Involvement of TBC1D1 and TBC1D4 in glucose and fatty acid metabolism – University of Copenhagen

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DEP. OF EXERCISE > Research > Ongoing research > Molecular Physiology > JTT Involvement

Involvement of TBC1D1 and TBC1D4 in glucose and fatty acid metabolism

Introduction

Type 2 (T2D) is one of the most rapidly growing diseases worldwide and are now being diagnosed in middle-aged and as well as young people. T2D is characterized by peripheral insulin resistance, primarily in skeletal muscle and adipose tissue, and hyperlipidaemia, and results from obesity and inactivity. Physical exercise can reduce or even reverse impaired insulin sensitivity by increasing the sensitivity towards insulin and thus contribute to the removal of glucose from the bloodstream without the involvement of insulin.

Even though we have known the beneficial effects of exercise for decades we still do not completely know the signaling pathways in skeletal muscle which are important for glucose and fatty acid uptake in response to muscle contraction and insulin stimulation. Therefore, we need treatment strategies for people with impaired insulin sensitivity that are based on results obtained from proper scientific experiments.

At the cellular level, energy stress induced by elevated rates of energy consuming processes increases the carrier mediated uptake of substrates into cells. Recent evidence suggests this process involves activation of the signaling molecule AMP-activated protein kinase (AMPK) (2). In multi-cellular organisms hormones are also important stimuli acting via membrane embedded receptors simulating uptake of substrates, often through signaling cascades involving the phosphatidyl inositol 3 kinase and Akt.

For years, the puzzle as to how different stimuli acting via different signal pathways regulate the same pool or type of vesicles has been unresolved. Interestingly, recent investigations have revealed that several of these signaling pathways intercept at the level of two Rab GTPase-activating proteins (GAPs) TBC1D1 and TBC1D4 (10). Such molecules facilitate the turnover of GTP to GDP bound to Rab GTPases, which are molecular switches in vesicular trafficking.

We have successfully explored TBC1D1 and TBC1D4 in skeletal muscle, and it is our goal to refine our knowledge on the regulation of these GAPs in intact tissues and to identify the downstream Rab GTPases targeted by TBC1D1/4. This should provide insight into vesicular trafficking and cell surface membrane transport capacity for various substrates; e.g. glucose and free fatty acids.

Objectives

  1. To test the hypothesis that regulation of TBC1D1 and TBC1D4 via site specific phosphorylation is necessary for trafficking of vesicles containing transport proteins involved in cellular metabolism, e.g. Glut4 and CD36 in skeletal muscle.
  2. To test the hypothesis that the GTPase activating effects of TBC1D1 and TBC1D4 are directed towards specific Rab proteins involved in vesicular trafficking.

Background

A family of proteins called Rab GTPases cycles between a GTP-bound (active) form and a GDP-bound (inactive) form. Rab GTPases are ineffective in exchanging and hydrolyzing nucleotides. However, the intrinsic GTPase activity can be greatly enhanced by specific guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) (1).

Rab GTPases link signal transduction cascades and molecular effectors regulating various steps in membrane traffic (15). Recently, ~20 Rab GTPases in human skeletal muscle were identified (3; 7). Of these, Rab2A, 5A, 5B, 5C, 8A, 10, 11A, 11B, and 14 have been suggested to be involved in trafficking of vesicles containing the glucose transporter (GLUT) 4 in various in vitro cell systems. As GLUT4 is the major regulated glucose transporter in skeletal muscle, heart, and adipose tissue the specific Rab GTPases associated with GLUT4 are obvious research targets in such tissues. Whether specific Rab GTPases are involved in vesicular trafficking containing other transporters e.g. the free fatty acid transporter, CD36, is at present not known.

Rab-GAPs are characterized by the presence of a conserved TBC (tre-2/USP6, BUB2, cdc16) domain (1). In addition, these GAPs contain additional known protein motifs such as PTB, SH3, and PH (4). Two TBC domain-containing proteins, TBC1D1 and TBC1D4 (5; 6; 10; 12), have GAP activity towards Rab2A, 8A, 10, and 14 in vitro (9). Interestingly, in 3T3-L1 adipocytes, GLUT4 translocation to the plasma membrane is affected only when Rab10 is knocked down (11).

In contrast, Rab8A and Rab14 appear to be important for insulin-stimulated GLUT4 translocation in L6 muscle cells. Thus, depending on cell type, GAPs appear to control specific Rab GTPases. Further investigation into whether results from cell culture studies can be transferred directly into intact mature tissues like skeletal muscle remains to be investigated. Obviously, Rab8A, Rab10, and Rab14 would be the first candidates to test.

Recently, we described regulation of TBC1D1/4 in human and rodent skeletal muscle using novel phospho-specific antibodies developed in our laboratory (8; 13). Interestingly, these GAPs were phosphorylated not only upon Akt activation but also by signaling elicited by muscle contraction and pharmacological agents (e.g. AICAR) known to regulate trafficking of vesicles containing GLUT4 and CD36.

In cell free assays we observed that these GAPs were phosphorylated by AMPK, and subsequently we provided genetic evidence that AMPK targets TBC1D1 and TBC1D4 in skeletal muscle (8; 14). Thus, we have shown that TBC1D1 and TBC1D4 multi kinase substrates, and we have described the specific amino acid residues phosphorylated in response to stimuli acting through both receptor and non-receptor mediated signaling.

It is now believed that phosphorylation of GAPs leads to regulation of the Rab-GAP activity either directly or through association with binding proteins such as IRAP, 14-3-3, or RUVBL2 (10; 16) (Figure 1). Although studies have revealed a significant role for both TBC1D1 and TBC1D4 in regulating vesicular trafficking, the importance of the individual phosphorylation sites is at present unknown.

Figure 1
A cartoon illustrating the current hypothesis regarding the regulation of vesicle trafficking; exemplified by the glucose transporter, GLUT4. The GTPase activating proteins TBC1D1/4, are regulated by association with various binding proteins and by phosphorylation by various upstream kinases through receptor and non-receptor dependent pathways. The relative importance of these events for the GTPase activating effects of the GAPs and identity of the Rab proteins targeted by these GAPs are largely unknown in mature intact tissues.

References 

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  2. Hardie DG. AMPK: a key regulator of energy balance in the single cell and the whole organism. Int J Obes (Lond) 32 Suppl 4: S7-12, 2008.
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  4. Itoh T, Satoh M, Kanno E and Fukuda M. Screening for target Rabs of TBC (Tre-2/Bub2/Cdc16) domain-containing proteins based on their Rab-binding activity. Genes Cells 11: 1023-1037, 2006.
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  8. Pehmoller C, Treebak JT, Birk JB, Chen S, MacKintosh C, Hardie DG, Richter EA and Wojtaszewski JF. Genetic disruption of AMPK signaling abolishes both contraction- and insulin-stimulated TBC1D1 phosphorylation and 14-3-3 binding in mouse skeletal muscle. Am J Physiol Endocrinol Metab 297: E665-E675, 2009.
  9. Pereira-Leal JB and Seabra MC. Evolution of the Rab family of small GTP-binding proteins. J Mol Biol 313: 889-901, 2001.
  10. Sakamoto K and Holman GD. Emerging role for AS160/TBC1D4 and TBC1D1 in the regulation of GLUT4 traffic. Am J Physiol Endocrinol Metab 295: E29-E37, 2008.
  11. Sano H, Roach WG, Peck GR, Fukuda M and Lienhard GE. Rab10 in insulin-stimulated GLUT4 translocation. Biochem J 411: 89-95, 2008.
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  13. Treebak JT, Birk JB, Hansen BF, Olsen GS and Wojtaszewski JF. A-769662 activates AMPK {beta}1-containing complexes but induces glucose uptake through a PI3 kinase-dependent pathway in mouse skeletal muscle. Am J Physiol Cell Physiol 2009.
  14. Treebak JT, Glund S, Deshmukh A, Klein DK, Long YC, Jensen TE, Jorgensen SB, Viollet B, Andersson L, Neumann D, Wallimann T, Richter EA, Chibalin AV, Zierath JR and Wojtaszewski JF. AMPK-mediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic and regulatory subunits. Diabetes 55: 2051-2058, 2006.
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  16. Xie X, Chen Y, Xue P, Fan Y, Deng Y, Peng G, Yang F and Xu T. RUVBL2, a novel AS160-binding protein, regulates insulin-stimulated GLUT4 translocation. Cell Res 2009.

Head of project: Jonas Thue Treebak