Disruption of the AMPK–TBC1D1 nexus increaseslipogenic gene expression and causes obesity inmice via promoting IGF1 secretionLiang Chena,1, Qiaoli Chena,1, Bingxian Xiea,1, Chao Quana, Yang Shenga, Sangsang Zhua, Ping Ronga, Shuilian Zhoua,Kei Sakamotob, Carol MacKintoshc, Hong Yu Wanga,d,2,3, and Shuai Chena,d,2,3

aState Key Laboratory of Pharmaceutical Biotechnology and Ministry of Education Key Laboratory of Model Animal for Disease Study, Model AnimalResearch Center, Nanjing University, Nanjing 210061, China; bMedical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of LifeSciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom; cDivision of Cell and Developmental Biology, College of Life Sciences, Universityof Dundee, Dundee DD1 5EH, Scotland, United Kingdom; and dCollaborative Innovation Center of Genetics and Development, Shanghai 200438, China

Edited by Marc Montminy, The Salk Institute for Biological Studies, La Jolla, CA, and approved May 16, 2016 (received for review January 12, 2016)

Tre-2/USP6, BUB2, cdc16 domain family member 1 (the TBC domainis the GTPase activating protein domain) (TBC1D1) is a Rab GTPaseactivating protein that is phosphorylated on Ser231 by the AMP-acti-vated protein kinase (AMPK) in response to intracellular energystress. However, the in vivo role and importance of this phosphoryla-tion event remains unknown. To address this question, we generateda mouse model harboring a TBC1D1Ser231Ala knockin (KI) mutation andfound that the KI mice developed obesity on a normal chow diet.Mechanistically, TBC1D1 is located on insulin-like growth factor 1(IGF1) storage vesicles, and the KI mutation increases endocrinaland paracrinal/autocrinal IGF1 secretion in an Rab8a-dependentmanner. Hypersecretion of IGF1 causes increased expression oflipogenic genes via activating the protein kinase B (PKB; alsoknown as Akt)–mammalian target of rapamycin (mTOR) pathwayin adipose tissues, which contributes to the development of obesity,diabetes, and hepatic steatosis as the KI mice age. Collectively,these findings demonstrate that the AMPK–TBC1D1 signaling nexusinteracts with the PKB–mTOR pathway via IGF1 secretion, whichconsequently controls expression of lipogenic genes in the adiposetissue. These findings also have implications for drug discovery tocombat obesity.

AMPK | TBC1D1 | phosphorylation | IGF1 secretion | obesity

Due to changes in diet and lifestyle, metabolic syndrome in-cluding obesity, type 2 diabetes, and nonalcoholic fatty liver

disease is increasing in prevalence worldwide, putting a hugeburden on modern society and motivating us to get better un-derstanding of these diseases and how to combat them.High energy intake and lack of physical activity can result in

imbalances of energy metabolism and changed energy status ofthe body, which is monitored by the energy-sensing kinase AMP-activated protein kinase (AMPK) (1). The AMPK holoenzyme isa heterotrimer, consisting of catalytic α (α1 and α2), regulatory β(β1 and β2), and γ (γ1, γ2 and γ3) subunits (1). AMPK is mainlyactivated through Thr172 phosphorylation in its catalytic domain bythe upstream liver kinase B1 (LKB1) under energy stress conditionsthat increase the cellular AMP:ATP ratio (2, 3). AMPK can regu-late both glucose and lipid metabolism by its phosphorylation ofmultiple substrates (4). For instance, acetyl-CoA carboxylase (ACC),an enzyme important for fatty acid synthesis and oxidation, is reg-ulated through an inhibitory phosphorylation by AMPK, which isessential for the control of lipid metabolism (5). The LKB1–AMPKpathway can also control glucose uptake into skeletal muscle bypromoting translocation of the glucose transporter 4 (GLUT4) fromits intracellular storage sites onto plasma membrane in response to apharmacological AMPK activator, 5-aminoimidazole-4-carboxamideriboside (AICAR), or muscle contraction (6). However, the mech-anism by which AMPK promotes translocation of GLUT4 ontoplasma membrane is not well understood.Tre-2/USP6, BUB2, cdc16 domain family member 1 [the TBC

domain is the GTPase activating protein (GAP) domain] (TBC1D1)

is an Rab GTPase activating protein (RabGAP), and an R125Wmutation on this protein has been linked to familial female obesity(7). A truncation or KO of TBC1D1 confers leanness to mice fedon high fat diet with increased fatty acid oxidation in skeletal muscle(8, 9). TBC1D1 has also been implicated in regulating muscle glu-cose uptake (10), whereas its deficiency causes a decreased ex-pression level of GLUT4 in skeletal muscle (8, 9, 11).We previously identified TBC1D1 as an AMPK substrate that

is phosphorylated on Ser231 by AMPK and interacts with 14-3-3proteins on Ser231 phosphorylation (12). However, the physio-logical role of this AMPK–TBC1D1 signal nexus remains elusive.In this study, we generated a TBC1D1Ser231Ala knockin (KI)mouse model to address the functions of TBC1D1 Ser231 phos-phorylation in vivo.

ResultsGeneration and Characterization of the TBC1D1 KI Mice.The TBC1D1KI mice were generated using the gene targeting strategy illus-trated in SI Appendix, Fig. S1A. The expression levels of theTBC1D1Ser231Ala mutant proteins were normal, and there wasno detectable alteration in the expression of the related RabGAPAS160 (also known as TBC1D4) in various tissues from the KImice. As expected, the phosphorylation of Ser231 on TBC1D1 was

Significance

Excess energy intake and physical inactivity are two major factorscausing obesity, but the underlying mechanisms have not beenfully understood. Both excess energy intake and physical inactivityincrease cellular energy status that is monitored by the energy-sensing AMP-activated protein kinase (AMPK). We demonstratethat the AMPK–tre-2/USP6, BUB2, cdc16 domain family member 1(the TBC domain is the GTPase activating protein domain) (TBC1D1)signaling nexus regulates insulin-like growth factor 1 (IGF1) secre-tion. Disruption of this AMPK–TBC1D1 signaling nexus in mice en-hances lipogenic gene expression via promoting IGF1 secretion,causes obesity, and eventually leads to development of metabolicsyndrome. These findings reveal a novel regulatory mechanismthat links energy status to development of obesity via control ofIGF1 secretion.

Author contributions: H.Y.W. and S.C. designed research; L.C., Q.C., B.X., C.Q., Y.S., S. Zhu,P.R., S. Zhou, H.Y.W., and S.C. performed research; L.C., Q.C., B.X., C.Q., Y.S., S. Zhu, P.R.,S. Zhou, H.Y.W., and S.C. analyzed data; and K.S., C.M., H.Y.W., and S.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1L.C., Q.C., and B.X. contributed equally to this work.2H.Y.W. and S.C. contributed equally to this work.3To whom correspondence may be addressed. Email: wanghy@nicemice.cn or schen6@163.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1600581113/-/DCSupplemental.

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not detectable in the tissues from the KI mice (SI Appendix, Fig.S1B). The pharmacological AMPK activator metformin inducedphosphorylation of AMPK in primary hepatocytes from the KImice to a similar extent as in WT cells. In contrast and as an-ticipated, metformin-stimulated TBC1D1 Ser231 phosphorylationwas only detected in the lysates of WT, but not KI, hepatocytes.The metformin-stimulated 14-3-3–TBC1D1 interaction assessedby 14-3-3 overlay assays was diminished in the TBC1D1 immu-noprecipitates from the KI cell lysates compared with the WTcontrols (Fig. 1A). Taken together, these data validate the suit-ability of the TBC1D1 KI mice and their derived cells for studyingthe specific in vivo and in vitro function of TBC1D1 Ser231

phosphorylation.

The TBC1D1 KI Mice Developed Obesity and Displayed Characteristicsof Metabolic Syndrome When They Aged. We next monitored thegrowth of the KI mice and found that they became significantlyheavier than the WT littermates from 5 wk after birth (SI Ap-pendix, Fig. S1C). The KI mice had longer bodies and tibias thanWTs, and their growth plates of tibia were significantly elongatedin the zones of proliferation and of hypertrophy (SI Appendix,Fig. S1 D–G). The increased body mass of the young KIs (lessthan 4 mo old) was accounted for by their significantly increasedlean mass. In contrast, the fat mass in the young KI mice wascomparable to that of the WT littermates (Fig. 1B and SI Ap-pendix, Fig. S2 A and B). However, in contrast to the normal fatmass and enhanced lean mass of young KI mice, older KI micebecame fatter, and had higher lean and fat masses than WTs(Fig. 1 C andD and SI Appendix, Fig. S2 C andD). Around 5 mo of

age was a transition period for increased fat mass in the KI mice.Consistent with the increased fat mass, adipocytes were markedlyenlarged in the older KI mice (Fig. 1 E–H). Obesity is a high riskfactor for development of type II diabetes and hepatic steatosis.The young KI mice (4.5 mo old) had normal blood chemistry pa-rameters including blood glucose, free fatty acid, triglyceride, totalcholesterol, and plasma insulin and displayed normal glucose tol-erance. However, as these animals aged (∼1 y old), they developedhyperglycemia, hyperinsulinemia, and hypercholesterolemia andbecame glucose intolerant and insulin resistant (SI Appendix, Fig.S1 H–N). Furthermore, these mice developed hepatic steatosis,and their livers were significantly enlarged and contained largerlipid droplets (SI Appendix, Fig. S1 O–Q).

Lipogenic Gene Expression Was Increased in the Adipose of theTBC1D1 KI Mice. Although TBC1D1 has been implicated in regu-lating muscle glucose uptake (10), we found that GLUT4 expres-sion and glucose uptake was normal in multiple tissues/primarycells and insulin-stimulated GLUT4 translocation was not alteredin primary adipocytes from the KI mice (SI Appendix, Fig. S3),suggesting that glucose uptake did not account for the obesephenotype of these mice.We then investigated whether altered adipogenesis, lipogen-

esis, or lipolysis in the adipose might account for the obesephenotype of the KI mice. To this end, we analyzed expression ofkey regulators for these processes in the adipose of young KImice (4 mo old). Both lipolysis marker [adipose triglyceride li-pase (ATGL)] and adipogenesis markers [CAAT/enhancer-binding protein α (CEBPα) and peroxisome proliferator-activated receptor γ (PPARγ)] remained normal in the adipose ofthe KI mice (Fig. 2A and SI Appendix, Fig. S4A). In contrast, theexpression of key enzymes for lipogenesis including fatty acidsynthase (FASN), ATP-citrate lyase (ACL), cytoplasmic acetyl-CoA synthetase (AceCS1), acetyl-CoA carboxylase (ACC1), andstearoyl-CoA desaturase (SCD) was significantly up-regulated inthe KI adipose tissue, despite the normal mRNA levels of sterolregulatory element-binding protein 1 (SREBP1), which is an up-stream transcriptional regulator for these lipogenic genes(Fig. 2 A and B and SI Appendix, Fig. S4B). Full-length SREBP1(flSREBP1) resides on the endoplasmic reticulum (ER), and itsN-terminal fragment (nSREBP1) enters the nucleus after cleavageand regulates lipogenic gene expression (13). Interestingly, thenSREBP1 levels were significantly increased in the adipose of theKI mice (Fig. 2B and SI Appendix, Fig. S4B). The cleavage ofSREBP1 on the ER is under control of mammalian target ofrapamycin (mTOR) (14). Consistently, phosphorylation of proteinkinase B (PKB), tuberous sclerosis 2 (TSC2, also known astuberin), mTOR, and downstream mTOR targets S6K and 4EBP1were all increased in the KI adipose tissue (Fig. 2C and SI Ap-pendix, Fig. S2 A–C). Lipin1, a downstream target of mTOR, candown-regulate SREBP1 activity in the nucleus and consequentlycontrol the expression of lipogenic genes (15). The entry of lipin1into the nucleus is under control of its phosphorylation by mTOR,and this phosphorylation promotes lipogenesis by releasing theinhibitory effect of lipin1 on SREBP1 and consequently up-regulates lipogenic gene expression (15). Consistent with mTORactivation, the phosphorylation of lipin1 was significantly up-regulated in the KI adipose tissue (Fig. 2C and SI Appendix,Fig. S4C). Collectively, these data suggest that the activation ofPKB–mTOR pathway in the KI mice may up-regulate lipogenicgenes via elevation of nSREBP1 levels and relief of the inhibitoryeffect of lipin1. We further found that the phosphorylation ofPKB, TSC2, mTOR, S6K and 4EBP1 was also up-regulated inskeletal muscle and liver of the KI mice (SI Appendix, Fig. S4D–I). Although the PKB activation in the KI mice expectedlyincreased the phosphorylation of its downstream targets in-cluding TSC2, GSK3, and FOXO1, the phosphorylation ofAS160, a key regulator for insulin-stimulated glucose uptake,was not increased in the adipose and skeletal muscle of the KImice (SI Appendix, Fig. S4).

Fig. 1. Generation and characterization of the TBC1D1 KI mice. (A) Ser231

phosphorylation and 14-3-3 binding of TBC1D1 isolated from primary hepa-tocytes that had been treated with metformin (Met) for 1 h. (B–D) Bodycomposition of the male TBC1D1Ser231Ala KI mice measured at the age of 14 wk(B, WT: n = 18; KI: n = 19), 20–22 wk (C, WT: n = 15; KI: n = 15), and 52 wk (D,WT: n = 8; KI: n = 9). (E–H) Histology (E) and cell number (F) and size (G and H)of the adipose from the WT and TBC1D1 KI male mice. Bars indicate 50 μm inlength. Six sections per genotype were analyzed to measure cell numbers. Thenumbers shown in the bar graphs refer to the number of cells measured in theexperiments. The data are given as the mean ± SEM, and the asterisk indicatesP < 0.05.

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Plasma Insulin-Like Growth Factor 1 Levels Were Elevated in theTBC1D1 KI Mice. We next sought to find out how the KI muta-tion causes the activation of the PKB–TSC2–mTOR pathway.Considering the overgrowth phenotype of the KI mice, we sur-mised that plasma levels of insulin-like growth factor 1 (IGF1), akey growth promoter that activates the PKB–mTOR pathway inmultiple tissues, might be elevated. Indeed, plasma IGF1 levelswere significantly higher in the KI mice (Fig. 3A and SI Appendix,Fig. S2G). Consistently, activation of the IGF1 receptor (IGF1R)was significantly enhanced in the KI mice (Fig. 2C and SI Appendix,Fig. S4). In contrast to IGF1, plasma IGF2 and IGF1-bindingprotein IGFBP3 levels were normal in the KI mice (SI Appendix,Figs. S1 R and S, and S2H). Expression of IGF1, IGF2, andIGFBPs were normal in the liver of the KI mice (SI Appendix, Fig.S5). IGF1 expression in the liver is under control of growth hor-mone (GH), whose levels in the plasma were normal in the KImice with unaltered hepatic GH signaling (SI Appendix, Figs. S1T,S2I, and S5H). These data show that plasma IGF1 levels wereselectively elevated in the KI mice.

TBC1D1 Colocalizes with IGF1 Vesicles and Regulates IGF1 Secretion.IGF1 is synthesized and temporarily stored within vesicles inhepatocytes and then secreted into the bloodstream. Interestingly,when TBC1D1 and IGF1 were coexpressed in human liver car-cinoma HepG2 cells, the two proteins largely colocalized witheach other (SI Appendix, Fig. S6A). We further found that en-dogenous TBC1D1 was localized on the IGF1 storage vesicles inprimary hepatocytes (Fig. 3B) and in myotubes (SI Appendix, Fig.S6B). Together, these data suggest that TBC1D1 might be in-volved in the regulation of IGF1 secretion. Indeed, knockdownof TBC1D1 via small interference RNA (siRNA) significantlydecreased IGF1 secretion rates in primary hepatocytes with noalteration in IGF1 expression. Furthermore, in primary hepato-cytes from our previously reported TBC1D1 KO mice (16), IGF1secretion rates were also significantly lower than those of the WTcontrols, despite normal hepatic IGF1 expression (Fig. 3 C–F).

The TBC1D1Ser231Ala KI Mutation Increases both Endocrinal andParacrinal/autocrinal IGF1 Secretion. Consistent with the elevatedlevels of plasma IGF1 in the KI mice, IGF1 secretion rates ofprimary hepatocytes from these mice were significantly higherthan those of WT cells under unstimulated conditions (Fig. 3G).

The widely used AMPK activators, metformin and AICAR, sig-nificantly decreased IGF1 secretion rates of WT hepatocytes. Al-though the rates of IGF1 secretion by TBC1D1 KI hepatocytesdecreased in response to metformin or AICAR, these secretionrates still remained significantly higher than those of WT cellstreated with these drugs. We confirmed that the activation ofAMPK by AICAR and metformin was comparable in the twogenotypes of primary hepatocytes, with no effect on IGF1 proteinexpression (SI Appendix, Fig. S6C). We further found that the KImutation also enhanced paracrinal/autocrinal IGF1 secretion inprimary adipocytes (Fig. 3H), primary chondrocytes (SI Appendix,Fig. S6 C and D), and isolated skeletal muscle ex vivo (SI Appendix,Fig. S6F). Consistent with normal plasma levels of IGFBP3,secretion of IGFBP3 was unaltered in primary hepatocytes fromthe KI mice (SI Appendix, Fig. S5 L andM). These data show thatthe KI mutation selectively affects IGF1 secretion. Consistentwith the hypersecretion of IGF1, the PKB–TSC2–mTOR pathwaywas activated in primary adipocytes and hepatocytes isolated fromthe KI mice (SI Appendix, Figs. S7 A and B, and S8 A–C).

Inhibition of IGF1 Secretion or Action Blunted the Hyperactivation ofthe PKB Pathway in Primary Cells from the TBC1D1 KI Mice. To es-tablish a causal relationship between the hypersecretion of IGF1and the activation of the PKB–TSC2–mTOR pathway in the KImice, we used an IGF1R inhibitor, picropodophyllin (PPP) (17),to treat primary adipocytes and hepatocytes from the KI mice. We

Fig. 2. Lipogenenic gene expression in the adipose of the TBC1D1 KI mice.(A) mRNA expression of indicated genes in the epididymal fat of the WT andKI male mice (16 wk old). n = 5. (B) Expression of indicated proteins weremeasured in the adipose from the WT and KI male mice (16 wk old) usingGAPDH as a loading control. (C) Phosphorylated and total lipin1, mTOR,TSC2, PKB, and IGF1R were measured in the adipose from the KI mice andWT littermates (16 wk old). The data are given as the mean ± SEM, and theasterisk indicates P < 0.05.

Fig. 3. Plasma IGF1 levels and IGF1 secretion in primary cells from the TBC1D1KI mice. (A) Plasma levels of IGF1 in the male WT and KI mice (8 wk old). n = 7.(B) Colocalization of TBC1D1 and IGF1 in primary hepatocytes. Bars indicate10 μm in length. (C and D) Expression of TBC1D1 and IGF1 in TBC1D1-depletedprimary hepatocytes via siRNA (C) or in primary hepatocytes from the TBC1D1KO mice (D). NC, negative control. (E and F) IGF1 secretion in TBC1D1-depletedprimary hepatocytes (E) or in primary hepatocytes from the TBC1D1 KO mice(F). NC, negative control. n = 6. (G) IGF1 secretion in primary hepatocytes fromthe WT and TBC1D1 KI mice. aP < 0.05 (WT metformin or AICAR vs. WT un-treated) and bP < 0.05 (KI metformin or AICAR vs. KI untreated). n = 8. (H) IGF1secretion in primary adipocytes from the KI mice. WT: n = 5; KI: n = 6. The dataare given as the mean ± SEM, and the asterisk indicates P < 0.05. Statisticalanalysis for G was carried out using two-way ANOVA.

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found that blocking the IGF1 receptor with PPP largely normal-ized phosphorylation of the signaling proteins in the IGF1R–PKB–mTOR pathway in TBC1D1 KI adipocytes. Moreover, thelevels of nSREBP1 were significantly increased in the KI adipo-cytes under untreated conditions, and PPP treatment diminishedthis increase (Fig. 4A and SI Appendix, Fig. S7C). Similar effectson PKB activation by PPP were observed in primary hepatocytes(SI Appendix, Fig. S8 D and E). We examined the possibility thatWT and TBC1D1 KI cells might differ in their sensitivities toIGF1 or PPP. We found treatment with exogenous IGF1 activatedPKB in the two genotypes of cells to similar extents, and PPPtreatment could inhibit PKB activation elicited by exogenousIGF1 in cells from both genotypes of mice to similar levels (SIAppendix, Figs. S7D and S8 F and G). These findings suggest thatthe hyperactivation of PKB in KI adipocytes and hepatocytes wasmost likely due to hypersecretion of IGF1. It was still possible thatthe KI mutation affected the IGF1R independent of IGF1 se-cretion. To examine this possibility, we down-regulated IGF1 viasiRNA in primary hepatocytes from the WT and KI mice. IGF1Rexpression was normal in the KI hepatocytes, whereas its phos-phorylation was significantly increased in parallel with PKBphosphorylation in these cells (SI Appendix, Fig. S8 H and I). Onknockdown of IGF1, IGF1 secretion rates were decreased in theKI hepatocytes, which largely normalized IGF1R and PKB acti-vation (SI Appendix, Fig. S8H and I), suggesting that the effects ofthe KI mutation on the IGF1R–PKB pathway requires IGF1 se-cretion. Together, these data strongly suggest a causal relationshipbetween hypersecretion of IGF1 and activation of the PKB–TSC2–mTOR pathway in the TBC1D1 KI mice.

Inhibition of the IGF1–mTOR Pathway Normalized the Expression ofLipogenic Genes in Primary Adipocytes from the TBC1D1 KI Mice.IGF1 can stimulate lipogenesis in porcine adipose tissue ex vivo(18), and it can also increase lipogenesis via activation of the PKBpathway in SEB-1 sebocytes (19). Similarly, treatment of WTprimary adipocytes with IGF1 activated the PKB–mTOR pathwayand strongly increased lipogenic gene expression (SI Appendix,Fig. S9). Again, we used primary adipocytes from the KI mice toinvestigate the relationship between the hypersecretion of IGF1and lipogenic gene expression. The mRNA levels of lipogenicgenes were significantly increased in primary KI adipocytes underbasal conditions, which were significantly inhibited on PPP treat-ment (Fig. 4B), suggesting that the increased lipogenic gene ex-pression was most likely due to the hypersecretion of IGF1 andconsequent hyperactivation of its receptor. Moreover, an mTORinhibitor rapamycin inhibited the expression of these lipogenicgenes in the KI adipocytes (Fig. 4B), which further supports ourproposal that hyperactivation of PKB–mTOR pathway down-stream of the IGF1R is responsible for the induction of lipogenicgenes in the adipose of the KI mice.

Rab8a Plays a Key Role Downstream of the TBC1D1 in Regulating IGF1Secretion.We then sought to find out how TBC1D1 and its Ser231phosphorylation regulate IGF1 secretion. IGF1 secretion rateswere largely restored when a human recombinant TBC1D1(hTBC1D1) or an hTBC1D1Ser237Ala mutant protein (Ser237 onhuman TBC1D1 corresponds to Ser231 on the mouse protein)were expressed in the TBC1D1 KO hepatocytes (SI Appendix,Fig. S10). Furthermore, metformin and AICAR could also sig-nificantly decrease IGF1 secretion rates in these hepatocytesexpressing hTBC1D1 or hTBC1D1Ser237Ala mutant proteins. In-terestingly, the expression of a GAP-deficient TBC1D1 mutant(20) could not restore IGF1 secretion rates, suggesting that theGAP activity of TBC1D1 is required for IGF1 secretion. We nextinvestigated which Rab(s) downstream of TBC1D1 mediatesIGF1 secretion by downregulating four known in vitro TBC1D1substrates, namely Rab2a, Rab8a, Rab10, and Rab14 (20).Knockdown of Rab8a caused a significant decrease in IGF1 se-cretion rates in WT primary hepatocytes, whereas down-regu-lation of Rab2a, Rab10, and Rab14 had no significant effect (SIAppendix, Fig. S11 A and B). We also found that Rab8a largely

colocalized with IGF1 in primary hepatocytes (Fig. 5A).Furthermore, knockdown of Rab8a normalized IGF1 secre-tion in primary hepatocytes from the TBC1D1 KI mice (Fig.5 B and C), suggesting that regulation of IGF1 secretion bythe TBC1D1 Ser231 phosphorylation requires Rab8a. Over-expression of the WT Rab8a, but not a GTP-bound Rab8aQ67L

mutant, could restore IGF1 secretion in the TBC1D1 KO he-patocytes (Fig. 5 D and E). Although a GDP-bound Rab8aT22N

mutant was expressed at a much lower level, it could still partiallyrestore IGF1 secretion in the TBC1D1 KO hepatocytes (Fig. 5 Dand E). In an in vitro assay, immunoprecipitated recombinanthTBC1D1 displayed significant GAP activity toward Rab8a andenhanced the GTPase activity of Rab8a. The GAP activity ofhTBC1D1 toward Rab8a was inhibited by its cellular phosphory-lation in response to AMPK activator phenformin (SI Appendix,Fig. S11C) and, in contrast, was enhanced by Ser237Ala mutation(Fig. 5F). Moreover, more GDP-bound Rab8a and less GTP-bound Rab8a were found in the adipose of the TBC1D1 KI mice inpulldown assays (Fig. 5G). These data suggest that the GDP-boundRab8a promotes IGF1 secretion downstream of TBC1D1. Con-sistent with this proposal, we found that knockdown of Rabin8, aguanine nucleotide exchange factor (GEF) for Rab8a (21), pro-moted IGF1 secretion in primary hepatocytes (Fig. 5 H and I).Together, these data show that TBC1D1 and its Ser231 phosphory-lation control IGF1 secretion by regulating Rab8a.

DiscussionOur findings shed light on how cellular energy status regulateslipogenesis and are consistent with a model in which the AMPK–TBC1D1 signaling nexus controls lipogenic gene expression byregulating endocrinal and paracrinal/autocrinal IGF1 secretion

Fig. 4. Effects of IGF1R inhibition on activation of the PKB–TSC2–mTORpathway and lipogenic gene expression in primary adipocytes from theTBC1D1 KI mice. (A) Phosphorylated and total forms of indicated proteins,and nSREBP1 and flSREBP1 were measured in PPP treated or untreatedprimary adipocytes from the TBC1D1 KI and WT mice using GAPDH as aloading control. (B) mRNA expression of lipogenic genes in primary adipo-cytes treated with or without PPP or rapamycin. n = 3. The data are given asthe mean ± SEM. Statistical analyses were carried out using two-wayANOVA. The asterisk indicates P < 0.05 (basal vs. PPP or rapamycin). aP < 0.05(KI basal vs. WT basal).

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(SI Appendix, Fig. S11D). Inhibition of TBC1D1 Ser231 phos-phorylation increases expression of lipogenic genes in the adi-pose and consequently causes obesity in mice most likely due tohypersecretion of IGF1 and hyperactivation of the IGF1R–PKB–TSC2–mTOR pathway.It has long been recognized that the energy and nutrient

sensors, AMPK and mTOR, play opposing roles in governingcell growth in response to varying energy status (22, 23). Energydeficiency activates AMPK, which in turn phosphorylates pro-teins including TSC2 and raptor. Phosphorylation of TSC2 leadsto inhibition of the small GTPase Rheb, whereas phosphorylatedraptor binds to the dimeric phosphoprotein-binding 14-3-3 pro-teins, and both of these mechanisms lead to inhibition of mTOR.The AMPK–TSC2–Rheb–mTOR and AMPK–raptor–mTORpathways restrict cell growth in response to energy shortage (22,24). Besides this opposing effect on growth, both AMPK andmTOR regulate lipogenesis. AMPK phosphorylates ACC andthereby inhibits fatty acid synthesis (5). In contrast, mTOR phos-phorylates lipin1 and the latter promotes lipogenesis by enhancingSREBP1 activity (15).Our findings reveal a previously unrecognized layer of regu-

lation, involving IGF1 as a systemic signal that links these twosignaling pathways to control lipogenesis in response to energystatus. In our model, we propose that energy deficiency activatesAMPK, which inhibits both endocrinal and paracrinal/autocrinal

IGF1 secretion via phosphorylation of TBC1D1 and possiblyother regulators of IGF1 secretion. Decreased endocrinal andparacrinal/autocrinal IGF1 secretion results in hypoactivation ofthe PKB–mTOR pathway, which may contribute to a decline inlipogenesis in adipose tissues. When energy is sufficient, AMPKactivity and TBC1D1 phosphorylation decrease in the body,which promotes endocrinal and paracrinal/autocrinal IGF1 se-cretion. The elevated IGF1 activates PKB–mTOR signaling,which may promote lipogenesis in adipose tissues. Our findingsnot only shed light on the complexity of the regulatory mecha-nism linking energy status with lipogenesis but also provide po-tential targets for drug discovery to combat obesity. Thesefindings may also help to elucidate the therapeutic mechanismsfor the anti-diabetic drug metformin, which may regulate lipo-genesis in the adipose through modulating IGF1 secretion.Genetic evidence has suggested that TBC1D1 is linked to obe-

sity although the molecular mechanisms are not fully understood.For instance, the TBC1D1R125W mutation is a candidate for obesitysusceptibility in human patients (7), whereas deficiency of TBC1D1protects mice from diet-induced obesity (8, 9). Our findings pro-vide one mechanism linking TBC1D1 Ser231 phosphorylation tolipogenesis by controlling IGF1 secretion and consequent activa-tion of the IGF1R–PKB–mTOR pathway. Genetic polymorphismsof TBC1D1 have also been associated with growth traits in otheranimals including pigs (25) and rabbits (26). Hypersecretion of

Fig. 5. Rab8a and IGF1 secretion in primary hepatocytes. (A) Colocalization of Rab8a and IGF1 in primary hepatocytes. Bars indicate 10 μm in length. (B and C)Down-regulation of Rab8a in primary hepatocytes from the WT and TBC1D1 KI mice via siRNA (B), and IGF1 secretion in Rab8a-depleted primary hepatocytes (C).n = 6. (D and E) Expression of indicated proteins (D) and IGF1 secretion (E) in TBC1D1 KO primary hepatocytes expressing Rab8a WT and mutant proteins. n = 6.(F) The GTPase activity of recombinant GST-Rab8a was measured in the presence of immunoprecipitated WT or hTBC1D1S237A mutant protein. n = 5. (G) Levels ofGDP-bound Rab8a and GTP-bound Rab8a in the epididymal fat of the WT and KI male mice (5 mo old) were determined using pulldown assays. (H and I) Down-regulation of Rabin8 in primary hepatocytes via siRNA (H) and IGF1 secretion in Rabin8-depleted primary hepatocytes (I). NC, negative control. n = 6.

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IGF1 activated the IGF1R–PKB–mTOR pathway and phosphor-ylated classical mTOR substrates S6K and 4EBP1 in the liver,skeletal muscle and possible other tissues, which may regulateprotein synthesis and contribute to accelerated body growth. Thus,our data also suggest a link between TBC1D1 and body growthpossibly via endocrinal and paracrinal/autocrinal IGF1 secretion.Based on our current data, we propose a possible sequence of thechanges in the TBC1D1 KI mice as follows. The KI mutation in-creases IGF1 secretion, which promotes body growth and enhanceslipogenic gene expression via activation of the IGF1R–PKB–mTOR pathway in young mice (less than 4 mo old). The inductionof lipogenic genes may increase lipogenesis and possibly lead to fataccumulation over time, which consequently contributes to thedevelopment of obesity in older mice (over 5 mo old). Obesity mayfurther lead to the development of type II diabetes and hepaticsteatosis as the KI mice age (from 8 mo on). Although expressionof lipogenic genes strongly suggests an increase of de novo lipo-genesis in the TBC1D1 KI mice, further experimentation is stillneeded to firmly establish this in vivo.Clearly, TBC1D1 has other functions, and regulates GLUT4

expression and fatty acid oxidation in skeletal muscle (8, 9, 11).TBC1D1 can be phosphorylated on multiple sites besidesSer231 (12), and electroporation of a TBC1D1-4P mutant (in whichSer231, Thr499, Thr590, and Ser621 are mutated to Ala) into skeletalmuscle decreases contraction-stimulated glucose uptake (10). Be-cause our study does not support a role for TBC1D1 Ser231phosphorylation in regulating GLUT4 expression or traffickingunder the conditions tested here, it is possible that other phos-phorylation sites on TBC1D1 might be important for contraction-stimulated muscle glucose uptake.Our knowledge concerning the regulation of IGF1 secretion is

surprisingly sparse. Besides the regulatory mechanism we describehere, the only previously known mechanism involves a calciumsensor synaptotagmin-10 that regulates activity-dependent IGF1secretion in olfactory bulb neurons (27). Given the importantfunctions of IGF1 and potential for modulating its release for

therapeutic purposes, we must elucidate the regulation of IGF1 se-cretion in more detail in future. One open question is whether thereare additional kinase(s) besides AMPK that may be able to phos-phorylate TBC1D1-Ser231 and regulate IGF1 secretion in vivo,although we established AMPK being the upstream kinase forphosphorylation of Ser231 on TBC1D1 (12). It is also possible thatalanine substitution of Ser231 could have gain of function ratherthan merely mimic a nonphosphorylation state. Therefore, moreexperimentation is needed in future to further demonstrate thatAMPK regulates IGF1 secretion. Another open question stem-ming from our model is how Rab8a regulates IGF1 secretion in amanner that is dependent on its GDP-bound form. Although Rabsusually regulate their effector proteins in their GTP-bound forms,there are precedents for GDP-bound forms of Rabs having bi-ological functions. For instance, Rab8a has recently been shown topromote fusion of lipid droplets in its GDP-bound form (28).Identification of downstream effector(s) for the GDP-bound formof Rab8a will help to elucidate the mechanism how Rab8a regu-lates IGF1 secretion and deepen our understanding of this process.

Materials and MethodsDetails for reagents, generation of the TBC1D1 KI mice, body compositionmeasurement, cell culture and transfection, primary cell isolation and IGF1secretion assay, glucose uptake, imaging, tissue/cell lysis, and Western blotare described in SI Appendix, SI Materials and Methods. The animal facility atNanjing University is accredited by the Association for Assessment and Ac-creditation of Laboratory Animal Care International. All animal protocolswere approved by the Ethics Committees initially at University of Dundeeand then at Nanjing University.

ACKNOWLEDGMENTS. We thank Yanqiu Ji and Gail Fraser for assistancewith genotyping of mice and members of the resource units in Nanjing andDundee for technical assistance. We thank Drs. Rachel Toth and SimonArthur (University of Dundee) for generating constructs for the TBC1D1 KImouse. We thank Prof. Thomas C. Südhof (Stanford University) for the IGF1cDNA and Prof. Peng Li (Tsinghua University) for the Rab8a, Fsp27, and OPTNcDNAs. Funding in support of this work is described in SI Appendix.

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