Acute exercise and physiological insulin induce distinct phosphorylation signatures on TBC1D1 and TBC1D4 proteins in human skeletal muscle.

We investigated the phosphorylation signatures of two Rab-GTPase activating proteins TBC1D1 and TBC1D4 in human skeletal muscle in response to physical exercise and physiological insulin levels induced by a carbohydrate rich meal using a paired experimental design. Eight healthy male volunteers exercised in the fasted or fed state and muscle biopsies were taken before and immediately after exercise. We identified TBC1D1/4 phospho-sites that (1) did not respond to exercise or postprandial increase in insulin (TBC1D4: S666), (2) responded to insulin only (TBC1D4: S318), (3) responded to exercise only (TBC1D1: S237, S660, S700; TBC1D4: S588, S751), and (4) responded to both insulin and exercise (TBC1D1: T596; TBC1D4: S341, T642, S704). In the insulin-stimulated leg, Akt phosphorylation of both T308 and S473 correlated significantly with multiple sites on both TBC1D1 (T596) and TBC1D4 (S318, S341, S704). Interestingly, in the exercised leg in the fasted state TBC1D1 phosphorylation (S237, T596) correlated significantly with the activity of the α2/β2/γ3 AMPK trimer, whereas TBC1D4 phosphorylation (S341, S704) correlated with the activity of the α2/β2/γ1 AMPK trimer. Our data show differential phosphorylation of TBC1D1 and TBC1D4 in response to physiological stimuli in human skeletal muscle and support the idea that Akt and AMPK are upstream kinases. TBC1D1 phosphorylation signatures were comparable between in vitro contracted mouse skeletal muscle and exercised human muscle, and we show that AMPK regulated phosphorylation of these sites in mouse muscle. Contraction and exercise elicited a different phosphorylation pattern of TBC1D4 in mouse compared with human muscle, and although different circumstances in our experimental setup may contribute to this difference, the observation exemplifies that transferring findings between species is problematic. Key points Phosphorylation signature patterns on TBC1D1 and TBC1D4 proteins in the insulin–glucose pathway were investigated in human skeletal muscle in response to physiological insulin and exercise. In response to postprandial increase in insulin, Akt phosphorylation of T308 and S473 correlated significantly with sites on TBC1D1 (T596) and TBC1D4 (S318, S341, S704). Exercise induced phosphorylation of TBC1D1 (S237, T596) that correlated significantly with activity of the α2/β2/γ3 AMPK trimer, whereas TBC1D4 phosphorylation (S341, S704) with exercise correlated with activity of the α2/β2/γ1 AMPK trimer. TBC1D1 phosphorylation signatures with exercise/muscle contraction were comparable between human and mouse skeletal muscle, and AMPK regulated phosphorylation of these sites in mouse muscle, whereas contraction and exercise elicited different TBC1D4 phosphorylation patterns in mouse compared with human muscle. Our results show differential phosphorylation of TBC1D1 and TBC1D4 in response to physiological stimuli in human skeletal muscle and indicate that Akt and AMPK may be upstream kinases. Introduction Skeletal muscles constitute a major sink for whole body glucose disposal as evidenced by glucose uptake measurements during hyperinsulinaemic euglycaemic clamp conditions in non-insulin resistant individuals (DeFronzo et al. 1981; Richter et al. 1989). GLUT4 is the main glucose transporter in skeletal muscle and adipose tissue located within the endosomal and the trans-Golgi network compartments and to small tubule–vesicular structures (Slot et al. 1991; Ploug et al. 1998). In response to certain stimuli (e.g. muscle contraction, exercise, insulin, or hypoxia; Klip et al. 1987; Douen et al. 1989; Cartee et al. 1991; Kristiansen et al. 1996) these transporters are mobilized to the sarcolemma and/or T-tubules to facilitate glucose uptake. Skeletal muscle thus represents an important tissue for studying the intracellular signalling pathways leading to incorporation of GLUT4 molecules into the cell surface membrane. The signalling pathway in skeletal muscle leading to glucose uptake in response to contraction is not completely understood. It is thought to involve increases in intracellular Ca2+ and activation of kinases downstream of LKB1 (i.e. 5′-AMP activated protein kinase (AMPK) and sucrose nonfermenting AMPK-related kinase (SNARK); Friedrichsen et al. 2012; Jensen & Richter, 2012). The signalling pathway whereby insulin stimulates glucose uptake has been resolved in greater detail, and is known to involve the insulin receptor – insulin receptor substrate – phosphatidylinositol 3 kinase (IR-IRS-PI3K)–Akt2 axis (Huang & Czech, 2007; Ishikura et al. 2008). Akt2 activation is necessary for insulin-stimulated GLUT4 translocation to the plasma membrane in adipocytes (Hill et al. 1999) and for insulin-stimulated glucose uptake in skeletal muscle (Cho et al. 2001; Bouzakri et al. 2006). In the search for novel Akt targets involved in these processes, an Akt substrate of 160 kDa (AS160), known as TBC1D4 (Nagase et al. 1998), was identified as a Rab-GTPase activating protein (Rab-GAP) (Sano et al. 2003). It is now evident that TBC1D4 has multiple upstream kinases also including AMPK (Kramer et al. 2006a; Treebak et al. 2010) and potentially protein kinase Cζ (Ng et al. 2010). The current thinking is that Rab-GAPs in the signalling pathways to stimulate glucose uptake repress Rab-GTPase function in the basal state by inhibiting the exchange of GDP to GTP leading to intracellular retention of GLUT4 vesicles (Kane et al. 2002; Sakamoto & Holman, 2008). In response to stimulation Rab-GAPs are phosphorylated, for instance by Akt or AMPK, allowing for the active exchange of GDP to GTP in the Rab-GTPase and thus stimulating GLUT4 mobilization to the plasma membrane (Hoffman & Elmendorf, 2011). In addition to TBC1D4, the TBC1D4 paralogue TBC1D1 has been identified as a functional Rab-GAP. Similarly to TBC1D4, TBC1D1 has inhibitory effects on GLUT4 translocation in 3T3-L1 adipocytes (Roach et al. 2007) and skeletal muscle (An et al. 2010). TBC1D4 and TBC1D1 are involved in regulation of both insulin- and contraction-induced glucose uptake in intact mouse skeletal muscle (Kramer et al. 2006b; An et al. 2010), and TBC1D4 has emerged as a nexus for insulin- and contraction-responsive signals, potentially mediating enhanced insulin action in human and rat skeletal muscle after exercise (Arias et al. 2007; Funai et al. 2009; Treebak et al. 2009b; Pehmoller et al. 2012). The phosphorylation status of TBC1D1 and TBC1D4 in response to insulin stimulation in human skeletal muscle has been studied only during hyperinsulinaemic euglyacemic clamp conditions and most studies have used the phospho-Akt substrate (PAS) antibody (Karlsson et al. 2005a,b2005b; O'Gorman et al. 2006; Frosig et al. 2007; Howlett et al. 2007, 2008; Hojlund et al. 2008; Hoeg et al. 2011), whereas other studies (Treebak et al. 2009b; Vind et al. 2011; Pehmoller et al. 2012; Vendelbo et al. 2012; Consitt et al. 2013) have used site- and phospho-specific antibodies. No study has investigated whether physiological insulin levels (i.e. insulin levels obtained after a meal) would induce changes in phosphorylation status of the two proteins similarly to those observed during sustained elevated insulin levels as applied during the hyperinsulinaemic clamp condition. Moreover, acute exercise in humans is known to induce changes in phosphorylation status of both TBC1D1 and TBC1D4 (Frosig et al. 2010; Jessen et al. 2011), but the phosphorylation response to physiological insulin stimulation combined with acute exercise remains to be established. Here we employed the one-legged knee extensor exercise model (Andersen et al. 1985) to investigate skeletal muscle site-specific phosphorylation signature patterns of TBC1D1 and TBC1D4 in response to exercise in the fasted or fed condition of healthy human subjects. This design allows for a paired evaluation of the phosphorylation signatures in skeletal muscle at rest and in response to acute exercise alone, physiological insulin stimulation alone, and acute exercise combined with physiological insulin stimulation. We hypothesized the presence of stimuli-dependent phosphorylation signatures on TBC1D1 and TBC1D4. Identification of these signatures in response to physiological stimuli will increase the understanding of how TBC1D1 and TBC1D4 are regulated and how these proteins may impact on metabolism in human skeletal muscle.