Inhibition of GLUT4 translocation by Tbc1d1, a Rab GTPase-activating protein abundant in skeletal muscle, is partially relieved by AMP-activated protein kinase activation

Abstract

Insulin increases glucose transport by stimulating the trafficking of intracellular GLUT4 to the cell surface, a process known as GLUT4 translocation. A key protein in signaling this process is AS160, a Rab GTPase-activating protein (GAP) whose activity appears to be suppressed by Akt phosphorylation. Tbc1d1 is a Rab GAP with a sequence highly similar to that of AS160 and with the same Rab specificity as that of AS160. The role of Tbc1d1 in regulating GLUT4 trafficking has been unclear. Our previous study showed that overexpressed Tbc1d1 inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, even though insulin caused phosphorylation on its single canonical Akt motif. In the present study, we show in 3T3-L1 adipocytes that Tbc1d1 is only 1/20 as abundant as AS160, that knockdown of Tbc1d1 has no effect on insulin-stimulated GLUT4 translocation, and that overexpressed Tbc1d1 also inhibits GLUT4 translocation elicited by activated Akt expression. These results indicate that endogenous Tbc1d1 does not participate in insulin-regulated GLUT4 translocation in adipocytes and suggest that the GAP activity of Tbc1d1 is not suppressed by Akt phosphorylation. In addition, we discovered that Tbc1d1 is much more highly expressed in skeletal muscle than fat and that the AMP-activated protein kinase (AMPK) activator 5'-aminoimidazole-4-carboxamide ribonucleoside partially reversed the inhibition of insulin-stimulated GLUT4 translocation by overexpressed Tbc1d1 in 3T3-L1 adipocytes. 5'-Aminoimidazole-4-carboxamide ribonucleoside activation of the kinase AMPK is known to cause GLUT4 translocation in muscle. The above findings strongly suggest that Tbc1d1 is a component in the signal transduction pathway leading to AMPK-stimulated GLUT4 translocation in muscle.

FIGURE 1.

Expression of Tbc1d1 and AS160 during 3T3-L1 adipocyte differentiation. SDS samples of 3T3-L1 adipocytes as confluent fibroblasts (day 0) and on days 2, 4, 6, and 8 following the initiation of differentiation were immunoblotted for Tbc1d1 and AS160. The loads/lane were 100 μg for the Tbc1d1 blot and 50 μg for the AS160 blot. Known amounts (ng) of recombinant 3× FLAG-tagged mouse Tbc1d1 and human AS160 were included as standards. SDS samples of adipocytes on day 5 of differentiation expressing control (Con) shRNA or shRNA for Tbc1d1 or AS160 (KD) were also included (see Fig. 2 and (4), respectively, for the generation of these samples). Immunoblotting for AS160 was done with an affinity-purified antibody against amino acids 876–891 of mouse AS160, which has the same sequence in human and mouse AS160. A repetition of this experiment gave similar results.

FIGURE 2.

Effect of Tbc1d1 knockdown on cell surface GLUT4. A, the relative amount of HA-GLUT4-GFP at the cell surface in basal and insulin-stimulated adipocytes at day 5 of differentiation expressing control shRNA or Tbc1d1 shRNA (KD) was measured as described under “Experimental Procedures.” The results are the averages ± S.E. from three measurements. B, Tbc1d1 was immunoprecipitated from an SDS/C12E9 lysates of day 5 adipocytes expressing control shRNA or Tbc1d1 shRNA, and equal portions of the immunoprecipitates were immunoblotted for Tbc1d1. A repetition of the experiments in A and B starting with the generation of the cells expressing shRNAs gave similar results.

FIGURE 3.

Effect of myrAkt on cell surface GLUT4 and Tbc1d1 phosphorylation. A, 3T3-L1 adipocytes were co-transfected with the plasmid for HA-GLUT4-GFP and the plasmids for myrAkt1 with a carboxyl-terminal Myc tag (Akt) and 3× FLAG-tagged human Tbc1d1 (Tbc) or for the corresponding empty vectors (AV, TV). Cell surface HA-GLUT4-GFP was measured as described under “Experimental Procedures.” The values are the averages ± S.E. for four experiments. The values in each experiment were normalized to 1.0 for the AV, TV control in the insulin state. B, immunoblots of the SDS samples of the transfected cells in A for myrAkt with anti-Myc tag and for Tbc1d1 with anti-FLAG tag. The 1× load contained 20 μg of protein. The anti-Myc also cross-reacted with a protein that migrated just below myrAkt, which serves as a loading control. C, SDS/C12E9 lysates of the transfected cells in A were immunoprecipitated with anti-FLAG agarose. The immunoadsorbates were immunoblotted for phosphorylation of Tbc1d1 with the PAS antibody (upper panel) and for Tbc1d1 with anti-FLAG (lower panel). A repetition of B and C with a second set of samples gave similar results.

FIGURE 4.

Tbc1d1 expression in tissues and L6 cells. A, SDS samples of tissues from white adipose tissue (WAT), heart, and gastrocnemius (Gastroc) muscle from wild-type (WT) and AS160 knock-out (KO) mice were immunoblotted for Tbc1d1 and AS160. The 1× load was 100 μg of protein. Two replicates of these blots with tissue samples from two other littermate sets of wild-type and AS160 knock-out mice gave similar results. B, SDS samples of L6 cells on the stated days after initiation of differentiation were immunoblotted for Tbc1d1. The 1× load was 100 μg of protein. The blot shown was probed with the NT antibody against Tbc1d1. A similar pattern was obtained when the samples were immunoblotted with the PG antibody.

FIGURE 5.

Effect of AICAR on Tbc1d1 inhibition of GLUT4 translocation. 3T3-L1 adipocytes were electroporated with HA-GLUT4-GFP and either control vector plasmid (V) or the plasmid for 3× FLAG-tagged human Tbc1d1. The cells were untreated (basal, B) or treated with insulin (I), 1 mm AICAR (A), or 1 mm AICAR plus insulin (Ins). Treatment with AICAR was 70 min. Insulin was added during the final 30 min. A, cell surface HA-GLUT4-GFP was measured as described under “Experimental Procedures.” The values are the averages ± S.E. for four independent experiments. They have been normalized to a value of 1.0 for the vector (Vec) control in the insulin state. The significance of the difference between surface GLUT4 in the presence of insulin versus AICAR plus insulin for cells overexpressing Tbc1d1 was calculated by the Student's two-tailed, paired t test. B, SDS samples of the cells in A were immunoblotted for pThr172 AMPK, AMPK, pThr56 eEF2, eEF2, and 3× FLAG-tagged Tbc1d1. The 1× load contained 30 μg of protein. A repetition of these blots with the samples from a second experiment gave similar results.

FIGURE 6.

Effect of phosphorylation of Ser²³⁷ of Tbc1d1. A, the relative amount of HA-GLUT4-GFP at the cell surface in adipocytes expressing FLAG-tagged wild-type Tbc1d1 (WT), T596A Tbc1d1 (TA), or S237A Tbc1d1 (SA) in the presence of insulin (Ins) or AICAR plus insulin was determined as described in the legend to Fig. 5. The values are the averages ± S.E. for four independent experiments. They have been normalized to 1.0 for the wild type in the insulin state. For each Tbc1d1 construct the difference between insulin and AICAR plus insulin was significant at p < 0.05, whereas the difference between the AICAR plus insulin for SA compared with AICAR plus insulin for WT and TA was not significant at p < 0.05. B, 3T3-L1 adipocytes were electroporated with vector (V) or the plasmids for FLAG-tagged wild-type, T596A, or S237A Tbc1d1 and treated with insulin, AICAR, or AICAR plus insulin as described in the legend to Fig. 5. The samples were immunoblotted for phospho-Ser²³⁷, FLAG-tagged Tbc1d1 (anti-FLAG), peEF2, and, as a loading control, AMPK. The 1× load was 50 μg of protein. A replicate of this experiment gave similar results.