Introduction
Insulin regulates glucose uptake into both muscle and fat cells by stimulating the movement of the facilitative glucose transporter GLUT4 from intracellular storage vesicles called GLUT4 storage vesicle (GSV)
3The abbreviations used are:
GSV
GLUT4 storage vesicle
PARsylation
poly(ADP)-ribosylation
PARP
poly(ADP-ribose) polymerase
PAR
poly(ADP-ribose)
2-DG
2-deoxyglucose
TEAB
triethylammonium bicarbonate
VAMP
vesicle-associated membrane protein
SNARE
soluble NSF attachment protein receptor
HFHSD
high-fat/high-sucrose diet
GTT
glucose tolerance test
α-MEM
α-minimum essential medium
PM
plasma membrane.
to the plasma membrane (PM) (
1- Stöckli J.
- Fazakerley D.J.
- James D.E.
GLUT4 exocytosis.
). Insulin triggers GLUT4 translocation via the canonical phosphatidylinositol 3-kinase/Akt signal transduction pathway, including the phosphorylation of the Akt substrate AS160, a Rab GTPase-activating protein (RabGAP) that is localized to GSVs (
1- Stöckli J.
- Fazakerley D.J.
- James D.E.
GLUT4 exocytosis.
2- Sano H.
- Kane S.
- Sano E.
- Mîinea C.P.
- Asara J.M.
- Lane W.S.
- Garner C.W.
- Lienhard G.E.
Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation.
,
3- Larance M.
- Ramm G.
- Stöckli J.
- van Dam E.M.
- Winata S.
- Wasinger V.
- Simpson F.
- Graham M.
- Junutula J.R.
- Guilhaus M.
- James D.E.
Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking.
,
4- Mîinea C.P.
- Sano H.
- Kane S.
- Sano E.
- Fukuda M.
- Peränen J.
- Lane W.S.
- Lienhard G.E.
AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain.
5- Peck G.R.
- Ye S.
- Pham V.
- Fernando R.N.
- Macaulay S.L.
- Chai S.Y.
- Albiston A.L.
Interaction of the Akt substrate, AS160, with the glucose transporter 4 vesicle marker protein, insulin-regulated aminopeptidase.
). Phosphorylation of AS160 is thought to regulate the GTP loading of the cognate Rab, RAB10, to initiate translocation of GSVs to and fusion with the PM (
1- Stöckli J.
- Fazakerley D.J.
- James D.E.
GLUT4 exocytosis.
,
6- Ramm G.
- Larance M.
- Guilhaus M.
- James D.E.
A role for 14-3-3 in insulin-stimulated GLUT4 translocation through its interaction with the RabGAP AS160.
,
7- Sano H.
- Eguez L.
- Teruel M.N.
- Fukuda M.
- Chuang T.D.
- Chavez J.A.
- Lienhard G.E.
- McGraw T.E.
Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane.
8- Sano H.
- Roach W.G.
- Peck G.R.
- Fukuda M.
- Lienhard G.E.
Rab10 in insulin-stimulated GLUT4 translocation.
).
Detailed molecular analyses of GLUT4 translocation have focused on the molecular composition of the GSVs in which GLUT4 is stored under basal conditions. This has led to the identification of the aminopeptidase IRAP, the sorting protein sortilin that plays a key role in the biogenesis of GSVs (
9- Kandror K.V.
- Yu L.
- Pilch P.F.
The major protein of GLUT4-containing vesicles, gp160, has aminopeptidase activity.
10- Lin B.Z.
- Pilch P.F.
- Kandror K.V.
Sortilin is a major protein component of Glut4-containing vesicles.
,
11Sortilin is essential and sufficient for the formation of Glut4 storage vesicles in 3T3-L1 adipocytes.
,
12- Jedrychowski M.P.
- Gartner C.A.
- Gygi S.P.
- Zhou L.
- Herz J.
- Kandror K.V.
- Pilch P.F.
Proteomic analysis of GLUT4 storage vesicles reveals LRP1 to be an important vesicle component and target of insulin signaling.
,
13- Huang G.
- Buckler-Pena D.
- Nauta T.
- Singh M.
- Asmar A.
- Shi J.
- Kim J.Y.
- Kandror K.V.
Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin.
,
14- Keller S.R.
- Scott H.M.
- Mastick C.C.
- Aebersold R.
- Lienhard G.E.
Cloning and characterization of a novel insulin-regulated membrane aminopeptidase from Glut4 vesicles.
15- Mastick C.C.
- Aebersold R.
- Lienhard G.E.
Characterization of a major protein in GLUT4 vesicles: concentration in the vesicles and insulin-stimulated translocation to the plasma membrane.
), the v-SNAREs VAMP2, VAMP3, and VAMP8 that play a role in docking and fusion of GSVs with the PM (
16- Cain C.C.
- Trimble W.S.
- Lienhard G.E.
Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes.
,
17- Olson A.L.
- Knight J.B.
- Pessin J.E.
Syntaxin 4, VAMP2, and/or VAMP3/cellubrevin are functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes.
18- Zhao P.
- Yang L.
- Lopez J.A.
- Fan J.
- Burchfield J.G.
- Bai L.
- Hong W.
- Xu T.
- James D.E.
Variations in the requirement for v-SNAREs in GLUT4 trafficking in adipocytes.
) and several Rab GTPases including Rab10 (
3- Larance M.
- Ramm G.
- Stöckli J.
- van Dam E.M.
- Winata S.
- Wasinger V.
- Simpson F.
- Graham M.
- Junutula J.R.
- Guilhaus M.
- James D.E.
Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking.
).
Another protein found to be associated with GSVs via its interaction with IRAP is tankyrase (
19Tankyrase is a golgi-associated mitogen-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles.
). Tankyrase 1/2 are members of the poly(ADP-ribose)polymerases (PARPs) that catalyze poly(ADP)-ribosylation or PARsylation, a protein post-translational modification involving addition of a large number of linear and/or branched ADP-riboses onto target proteins (
20- Haikarainen T.
- Krauss S.
- Lehtio L.
Tankyrases: structure, function and therapeutic implications in cancer.
). PARsylated proteins can be targeted for ubiquitination and proteasomal degradation by the E3 ubiquitin ligase RNF146 that interacts with and is allosterically activated by the PAR chain (
21- Zhang Y.
- Liu S.
- Mickanin C.
- Feng Y.
- Charlat O.
- Michaud G.A.
- Schirle M.
- Shi X.
- Hild M.
- Bauer A.
- Myer V.E.
- Finan P.M.
- Porter J.A.
- Huang S.M.
- Cong F.
RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling.
,
22- DaRosa P.A.
- Wang Z.
- Jiang X.
- Pruneda J.N.
- Cong F.
- Klevit R.E.
- Xu W.
Allosteric activation of the RNF146 ubiquitin ligase by a poly(ADP-ribosyl)ation signal.
).
Tankyrase 1/2 have been implicated in insulin-stimulated glucose uptake in adipocytes (
23- Yeh T.Y.
- Sbodio J.I.
- Tsun Z.Y.
- Luo B.
- Chi N.W.
Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase.
). Insulin enhances tankyrase activity possibly via mitogen-activated protein kinase-mediated phosphorylation (
19Tankyrase is a golgi-associated mitogen-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles.
). Tankyrase 1 knockdown or tankyrase inhibition using the broad-range PARP inhibitor PJ34 resulted in impaired insulin-stimulated GLUT4 translocation to the PM or insulin resistance in adipocytes (
23- Yeh T.Y.
- Sbodio J.I.
- Tsun Z.Y.
- Luo B.
- Chi N.W.
Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase.
). The mechanism by which tankyrase affects insulin-stimulated GLUT4 translocation has not been established (
23- Yeh T.Y.
- Sbodio J.I.
- Tsun Z.Y.
- Luo B.
- Chi N.W.
Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase.
). Furthermore, the role of tankyrase in skeletal muscle, the tissue that plays a central role in whole body insulin-mediated glucose uptake, is not known.
The development of more selective tankyrase inhibitors, such as XAV939 (
24- Wahlberg E.
- Karlberg T.
- Kouznetsova E.
- Markova N.
- Macchiarulo A.
- Thorsell A.G.
- Pol E.
- Frostell Å.
- Ekblad T.
- Öncü D.
- Kull B.
- Robertson G.M.
- Pellicciari R.
- Schüler H.
- Weigelt J.
Family-wide chemical profiling and structural analysis of PARP and tankyrase inhibitors.
,
25- Huang S.M.
- Mishina Y.M.
- Liu S.
- Cheung A.
- Stegmeier F.
- Michaud G.A.
- Charlat O.
- Wiellette E.
- Zhang Y.
- Wiessner S.
- Hild M.
- Shi X.
- Wilson C.J.
- Mickanin C.
- Myer V.
- et al.
Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.
), has been the focus of anti-cancer therapeutics in an effort to inhibit aberrant Wnt/β-catenin signaling (
26Targeting tankyrase to fight WNT-dependent tumours.
). Tankyrase PARsylates AXIN, which is part of the β-catenin destruction complex, and targets it for degradation, thereby leading to the stabilization and activation of the β-catenin pathway (
25- Huang S.M.
- Mishina Y.M.
- Liu S.
- Cheung A.
- Stegmeier F.
- Michaud G.A.
- Charlat O.
- Wiellette E.
- Zhang Y.
- Wiessner S.
- Hild M.
- Shi X.
- Wilson C.J.
- Mickanin C.
- Myer V.
- et al.
Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.
).
In this study, we utilized XAV939 (
24- Wahlberg E.
- Karlberg T.
- Kouznetsova E.
- Markova N.
- Macchiarulo A.
- Thorsell A.G.
- Pol E.
- Frostell Å.
- Ekblad T.
- Öncü D.
- Kull B.
- Robertson G.M.
- Pellicciari R.
- Schüler H.
- Weigelt J.
Family-wide chemical profiling and structural analysis of PARP and tankyrase inhibitors.
,
25- Huang S.M.
- Mishina Y.M.
- Liu S.
- Cheung A.
- Stegmeier F.
- Michaud G.A.
- Charlat O.
- Wiellette E.
- Zhang Y.
- Wiessner S.
- Hild M.
- Shi X.
- Wilson C.J.
- Mickanin C.
- Myer V.
- et al.
Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.
) to inhibit tankyrase 1/2-mediated PARsylation in L6 myotubes and showed that this impaired insulin-stimulated glucose uptake. We applied label-free quantitative proteomics (
27- Cox J.
- Hein M.Y.
- Luber C.A.
- Paron I.
- Nagaraj N.
- Mann M.
Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.
) to determine XAV939-mediated changes in the proteome and discovered that tankyrase inhibition resulted in a reduction in protein levels of a number of GSV proteins. A similar reduction in GSV proteins was observed when tankyrase 1 was knocked down using a tankyrase 1-specific siRNA. Proteasome inhibition reversed the XAV939-mediated effect on glucose uptake and GSV protein levels in L6 myotubes. These data show an important role for tankyrase in glucose metabolism in skeletal muscle cells and provide new insights into the mechanism of insulin resistance mediated by tankyrase dysfunction.
Discussion
Over the years, tankyrase has been implicated in many biological processes including carbohydrate metabolism. However, it remains unclear exactly how tankyrase mediates these effects on glucose homeostasis or if it does so in skeletal muscle. In the present study, we used two different pharmacologic inhibitors of tankyrase (XAV939, PJ34) as well as siRNA knockdown of TNKS1 to show that in L6 myotubes, tankyrase has a profound effect on regulating insulin sensitivity. Although these 3 ways of modulating tankyrase differentially affected insulin signaling, they all had in common the targeted degradation of GSV proteins that include GLUT4 itself and RAB10, and in some cases also included SORT1 and VAMP8. The proteasome appears to play a role in this effect as inhibition of the proteasome rescued the impairments in glucose uptake and GSV protein levels that were triggered upon tankyrase inhibition.
In adipocytes, PARP inhibition (PJ34) or TNKS1 knockdown also resulted in reduced insulin-stimulated GLUT4 translocation to the PM (
23- Yeh T.Y.
- Sbodio J.I.
- Tsun Z.Y.
- Luo B.
- Chi N.W.
Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase.
), indicating a similar role for tankyrase in both insulin-sensitive cell types. However, whereas in muscle cells the primary defect appeared to be on reduced levels of GSV proteins, in adipocytes there was a redistribution of GLUT4 within the cell possibly into a non-insulin–responsive compartment. One possibility that we favor is that these two mechanisms may be related. One model that explains both observations is that tankyrase activity is required to maintain the integrity or stability of GSVs by, for example, blocking their fusion with other intracellular compartments such as endosomes or lysosomes. Inhibition of tankyrase may overcome this step leading initially to redistribution of GLUT4, the principle phenotype observed in adipocytes (
23- Yeh T.Y.
- Sbodio J.I.
- Tsun Z.Y.
- Luo B.
- Chi N.W.
Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase.
), commensurate with subsequent degradation of GSV proteins, the main effect seen in muscle cells. The difference in each cell type may simply reflect differences in the kinetics of transit through these different compartments in each cell type. It was intriguing that not all GSV proteins appeared to behave in the same way in muscle cells in that IRAP and VAMP2 did not undergo tankyrase-dependent degradation indicating that certain cargo may escape sorting into the degradative pathway.
The 4 GSV proteins that showed reduced protein levels upon tankyrase inhibition in muscle all play crucial roles in GLUT4 trafficking, aside from being localized to GSVs. GLUT4 itself is obviously the most important GSV protein and responsible for insulin-stimulated glucose uptake in myotubes. The vesicle-associated membrane protein (VAMP) VAMP8 is a v-SNARE that regulates fusion of vesicles with the PM and it has previously been implicated in insulin-regulated GLUT4 trafficking in adipocytes (
18- Zhao P.
- Yang L.
- Lopez J.A.
- Fan J.
- Burchfield J.G.
- Bai L.
- Hong W.
- Xu T.
- James D.E.
Variations in the requirement for v-SNAREs in GLUT4 trafficking in adipocytes.
). Most notably, it has been observed that simultaneous disruption in the expression of VAMP2, VAMP3, and VAMP8 is required to completely block insulin-stimulated GLUT4 translocation to the PM in adipocytes and this defect could be rescued by re-expression of any one of these three VAMPs (
18- Zhao P.
- Yang L.
- Lopez J.A.
- Fan J.
- Burchfield J.G.
- Bai L.
- Hong W.
- Xu T.
- James D.E.
Variations in the requirement for v-SNAREs in GLUT4 trafficking in adipocytes.
), indicating an important role for VAMP8 in this process. Furthermore, whole body deletion of VAMP8 affected glucose metabolism and glucose uptake in skeletal muscle in mice (
34- Zong H.
- Wang C.C.
- Vaitheesvaran B.
- Kurland I.J.
- Hong W.
- Pessin J.E.
Enhanced energy expenditure, glucose utilization, and insulin sensitivity in VAMP8 null mice.
). RAB10 is a GTPase localized on GSVs (
3- Larance M.
- Ramm G.
- Stöckli J.
- van Dam E.M.
- Winata S.
- Wasinger V.
- Simpson F.
- Graham M.
- Junutula J.R.
- Guilhaus M.
- James D.E.
Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking.
) that is a substrate for the RabGAP AS160 (
4- Mîinea C.P.
- Sano H.
- Kane S.
- Sano E.
- Fukuda M.
- Peränen J.
- Lane W.S.
- Lienhard G.E.
AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain.
) and its knockdown in adipocytes has a profound effect on insulin sensitivity (
7- Sano H.
- Eguez L.
- Teruel M.N.
- Fukuda M.
- Chuang T.D.
- Chavez J.A.
- Lienhard G.E.
- McGraw T.E.
Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane.
,
8- Sano H.
- Roach W.G.
- Peck G.R.
- Fukuda M.
- Lienhard G.E.
Rab10 in insulin-stimulated GLUT4 translocation.
,
35- Brewer P.D.
- Habtemichael E.N.
- Romenskaia I.
- Mastick C.C.
- Coster A.C.
Glut4 is sorted from a Rab10 GTPase-independent constitutive recycling pathway into a highly insulin-responsive Rab10 GTPase-dependent sequestration pathway after adipocyte differentiation.
). Furthermore, mice with adipose-specific deletion of RAB10 showed impaired whole body glucose homeostasis (
29- Vazirani R.P.
- Verma A.
- Sadacca L.A.
- Buckman M.S.
- Picatoste B.
- Beg M.
- Torsitano C.
- Bruno J.H.
- Patel R.T.
- Simonyte K.
- Camporez J.P.
- Moreira G.
- Falcone D.J.
- Accili D.
- Elemento O.
- Shulman G.I.
- Kahn B.B.
- McGraw T.E.
Disruption of adipose Rab10-dependent insulin signaling causes hepatic insulin resistance.
). Although other Rabs have been implicated in GLUT4 trafficking in L6 cells, including RAB8A, RAB13, and RAB14 (
36- Ishikura S.
- Bilan P.J.
- Klip A.
Rabs 8A and 14 are targets of the insulin-regulated Rab-GAP AS160 regulating GLUT4 traffic in muscle cells.
,
37- Sun Y.
- Bilan P.J.
- Liu Z.
- Klip A.
Rab8A and Rab13 are activated by insulin and regulate GLUT4 translocation in muscle cells.
), these studies were conducted in undifferentiated myoblasts and it is possible that RAB10 only has a crucial role in differentiated cells as is the case in adipocytes (
35- Brewer P.D.
- Habtemichael E.N.
- Romenskaia I.
- Mastick C.C.
- Coster A.C.
Glut4 is sorted from a Rab10 GTPase-independent constitutive recycling pathway into a highly insulin-responsive Rab10 GTPase-dependent sequestration pathway after adipocyte differentiation.
). SORT1 has been implicated in sorting of GLUT4 into GSVs in adipocytes (
11Sortilin is essential and sufficient for the formation of Glut4 storage vesicles in 3T3-L1 adipocytes.
,
13- Huang G.
- Buckler-Pena D.
- Nauta T.
- Singh M.
- Asmar A.
- Shi J.
- Kim J.Y.
- Kandror K.V.
Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin.
) and myotubes (
38- Tsuchiya Y.
- Hatakeyama H.
- Emoto N.
- Wagatsuma F.
- Matsushita S.
- Kanzaki M.
Palmitate-induced down-regulation of sortilin and impaired GLUT4 trafficking in C2C12 myotubes.
) as well as GLUT4 retrograde traffic from endosomes to the trans-Golgi network thereby preventing GLUT4 traffic to lysosomes and subsequent degradation (
39- Pan X.
- Zaarur N.
- Singh M.
- Morin P.
- Kandror K.V.
Sortilin and retromer mediate retrograde transport of Glut4 in 3T3-L1 adipocytes.
). So, the concerted loss of each of these proteins is likely to have a profound effect on insulin sensitivity.
It has been reported that the Wnt/β-catenin signaling pathway plays a role during myogenesis (
40- Cisternas P.
- Henriquez J.P.
- Brandan E.
- Inestrosa N.C.
Wnt signaling in skeletal muscle dynamics: myogenesis, neuromuscular synapse and fibrosis.
). Because β-catenin was markedly reduced in XAV939-treated myotubes, one possibility was that this affected myotube differentiation thereby causing the reduction in GSV protein expression. We therefore assessed whether any myogenesis or skeletal muscle proteins were changed in L6 myotubes in response to XAV939 treatment. Although some myogenesis markers were not identified in our dataset (Myf5, MyoD, and myogenin), a number of skeletal muscle-specific proteins were quantified in our dataset and were not significantly changed in response to XAV939 treatment, including skeletal muscle contractile apparatus proteins (Acta1, 1.11-fold change XAV939/control; Mylpf, 1.01; Tnni1, 1.10; Tnni2, 1.09; Tnnt3, 0.96) (
41Proteomic profiling of the contractile apparatus from skeletal muscle.
) as well as skeletal muscle glucose metabolism proteins (Gys1, 1.11; Pfkm, 1.05; Phka1, 0.90; Pygm, 0.87) (
42- Adeva-Andany M.M.
- González-Lucan M.
- Donapetry-García C.
- Fernández-Fernández C.
- Ameneiros-Rodríguez E.
Glycogen metabolism in humans.
). These data indicate that XAV939 treatment had no effect on myotube differentiation and the reduction in GSV proteins is due to direct or indirect effects of tankyrase inhibition.
One question that arises from these studies is how these GSV proteins are stabilized upon proteasome inhibition? It seems unlikely that the transmembrane domain containing GSV proteins such as GLUT4, SORT1, or VAMP8 undergo proteasomal degradation as at least GLUT4 is principally degraded via lysosomes (
43- Ma J.
- Nakagawa Y.
- Kojima I.
- Shibata H.
Prolonged insulin stimulation down-regulates GLUT4 through oxidative stress-mediated retromer inhibition by a protein kinase CK2-dependent mechanism in 3T3-L1 adipocytes.
,
44- Xie B.
- Chen Q.
- Chen L.
- Sheng Y.
- Wang H.Y.
- Chen S.
The inactivation of RabGAP function of AS160 promotes lysosomal degradation of GLUT4 and causes postprandial hyperglycemia and hyperinsulinemia.
). One possibility is that tankyrase inhibition induces the proteasomal degradation of just one of these proteins, like RAB10, which results in the mis-targeting of all GSV proteins and their lysosomal degradation. Notably, GLUT4 protein levels are reduced upon knockdown or deletion of several cytoplasmic proteins, including AS160, its close homologue TBC1D1 and the retromer complex (
39- Pan X.
- Zaarur N.
- Singh M.
- Morin P.
- Kandror K.V.
Sortilin and retromer mediate retrograde transport of Glut4 in 3T3-L1 adipocytes.
,
45- Stöckli J.
- Meoli C.C.
- Hoffman N.J.
- Fazakerley D.J.
- Pant H.
- Cleasby M.E.
- Ma X.
- Kleinert M.
- Brandon A.E.
- Lopez J.A.
- Cooney G.J.
- James D.E.
The RabGAP TBC1D1 plays a central role in exercise-regulated glucose metabolism in skeletal muscle.
,
46- Lansey M.N.
- Walker N.N.
- Hargett S.R.
- Stevens J.R.
- Keller S.R.
Deletion of Rab GAP AS160 modifies glucose uptake and GLUT4 translocation in primary skeletal muscles and adipocytes and impairs glucose homeostasis.
). In this instance, blockade of proteasomal activity would prevent degradation of these regulatory factor(s), thus overcoming this deleterious mechanism. It is unlikely that this regulatory factor is targeted for proteasomal degradation via tankyrase-mediated PARsylation because upon tankyrase inhibition the protein levels of tankyrase substrates would be increased rather than decreased. Altogether, these data suggest that the reduction in GSV protein levels upon tankyrase inhibition is likely due to an indirect effect of tankyrase inhibition involving proteasome activity. The slower reversal of insulin-regulated glucose uptake (6 h) compared with the GSV protein levels (2 h) likely reflects the slower reassembly of all GSV proteins into functional insulin-responsive vesicles, a process that may well require several hours under these conditions.
Given the prominent role of tankyrase in targeting its substrates for degradation, it was somewhat surprising that of the >200 regulated proteins identified upon XAV939 treatment, about 50% were down-regulated indicating indirect effects of tankyrase inhibition as is the case with β-catenin where upon tankyrase inhibition, its negative regulator Axin is stabilized, thereby leading to β-catenin degradation (
25- Huang S.M.
- Mishina Y.M.
- Liu S.
- Cheung A.
- Stegmeier F.
- Michaud G.A.
- Charlat O.
- Wiellette E.
- Zhang Y.
- Wiessner S.
- Hild M.
- Shi X.
- Wilson C.J.
- Mickanin C.
- Myer V.
- et al.
Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.
). Gene ontology enrichment of the XAV939-regulated proteins identified “cell proliferation” as the most significantly down-regulated process, which is consistent with the role of tankyrase in Wnt/β-catenin signaling and cell cycle, thus justifying the efforts spent on development of more specific tankyrase inhibitors for cancer treatment. Other proteins that were changed upon XAV939 treatment included proteins involved in various kinds of post-translational modifications,
e.g. MINK1, DDRGK1, AKAP11, MNAT1, and thioredoxin2 (TXN2). Changes in their protein expression may affect the downstream signal transduction cascades via phosphorylation or redox events, implying potential cross-talk between PARsylation and other post-translational modifications. This is interesting in light of the effects of tankyrase 1 knockdown on Akt signaling, showing reduced insulin-stimulated Akt and AS160 phosphorylation. This is in contrast to 3T3-L1 adipocytes, where tankyrase 1 knockdown resulted in impaired insulin-stimulated glucose uptake in the absence of a signaling defect (
23- Yeh T.Y.
- Sbodio J.I.
- Tsun Z.Y.
- Luo B.
- Chi N.W.
Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase.
). One reason for this difference might be that L6 myotubes are less insulin sensitive than 3T3-L1 adipocytes (
47- Robinson R.
- Robinson L.J.
- James D.E.
- Lawrence Jr., J.C.
Glucose transport in L6 myoblasts overexpressing GLUT1 and GLUT4.
,
48- Ng Y.
- Ramm G.
- Burchfield J.G.
- Coster A.C.
- Stöckli J.
- James D.E.
Cluster analysis of insulin action in adipocytes reveals a key role for Akt at the plasma membrane.
). Tankyrase knockdown was the only tankyrase modulating approach that showed impaired Akt activity as neither of the inhibitors showed a defect in phosphorylation of the Akt substrate AS160. The reduction in Akt phosphorylation observed with PJ34 had no effect on Akt activity consistent with the fact that little Akt phosphorylation is required for maximal phosphorylation of Akt substrates (
32- Tan S.X.
- Ng Y.
- Meoli C.C.
- Kumar A.
- Khoo P.S.
- Fazakerley D.J.
- Junutula J.R.
- Vali S.
- James D.E.
- Stöckli J.
Amplification and demultiplexing in the insulin-regulated Akt pathway in adipocytes.
). The reason for the discrepancy in insulin signaling between tankyrase inhibition and knockdown might be that in the case of the inhibitors, the tankyrase protein, whereas inactive, is still present or rather increased, suggesting that tankyrase may have activity-independent effects on insulin signaling at least in L6 myotubes. Even though the different approaches of tankyrase modulation (knockdown and 2 inhibitors) showed different effects on Akt signaling, all 3 approaches converged on insulin resistance as shown by reduced 2-DG uptake as well as a reduction in GSV protein levels, the latter being the likely cause of the observed insulin resistance upon tankyrase inhibition in these cells.
Although GLUT4 levels are reduced in adipose tissue of insulin-resistant mice as well as of humans with T2D (
49- Pedersen O.
- Kahn C.R.
- Flier J.S.
- Kahn B.B.
High fat feeding causes insulin resistance and a marked decrease in the expression of glucose transporters (Glut 4) in fat cells of rats.
,
50- Sinha M.K.
- Raineri-Maldonado C.
- Buchanan C.
- Pories W.J.
- Carter-Su C.
- Pilch P.F.
- Caro J.F.
Adipose tissue glucose transporters in NIDDM: decreased levels of muscle/fat isoform.
) this is not the case in skeletal muscle (
51- Pedersen O.
- Bak J.F.
- Andersen P.H.
- Lund S.
- Moller D.E.
- Flier J.S.
- Kahn B.B.
Evidence against altered expression of GLUT1 or GLUT4 in skeletal muscle of patients with obesity or NIDDM.
). Consistent with this GLUT4 protein levels were normal in insulin-resistant muscle from HFHSD-fed mice. Interestingly, tankyrase 1 protein levels were significantly reduced in insulin-resistant muscle, albeit with no effect on protein levels of GSV proteins or β-catenin, suggesting that the reduction in tankyrase 1 was not sufficient for downstream effects. Nevertheless, these data show that tankyrase 1 protein levels were impaired in insulin-resistant muscle tissue. Notably, β-catenin has previously been implicated in insulin resistance. It was part of a human skeletal muscle gene expression signature that was diagnostic for insulin resistance and inhibition of β-catenin inhibited insulin-stimulated glucose uptake in L6 myotubes (
52- Chaudhuri R.
- Khoo P.S.
- Tonks K.
- Junutula J.R.
- Kolumam G.
- Modrusan Z.
- Samocha-Bonet D.
- Meoli C.C.
- Hocking S.
- Fazakerley D.J.
- Stöckli J.
- Hoehn K.L.
- Greenfield J.R.
- Yang J.Y.H.
- James D.E.
Cross-species gene expression analysis identifies a novel set of genes implicated in human insulin sensitivity.
) and in 3T3-L1 adipocytes (
53- Dissanayake W.C.
- Sorrenson B.
- Cognard E.
- Hughes W.E.
- Shepherd P.R.
β-Catenin is important for the development of an insulin responsive pool of GLUT4 glucose transporters in 3T3-L1 adipocytes.
). Consistent with a role for β-catenin in insulin sensitivity, drug-induced β-catenin stabilization enhances insulin-mediated glucose uptake in adipocytes (
53- Dissanayake W.C.
- Sorrenson B.
- Cognard E.
- Hughes W.E.
- Shepherd P.R.
β-Catenin is important for the development of an insulin responsive pool of GLUT4 glucose transporters in 3T3-L1 adipocytes.
). As β-catenin stabilization also occurs as a consequence of tankyrase activity (
25- Huang S.M.
- Mishina Y.M.
- Liu S.
- Cheung A.
- Stegmeier F.
- Michaud G.A.
- Charlat O.
- Wiellette E.
- Zhang Y.
- Wiessner S.
- Hild M.
- Shi X.
- Wilson C.J.
- Mickanin C.
- Myer V.
- et al.
Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.
), it is conceivable that tankyrase activation, which reportedly occurs in response to insulin (
19Tankyrase is a golgi-associated mitogen-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles.
), may in fact enhance insulin sensitivity. It is also possible that tankyrase activity is involved in GSV protein stabilization. Hence, it will be of interest to determine whether dysregulated β-catenin activity contributes to the altered stability of GSV proteins or if this represents a separate regulatory pathway that contributes to insulin resistance in parallel.
Taken together, this study for the first time unveils the total proteome regulation of tankyrase inhibition in skeletal muscle cells, which paves the way for expanding our knowledge of tankyrase dysfunction-induced insulin resistance. This study will also likely have an impact on the use of tankyrase inhibitors in humans as this treatment might result in insulin resistance.
Experimental procedures
Antibodies
Antibodies were purchased from Santa Cruz Biotechnology (tankyrase 1/2, 14-3-3), Trevigen (anti-PAR antibody), Sigma (TFRC, α-Tubulin), BD Biosciences Pharmingen (Vti1b), Synaptic Systems (VAMP2, VAMP8), and Cell Signaling Technology (PTEN, β-Catenin, AKT-pThr-308, AKT-pSer-473, AKT, AS160-pThr-642, RAB10). Antibodies against total AS160 (
3- Larance M.
- Ramm G.
- Stöckli J.
- van Dam E.M.
- Winata S.
- Wasinger V.
- Simpson F.
- Graham M.
- Junutula J.R.
- Guilhaus M.
- James D.E.
Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking.
), GLUT4 (
54- James D.E.
- Brown R.
- Navarro J.
- Pilch P.F.
Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein.
), and IRAP (
55- Shewan A.M.
- van Dam E.M.
- Martin S.
- Luen T.B.
- Hong W.
- Bryant N.J.
- James D.E.
GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in Syntaxins 6 and 16 but not TGN38: involvement of an acidic targeting motif.
) were previously described. Some antibodies were kindly provided by Dr. Gus Lienhard (SORT1 (
56- Morris N.J.
- Ross S.A.
- Lane W.S.
- Moestrup S.K.
- Petersen C.M.
- Keller S.R.
- Lienhard G.E.
Sortilin is the major 110-kDa protein in GLUT4 vesicles from adipocytes.
), Dartmouth Medical School, NH), Dr. Jagath Junutula (Rab14 (
57- Junutula J.R.
- De Maziére A.M.
- Peden A.A.
- Ervin K.E.
- Advani R.J.
- van Dijk S.M.
- Klumperman J.
- Scheller R.H.
Rab14 is involved in membrane trafficking between the Golgi complex and endosomes.
), Genentech Inc., CA), and Dr. Robert Parton (Rab11 (
58- Urbé S.
- Huber L.A.
- Zerial M.
- Tooze S.A.
- Parton R.G.
Rab11, a small GTPase associated with both constitutive and regulated secretory pathways in PC12 cells.
), University of Queensland, Brisbane, Australia).
Cell culture and treatment
L6 rat myoblasts were grown in α-minimum essential medium (α-MEM) containing 10% fetal bovine serum in 10% CO2 at 37 °C. L6 myoblasts were differentiated into myotubes using α-MEM with 2% fetal bovine serum when myoblasts reached 90–95% confluence. All L6 studies used myotubes between 6 and 8 days after differentiation. L6 myotubes were treated with 0.1, 1, or 10 μm XAV939 for 6, 24, or 48 h, respectively, prior to further experiments.
siRNA transfection
Three independent siRNA oligonucleotide sequences were designed against rat tankyrase 1 (Shanghai GenePharma Co., Ltd.) with the following sequences: GCAGCGAACGUGAAUGCAATT, GCGAAAGUCUACUCCGUUATT, and GCGUCGAAGUCUGUUCUUUTT. A scrambled siRNA oligonucleotide (UUCUCCGAACGUGUCACGUTT) was used as a negative control. Differentiated L6 myotubes (days 5 or 6) were transfected with TNKS1 or scrambled siRNA using Lipofectamine 2000 (Invitrogen) for 24 and 48 h, respectively, and experiments were performed on day 7 of differentiation.
PARsylated protein enrichment
PARsylated proteins were enriched as previously described (
59- Zhang Y.
- Wang J.
- Ding M.
- Yu Y.
Site-specific characterization of the Asp- and Glu-ADP-ribosylated proteome.
). Briefly, cells were lysed in SDS lysis buffer (1% SDS, 10 m
m HEPES, pH 8.5). Lysates were mixed with
m-aminophenylboronic acid-agarose. After 1 h incubation at room temperature, beads were washed with SDS wash buffer (1% SDS, 100 m
m HEPES, 150 m
m NaCl, pH 8.5) and subsequently with wash buffer containing 100 m
m HEPES and 150 m
m NaCl (pH 8.5). Beads were mixed with 3
m ammonium acetate (pH 5.0) for 1.5 h and washed once with SDS lysis buffer. Beads were then incubated with 4× SDS-PAGE loading buffer at 95 °C for 10 min and the eluate was subjected to immunoblotting analysis.
SDS-PAGE and Western blotting
Protein concentrations were determined via BCA assay and 10 μg of proteins were resolved by SDS-PAGE. The gel was transferred to PVDF membranes. Membranes were blocked in 5% skim milk powder in TBST (0.1% Tween 20 in TBS) for 2 h followed by an overnight incubation at 4 °C with the indicated primary antibody solutions. Membranes were incubated with an appropriate secondary antibody at room temperature for 1 h before signals were detected using a Chemidoc. Immunoblots were quantified by ImageJ software.
[3H]2-DG uptake assay
This assay was performed as previously described (
30- Fazakerley D.J.
- Naghiloo S.
- Chaudhuri R.
- Koumanov F.
- Burchfield J.G.
- Thomas K.C.
- Krycer J.R.
- Prior M.J.
- Parker B.L.
- Murrow B.A.
- Stöckli J.
- Meoli C.C.
- Holman G.D.
- James D.E.
Proteomic analysis of GLUT4 storage vesicles reveals tumor suppressor candidate 5 (TUSC5) as a novel regulator of insulin action in adipocytes.
). Briefly, L6 myotubes grown in 24-well plates were serum starved for 2 h in basal α-MEM and washed with 37 °C Krebs-Ringer buffer with 0.2% BSA. Cells were then stimulated with 100 n
m insulin for 20 min in Krebs-Ringer buffer with 0.2% BSA. To determine nonspecific glucose uptake, 25 μ
m cytochalasin B was added to control wells prior to addition of [
3H]2-DG (0.125 μCi/well, 50 μ
m unlabeled 2-DG) during the final 5 min of insulin stimulation. Following rapid washes with ice-cold PBS, cells were solubilized in 1% Triton X-100 in PBS on a shaker for 1 h and assessed for radioactivity by scintillation counting using a β-scintillation counter. All data were normalized to protein concentration.
Mouse experiments
All experiments were approved by the University of Sydney Animal Ethics Committee. Male C57Bl/6J mice were group-housed on a 12-h light/dark cycle with free access to chow diet (13% calories from fat, 65% carbohydrate, 22% protein) or HFHSD (47% fat (7:1 lard to safflower oil ratio), 32% carbohydrate, 21% protein), and water. Mice were subjected to an intraperitoneal glucose tolerance test (GTT, 2 g/kg lean mass) after 5 weeks on either chow or HFHSD. Mice were euthanized after 6 weeks on the diet, quadriceps muscle was removed, freeze-clamped, frozen and powdered, followed by immunoblotting.
Sample preparation for MS analysis
L6 myotubes were washed with ice-cold PBS and harvested in lysis buffer (6 m urea, 2 m thiourea, 25 mm TEAB, 0.1% SDS). After sonication, cell lysates were centrifuged at 15,000 × g for 30 min at room temperature. The supernatant was precipitated using pre-chilled acetone overnight. Protein pellets were resuspended in buffer containing 6 m urea, 2 m thiourea, 25 mm TEAB. After reduction with 10 mm DTT at room temperature for 60 min and alkylation with 25 mm iodoacetamide at room temperature for 30 min in the dark, the protein mixture was digested using LysC (enzyme:substrate, 1:100) at 30 °C for 2 h. The protein solution was then diluted 1:5 using 25 mm TEAB and further digested with trypsin (enzyme:substrate 1:50) at 37 °C overnight. The peptide mixture was desalted using stage-tips and dried by vacuum centrifugation for total proteome analysis.
Mass spectrometry analysis and data processing
MS-based total proteome analysis was performed on a Dionex UltiMate 3000 coupled to a Q-Exactive Plus in positive polarity mode. Peptides were separated using an in-house packed 75 μm × 40-cm column (1.9 μm particle size, C18AQ) with a gradient of 10–35% acetonitrile containing 0.1% formic acid over 360 min at 250 nl/min at 55 °C. The MS1 scan was acquired from 350 to 1550 m/z (70,000 resolution, 3e6 automatic gain control, 100 ms injection time) followed by MS/MS data-dependent acquisition of the top 20 ions with higher-energy collisional dissociation (17,500 resolution, 1e5 automatic gain control, 60 ms injection time, 27 NCE, 1.2 m/z isolation width).
Raw data were processed using MaxQuant (version 1.5.3.24) (
60MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
) against a Uniprot rat database (May 2016, 37,402 entries) with default settings and minor changes. Oxidation of methionine and acetylation of protein N terminus were set as variable modifications and carbamidomethylation of cysteine was set as a fixed modification, “re-quantify,” “second peptides searching,” and “match between runs” were enabled. Both peptide spectral match and protein false discovery rate were set to 1%. “Label-free quantification method” (
27- Cox J.
- Hein M.Y.
- Luber C.A.
- Paron I.
- Nagaraj N.
- Mann M.
Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.
) was used for protein quantification and normalization. The data were filtered to retain proteins quantified in at least 2 samples in each of the control and XAV939-treated conditions. Bioinformatics analysis was mainly performed using the LIMMA package (
61- Ritchie M.E.
- Phipson B.
- Wu D.
- Hu Y.
- Law C.W.
- Shi W.
- Smyth G.K.
limma powers differential expression analyses for RNA-sequencing and microarray studies.
) in the R programming environment to identify proteins differentially regulated with XAV939 treatment. Functional annotation was done by David version 6.8β (released in May 2016,
https://david-d.ncifcrf.gov/)
4Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
(
62- Huang da W.
- Sherman B.T.
- Lempicki R.A.
Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
). The MS proteomics data have been deposited at the ProteomeXchange Consortium with identifier PXD007182.
Statistical analysis
Data are presented as mean ± S.E., unless otherwise indicated. Statistical analyses were performed using t test or analysis of variance using GraphPad Prism software. Significance was set at p < 0.05 and p values are indicated.
Article info
Publication history
Published online: April 18, 2018
Received in revised form:
April 5,
2018
Received:
November 21,
2017
Edited by Jeffrey E. Pessin
Footnotes
This work was supported in part by National Health and Medical Research Council (NHMRC) project Grants GNT1061122 (to D. E. J.) and GNT1068469 (to J. S). The contents of the published material are solely the responsibility of the individual authors and do not reflect the view of NHMRC. The authors declare that they have no conflicts of interest with the contents of this article.
This article contains Tables S1–S5.
The MS proteomics data have been deposited at the ProteomeXchange Consortium with identifier PXD007182.
Copyright
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.