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

Abstract

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.

Figure 1

Plasma levels of glucose, lactate and insulin as well as muscle glycogen content

Figure 2

Acute exercise and physiological insulin stimulation affect AMPK and Akt signalling in human skeletal muscle

Figure 3

Acute exercise increases AMPK trimer-specific activity in human skeletal muscle

Figure 4

Phosphorylation signatures of TBC1D1 in human skeletal muscle in response to exercise and physiological insulin stimulation

Figure 5

TBC1D1 phosphorylation signature in response to in vitro contraction in mouse skeletal muscle

Figure 6

In vitro contraction of mouse skeletal muscle activates γ1 and γ3 AMPK trimers

Figure 7

Phosphorylation signatures of TBC1D4 in human skeletal muscle in response to exercise and physiological insulin stimulation

Figure 8

Representative Western blots for the data presented in Fig. 7 with molecular weight marker indications

Figure 9

AMPK trimer activity correlates with phosphorylation of TBC1D1/4 on specific sites in human skeletal muscle

Figure 10

TBC1D4 phosphorylation signatures in response to in vitro contraction in mouse skeletal muscle

Figure 11

In vitro contraction of mouse skeletal muscle activates γ1 and γ3 AMPK trimers

Figure 12

Exercise-induced phosphorylation of AMPK, ACC and Akt in mouse soleus and EDL muscles

Figure 13

Exercise-induced phosphorylation signatures of TBC1D4 and TBC1D1 in mouse soleus and EDL muscles

Figure 14

Exercise and physiological insulin stimulation results in differential 14-3-3 binding capacity to TBC1D1 and TBC1D4

Figure 15

Akt phosphorylation status on T308 correlates with phosphorylation of TBC1D1/4 on specific sites in human skeletal muscle

Figure 16

Akt phosphorylation status on S473 correlates with phosphorylation of TBC1D1/4 on specific sites in human skeletal muscle