AKT2 is the predominant AKT isoform expressed in human skeletal muscle.

Skeletal muscle physiology and metabolism are regulated by complex networks of intracellular signaling pathways. Among many of these pathways, the protein kinase AKT plays a prominent role. While three AKT isoforms have been identified (AKT1, AKT2, and AKT3), surprisingly little is known regarding isoform-specific expression of AKT in human skeletal muscle. To address this, we examined the expressions of each AKT isoform in muscle biopsy samples collected from the vastus lateralis of healthy male adults at rest. In muscle, AKT2 was the most highly expressed AKT transcript, exhibiting a 15.4-fold increase over AKT1 and AKT3 transcripts. Next, the abundance of AKT protein isoforms was determined using antibody immunoprecipitation followed by Liquid Chromatography-Parallel Reaction Monitoring/Mass Spectrometry. Immunoprecipitation was performed using either mouse or rabbit pan AKT antibodies that were immunoreactive with all three AKT isoforms. We found that AKT2 was the most abundant AKT isoform in human skeletal muscle (4.2-fold greater than AKT1 using the rabbit antibody and 1.6-fold greater than AKT1 using the mouse antibody). AKT3 was virtually undetectable. Next, cultured primary human myoblasts were virally-transduced with cDNAs encoding either wild-type (WT) or kinase-inactive AKT1 (AKT1-K179M) or AKT2 (AKT2-K181M) and allowed to terminally differentiate. Myotubes expressing WT-AKT1 or WT-AKT2 showed enhanced fusion compared to control myotubes, while myotubes expressing AKT1-K179M showed a 14% reduction in fusion. Myotubes expressing AKT2-K181M displayed 63% decreased fusion compared to control. Together, these data identify AKT2 as the most highly-expressed AKT isoform in human skeletal muscle and as the principal AKT isoform regulating human myoblast differentiation.

Skeletal muscle PI3K p110ß regulates expression of AMP-activated protein kinase.

Skeletal muscle metabolic homeostasis is maintained through numerous biochemical and physiological processes. Two principal molecular regulators of skeletal muscle metabolism include AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K); however, PI3K exists as multiple isoforms, and specific metabolic actions of each isoform have not yet been fully elucidated in skeletal muscle. Given this lack of knowledge, we performed a series of experiments to define the extent to which PI3K p110ß mediated expression and (or) activation of AMPK in skeletal muscle. To determine the effect of p110ß inhibition on AMPK expression and phosphorylation in cultured cells, C2C12 myoblasts were treated with a pharmacological inhibitor of p110ß (TGX-221), siRNA against p110ß, or overexpression of kinase-dead p110ß. Expression and phosphorylation of AMPK were unaffected in myoblasts treated with TGX-221 or expressing kinase-dead p110ß. However, expressions of total and phosphorylated AMPK at T172 were reduced in myoblasts treated with p110ß siRNA. When normalized to expression of total AMPK, phosphorylation of AMPK S485/491 was elevated in p110ß-deficient myoblasts. Similar results were observed in tibialis anterior muscle from mice with conditional deletion of p110ß (p110ß-mKO mice). Analysis of AMPK transcript expression revealed decreased expression of Prkaa2 in p110ß-deficient myoblasts and in p110ß-mKO muscle. Loss of p110ß had no effect on oligomycin-stimulated phosphorylation of AMPK or phosphorylated Acetyl-CoA carboxylase (ACC), although oligomycin-induced AMPK and ACC phosphorylation were increased in p110ß-deficient myoblasts compared to oligomycin-stimulated control myoblasts when normalized to levels of total AMPK or ACC. Overall, these results suggest that p110ß positively regulates expression of AMPK in cultured myoblasts and in skeletal muscle in vivo; moreover, despite the reduced abundance of AMPK in p110ß-deficient myoblasts, loss of p110ß does not appear to impair AMPK activation following stimulus. These findings thus reveal a novel role for p110ß in mediating skeletal muscle metabolic signaling.

RNA transcript expression of IGF-I/PI3K pathway components in regenerating skeletal muscle is sensitive to initial injury intensity.

OBJECTIVE: Skeletal muscle regeneration is a complex process involving the coordinated input from multiple stimuli. Of these processes, actions of the insulin-like growth factor-I (IGF-I) and phosphoinositide 3-kinase (PI3K) pathways are vital; however, whether IGF-I or PI3K expression is modified during regeneration relative to initial damage intensity is unknown. The objective of this study was to determine whether mRNA expression of IGF-I/PI3K pathway components was differentially regulated during muscle regeneration in mice in response to traumatic injury induced by freezing of two different durations. DESIGN: Traumatic injury was imposed by applying a 6-mm diameter cylindrical steel probe, cooled to the temperature of dry ice (-79°C), to the belly of the left tibialis anterior muscle of 12-week-old C57BL/6J mice for either 5s (5s) or 10s (10s). The right leg served as the uninjured control. RNA was obtained from injured and control muscles following 3, 7, and 21days recovery and examined by real-time PCR. Expression of transcripts within the IGF, PI3K, and Akt families, as well as for myogenic regulatory factors and micro-RNAs were studied. RESULTS: Three days following injury, there was significantly increased expression of Igf1, Igf2, Igf1r, Igf2r, Pik3cb, Pik3cd, Pik3cg, Pik3r1, Pik3r5, Akt1, and Akt3 in response to either 5s or 10s injury compared to uninjured control muscle. There was a significantly greater expression of Pik3cb, Pik3cd, Pik3cg, Pik3r5, Akt1, and Akt3 in 10s injured muscle compared to 5s injured muscle. Seven days following injury, we observed significantly increased expression of Igf1, Igf2, Pik3cd, and Pik3cg in injured muscle compared to control muscle in response to 10s freeze injury. We also observed significantly reduced expression of Igf1r and miR-133a in response to 5s freeze injury compared to control muscle, and significantly reduced expression of Ckm, miR-1 and miR-133a in response to 10s freeze injury as compared to control. Twenty-one days following injury, 5s freeze-injured muscle exhibited significantly increased expression of Igf2, Igf2r, Pik3cg, Akt3, Myod1, Myog, Myf5, and miR-206 compared to control muscle, while 10s freeze-injured muscles showed significantly increased expression of Igf2, Igf2r, Pik3cb, Pik3cd, Pik3r5, Akt1, Akt3, and Myog compared to control. Expression of miR-1 was significantly reduced in 10s freeze-injured muscle compared to control muscle at this time. There were no significant differences in RNA expression between 5s and 10s injury at either 7d or 21d recovery in any transcript examined. CONCLUSIONS: During early skeletal muscle regeneration in mice, transcript expressions for some components of the IGF-I/PI3K pathway are sensitive to initial injury intensity induced by freeze damage.

Phosphatidylinositol 3-kinase p110α mediates phosphorylation of AMP-activated protein kinase in myoblasts.

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor of intracellular energy. Activation of AMPK is associated with increased phosphorylation of the α-subunit at threonine 172 (T172) and decreased phosphorylation at serine 485 in AMPKα1 and serine 491 in AMPKα2 (S485/491). One potential mediator of AMPK phosphorylation is phosphatidylinositol 3-kinase (PI3K); however, the mechanism and the identities of the specific PI3K isoforms that regulate AMPK activation are not known. To determine whether PI3K p110α regulated AMPK activation in muscle cells, C2C12 myoblasts were subjected to pharmacological inhibition of p110α, siRNA directed against p110α, or overexpression of constitutively-active or dominant negative p110α. Chemical inhibition, siRNA, and expression of dominant-negative p110α were all associated with increased AMPK T172 phosphorylation, whereas expression of constitutively-active p110α reduced T172 phosphorylation. Conversely, pharmacological inhibition of p110α reduced AMPK S485/491 phosphorylation, while constitutively-active p110α increased AMPK S485/491 phosphorylation. This p110α-mediated increase in AMPK S485/491 phosphorylation was eliminated in the presence of the Akt inhibitor MK2206, suggesting that p110α-mediated phosphorylation of AMPKα at S485/491 is Akt-dependent. In response to oligomycin or serum-starvation, AMPK T172 phosphorylation was elevated in p110α-deficient myoblasts compared to control myoblasts. Overall, our findings identify PI3K p110α as a mediator of AMPK phosphorylation in myoblasts.

Role of phosphoinositide 3-OH kinase p110ß in skeletal myogenesis.

Phosphoinositide 3-OH kinase (PI3K) regulates a number of developmental and physiologic processes in skeletal muscle; however, the contributions of individual PI3K p110 catalytic subunits to these processes are not well-defined. To address this question, we investigated the role of the 110-kDa PI3K catalytic subunit ß (p110ß) in myogenesis and metabolism. In C2C12 cells, pharmacological inhibition of p110ß delayed differentiation. We next generated mice with conditional deletion of p110ß in skeletal muscle (p110ß muscle knockout [p110ß-mKO] mice). While young p110ß-mKO mice possessed a lower quadriceps mass and exhibited less strength than control littermates, no differences in muscle mass or strength were observed between genotypes in old mice. However, old p110ß-mKO mice were less glucose tolerant than old control mice. Overexpression of p110ß accelerated differentiation in C2C12 cells and primary human myoblasts through an Akt-dependent mechanism, while expression of kinase-inactive p110ß had the opposite effect. p110ß overexpression was unable to promote myoblast differentiation under conditions of p110α inhibition, but expression of p110α was able to promote differentiation under conditions of p110ß inhibition. These findings reveal a role for p110ß during myogenesis and demonstrate that long-term reduction of skeletal muscle p110ß impairs whole-body glucose tolerance without affecting skeletal muscle size or strength in old mice.

Lysophosphatidic acid-stimulated phosphorylation of PKD2 is mediated by PI3K p110ß and PKCδ in myoblasts.

CONTEXT: G-protein coupled receptor (GPCR) signaling in skeletal muscle is incompletely understood; in particular, the signaling pathways that regulate GPCR-mediated signaling in skeletal muscle are only beginning to be established. Lysophosphatidic acid (LPA) is a GPCR agonist that has previously been shown to activate protein kinase D (PKD) in non-muscle cells; however, whether PKD is activated in response to LPA in skeletal muscle myoblasts, and the identities of signaling intermediates that regulate this activation, have not been defined. OBJECTIVE: To determine whether PKD is activated in response to LPA administration in myoblasts, and to define the signaling pathways that mediate LPA-stimulated PKD phosphorylation. METHODS: C2C12 myoblasts were treated with LPA and signaling pathways examined by means of Western immunoblotting and real-time PCR (RT-PCR). Pharmacological inhibition and RNA-interference were used to target specific molecules to determine their involvement in LPA-induced PKD phosphorylation. RESULTS: Treatment of myoblasts with exogenous LPA revealed that PI3K p110ß mediated PKD phosphorylation at Ser 748 and at Ser 916 through kinase-dependent and kinase-independent mechanisms. Loss of PKCδ, but not the loss of PKCα, prevented LPA-induced PKD phosphorylation. The PKD isoform responsive to LPA treatment was identified as PKD2. CONCLUSION: These results indicate that LPA-stimulated PKD2 phosphorylation requires PKCδ and non-catalytic actions of PI3K p110ß, and provide new information with respect to GPCR-mediated signal transduction in myoblasts.

Enhanced Akt phosphorylation and myogenic differentiation in PI3K p110ß-deficient myoblasts is mediated by PI3K p110α and mTORC2.

Phosphoinositide 3-kinase (PI3K) is a principal regulator of Akt activation and myogenesis; however, the function of PI3K p110ß in these processes is not well defined. To address this, we investigated the role of p110ß in Akt activation and skeletal muscle cell differentiation. We found that Akt phosphorylation was enhanced in p110ß-deficient myoblasts in response to Insulin-like Growth Factor-I (IGF-I), epidermal growth factor, or p110α overexpression, as compared to p110ß-sufficient cells. This effect was associated with increased mammalian target of rapamycin complex 2 activation, even in myoblasts deficient in mSin1 and rictor. Conversely, in response to the G-protein-coupled receptor agonist lysophosphatidic acid, Akt phosphorylation was attenuated in p110ß-deficient myoblasts. Loss of p110ß also enhanced the expression of myogenic markers at the myoblast stage and during the first 48 h of differentiation. These data demonstrate that reductions in p110ß are associated with agonist-specific Akt hyperactivation and accelerated myogenesis, thus revealing a negative role for p110ß in Akt activation and during myoblast differentiation.