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 AMPKstimulated GLUT4 translocation in muscle.Insulin rapidly stimulates glucose transport into adipose tissue, skeletal muscle, and heart. The basis for this effect is a rapid increase in the amount of the glucose transporter GLUT4 in the plasma membrane. These cell types contain specialized intracellular vesicles enriched in GLUT4. Insulin stimulates the movement of the GLUT4 vesicles to the plasma membrane and their fusion with it (1). This process is known as insulin-stimulated GLUT4 translocation. A key signal transduction pathway for GLUT4 translocation proceeds from the insulin receptor to activation of the protein kinase Akt (1). One way in which Akt controls GLUT4 translocation is through phosphorylation of the Rab GAP 3 AS160 (also known as Tbc1d4). Current evidence supports the following scheme (2-6). In the absence of insulin AS160 keeps Rab10 and possibly other Rabs in their inactive GDP form; insulin treatment causes Akt phosphorylation of AS160; phosphorylation suppresses its GAP activity and thereby results in an elevation of the active GTP form of Rab10 and possibly other Rabs; the GTP form of the Rab(s) triggers the machinery for the docking of GLUT4 vesicles to the plasma membrane, which then leads to the fusion with the membrane.We recently partially characterized a Rab GAP known as T...

RUVBL2, a novel AS160-binding protein, regulates insulin-stimulated GLUT4 translocation

“…As shown in Figure 2A, as differentiation progressed, RUVBL2 was increasingly upregulated (Supplementary information, Figure S1). However, the expression of AS160 reached a plateau at day 4 of differentiation and declined thereafter, consistent with a previous report [11]. RUVBL2 expression levels in epididymal fat tissues of obese insulin-resistant diabetic KKAy mice and C57BL/6J control mice were compared, and RUVBL2 expression was found to be markedly reduced in fat tissues of obese insulin-resistant diabetic KKAy mice (Figure 2B).…”

AMP-activated protein kinase is activated in adipose tissue of individuals with type 2 diabetes treated with metformin: a randomised glycaemia-controlled crossover study

“…Prolonged AMPK activation has been reported to inhibit differentiation of pre-adipocytes into mature adipocytes with an associated reduced protein production of several enzymes concerned with lipid metabolism, namely ACC, fatty acid synthase (FAS) and peroxisome proliferator-activated receptor (PPAR)γ [13]. In addition, we and others have reported that AMPK activation is associated with reduced insulinstimulated glucose transport in adipocytes [14][15][16], although a conflicting study has reported no effect of AMPK stimulation when assessing translocation of the insulinsensitive glucose transporter, GLUT4 [17]. Several physiological and pharmacological stimuli have been reported to stimulate rodent adipose tissue AMPK, including exercise, starvation, troglitazone and adrenaline (epinephrine) [10,[18][19][20], whereas AMPK is inhibited in response to ghrelin, corticosterone and a high-fat diet [21][22][23] in vivo.…”

Reduced Activity of AMP-Activated Protein Kinase Protects against Genetic Models of Motor Neuron Disease

“…TBC1D1 is a Rab-GTPase activating protein that has been linked to obesity and its inactivation leads to a "lean" phenotype with increased glucose transport and fatty acid oxidation in tissues, especially skeletal muscle (Chadt et al, 2008;Frosig et al, 2010). TBC1D1 is also known to be phosphorylated at serine 237 by AMPK (Chavez et al, 2008), although the functional significance of AMPKdependent phosphorylation on TBC1D1 activities, such as glucose transport, is unknown. Considering the metabolic similarities between mice with conditional postnatal ablation of TDP-43 and mSOD1 transgenic mice, we wondered whether TBC1D1 activity is decreased in our mSOD1 models and whether the beneficial effects of reduced AMPK activity are associated with alterations in TBC1D1 activity.…”