Thr649Ala-AS160 knock-in mutation does not impair contraction/AICAR-induced glucose transport in mouse muscle

Abstract

AS160 and its closely related protein TBC1D1 have emerged as key mediators for both insulin- and contraction-stimulated muscle glucose uptake through regulating GLUT4 trafficking. Insulin increases AS160 phosphorylation at multiple Akt/PKB consensus sites, including Thr(649), and promotes its binding to 14-3-3 proteins through phospho-Thr(649). We recently provided genetic evidence that AS160-Thr(649) phosphorylation/14-3-3 binding plays a key role in mediating insulin-stimulated glucose uptake in muscle. Contraction has also been proposed to increase phosphorylation of AS160 and TBC1D1 via AMPK, which could be detected by a generic phospho-Akt substrate (PAS) antibody. Here, analysis of AS160 immunoprecipitates from muscle extracts with site-specific phospho-antibodies revealed that contraction and AICAR caused no increase but rather a slight decrease in phosphorylation of the major PAS recognition site AS160-Thr(649). In line with this, contraction failed to enhance 14-3-3 binding to AS160. Consistent with previous reports, we also observed that in situ contraction stimulated the signal intensity of PAS antibody immunoreactive protein of ∼150-160 kDa in muscle extracts. Using a TBC1D1 deletion mutant mouse, we showed that TBC1D1 protein accounted for the majority of the PAS antibody immunoreactive signals of ∼150-160 kDa in extracts of contracted muscles. Consistent with the proposed role of AS160-Thr(649) phosphorylation/14-3-3 binding in mediating glucose uptake, AS160-Thr(649)Ala knock-in mice displayed normal glucose uptake upon contraction and AICAR in isolated muscles. We conclude that the previously reported PAS antibody immunoreactive band ∼150-160 kDa, which were increased upon contraction, does not represent AS160 but TBC1D1, and that AS160-Thr(649)Ala substitution impairs insulin- but neither contraction- nor AICAR-stimulated glucose uptake in mouse skeletal muscle.

Fig. 1.

Phosphorylation of Akt substrate of 160 kDa (AS160) and TBC1D1 in gastrocnemius muscles from wild-type (WT) and AS160-Thr⁶⁴⁹Ala knock-in (KI) mice upon in situ contraction. In situ contraction of hindlimb muscles via sciatic nerve was performed for 10 min on one leg (Ctxn) from anesthetized KI mice and WT littermates (13–14 wk old), and the other leg served as sham-operated control (basal). For insulin stimulation, KI mice and WT littermates (12 wk old) were anesthetized, injected with 150 mU/g ip insulin (Ins) or saline (basal), and gastrocnemius (GAS) muscle was removed and snap-frozen 20 min after injection. AS160 protein was immunoprecipitated from 3 mg of extracts of GAS muscles and their phosphorylation determined using indicated site-specific phospho-antibodies. 14-3-3 Proteins were detected using 14-3-3 K19 antibody in AS160 immunoprecipitates. Total and phosphorylated AMPK were determined in 40 μg of muscle extracts using anti-AMPKα2 and anti-pT172AMPK antibodies, respectively. A: representative blots showing total and phosphorylated AS160, AMPK, and 14-3-3. B: quantitation of AS160 Thr⁶⁴⁹/PAS phosphorylation; n = 5. *P < 0.05; ^aP < 0.05 vs. WT (basal); ^bP < 0.05 vs. WT (Ctxn).

Fig. 2.

Phosphorylation of AS160 and TBC1D1 in tibialis anterior (TA) muscles from WT and KI mice upon in situ contraction. Hindlimb muscles were contracted in situ via sciatic nerve stimulation for 10 min on one leg (Ctxn) from anesthetized KI mice and WT littermates (13–14 wk old), and the other leg served as sham-operated control (basal). AS160 and TBC1D1 proteins were sequentially immunoprecipitated from 2 mg of extracts of TA muscles and their phosphorylation determined using indicated site-specific phospho-antibodies. Total and phosphorylated AMPK were determined in 40 μg of muscle extracts using anti-AMPKα2 and anti-pT172AMPK antibodies, respectively. phospho-Akt substrate (PAS) signal ∼160 kDa was detected in muscle extract using PAS antibody. A: representative blots showing total and phosphorylated AS160, TBC1D1, and AMPK. B: quantitation of AS160 Thr⁶⁴⁹/PAS phosphorylation; n = 5. *P < 0.05; ^aP < 0.05 vs. WT (basal); ^bP < 0.05 vs. WT (Ctxn).

Fig. 3.

Phosphorylation of TBC1D1 in TA muscles from WT and TBC1D1 deletion mutant mice upon in situ contraction. Hindlimb muscles were contracted in situ via sciatic nerve stimulation for 10 min on one leg (Ctxn) from anesthetized TBC1D1 deletion mutant mice and WT littermates (8 wk old), and the other leg served as sham-operated control (basal). Total and phosphorylated TBC1D1 and AMPK were determined in 40 μg of muscle extracts using anti-TBC1D1, anti-AMPKα2, and anti-pT172AMPK antibodies, respectively. PAS signal at ∼150–160 kDa was detected in muscle extract using PAS antibody.

Fig. 4.

Glucose uptake in EDL muscle and glycogen content in TA muscle from WT and KI mice upon in situ contraction. In situ contraction of hindlimb muscles via sciatic nerve was performed for 5 min on one leg (Ctxn) from anesthetized KI mice and WT littermates (8 wk old), and the other leg served as sham-operated control (basal). After contraction, EDL and TA muscles were removed from both legs, and glucose uptake (A) determined in EDL muscle and muscle glycogen content (B) determined in TA muscle as described in materials and methods; n = 5. *P < 0.05.

Fig. 5.

Phosphorylation of AS160 Thr⁶⁴⁹ in isolated soleus and EDL muscles from WT and KI mice upon AICAR stimulation ex vivo. Soleus and EDL muscles from KI mice and WT littermates (10–14 wk old) were incubated with or without 2 mM AICAR ex vivo. AS160 was immunoprecipitated from 200 μg of muscle extracts, and Thr⁶⁴⁹ phosphorylation was determined using a site-specific phospho-antibody. Phosphorylated ACC and AMPK were determined in 40 μg of muscle extracts using antibodies that recognize phosphorylated ACC-Ser²¹² and AMPK-Thr¹⁷². Total ACC and AMPKα were determined in 40 μg of muscle extracts. A: quantitation of AS160 Thr⁶⁴⁹ phosphorylation in soleus muscles; n = 5. B: representative blots showing total and phosphorylated AS160 on immunoprecipitated AS160 and phosphorylated ACC and AMPK in muscle extracts. C: quantitation of AS160 Thr⁶⁴⁹ phosphorylation in EDL muscles; n = 5. D: representative blots showing total and phosphorylated AS160 on immunoprecipitated AS160 and total and phosphorylated ACC and AMPK.

Fig. 6.

Glucose uptake in isolated EDL muscle upon AICAR stimulation, and AICAR tolerance test in WT and KI mice. A: EDL muscle from AS160-Thr⁶⁴⁹Ala knock-in mice and WT littermates (10–12 wk old) was incubated with or without 2 mM AICAR ex vivo. After incubation, muscle glucose uptake was determined as described in materials and methods; n = 6–7. *P < 0.05. B: KI mice and WT littermates (8 wk old) were partially fasted for 4 h before injection with AICAR (250 mg/kg body wt ip). Blood glucose levels were monitored before and after AICAR injection at indicated time points; n = 4–5.