Disruption of the AMPK–TBC1D1 Nexus Increases Lipogenic Gene Expression and Causes Obesity in Mice Via Promoting IGF1 Secretion

Disruption of the AMPK–TBC1D1 nexus increases lipogenic gene expression and causes obesity in mice via promoting IGF1 secretion

Liang 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 Animal Research Center, Nanjing University, Nanjing 210061, China; bMedical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom; cDivision of Cell and Developmental Biology, College of Life Sciences, University of 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 domain is an Rab GTPase activating protein (RabGAP), and an R125W is the GTPase activating protein domain) (TBC1D1) is a Rab GTPase mutation on this protein has been linked to familial female obesity activating protein that is phosphorylated on Ser231 by the AMP-acti- (7). A truncation or KO of TBC1D1 confers leanness to mice fed vated protein kinase (AMPK) in response to intracellular energy on high fat diet with increased fatty acid oxidation in skeletal muscle stress. However, the in vivo role and importance of this phosphoryla- (8, 9). TBC1D1 has also been implicated in regulating muscle glu- tion event remains unknown. To address this question, we generated cose uptake (10), whereas its deficiency causes a decreased ex- a mouse model harboring a TBC1D1Ser231Ala knockin (KI) mutation and pression level of GLUT4 in skeletal muscle (8, 9, 11). found that the KI mice developed obesity on a normal chow diet. We previously identified TBC1D1 as an AMPK substrate that 231 Mechanistically, TBC1D1 is located on insulin-like growth factor 1 is phosphorylated on Ser by AMPK and interacts with 14-3-3 231 (IGF1) storage vesicles, and the KI mutation increases endocrinal proteins on Ser phosphorylation (12). However, the physio- – and paracrinal/autocrinal IGF1 secretion in an Rab8a-dependent logical role of this AMPK TBC1D1 signal nexus remains elusive. Ser231Ala manner. Hypersecretion of IGF1 causes increased expression of In this study, we generated a TBC1D1 knockin (KI) 231 lipogenic genes via activating the protein kinase B (PKB; also mouse model to address the functions of TBC1D1 Ser phos- known as Akt)–mammalian target of rapamycin (mTOR) pathway phorylation in vivo. in adipose tissues, which contributes to the development of obesity, Results diabetes, and hepatic steatosis as the KI mice age. Collectively, these findings demonstrate that the AMPK–TBC1D1 signaling nexus Generation and Characterization of the TBC1D1 KI Mice. The TBC1D1 interacts with the PKB–mTOR pathway via IGF1 secretion, which KI mice were generated using the gene targeting strategy illus- trated in SI Appendix,Fig.S1A. The expression levels of the consequently controls expression of lipogenic genes in the adipose Ser231Ala tissue. These findings also have implications for drug discovery to TBC1D1 mutant proteins were normal, and there was combat obesity. no detectable alteration in the expression of the related RabGAP AS160 (also known as TBC1D4) in various tissues from the KI 231 AMPK | TBC1D1 | phosphorylation | IGF1 secretion | obesity mice. As expected, the phosphorylation of Ser on TBC1D1 was

Significance ue to changes in diet and lifestyle, metabolic syndrome in- Dcluding obesity, type 2 diabetes, and nonalcoholic fatty liver disease is increasing in prevalence worldwide, putting a huge Excess energy intake and physical inactivity are two major factors burden on modern society and motivating us to get better un- causing obesity, but the underlying mechanisms have not been derstanding of these diseases and how to combat them. fully understood. Both excess energy intake and physical inactivity High energy intake and lack of physical activity can result in increase cellular energy status that is monitored by the energy- imbalances of energy metabolism and changed energy status of sensing AMP-activated protein kinase (AMPK). We demonstrate the body, which is monitored by the energy-sensing kinase AMP- that the AMPK–tre-2/USP6, BUB2, cdc16 domain family member 1 activated protein kinase (AMPK) (1). The AMPK holoenzyme is (the TBC domain is the GTPase activating protein domain) (TBC1D1) a heterotrimer, consisting of catalytic α (α1 and α2), regulatory β signaling nexus regulates insulin-like growth factor 1 (IGF1) secre- (β1 and β2), and γ (γ1, γ2 and γ3) subunits (1). AMPK is mainly tion. Disruption of this AMPK–TBC1D1 signaling nexus in mice en- activated through Thr172 phosphorylation in its catalytic domain by hances lipogenic gene expression via promoting IGF1 secretion, MEDICAL SCIENCES the upstream liver kinase B1 (LKB1) under energy stress conditions causes obesity, and eventually leads to development of metabolic that increase the cellular AMP:ATP ratio (2, 3). AMPK can regu- syndrome. These findings reveal a novel regulatory mechanism late both glucose and lipid metabolism by its phosphorylation of that links energy status to development of obesity via control of multiple substrates (4). For instance, acetyl-CoA carboxylase (ACC), IGF1 secretion. an enzyme important for fatty acid synthesis and oxidation, is regulated through an inhibitory phosphorylation by AMPK, which is Author contributions: H.Y.W. and S.C. designed research; L.C., Q.C., B.X., C.Q., Y.S., S. Zhu, essential for the control of lipid metabolism (5). The LKB1–AMPK 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., pathway can also control glucose uptake into skeletal muscle by S. Zhou, H.Y.W., and S.C. analyzed data; and K.S., C.M., H.Y.W., and S.C. wrote the paper. promoting translocation of the glucose transporter 4 (GLUT4) from The authors declare no conflict of interest. its intracellular storage sites onto plasma membrane in response to a This article is a PNAS Direct Submission. pharmacological AMPK activator, 5-aminoimidazole-4-carboxamide 1L.C., Q.C., and B.X. contributed equally to this work. riboside (AICAR), or muscle contraction (6). However, the mech- 2H.Y.W. and S.C. contributed equally to this work. anism by which AMPK promotes translocation of GLUT4 onto 3To whom correspondence may be addressed. Email: [email protected] or schen6@ plasma membrane is not well understood. 163.com. Tre-2/USP6, BUB2, cdc16 domain family member 1 [the TBC This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. domain is the GTPase activating protein (GAP) domain] (TBC1D1) 1073/pnas.1600581113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1600581113 PNAS | June 28, 2016 | vol. 113 | no. 26 | 7219–7224 Downloaded by guest on October 1, 2021 not detectable in the tissues from the KI mice (SI Appendix,Fig. age was a transition period for increased fat mass in the KI mice. S1B). The pharmacological AMPK activator metformin induced Consistent with the increased fat mass, adipocytes were markedly phosphorylation of AMPK in primary hepatocytes from the KI enlarged in the older KI mice (Fig. 1 E–H). Obesity is a high risk mice to a similar extent as in WT cells. In contrast and as an- factor for development of type II diabetes and hepatic steatosis. ticipated, metformin-stimulated TBC1D1 Ser231 phosphorylation The young KI mice (4.5 mo old) had normal blood chemistry pa- was only detected in the lysates of WT, but not KI, hepatocytes. rameters including blood glucose, free fatty acid, triglyceride, total The metformin-stimulated 14-3-3–TBC1D1 interaction assessed cholesterol, and plasma insulin and displayed normal glucose tol- by 14-3-3 overlay assays was diminished in the TBC1D1 immu- erance. However, as these animals aged (∼1 y old), they developed noprecipitates from the KI cell lysates compared with the WT hyperglycemia, hyperinsulinemia, and hypercholesterolemia and controls (Fig. 1A). Taken together, these data validate the suit- became glucose intolerant and insulin resistant (SI Appendix,Fig. ability of the TBC1D1 KI mice and their derived cells for studying S1 H–N). Furthermore, these mice developed hepatic steatosis, the specific in vivo and in vitro function of TBC1D1 Ser231 and their livers were significantly enlarged and contained larger phosphorylation. lipid droplets (SI Appendix,Fig.S1O–Q).

The TBC1D1 KI Mice Developed Obesity and Displayed Characteristics Lipogenic Gene Expression Was Increased in the Adipose of the of Metabolic Syndrome When They Aged. We next monitored the TBC1D1 KI Mice. Although TBC1D1 has been implicated in regu- growth of the KI mice and found that they became significantly lating muscle glucose uptake (10), we found that GLUT4 expres- heavier than the WT littermates from 5 wk after birth (SI Ap- sion and glucose uptake was normal in multiple tissues/primary pendix, Fig. S1C). The KI mice had longer bodies and tibias than cells and insulin-stimulated GLUT4 translocation was not altered WTs, and their growth plates of tibia were significantly elongated in primary adipocytes from the KI mice (SI Appendix,Fig.S3), in the zones of proliferation and of hypertrophy (SI Appendix, suggesting that glucose uptake did not account for the obese Fig. S1 D–G). The increased body mass of the young KIs (less phenotype of these mice. than 4 mo old) was accounted for by their significantly increased We then investigated whether altered adipogenesis, lipogen- lean mass. In contrast, the fat mass in the young KI mice was esis, or lipolysis in the adipose might account for the obese comparable to that of the WT littermates (Fig. 1B and SI Ap- phenotype of the KI mice. To this end, we analyzed expression of pendix, Fig. S2 A and B). However, in contrast to the normal fat key regulators for these processes in the adipose of young KI mass and enhanced lean mass of young KI mice, older KI mice mice (4 mo old). Both lipolysis marker [adipose triglyceride li- became fatter, and had higher lean and fat masses than WTs pase (ATGL)] and adipogenesis markers [CAAT/enhancer- (Fig. 1 C and D and SI Appendix,Fig.S2C and D). Around 5 mo of binding protein α (CEBPα) and peroxisome proliferator- activated receptor γ (PPARγ)] remained normal in the adipose of theKImice(Fig.2A and SI Appendix, Fig. S4A). In contrast, the expression of key enzymes for lipogenesis including fatty acid synthase (FASN), ATP-citrate lyase (ACL), cytoplasmic acetyl- CoA synthetase (AceCS1), acetyl-CoA carboxylase (ACC1), and stearoyl-CoA desaturase (SCD) was significantly up-regulated in the KI adipose tissue, despite the normal mRNA levels of sterol regulatory element-binding protein 1 (SREBP1), which is an upstream 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 its N-terminal fragment (nSREBP1) enters the nucleus after cleavage and regulates lipogenic gene expression (13). Interestingly, the nSREBP1 levels were significantly increased in the adipose of the KI mice (Fig. 2B and SI Appendix,Fig.S4B). The cleavage of SREBP1 on the ER is under control of mammalian target of rapamycin (mTOR) (14). Consistently, phosphorylation of protein kinase B (PKB), tuberous sclerosis 2 (TSC2, also known as tuberin), mTOR, and downstream mTOR targets S6K and 4EBP1 were all increased in the KI adipose tissue (Fig. 2C and SI Ap- pendix,Fig.S2A–C). Lipin1, a downstream target of mTOR, can down-regulate SREBP1 activity in the nucleus and consequently control the expression of lipogenic genes (15). The entry of lipin1 into the nucleus is under control of its phosphorylation by mTOR, and this phosphorylation promotes lipogenesis by releasing the inhibitory effect of lipin1 on SREBP1 and consequently up- regulates lipogenic gene expression (15). Consistent with mTOR activation, the phosphorylation of lipin1 was significantly up- regulated in the KI adipose tissue (Fig. 2C and SI Appendix, Fig. S4C). Collectively, these data suggestthattheactivationof PKB–mTOR pathway in the KI mice may up-regulate lipogenic 231 Fig. 1. Generation and characterization of the TBC1D1 KI mice. (A)Ser genes via elevation of nSREBP1 levels and relief of the inhibitory phosphorylation and 14-3-3 binding of TBC1D1 isolated from primary hepa- effect of lipin1. We further found that the phosphorylation of tocytes that had been treated with metformin (Met) for 1 h. (B–D)Body Ser231Ala PKB, TSC2, mTOR, S6K and 4EBP1 was also up-regulated in composition of the male TBC1D1 KI mice measured at the age of 14 wk skeletal muscle and liver of the KI mice (SI Appendix,Fig.S4 (B,WT:n = 18; KI: n = 19), 20–22 wk (C,WT:n = 15; KI: n = 15), and 52 wk (D, D–I WT: n = 8; KI: n = 9). (E–H) Histology (E) and cell number (F) and size (G and H) ). Although the PKB activation in the KI mice expectedly of the adipose from the WT and TBC1D1 KI male mice. Bars indicate 50 μmin increased the phosphorylation of its downstream targets in- length. Six sections per genotype were analyzed to measure cell numbers. The cluding TSC2, GSK3, and FOXO1, the phosphorylation of numbers shown in the bar graphs refer to the number of cells measured in the AS160, a key regulator for insulin-stimulated glucose uptake, experiments. The data are given as the mean ± SEM, and the asterisk indicates was not increased in the adipose and skeletal muscle of the KI P < 0.05. mice (SI Appendix,Fig.S4).

7220 | www.pnas.org/cgi/doi/10.1073/pnas.1600581113 Chen et al. Downloaded by guest on October 1, 2021 The widely used AMPK activators, metformin and AICAR, significantly decreased IGF1 secretion rates of WT hepatocytes. Al- though the rates of IGF1 secretion by TBC1D1 KI hepatocytes decreased in response to metformin or AICAR, these secretion rates still remained significantly higher than those of WT cells treated with these drugs. We confirmed that the activation of AMPK by AICAR and metformin was comparable in the two genotypes of primary hepatocytes,withnoeffectonIGF1protein expression (SI Appendix,Fig.S6C). We further found that the KI mutation also enhanced paracrinal/autocrinal IGF1 secretion in primary 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 from the KI mice (SI Appendix,Fig.S5L and M). These data show that the KI mutation selectively affects IGF1 secretion. Consistent with the hypersecretion of IGF1, the PKB–TSC2–mTOR pathway was activated in primary adipocytes and hepatocytes isolated from the KI mice (SI Appendix, Figs. S7 A and B, and S8 A–C). 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 and Inhibition of IGF1 Secretion or Action Blunted the Hyperactivation of = KI male mice (16 wk old). n 5. (B) Expression of indicated proteins were the PKB Pathway in Primary Cells from the TBC1D1 KI Mice. To es- measured in the adipose from the WT and KI male mice (16 wk old) using tablish a causal relationship between the hypersecretion of IGF1 GAPDH as a loading control. (C) Phosphorylated and total lipin1, mTOR, – – TSC2, PKB, and IGF1R were measured in the adipose from the KI mice and and the activation of the PKB TSC2 mTOR pathway in the KI WT littermates (16 wk old). The data are given as the mean ± SEM, and the mice, we used an IGF1R inhibitor, picropodophyllin (PPP) (17), asterisk indicates P < 0.05. to treat primary adipocytes and hepatocytes from the KI mice. We

Plasma Insulin-Like Growth Factor 1 Levels Were Elevated in the TBC1D1 KI Mice. We next sought to find out how the KI mutation 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), a key growth promoter that activates the PKB–mTOR pathway in multiple tissues, might be elevated. Indeed, plasma IGF1 levels were 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-binding protein IGFBP3 levels were normal in the KI mice (SI Appendix, Figs. S1 R and S,andS2H). Expression of IGF1, IGF2, and IGFBPs 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 KI mice with unaltered hepatic GH signaling (SI Appendix,Figs.S1T, S2I, and S5H). These data show that plasma IGF1 levels were selectively elevated in the KI mice.

TBC1D1 Colocalizes with IGF1 Vesicles and Regulates IGF1 Secretion. IGF1 is synthesized and temporarily stored within vesicles in hepatocytes 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 with each other (SI Appendix,Fig.S6A). We further found that en- MEDICAL SCIENCES dogenous TBC1D1 was localized on the IGF1 storage vesicles in primary hepatocytes (Fig. 3B) and in myotubes (SI Appendix, Fig. S6B). Together, these data suggest that TBC1D1 might be involved in the regulation of IGF1 secretion. Indeed, knockdown of TBC1D1 via small interference RNA (siRNA) significantly Fig. 3. Plasma IGF1 levels and IGF1 secretion in primary cells from the TBC1D1 decreased IGF1 secretion rates in primary hepatocytes with no KI mice. (A) Plasma levels of IGF1 in the male WT and KI mice (8 wk old). n = 7. alteration in IGF1 expression. Furthermore, in primary hepato- (B) Colocalization of TBC1D1 and IGF1 in primary hepatocytes. Bars indicate cytes from our previously reported TBC1D1 KO mice (16), IGF1 10 μminlength.(C and D) Expression of TBC1D1 and IGF1 in TBC1D1-depleted secretion rates were also significantly lower than those of the WT primary hepatocytes via siRNA (C) or in primary hepatocytes from the TBC1D1 controls, despite normal hepatic IGF1 expression (Fig. 3 C–F). KO mice (D). NC, negative control. (E and F) IGF1 secretion in TBC1D1-depleted primary hepatocytes (E) or in primary hepatocytes from the TBC1D1 KO mice = Ser231Ala (F). NC, negative control. n 6. (G) IGF1 secretion in primary hepatocytes from The TBC1D1 KI Mutation Increases both Endocrinal and the WT and TBC1D1 KI mice. aP < 0.05 (WT metformin or AICAR vs. WT un- Paracrinal/autocrinal IGF1 Secretion. Consistent with the elevated treated) and bP < 0.05 (KI metformin or AICAR vs. KI untreated). n = 8. (H)IGF1 levels of plasma IGF1 in the KI mice, IGF1 secretion rates of secretion in primary adipocytes from the KI mice. WT: n = 5; KI: n = 6. The data primary hepatocytes from these mice were significantly higher are given as the mean ± SEM, and the asterisk indicates P < 0.05. Statistical than those of WT cells under unstimulated conditions (Fig. 3G). analysis for G was carried out using two-way ANOVA.

Chen et al. PNAS | June 28, 2016 | vol. 113 | no. 26 | 7221 Downloaded by guest on October 1, 2021 found that blocking the IGF1 receptor with PPP largely normalized phosphorylation of the signaling proteins in the IGF1R– PKB–mTOR pathway in TBC1D1 KI adipocytes. Moreover, the levels of nSREBP1 were significantly increased in the KI adipocytes under untreated conditions, and PPP treatment diminished this increase (Fig. 4A and SI Appendix, Fig. S7C). Similar effects on PKB activation by PPP were observed in primary hepatocytes (SI Appendix,Fig.S8D and E). We examined the possibility that WT and TBC1D1 KI cells might differ in their sensitivities to IGF1 or PPP. We found treatment with exogenous IGF1 activated PKB in the two genotypes of cells to similar extents, and PPP treatment could inhibit PKB activation elicited by exogenous IGF1 in cells from both genotypes of mice to similar levels (SI Appendix,Figs.S7D and S8 F and G). These findings suggest that the hyperactivation of PKB in KI adipocytes and hepatocytes was most likely due to hypersecretion of IGF1. It was still possible that the KI mutation affected the IGF1R independent of IGF1 secretion. To examine this possibility, we down-regulated IGF1 via siRNA in primary hepatocytes from the WT and KI mice. IGF1R expression was normal in the KI hepatocytes, whereas its phosphorylation was significantly increased in parallel with PKB phosphorylation in these cells (SI Appendix,Fig.S8H and I). On knockdown of IGF1, IGF1 secretion rates were decreased in the KI hepatocytes, which largely normalized IGF1R and PKB activation (SI Appendix,Fig.S8H and I), suggesting that the effects of the KI mutation on the IGF1R–PKB pathway requires IGF1 secretion. Together, these data strongly suggest a causal relationship between hypersecretion of IGF1 and activation of the PKB– – TSC2 mTOR pathway in the TBC1D1 KI mice. Fig. 4. Effects of IGF1R inhibition on activation of the PKB–TSC2–mTOR pathway and lipogenic gene expression in primary adipocytes from the Inhibition of the IGF1–mTOR Pathway Normalized the Expression of TBC1D1 KI mice. (A) Phosphorylated and total forms of indicated proteins, Lipogenic Genes in Primary Adipocytes from the TBC1D1 KI Mice. and nSREBP1 and flSREBP1 were measured in PPP treated or untreated IGF1 can stimulate lipogenesis in porcine adipose tissue ex vivo primary adipocytes from the TBC1D1 KI and WT mice using GAPDH as a (18), and it can also increase lipogenesis via activation of the PKB loading control. (B) mRNA expression of lipogenic genes in primary adipo- pathway in SEB-1 sebocytes (19). Similarly, treatment of WT cytes treated with or without PPP or rapamycin. n = 3. The data are given as primary adipocytes with IGF1 activated the PKB–mTOR pathway the mean ± SEM. Statistical analyses were carried out using two-way and strongly increased lipogenic gene expression (SI Appendix, ANOVA. The asterisk indicates P < 0.05 (basal vs. PPP or rapamycin). aP < 0.05 Fig. S9). Again, we used primary adipocytes from the KI mice to (KI basal vs. WT basal). investigate the relationship between the hypersecretion of IGF1 and lipogenic gene expression. The mRNA levels of lipogenic A genes were significantly increased in primary KI adipocytes under colocalized with IGF1 in primary hepatocytes (Fig. 5 ). basal conditions, which were significantly inhibited on PPP treat- Furthermore, knockdown of Rab8a normalized IGF1 secre- ment (Fig. 4B), suggesting that the increased lipogenic gene ex- tion in primary hepatocytes from the TBC1D1 KI mice (Fig. B C pression was most likely due to the hypersecretion of IGF1 and 5 and ), suggesting that regulation of IGF1 secretion by the TBC1D1 Ser231 phosphorylation requires Rab8a. Over- consequent hyperactivation of its receptor. Moreover, an mTOR Q67L inhibitor rapamycin inhibited the expression of these lipogenic expression of the WT Rab8a, but not a GTP-bound Rab8a genes in the KI adipocytes (Fig. 4B), which further supports our mutant, could restore IGF1 secretion in the TBC1D1 KO he- D E T22N proposal that hyperactivation of PKB–mTOR pathway down- patocytes (Fig. 5 and ). Although a GDP-bound Rab8a stream of the IGF1R is responsible for the induction of lipogenic mutant was expressed at a much lower level, it could still partially genes in the adipose of the KI mice. restore IGF1 secretion in the TBC1D1 KO hepatocytes (Fig. 5 D and E). In an in vitro assay, immunoprecipitated recombinant Rab8a Plays a Key Role Downstream of the TBC1D1 in Regulating IGF1 hTBC1D1 displayed significant GAP activity toward Rab8a and Secretion. We then sought to find out how TBC1D1 and its Ser231 enhanced the GTPase activity of Rab8a. The GAP activity of phosphorylation regulate IGF1 secretion. IGF1 secretion rates hTBC1D1 toward Rab8a was inhibited by its cellular phosphory- were largely restored when a human recombinant TBC1D1 lation in response to AMPK activator phenformin (SI Appendix, (hTBC1D1) or an hTBC1D1Ser237Ala mutant protein (Ser237 on Fig. S11C) and, in contrast, was enhanced by Ser237Ala mutation human TBC1D1 corresponds to Ser231 on the mouse protein) (Fig. 5F). Moreover, more GDP-bound Rab8a and less GTP- were expressed in the TBC1D1 KO hepatocytes (SI Appendix, bound Rab8a were found in the adipose of the TBC1D1 KI mice in Fig. S10). Furthermore, metformin and AICAR could also sig- pulldown assays (Fig. 5G). These data suggest that the GDP-bound nificantly decrease IGF1 secretion rates in these hepatocytes Rab8a promotes IGF1 secretion downstream of TBC1D1. Con- expressing hTBC1D1 or hTBC1D1Ser237Ala mutant proteins. In- sistent with this proposal, we found that knockdown of Rabin8, a terestingly, the expression of a GAP-deficient TBC1D1 mutant guanine nucleotide exchange factor (GEF) for Rab8a (21), pro- (20) could not restore IGF1 secretion rates, suggesting that the moted IGF1 secretion in primary hepatocytes (Fig. 5 H and I). GAP activity of TBC1D1 is required for IGF1 secretion. We next Together, these data show that TBC1D1 and its Ser231 phosphory- investigated which Rab(s) downstream of TBC1D1 mediates lation control IGF1 secretion by regulating Rab8a. IGF1 secretion by downregulating four known in vitro TBC1D1 substrates, namely Rab2a, Rab8a, Rab10, and Rab14 (20). Discussion Knockdown of Rab8a caused a significant decrease in IGF1 se- Our findings shed light on how cellular energy status regulates cretion rates in WT primary hepatocytes, whereas down-regu- lipogenesis and are consistent with a model in which the AMPK– lation of Rab2a, Rab10, and Rab14 had no significant effect (SI TBC1D1 signaling nexus controls lipogenic gene expression by Appendix, Fig. S11 A and B). We also found that Rab8a largely regulating endocrinal and paracrinal/autocrinal IGF1 secretion

7222 | www.pnas.org/cgi/doi/10.1073/pnas.1600581113 Chen et al. Downloaded by guest on October 1, 2021 (SI Appendix, Fig. S11D). Inhibition of TBC1D1 Ser231 phos- IGF1 secretion via phosphorylation of TBC1D1 and possibly phorylation increases expression of lipogenic genes in the adi- other regulators of IGF1 secretion. Decreased endocrinal and pose and consequently causes obesity in mice most likely due to paracrinal/autocrinal IGF1 secretion results in hypoactivation of hypersecretion of IGF1 and hyperactivation of the IGF1R–PKB– the PKB–mTOR pathway, which may contribute to a decline in TSC2–mTOR pathway. lipogenesis in adipose tissues. When energy is sufficient, AMPK It has long been recognized that the energy and nutrient activity and TBC1D1 phosphorylation decrease in the body, sensors, AMPK and mTOR, play opposing roles in governing which promotes endocrinal and paracrinal/autocrinal IGF1 se- cell growth in response to varying energy status (22, 23). Energy cretion. The elevated IGF1 activates PKB–mTOR signaling, deficiency activates AMPK, which in turn phosphorylates pro- which may promote lipogenesis in adipose tissues. Our findings teins including TSC2 and raptor. Phosphorylation of TSC2 leads not only shed light on the complexity of the regulatory mecha- to inhibition of the small GTPase Rheb, whereas phosphorylated nism linking energy status with lipogenesis but also provide po- raptor binds to the dimeric phosphoprotein-binding 14-3-3 pro- tential targets for drug discovery to combat obesity. These teins, and both of these mechanisms lead to inhibition of mTOR. findings may also help to elucidate the therapeutic mechanisms The AMPK–TSC2–Rheb–mTOR and AMPK–raptor–mTOR for the anti-diabetic drug metformin, which may regulate lipo- pathways restrict cell growth in response to energy shortage (22, genesis in the adipose through modulating IGF1 secretion. 24). Besides this opposing effect on growth, both AMPK and Genetic evidence has suggested that TBC1D1 is linked to obe- mTOR regulate lipogenesis. AMPK phosphorylates ACC and sity although the molecular mechanisms are not fully understood. thereby inhibits fatty acid synthesis (5). In contrast, mTOR phos- For instance, the TBC1D1R125W mutation is a candidate for obesity phorylates lipin1 and the latter promotes lipogenesis by enhancing susceptibility in human patients (7), whereas deficiency of TBC1D1 SREBP1 activity (15). protects mice from diet-induced obesity (8, 9). Our findings pro- Our findings reveal a previously unrecognized layer of regu- vide one mechanism linking TBC1D1 Ser231 phosphorylation to lation, involving IGF1 as a systemic signal that links these two lipogenesis by controlling IGF1 secretion and consequent activa- signaling pathways to control lipogenesis in response to energy tion of the IGF1R–PKB–mTOR pathway. Genetic polymorphisms status. In our model, we propose that energy deficiency activates of TBC1D1 have also been associated with growth traits in other AMPK, which inhibits both endocrinal and paracrinal/autocrinal animals including pigs (25) and rabbits (26). Hypersecretion of MEDICAL SCIENCES

Fig. 5. Rab8a and IGF1 secretion in primary hepatocytes. (A) Colocalization of Rab8a and IGF1 in primary hepatocytes. Bars indicate 10 μminlength.(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)Levelsof GDP-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.

Chen et al. PNAS | June 28, 2016 | vol. 113 | no. 26 | 7223 Downloaded by guest on October 1, 2021 IGF1 activated the IGF1R–PKB–mTOR pathway and phosphor- therapeutic purposes, we must elucidate the regulation of IGF1 se- ylated classical mTOR substrates S6K and 4EBP1 in the liver, cretion in more detail in future. Oneopenquestioniswhetherthere skeletal muscle and possible other tissues, which may regulate are additional kinase(s) besides AMPK that may be able to phos- protein synthesis and contribute to accelerated body growth. Thus, phorylate TBC1D1-Ser231 and regulate IGF1 secretion in vivo, our data also suggest a link between TBC1D1 and body growth although we established AMPK being the upstream kinase for possibly via endocrinal and paracrinal/autocrinal IGF1 secretion. phosphorylation of Ser231 on TBC1D1 (12). It is also possible that Based on our current data, we propose a possible sequence of the alanine substitution of Ser231 could have gain of function rather changes in the TBC1D1 KI mice as follows. The KI mutation in- than merely mimic a nonphosphorylation state. Therefore, more creases IGF1 secretion, which promotes body growth and enhances experimentation is needed in future to further demonstrate that lipogenic gene expression via activation of the IGF1R–PKB– AMPK regulates IGF1 secretion. Another open question stem- mTOR pathway in young mice (less than 4 mo old). The induction ming from our model is how Rab8a regulates IGF1 secretion in a of lipogenic genes may increase lipogenesis and possibly lead to fat manner that is dependent on its GDP-bound form. Although Rabs accumulation over time, which consequently contributes to the usually regulate their effector proteins in their GTP-bound forms, development of obesity in older mice (over 5 mo old). Obesity may there are precedents for GDP-bound forms of Rabs having bi- further lead to the development of type II diabetes and hepatic ological functions. For instance, Rab8a has recently been shown to steatosis as the KI mice age (from 8 mo on). Although expression promote fusion of lipid droplets in its GDP-bound form (28). of lipogenic genes strongly suggests an increase of de novo lipo- Identification of downstream effector(s) for the GDP-bound form genesis in the TBC1D1 KI mice, further experimentation is still of Rab8a will help to elucidate the mechanism how Rab8a regu- needed to firmly establish this in vivo. lates IGF1 secretion and deepen our understanding of this process. Clearly, TBC1D1 has other functions, and regulates GLUT4 expression and fatty acid oxidation in skeletal muscle (8, 9, 11). Materials and Methods TBC1D1 can be phosphorylated on multiple sites besides 231 Details for reagents, generation of the TBC1D1 KI mice, body composition Ser (12), and electroporation of a TBC1D1-4P mutant (in which measurement, cell culture and transfection, primary cell isolation and IGF1 231 499 590 621 Ser ,Thr ,Thr ,andSer are mutated to Ala) into skeletal secretion assay, glucose uptake, imaging, tissue/cell lysis, and Western blot muscle decreases contraction-stimulated glucose uptake (10). Be- are described in SI Appendix, SI Materials and Methods. The animal facility at 231 cause our study does not support a role for TBC1D1 Ser Nanjing University is accredited by the Association for Assessment and Ac- phosphorylation in regulating GLUT4 expression or trafficking creditation of Laboratory Animal Care International. All animal protocols under the conditions tested here, it is possible that other phos- were approved by the Ethics Committees initially at University of Dundee phorylation sites on TBC1D1 might be important for contraction- and then at Nanjing University. stimulated muscle glucose uptake. Our knowledge concerning the regulation of IGF1 secretion is ACKNOWLEDGMENTS. We thank Yanqiu Ji and Gail Fraser for assistance surprisingly sparse. Besides the regulatory mechanism we describe with genotyping of mice and members of the resource units in Nanjing and here, the only previously known mechanism involves a calcium Dundee for technical assistance. We thank Drs. Rachel Toth and Simon Arthur (University of Dundee) for generating constructs for the TBC1D1 KI sensor synaptotagmin-10 that regulates activity-dependent IGF1 mouse. We thank Prof. Thomas C. Südhof (Stanford University) for the IGF1 secretion in olfactory bulb neurons (27). Given the important cDNA and Prof. Peng Li (Tsinghua University) for the Rab8a, Fsp27, and OPTN functions of IGF1 and potential for modulating its release for cDNAs. Funding in support of this work is described in SI Appendix.

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