Role of ataxia telangiectasia mutated in insulin signalling of muscle‐derived cell lines and mouse soleus

Abstract

Aim Ataxia telangiectasia mutated (ATM) reportedly plays a role in insulin-stimulated activation of Akt in some cell types but not others. The role of ATM in insulin signaling has not been firmly resolved for skeletal muscle cells, for which Akt phosphorylation is a pivotal step in stimulation of glucose transport. Accordingly, our aim was to determine the role of ATM in insulin effects for cell lines derived from skeletal muscle and for skeletal muscle. Methods: We examined insulin effects in L6 myotubes, mouse soleus, C2C12 myotubes, and differentiated rhabdomyosarcoma (RD) cells in the presence and absence of a low concentration (1 μM) of the ATM inhibitor KU55933. We also compared insulin signaling in C2C12 cells expressing shRNA against ATM and control cell lines (empty vector; cells expressing non-targeting shRNA). Results In L6 myotubes and mouse soleus muscle, KU55933 inhibited insulin-stimulated phosphorylation of the 160 kDa substrate of Akt (AS160) despite no effect on Akt. In contrast, KU55933 prevented insulin-stimulated Akt phosphorylation in C2C12 myotubes. Futhermore, C2C12 myotubes expressing shRNA against ATM displayed reduced insulin-stimulated Akt phosphorylation compared to controls. KU55933 also decreased insulin-stimulated Akt phosphorylation in differentiated RD cells. Conclusion These model-dependent differences in the role of ATM in insulin action demonstrate a role of ATM in insulin-stimulated phosphorylation of Akt (in C2C12 and RD cells) but also allow the elucidation of a novel, Akt-independent role of ATM (in L6 myotubes and mouse soleus, at the level of AS160) in insulin signaling.

ATM Protein Kinase Signaling, Type 2 Diabetes and Cardiovascular Disease

“…2) [44,45]. Consequently, reduced glucose uptake was observed, despite PKB/Akt and atypical PKC phosphorylation being unaffected in these cells [44]. ATM was found to play a role in glucose uptake mediated by both GLUT4 (insulin stimulated) and GLUT1 (basal).…”

“…Inhibition of ATM in L6 myotubes and mouse soleus muscle reduced insulin-stimulated phosphorylation of AS160, a Rab GTPase-activating protein which plays a key role in GLUT4 translocation to the cell surface (Fig. 2) [44,45]. Consequently, reduced glucose uptake was observed, despite PKB/Akt and atypical PKC phosphorylation being unaffected in these cells [44].…”

“…By regulating an as yet unidentified phosphatase (possibly PP2A), ATM increases the phosphorylation of PKB/Akt in response to insulin stimulation, leading to increased protein synthesis and glucose uptake [42,43]. ATM also directly phosphorylates 4E-BP1 in response to insulin stimulation, resulting in increased protein synthesis and it has also been shown that ATM mediates insulin stimulated glucose uptake by phosphorylating AS160, causing GLUT4 externalization [44,45]. Abbreviations: AS160 Akt substrate of 160 kDa, AMPK AMP-activated protein kinase, ATM ataxia telangiectasia mutated, eIF-4E eukaryotic translation initiation factor 4E, 4E-BP1 eukaryotic translation initiation factor 4E binding protein 1, GLUT4 glucose transporter 4, G6PD glucose-6-phosphotase dehydrogenase, Hsp27 heat shock protein 27, HIF-1α hypoxia inducible factor 1-alpha, LKB1 liver kinase B1, mTORC1 mammalian target of rapamycin complex 1, PPP pentose phosphate pathway, PKB/Akt protein kinase B/Akt, PP2A protein phosphatase 2A, REDD1 regulated in development and DNA damage response 1, TSC2 tuberous sclerosis 2 cytokines and reduced expression of antioxidants [35].…”

“…ATMdependent PKB/Akt phosphorylation at serine 473 is required for the subsequent threonine 308 phosphorylation, to achieve full activation of PKB/Akt [42,60]. However, the involvement of ATM in PKB/Akt activation is unsure as ATM was shown to play a role in insulin-stimulated PKB/ Akt activation in some cell types, but not in others [44]. In light of this, Ching and colleagues [62] hypothesized that the strength of the signal, which is weaker through IGF-IR and stronger through IR activation, determines whether or not ATM is required for full PKB/Akt activation.…”

Ataxia telangiectasia mutated influences cytochrome c oxidase activity

“…The 1 μM concentration of KU55933 is substantially lower than the IC50s for other members of the PI3K family kinases, including PI3K, mTOR, and ATR [9]. At 1 μM, KU55933 does not affect insulin-stimulated phosphorylation of Akt in mouse skeletal muscle [10], suggesting that it does not interfere with PI3K, which is upstream of Akt.…”