and activity after 2 min but not 10 min of contraction while strongly inhibiting contraction-stimulated 2-deoxyglucose uptake at both the 2- and 10-min time points. These data suggest inhibition of Ca(2+)/CaM-dependent signaling events upstream of AMPK, the most likely candidate being the novel AMPK kinase CaM-dependent protein kinase kinase (CaMKK). CaMKK protein expression was detected in mouse skeletal muscle. Similar to KN-93, the CaMKK inhibitor STO-609 strongly reduced AMPK phosphorylation and activity at 2 min and less potently at 10 min. Pretreatment with STO-609 inhibited contraction-stimulated glucose uptake at 2 min in soleus, but not EDL, and in both muscles after 10 min. Neither KN-93 nor STO-609 inhibited 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside-stimulated glucose uptake, AMPK phosphorylation, or recombinant LKB1 activity, suggestive of an LKB1-independent effect. Finally, neither KN-93 nor STO-609 had effects on the reductions in glucose uptake seen in mice overexpressing a kinase-dead AMPK construct, indicating that the effects of KN-93 and STO-609 on glucose uptake require inhibition of AMPK activity. We propose that CaMKKs act in mouse skeletal muscle regulating AMPK phosphorylation and glucose uptake at the onset of mild tetanic contraction and that an intensity- and/or time-dependent switch occurs in the relative importance of AMPKKs during contraction.
Previous studies have proposed that caffeine-induced activation of glucose transport in skeletal muscle is independent of AMP-activated protein kinase (AMPK) because alpha-AMPK Thr172 phosphorylation was not increased by caffeine. However, our previous studies, as well as the present, show that AMPK phosphorylation measured in whole muscle lysate is not a good indicator of AMPK activation in rodent skeletal muscle. In lysates from incubated rat soleus muscle, a predominant model in previous caffeine-studies, both acetyl-CoA carboxylase-beta (ACCbeta) Ser221 and immunoprecipitated alpha(1)-AMPK activity increased with caffeine incubation, without changes in AMPK phosphorylation or immunoprecipitated alpha(2)-AMPK activity. This pattern was also observed in mouse soleus muscle, where only ACCbeta and alpha(1)-AMPK phosphorylation were increased following caffeine treatment. Preincubation with the selective CaMKK inhibitor STO-609 (5 microM), the CaM-competitive inhibitor KN-93 (10 microM), or the SR Ca(2+) release blocking agent dantrolene (10 microM) all inhibited ACCbeta phosphorylation and alpha(1)-AMPK phosphorylation, suggesting that SR Ca(2+) release may work through a CaMKK-AMPK pathway. Caffeine-stimulated 2-deoxyglucose (2DG) uptake reflected the AMPK activation pattern, being increased with caffeine and inhibited by STO-609, KN-93, or dantrolene. The inhibition of 2DG uptake is likely causally linked to AMPK activation, since muscle-specific expression of a kinase-dead AMPK construct greatly reduced caffeine-stimulated 2DG uptake in mouse soleus. We conclude that a SR Ca(2+)-activated CaMKK may control alpha(1)-AMPK activation and be necessary for caffeine-stimulated glucose uptake in mouse soleus muscle.
Previous studies have shown that raising cytosolic calcium in myotubes induces increases in peroxisome proliferator-activated receptor gamma coactivator-1alpha expression and mitochondrial biogenesis. This finding suggests that the increases in cytosolic calcium in skeletal muscle during exercise may mediate the exercise-induced increase in mitochondria. The initial aim of this study was to determine whether raising calcium in skeletal muscle induces the same adaptations as in myotubes. We found that treatment of rat epitrochlearis muscles with a concentration of caffeine that raises cytosolic calcium to a concentration too low to cause contraction induces increases in peroxisome proliferator-activated receptor gamma coactivator-1alpha expression and mitochondrial biogenesis. Our second aim was to elucidate the pathway by which calcium induces these adaptations. Raising cytosolic calcium has been shown to activate calcium/calmodulin-dependent protein kinase in muscle. In the present study raising cytosolic calcium resulted in increases in phosphorylation of p38 mitogen-activated protein kinase and activating transcription factor-2, which were blocked by the calcium/calmodulin-dependent protein kinase inhibitor KN93 and by the p38 mitogen-activated protein kinase inhibitor SB202190. The increases in peroxisome proliferator-activated receptor gamma coactivator-1alpha expression and mitochondrial biogenesis were also prevented by inhibiting p38 activation. We interpret these findings as evidence that p38 mitogen-activated protein kinase is downstream of calcium/calmodulin-dependent protein kinase in a signaling pathway by which increases in cytosolic calcium lead to increases in peroxisome proliferator-activated receptor gamma coactivator-1alpha expression and mitochondrial biogenesis in muscle.
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.
We reported that one of the isoquinolinesulfonamide derivatives, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M. and Hidaka, H. (1990) J. Biol. Chem. 265, 4315-4320). We have now investigated the inhibitory property of a newly synthesized methoxybenzenesulfonamide, KN-93, on CaMKII activity in situ and in vitro. KN-93 elicited potent inhibitory effects on CaMKII phosphorylating activity with an inhibition constant of 0.37 microM but this compound had no significant effects on the catalytic activity of cAMP-dependent protein kinase, Ca2+/phospholipid dependent protein kinase, myosin light chain kinase and Ca(2+)-phosphodiesterase. KN-93 also inhibited the autophosphorylation of both the alpha- and beta-subunits of CaMKII. Kinetic analysis indicated that KN-93 inhibits CaMKII, in a competitive fashion against calmodulin. To evaluate the regulatory role of CaMKII on catecholamine metabolism, we examined the effect of KN-93 on dopamine (DA) levels in PC12h cells. The DA levels decreased in the presence of KN-93. Further, the tyrosine hydroxylase (TH) phosphorylation induced by KCl or acetylcholine was significantly suppressed by KN-93 in PC12h cells while events induced by forskolin or 8-Br-cAMP were not affected. These results suggest that KN-93 inhibits DA formation by modulating the reaction rate of TH to reduce the Ca(2+)-mediated phosphorylation levels of the TH molecule.
In this study we investigated the possibility that an increase in cytoplasmic Ca2+ concentration that is too low to cause muscle contraction can induce an increase in glucose transport activity in skeletal muscle. The compound N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which induces Ca2+ release from the sarcoplasmic reticulum (SR), caused a dose-dependent increase in tension in rat epitrochlearis muscles at concentrations more than approximately 200 microM. Although 100 microM W-7 did not increase muscle tension, it accelerated loss of preloaded 45Ca2+. Glucose transport activity, measured with the nonmetabolizable glucose analogue 3-O-methylglucose, increased sixfold in muscles treated for 100 min with 50 microM W-7 (P less than 0.001) and eightfold in response to 100 microM W-7 (P less than 0.001). The increase in glucose transport activity was completely blocked with 25 microM cytochalasin B. There was no decrease in ATP or creatine phosphate concentrations ([approximately P]) in muscles incubated with 50 microM W-7. Dantrolene (25 microM), which blocks Ca2+ release from the SR, blocked the effects of W-7 both on 45Ca2+ release and on glucose transport activity. 9-Aminoacridine, another inhibitor of Ca2+ release from the SR, also blocked the stimulation of hexose transport by W-7. Caffeine, a compound structurally unrelated to W-7 that also releases Ca2+ from the SR, also increased glucose transport activity. Incubation of muscles with 3 mM caffeine for 30 min, which did not cause contraction or lower [approximately P], induced a threefold increase in 3-O-methylglucose transport (P less than 0.001). These results provide evidence suggesting that an increase in cytoplasmic Ca2+ too low to cause contraction or [approximately P] depletion can bring about an increase in glucose transport activity in skeletal muscle.
The regulation of acetyl-CoA carboxylase and malonyl-CoA levels in skeletal muscle may involve a calcium-dependent mechanism. To examine the effects of increased free sarcoplasmic calcium concentrations on malonyl-CoA in skeletal muscle, isolated hindlimbs of rats were perfused for 30 min with a medium containing bovine red blood cells, bovine serum albumin, 200 microU/ml insulin, and 10 mM glucose in Krebs-Henseleit buffer and caffeine at 0, 0.12, 0.5, or 3 mM. Malonyl-CoA decreased from control (no caffeine) values of 1.34 +/- 0.9 to 0.95 +/- 0.12 pmol/mg in gastrocnemius-plantaris muscles perfused with 0.12 and 0.5 mM caffeine and to 0.63 +/- 0.07 pmol/mg in the muscles perfused with 3 mM caffeine. Adenosine 3',5'-cyclic monophosphate (cAMP) increased from 0.24 +/- 0.02 to 0.32 +/- 0.04 nmol/g, and AMP decreased from 83 +/- 8 to 53 +/- 3 nmol/g in response to 3 mM caffeine. Citrate and ATP were unaffected by caffeine. A decline in malonyl-CoA with 0.12 and 0.5 mM caffeine without an increase in cAMP supports the hypothesis that a calcium-dependent mechanisms of acetyl-CoA carboxylase and malonyl-CoA regulation exists, but a cAMP-dependent mechanism may also be involved with 3 mM caffeine.
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.
5-Aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR) is taken up by perfused skeletal muscle and phosphorylated to form 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuraosyl-5'-monopho sph ate (analog of 5'-AMP) with consequent activation of AMP-activated protein kinase, phosphorylation of acetyl-CoA carboxylase, decrease in malonyl-CoA, and increase in fatty acid oxidation. This study was designed to determine the effect of increasing levels of palmitate on the rate of fatty acid oxidation. Malonyl-CoA concentration was manipulated with AICAR at different palmitate concentrations. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine red cells, 200 microU/ml insulin, 10 mM glucose, and different concentrations of palmitate (0. 1-1.0 mM) without or with AICAR (2.0 mM). Perfusion with medium containing AICAR was found to activate AMP-activated protein kinase in skeletal muscle, inactivate acetyl-CoA carboxylase, and decrease malonyl-CoA at all concentrations of palmitate. The rate of palmitate oxidation increased as a function of palmitate concentration in both the presence and absence of AICAR but was always higher in the presence of AICAR. These results provide additional evidence that malonyl-CoA is an important regulator of the rate of fatty acid oxidation at palmitate concentrations in the physiological range.
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.
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.
To determine whether changes in long-chain fatty acid (LCFA) oxidative metabolism induced by elevated intracellular carbohydrate availability are due to changes in LCFA uptake or in mitochondrial transport capacity, rat hindquarters were perfused with 500 microM palmitate and [1-14C]palmitate or [1-14C]octanoate as well as with either low (LG) or high (HG) carbohydrate availability. Glucose uptake was higher in the HG vs. LG group (23.6 +/- 1.5 vs 4.7 +/- 0.9 micromol x g(-1) x h(-1), P < 0.05). Palmitate delivery was not significantly different between groups and averaged 97.1 +/- 4.6 nmol x min(-1) x g(-1) (P > 0.05). Fractional and total palmitate uptake values were 60% higher (P < 0.05) in the HG (0.125 +/- 0.012 and 7.4 +/- 1.2 nmol x min(-1) x g(-1)) vs. LG (0.079 +/- 0.009 and 11.8 +/- 1.5 nmol x min(-1) x g(-1)) group. Values of percent and total palmitate oxidized were significantly lower (P < 0.05) in the HG (9.1 +/- 1.1% and 1.31 +/- 0.16 nmol x min(-1) x g(-1)) vs. LG (23.4 +/- 5.2% and 0.76 +/- 0.08 nmol x min(-1) x g(-1)) group. Conversely, values of fractional uptake and percent oxidation of octanoate were not significantly different between groups (P > 0.05). Malonyl-CoA levels were inversely correlated with LCFA oxidation (P < 0.05). These results demonstrate that high carbohydrate availability in muscle is associated with a decrease in LCFA oxidation that is not due to a parallel decrease in LCFA uptake; rather, the decrease in LCFA oxidation could be due to malonyl-CoA inhibition of mitochondrial LCFA transport.
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.
Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca2+ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca2+ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro, but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca2+] ([Ca2+]i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca2+]i by ~25 %, but had no significant effect on the rate of SR Ca2+ uptake or the force-[Ca2+]i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca2+]i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca2+ release in the basal state and during repeated contractions, providing a positive feedback between [Ca2+]i and SR Ca2+ release.
Exercise enhances insulin-stimulated glucose transport (GT) in skeletal muscle. Evidence suggests that 5' AMP-activated protein kinase (AMPK) and glycogen may be important for enhanced insulin sensitivity. Our goals were to investigate the effect of various in situ muscle contraction protocols on insulin-stimulated GT and assess the relationship of contraction-induced changes in AMPK and glycogen with postcontraction improvement in insulin-stimulated GT. Rats were anesthetized, both ulnar nerves were exposed, and one nerve was electrically stimulated to contract forelimb muscles. We performed a series of five experiments, sequentially varying only one contraction parameter (train duration, train rate, pulse frequency, number of 5-min bouts, or pulse duration) while holding the others constant. Both epitrochlearis muscles were dissected out and incubated for 3.5 h before measurement of GT. For each contraction parameter studied, we identified an apparent threshold value that did not induce a significant increase in insulin-stimulated GT and an apparent peak value, above which there was a plateau or decline in insulin-stimulated GT. Using other rats, we evaluated muscle AMPK phosphorylation and glycogen concentration immediately postcontraction. AMPK phosphorylation and reduction in glycogen were increased compared with resting controls in each protocol, which had previously been shown to increase insulin-stimulated GT, as well as in several protocols that did not significantly increase insulin-stimulated GT. These data suggest that contraction-induced AMPK phosphorylation and decrease in glycogen may be necessary but are not sufficient for the postcontraction increase in insulin-stimulated GT in rat skeletal muscle.
There is evidence in rodents that Ca2+-calmodulin-dependent protein kinase II (CaMKII) activity is higher in contracting skeletal muscle, and this kinase may regulate skeletal muscle function and metabolism during exercise. To investigate the effect of exercise on CaMKII in human skeletal muscle, healthy men (n = 8) performed cycle ergometer exercise for 40 min at 76 +/- 1% peak pulmonary O2 uptake (VO2peak), with skeletal muscle samples taken at rest and after 5 and 40 min of exercise. CaMKII expression and activities were examined by immunoblotting and in vitro kinase assays, respectively. There were no differences in maximal (+ Ca2+, CaM) CaMKII activity during exercise compared with rest. Autonomous (- Ca2+, CaM) CaMKII activity was 9 +/- 1% of maximal at rest, remained unchanged at 5 min, and increased to 17 +/- 1% (P < 0.01) at 40 min. CaMKII autophosphorylation at Thr287 was 50-70% higher during exercise, with no differences in CaMKII expression. The effect of maximal aerobic exercise on CaMKII was also examined (n = 9), with 0.7- to 1.5-fold increases in autonomous CaMKII activity, but no change in maximal CaMKII activity. CaMKIV was not detected in human skeletal muscle. In summary, exercise increases the activity of CaMKII in skeletal muscle, suggesting that it may have a role in regulating skeletal muscle function and metabolism during exercise in humans.
Previous research has shown that the CAMK (calcium/calmodulin dependent protein kinase) inhibitor, KN62, can lead to reductions in insulin stimulated glucose transport. Although controversial, an L-type calcium channel mechanism has also been hypothesized to be involved in insulin stimulated glucose transport. The purpose of this report was to determine if 1) L-type calcium channels and CAMK are involved in a similar signaling pathway in the control of insulin stimulated glucose transport and 2) determine if insulin induces an increase in CAMKII phosphorylation through an L-type calcium channel dependent mechanism. Insulin stimulated glucose transport was significantly (p<0.05) inhibited to a similar extent ( approximately 30%) by both KN62 and nifedipine in rat soleus and epitrochelaris muscles. The new finding of these experiments was that the combined inhibitory effect of these two compounds was not greater than the effect of either inhibitor alone. To more accurately determine the interaction between CAMK and L-type calcium channels, we measured insulin induced changes in CAMKII phosphorylation using Western blot analysis. The novel finding of this set of experiments was that insulin induced an increase in phosphorylated CAMKII ( approximately 40%) in rat soleus muscle that was reversed in the presence of KN62 but not nifedipine. Taken together these results suggest that a CAMK signaling mechanism may be involved in insulin stimulated glucose transport in skeletal muscle through an L-type calcium channel independent mechanism.
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.
Increases in contraction-stimulated glucose transport in fast-twitch rat epitrochlearis muscle are mediated by AMPK- and Ca2+/calmodulin-dependent protein kinase (CAMK)-dependent signaling pathways. However, recent studies provide evidence suggesting that contraction-stimulated glucose transport in slow-twitch skeletal muscle is mediated through an AMPK-independent pathway. The purpose of the present study was to test the hypothesis that contraction-stimulated glucose transport in rat slow-twitch soleus muscle is mediated by an AMPK-independent/Ca2+-dependent pathway. Caffeine, a sarcoplasmic reticulum (SR) Ca2+-releasing agent, at a concentration that does not cause muscle contractions or decreases in high-energy phosphates, led to an approximately 2-fold increase in 2-deoxyglucose (2-DG) uptake in isolated split soleus muscles. This increase in glucose transport was prevented by the SR calcium channel blocker dantrolene and the CAMK inhibitor KN93. Conversely, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an AMPK activator, had no effect on 2-DG uptake in isolated split soleus muscles yet resulted in an approximately 2-fold increase in the phosphorylation of AMPK and its downstream substrate acetyl-CoA carboxylase. The hypoxia-induced increase in 2-DG uptake was prevented by dantrolene and KN93, whereas hypoxia-stimulated phosphorylation of AMPK was unaltered by these agents. Tetanic muscle contractions resulted in an approximately 3.5-fold increase in 2-DG uptake that was prevented by KN93, which did not prevent AMPK phosphorylation. Taken in concert, our results provide evidence that hypoxia- and contraction-stimulated glucose transport is mediated entirely through a Ca2+-dependent mechanism in rat slow-twitch muscle.
The purpose of this experiment was to investigate the role of extracellular signal-regulated kinase 1/2 (ERK1/2) signalling in the contraction-induced increase in muscle FA uptake.
Male Wistar rats (n = 41) were randomly assigned to either a resting or stimulated group. Within each group, animals were randomly assigned to receive PD-98059, an inhibitor of MAP/ERK kinase 1/2 (MEK1/2), a kinase upstream of ERK1/2 and perfused with 550 microM palmitate, [(14)C]palmitate, 7 mM glucose, and no insulin. In the stimulated group, electrical stimulation (ES) of supramaximal trains of 100 ms was delivered every 2 s for 20 min.
ERK1/2 phosphorylation was increased by 50% (P < 0.05) during ES but the contraction-induced increase was prevented by the addition of PD-98059. Glucose uptake increased by 3.6-fold (P < 0.05) from rest to ES in muscle perfused without PD-98059 and was not affected by the addition of PD-98059 either at rest (P > 0.05) or during ES (P > 0.05). For a matched palmitate delivery, ES increased palmitate uptake by 35% (P < 0.05). PD-98059 had no effect on palmitate uptake at rest but completely abolished the increase in palmitate uptake during ES. Plasma membrane FAT/CD36 protein content was increased by 38% during ES (P < 0.05) but the contraction-induced increase was prevented by the addition of PD-98059. AMPK activity was increased by ES (P < 0.05) but was unaffected by PD-98059.
These results show for the first time that the increase in FA uptake and in plasma membrane FAT/CD36 protein content is mediated, at least in part, by the ERK1/2 signalling pathway during muscle contraction.
The AMP-activated protein kinase (AMPK) is a critical regulator of energy balance at both the cellular and whole-body levels. Two upstream kinases have been reported to activate AMPK in cell-free assays, i.e., the tumor suppressor LKB1 and calmodulin-dependent protein kinase kinase. However, evidence that this is physiologically relevant currently only exists for LKB1. We now report that there is a significant basal activity and phosphorylation of AMPK in LKB1-deficient cells that can be stimulated by Ca2+ ionophores, and studies using the CaMKK inhibitor STO-609 and isoform-specific siRNAs show that CaMKKbeta is required for this effect. CaMKKbeta also activates AMPK much more rapidly than CaMKKalpha in cell-free assays. K(+)-induced depolarization in rat cerebrocortical slices, which increases intracellular Ca2+ without disturbing cellular adenine nucleotide levels, activates AMPK, and this is blocked by STO-609. Our results suggest a potential Ca(2+)-dependent neuroprotective pathway involving phosphorylation and activation of AMPK by CaMKKbeta.
ATP is released at the neuromuscular junction to regulate development and proliferation. The sequential expression of P2X and P2Y receptors has been correlated to these effects in many species and cell lines. We have therefore investigated ATP mediated signalling in differentiated primary human skeletal muscle cells. ATP was capable to trigger Ca2+ transients in these cells via P2Y receptors which were not attributable to Ca2+ influx via P2X receptors. Instead, ATP propagated the formation of inositol phosphate (IP) with an EC50 of 21.3 microM. The Ca2+ transient provoked by ATP was abrogated roughly 75% by the phospholipase C (PLC) inhibitor, U73122. Interestingly, the ryanodine sensitive Ca2+ pool was not involved in ATP triggered Ca2+ release. On mRNA level and by a pharmacological approach we confirmed the presence of the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Substantially, ATP activated IP formation via a P2Y1 receptor. In addition, ATP elicited extracellular signal regulated kinase (ERK)1/2 phosphorylation in a time and concentration dependent manner, again mainly via P2Y1 receptors. The ATP mediated ERK1/2 phosphorylation was strictly dependent on phospholipase C and PI3 kinase activity. Importantly, ATP mediated ERK1/2 phosphorylation was Ca2+ independent. This observation was corroborated by the finding that conventional protein kinase C inhibitors did not suppress ATP triggered ERK1/2 phosphorylation. Taken together, these observations highlight the importance of ATP as a co-neurotransmitter at the neuromuscular junction via dual signalling, i.e. IP3 receptor mediated Ca2+ transients and Ca2+ insensitive phosphorylation of ERK1/2.
Ca2+ signalling is proposed to play an important role in skeletal muscle function during exercise. Here, we examined the expression of multifunctional Ca2+-calmodulin-dependent protein kinases (CaMK) in human skeletal muscle and show that CaMKII and CaMKK, but not CaMKI or CaMKIV, are expressed. Furthermore, the effect of exercise duration and intensity on skeletal muscle CaMKII activity and phosphorylation of downstream targets was examined. Eight healthy men exercised at approximately 67% of peak pulmonary O2 uptake(VO2peak) with muscle samples taken at rest and after 1, 10, 30, 60 and 90 min of exercise. Ten other men exercised for three consecutive 10 min bouts at 35%, 60% and 85% VO2peak with muscle samples taken at rest, at the end of each interval and 30 min post-exercise. There was a rapid and transient increase in autonomous CaMKII activity and CaMKII phosphorylation at Thr287 in skeletal muscle during exercise. Furthermore, the phosphorylation of phospholamban (PLN) at Thr17, which was identified as a CaMKII substrate in skeletal muscle, was rapidly (< 1 min) increased by exercise, and remained phosphorylated 5-fold above basal level during 90 min of exercise. The phosphorylation of serum response factor at Ser103, a putative CaMKII substrate, was higher after 30 min of exercise. PLN phosphorylation at Thr17 was higher with increasing exercise intensities. These data indicate that CaMKII is the major multifunctional CaMK in skeletal muscle and its activation occurs rapidly and is sustained during continuous exercise, with the activation being greater during intense exercise.
Muscle contraction activates AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK1/2), two signaling molecules involved in the regulation of muscle metabolism. The purpose of this study was to determine whether activation of AMPK and/or ERK1/2 contributes to the regulation of muscle fatty acid (FA) uptake and oxidation in contracting muscle. Rat hindquarters were perfused during rest (R) or electrical stimulation (E) of increasing intensity by manipulating train duration (E1 = 25 ms, E2 = 50 ms, E3 = 100 ms, E4 = 200 ms). For matched FA delivery, FA uptake was significantly greater than R during E1, E2, and E3 (7.8 +/- 0.7 vs. 14.4 +/- 0.3, 16.9 +/- 0.8, 15.2 +/- 0.5 nmol.min(-1).g(-1), respectively, P < 0.05), but not during E4 (8.3 +/- 0.3 nmol.min(-1).g(-1), P > 0.05). FA oxidation was significantly greater than R during E1 and E2 (1.5 +/- 0.1 vs. 2.3 +/- 0.2, 2.5 +/- 0.2 nmol.min(-1).g(-1), P < 0.05) before returning to resting levels for E3 and E4 (1.8 +/- 0.1 and 1.5 +/- 0.2 nmol.min(-1).g(-1), P > 0.05). A positive correlation was found between FA uptake and ERK1/2 phosphorylation from R to E3 (R(2) = 0.55, P < 0.05) and between FA oxidation and ERK1/2 phosphorylation from R to E2 (R(2) = 0.76, P < 0.05), correlations that were not maintained when the data for E4 and E3 and E4, respectively, were included in the analysis (R(2) = 0.04 and R(2) = 0.03, P > 0.05). A positive correlation was also found between FA uptake and FA oxidation and AMPK activity for all exercise intensities (R(2) = 0.57, R(2) = 0.65 respectively, P < 0.05). These results, in combination with previous data from our laboratory, suggest that ERK1/2 and AMPK are the predominant signaling molecules regulating FA uptake and oxidation during low- to moderate-intensity muscle contraction and during moderate- to high-intensity muscle contraction, respectively.
Data show that extracellular signal-regulated kinase 1/2 (ERK1/2) may be involved in the regulation of fatty acid (FA) uptake during muscle contraction via stimulation of CD36 translocation to the plasma membrane. The perfused hind limb model was used to determine (1) the importance of ERK1/2 signaling on contraction-induced FA uptake and (2) the effect of ERK1/2-mediated FA uptake on contraction-induced FA oxidation. We perfused rat hind limbs with 8 mmol/L glucose, 550 micromol/L palmitate, and no insulin at rest in the absence of inhibitor and during moderate-intensity electrical stimulation and dose-dependent pharmacologic inhibition of ERK1/2 using increasing concentrations of PD98059 (P1 = none, P2 = 10 micromol/L, P3 = 20 micromol/L, P4 = 50 micromol/L). Increasing PD98059 concentration resulted in a gradual decrease in contraction-induced ERK1/2 phosphorylation, and this was accompanied by a decrease in contraction-induced FA uptake (concentration required for 50% inhibition [IC(50)] = 15.8 +/- 1.6 mumol/L) and in plasma membrane CD36 content (IC(50) = 8.7 +/- 0.3 micromol/L) (P < .05). Percent FA oxidation was significantly lower in P3 and P4 compared with P1 and P2. Based on IC(50) values, FA oxidation demonstrated a greater sensitivity than FA uptake to changes in ERK1/2 phosphorylation (IC(50) = 5.4 +/- 0.3 micromol/L) (P < .05). A positive correlation was found between FA uptake and plasma membrane CD36 content (R(2) = 0.85, P < .05). Plasma membrane CD36 content, FA uptake, and FA oxidation each shared a positive correlation with ERK1/2 phosphorylation (R(2) = 0.64, 0.66, and 0.71, respectively; P < .05). These results suggest that during moderate-intensity muscle contraction, ERK1/2 phosphorylation is required for translocation of CD36 to the plasma membrane and the subsequent increase in FA uptake. In addition, these data suggest that ERK1/2 signaling may be involved in the regulation of FA oxidation independently of its effects on FA uptake.
The newly synthesized selective Ca 2ϩ /calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells
- M Sumi
- K Kiuchi
- T Ishikawa
- A Ishii
- M Hagiwara
- T Nagatsu
- H Hidaka
Sumi M, Kiuchi K, Ishikawa T, Ishii A, Hagiwara M, Nagatsu T,
Hidaka H. The newly synthesized selective Ca 2ϩ /calmodulin dependent
protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h
cells. Biochem Biophys Res Commun 181: 968 -975, 1991.