Sucrose nonfermenting AMPK-related kinase (SNARK) mediates contraction-stimulated glucose transport in mouse skeletal muscle

Abstract

The signaling mechanisms that mediate the important effects of contraction to increase glucose transport in skeletal muscle are not well understood, but are known to occur through an insulin-independent mechanism. Muscle-specific knockout of LKB1, an upstream kinase for AMPK and AMPK-related protein kinases, significantly inhibited contraction-stimulated glucose transport. This finding, in conjunction with previous studies of ablated AMPKalpha2 activity showing no effect on contraction-stimulated glucose transport, suggests that one or more AMPK-related protein kinases are important for this process. Muscle contraction increased sucrose nonfermenting AMPK-related kinase (SNARK) activity, an effect blunted in the muscle-specific LKB1 knockout mice. Expression of a mutant SNARK in mouse tibialis anterior muscle impaired contraction-stimulated, but not insulin-stimulated, glucose transport. Whole-body SNARK heterozygotic knockout mice also had impaired contraction-stimulated glucose transport in skeletal muscle, and knockdown of SNARK in C2C12 muscle cells impaired sorbitol-stimulated glucose transport. SNARK is activated by muscle contraction and is a unique mediator of contraction-stimulated glucose transport in skeletal muscle.

Fig. 1.

Contraction-stimulated glucose transport is impaired in muscle-specific LKB1 knockout (MLKB1KO) mice. (A) Soleus muscles from MLKB1KO and littermate controls were contracted to measure glucose transport. (B) In vivo glucose transport was measured over the 15 min of contraction and the subsequent 30 min in tibialis anterior muscles. (C and D) Muscle lysates were obtained from gastrocnemius muscle, and PAS phosphorylation of AS160/TBC1D1 (C) and ERK phosphorylation (D) were determined by Western blot. Data are means ± SEM, n = 6/group for A–C, n = 5–12/group for D. **P < 0.01 and ***P < 0.001 vs. basal of the same genotype. ^††P < 0.01 and ^†††P < 0.001 vs. corresponding control.

Fig. 2.

SNARK expression and activity in mouse and human skeletal muscle. (A) Tibialis anterior muscles from MLKB1KO and littermate controls were contracted in situ for 15 min. ARK5/SNARK activity was measured using an immune complex assay with an antibody that recognizes both ARK5 and SNARK. Relative SNARK protein expression in various (B) mouse tissues and (C) mouse muscles (TA, tibialis anterior; EDL, extensor digitorium longus; WG, white gastrocnemius; RG, red gastrocnemius; SOL, soleus; PC, positive control). (D) Relative SNARK mRNA expression in various mouse tissues. (E) SNARK activity in tibialis anterior muscles from MLKB1KO and littermate controls was measured using an immune complex assay with SNARK antibody. (F) Time course of SNARK activity in tibialis anterior muscles from mice exercised on a treadmill at 22 m/min, 12% incline for 15, 30, and 60 min. (G) EDL muscles were isolated and incubated in KRB buffer. Muscles were either electrically stimulated to contract for 10 min or stimulated with insulin (50 mU/mL) for 40 min. (H and I) Healthy volunteers performed cycle exercise at 70% of peak work rate for 20 min (H) or 110% of peak work rate for 2 min (I). Data are means ± SEM, n = 5–11/group. *P < 0.05 and **P < 0.01 compared with control. ^†P < 0.05 vs. corresponding control.

Fig. 3.

Effects of SNARK inhibition in glucose transport in response to sorbitol and insulin. Retroviruses containing empty vector (EV), scrambled shRNA (Sc), and shRNA for SNARK (ShRNA) were infected in C2C12 cells. (A) SNARK expression was determined by immunoblot analysis. (B and C) Myotubes infected with Sc or ShRNA were incubated in the absence (black bars) or presence (grey bars) of sorbitol (300 mM) for 30 min. Glucose transport (B) and AMPK phosphorylation (C) were determined by 2-DG glucose transport measurement and Western blot analysis, respectively. (D) Cells were treated with insulin (100 nM) for 20 min and glucose transport was measured. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control. ^†P < 0.01 vs. corresponding control.

Fig. 4.

Overexpression of mutant SNARK (mtSNARK) and knockdown of SNARK impaired contraction-stimulated glucose transport and phosphorylation of AS160 and TBC1D1. (A–D) mtSNARK replacing Thr²⁰⁸ to Ala was generated, and the cDNA construct was injected and electroporated into tibialis anterior muscles. Muscles were studied 10 d after injection. (A) SNARK activity was measured in tibialis anterior muscle lysates after 10 min of in situ contraction. (B) In vivo glucose transport in response to 15 min of in situ muscle contraction was measured in tibialis anterior muscles overexpressing either empty vector (EV) or mtSNARK. (C) Contraction-stimulated PAS phosphorylation of AS160 and TBC1D1 was measured by Western blot. Protein levels of AS160 and TBC1D1 were also determined by Western blot. (D) In vivo insulin-stimulated 2-DG glucose transport in skeletal muscle was measured by i.v. injection of a glucose bolus (1 mg/g). (E) Soleus muscles from SNARK ^(+/−) or control littermates were dissected and contracted in vitro for 10 min, and glucose transport was measured. (F) Force production was measured as described in Fig. S1A. (G) Contraction-stimulated AMPK Thr¹⁷² phosphorylation was determined by Western blot. (H) Contraction-stimulated phosphorylation of AS160 and TBC1D1 was determined by Western blot using PAS antibody. Data are means ± SEM, n = 5–6/group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. basal in the same group. ^†P < 0.05 and ^††P < 0.01 vs. corresponding control.