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The multifaceted role of mTORC1 in the control of lipid metabolism

The multifaceted role of mTORC1 in the control of lipid metabolism review review The multifaceted role of mTORC1 in the control of lipid metabolism Stéphane J.H. Ricoult & Brendan D. Manning Department of Genetics & Complex Diseases, Harvard School of Public Health, Boston, Massachusetts, USA The mechanistic target of rapamycin is a protein kinase that, as part mTOR is an evolutionarily conserved serine/threonine kinase of the mechanistic target of rapamycin complex 1 (mTORC1), senses that exists within two functionally distinct protein complexes, the both local nutrients and, through insulin signalling, systemic nutri­ mechanistic target of rapamycin complexes 1 (mTORC1) and 2 ents to control a myriad of cellular processes. Although roles for (mTORC2). mTORC1 senses and integrates a diverse array of cell­ mTORC1 in promoting protein synthesis and inhibiting autophagy in ular signals, with mTOR kinase activity within the complex being response to nutrients have been well established, it is emerging as a influenced by a variety of nutrients—for example, amino acids, central regulator of lipid homeostasis. Here, we discuss the growing glucose and oxygen, cellular energy levels, such as ATP, and many genetic and pharmacological evidence demonstrating the functional secreted growth factors, cytokines and hormones, including insu­ importance of its signalling in controlling mammalian lipid meta­ lin. All of these signals require the Ras­related small G protein bolism, including lipid synthesis, oxidation, transport, storage and Rheb, which on GTP­loading is an essential upstream activator lipolysis, as well as adipocyte differentiation and function. Defining of mTORC1 [1]. Many of the signals that regulate mTORC1 do so the role of mTORC1 signalling in these metabolic processes is crucial by altering the GTP­binding status of Rheb through activation or to understanding the pathophysiology of obesity and its relationship inhibition of a GTPase­activating protein complex, comprised of to complex diseases, including diabetes and cancer. TSC1, TSC2 and TBC1D7—the TSC–TBC complex [2]. For instance, Keywords: adipocytes; Akt; insulin; liver; mTOR insulin, IGF1 and other growth factors inhibit the complex to acti­ EMBO reports (2013) 14, 242–251; published online 12 February 2013; vate Rheb and mTORC1 through Akt­mediated phosphorylation doi:10.1038/embor.2013.5 of TSC2 [3,4]. By contrast, a decrease in cellular ATP, such as the See the Glossary for abbreviations used in this article. decrease that occurs during glucose depletion, activates the com­ plex to inhibit Rheb and mTORC1, at least in part, through the Introduction action of AMPK (Fig  1; [5–7]). On activation, mTORC1 directly Of the four main classes of biological macromolecule, our under­ phosphorylates S6K1 and S6K2, 4E­BP1 and 4E­BP2, and a grow ­ standing of the molecular mechanisms by which cellular signal­ ing number of other downstream targets [8]. Whilst the overall ling pathways regulate lipid metabolism has lagged behind that of effects of mTORC1 signalling differ in cells and tissues, it has an carbohydrates, proteins and nucleic acids. However, lipids are cru­ evolutionarily conserved role in promoting anabolic cell growth cially important both structurally and functionally in all living organ­ and inhibiting the catabolic process of autophagy. On the other isms. An obvious reason for this dependence is the lipid makeup of hand, mTORC2 seems to be regulated primarily by growth factor the plasma membrane and many subcellular organelles. Moreover, signalling and phosphorylates a conserved hydrophobic motif in lipids act as signalling molecules on both a cellular, for example phos­ the protein kinases Akt, SGK and some isoforms of PKC, thereby phoinositides, and organismal, for example steroid hormones, scale. increasing their kinase activity [9]. Through these targets, and prob­ Lipids are also used for energy storage, primarily as triacylglycerides in ably through others, mTORC2 signalling is believed to promote cell adipocytes, and as an alternative to glucose for catabolic meta bolism. survival, proliferation, metabolism and changes in the actin cyto­ Despite the dependence of living organisms on lipids, we know little skeleton. The two mTOR complexes can be distinguished from one about how lipid homeostasis is controlled by the intricate network of another by their differential sensitivity to rapamycin, an allosteric cellular signalling pathways that sense cellular growth conditions. As and partial inhibitor of mTOR (Sidebar A). detailed in this review, the mechanistic target of rapamycin (mTOR) Many studies in cell and mouse models, combined with pre­ protein kinase has emerged as a crucial link between cellular and clinical and clinical data on mTOR inhibitors, have revealed a systemic growth signals and the regulation of lipid metabolism. pivotal role for mTOR—particularly within mTORC1—in con­ trolling lipid homeostasis in many settings, both physiological and pathological. We review this evidence below, with a focus Department of Genetics & Complex Diseases, Harvard School of Public Health, on the key aspects of lipid synthesis, storage and mobilization. 665 Huntington Avenue, SPH2‑117, Boston, Massachusetts 02115, USA Corresponding author. Tel: +1 617 432 5614; Fax: +1 617 432 5236; The emerging picture is that, through a variety of molecular E‑mail: [email protected] mechanisms, mTORC1 signalling promotes processes to syn­ thesize and store  lipids, whilst inhibiting those leading to lipid Received 6 November 2012; accepted 16 January 2013; published online 12 February 2013 consumption (Fig 1). 242 EMBO reports VOL 14 | NO 3 | 2013 ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION m mTORC1 signalling regulates lipid metabolism review Sidebar A | mTORC1 versus mTORC2 and the differential effects Glucose Insulin / IGF1 (ATP) of mTOR inhibitors In studying the mechanistic target of rapamycin (mTOR) signalling network, or interpreting the mTOR literature, it is crucial to understand some of the basic complexities of mTOR signalling and inhibition. The AMPK AKT mechanistic target of rapamycin complex 1 (mTORC1) is composed of the core essential components mTOR, mTOR‑ associated protein, LST8 homologue (mLST8) and the regulatory‑ associated protein of mTOR TSC1 TSC2 (Raptor), whereas mTORC2 is composed of mTOR, mLST8, SAPK‑ TBC1D7 interacting protein 1 (SIN1) and the Raptor‑ independent companion of mTOR (Rictor). Although these complexes are functionally distinct, they can have an influence on each other’s activity. For instance, as mTORC2 stimulates an increase in Akt activity [84], it might influence its downstream Rheb signalling from mTORC1. On the other hand, several negative feedback GTP mechanisms are triggered by mTORC1 activation, which influences Amino mTORC2 activity, including one leading to direct phosphorylation of acids Rictor within mTORC2 by ribosomal S6 kinase 1 (S6K1) downstream from mTORC1 [85,86]. Regarding mTOR inhibitors, the widely used rapamycin mTOR and its many analogues, which on interaction with the ubiquitous protein Lipogenesis Lipolysis FK506 binding protein of 12 kDa (FKBP12) binds to an allosteric site amino Raptor mLST8 terminal to the mTOR kinase domain—the FKBP12‑ rapamycin binding β-oxidation Adipogenesis domain—only has access to mTOR within mTORC1. However, it is evident that in both cell culture and mice, prolonged exposure to rapamycin can Lipid storage Ketogenesis block the assembly of mTORC2 by sequestering uncomplexed mTOR [82,87]. Therefore, although rapamycin is specific to mTORC1 for acute inhibition and generally leads to an increase in upstream signalling from Fig 1 | Upstream regulation from the mTORC1 and its downstream functions mTORC2 and Akt by blocking negative feedback mechanisms, one must related to lipid metabolism. The presence of amino acids is required for the consider that the observed effects of long‑ term rapamycin treatment might activation of mTORC1 by GTP‑ bound Rheb. Upstream from Rheb, the TSC–TBC be due to loss of mTORC2 in some experimental systems, which affects the many processes downstream from Akt. Also, the development of mTOR complex receives signals about systemic and local nutrient and energy availability, kinase domain inhibitors, which completely block mTOR within both in part through AMPK and Akt. These signals either activate or inhibit the complexes, has revealed that rapamycin only partly inhibits mTORC1 ability of the TSC–TBC complex to act as a GAP for Rheb, thereby inhibiting activity. Whilst the nature of this differential sensitivity is unknown, or activating mTORC1, respectively. Activated mTORC1 leads to enhanced rapamycin strongly affects the phosphorylation of some mTORC1 targets phosphorylation of IRS1, which serves as negative feedback to dampen the insulin (for example, S6K1) but only modestly inhibits other targets (for example, response. mTORC1 has many roles in regulating lipid metabolism, including eIF4E‑ binding protein 1; [88]). the promotion of lipid synthesis and storage and inhibition of lipid release and consumption, which are detailed in the text. AMPK, adenosine monophosphate‑ Lipogenesis activated protein kinase; GAP, GTPase‑ activating protein; IRS1, insulin receptor The regulation of de  novo sterol and fatty acid synthesis by sig­ substrate 1; IGF1, insulin‑ like growth factor 1; mTORC1, mechanistic target of nalling pathways, especially insulin signalling, has garnered rapamycin complex 1; Raptor, regulatory‑ associated protein of mTOR; TSC, intense interest. Unlike most terminally differentiated cells, tuberous sclerosis complex; TBC, Tre‑ 2/Bub2/Cdc16 domain‑ containing protein. hepatocytes and adipocytes synthesize significant amounts of lipid de  novo through pathways in which cytosolic acetyl­ CoA, induces transcription from SREs within target genes. SREBP1a derived from glucose or amino acid catabolism, is used to form and 1c are products of alternative splicing of the SREBF1 gene and the hydro phobic carbon backbone of lipids. Acetyl­ CoA is have been primarily implicated in the control of genes involved either committed to sterol and isoprenoid biosynthesis through in fatty acid synthesis, although SREBP1a is thought to activate the action of HMG­ CoA synthase or to fatty acid biosynthesis most SRE­ containing genes [12]. SREBP2 is encoded by SREBF2 through acetyl­ CoA carboxylase. Both the sterol and fatty acid and is believed to have a more important role in the transcription synthesis branches comprise many steps requiring many specific of steroido genic genes, including those involved in cholesterol enzymes. Importantly, the SREBPs are transcription factors that synthesis in the liver [13,14]. Although the SREBPs preferentially stimulate the expression of genes encoding nearly all of these activate transcription of different sets of genes, there is substan­ lipogenic enzymes [10]. The three SREBP isoforms, encoded by tial overlap between the targets of the SREBP isoforms and the two genes, are produced as inactive transmembrane proteins at tissue specificity of these preferences, which has not been fully the endoplasmic reticulum (Fig 2). Under conditions of abundant established. Importantly, independent studies have identified sterols, full­ length SREBP, through its sterol­ sensing binding part­ the SREBPs as major transcriptional effectors of mTORC1 signal­ ner SCAP, is retained in the endoplasmic reticulum by the INSIG ling and have demonstrated that mTORC1 activation promotes proteins [11]. Depletion of intracellular sterols results in release lipogenesis through this family of transcription factors [15,16]. of the SREBP–SCAP complex from Insig and their transport to mTORC1 signalling promotes SREBP activation and lipo genesis the Golgi apparatus, in which two proteolytic cleavage events by in response to both physiological and genetic stimuli. In primary the site­ specific proteases S1P and S2P liberate the active amino­ rodent hepatocytes and the intact liver, insulin or feeding has terminus of SREBP. This fragment then enters the nucleus and been shown to increase the expression of the major liver isoform ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 14 | NO 3 | 2013 243 1 mTORC1 signalling regulates lipid metabolism review to promote the activation of hepatic SREBP1c by having an effect Glossary on its processing [20,21], and to affect the processing of SREBP2 4E‑BP1/2 eIF4E‑binding protein 1/2 in a hepatocellular carcinoma cell line [22]. mTORC1 signal­ AMPK adenosine monophosphate‑activated protein kinase ling has also been suggested to increase SREBP1 activation in an ATG5/7 autophagy‑related 5/7 S6K1­dependent manner in cultured myotubes [23]. ATGL adipose triglyceride lipase Genetic mouse models have demonstrated that mTORC1 C/EBP CCAAT/enhancer‑binding protein CPT1 carnitine palmitoyltransferase 1 activation is essential, but not sufficient, to stimulate hepatic DAG diacylglycerol SREBP1c and its lipogenic targets in response to feeding [18,24]. eIF4E eukaryotic translation initiation factor 4E Mice lacking mTORC1 in their liver, through liver­specific Raptor GSK3 glycogen synthase kinase 3 knockout, fail to induce SREBP1c and lipogenesis [24], and have HSL hormone‑sensitive lipase reduced levels of both liver triglycerides and circulating choles­ IDL intermediate density lipoprotein terol on a ‘Western’ diet [25]. However, characterization of mice IGF1 insulin‑like growth factor 1 with a liver­specific knockout of Tsc1 (LTsc1KO) and constitu­ Insig insulin‑induced gene tive activation of mTORC1, which is independent of insulin and LDL low density lipoprotein feeding, revealed that mTORC1 signalling, although essential, is LDLR LDL receptor not capable of activating SREBP1c and hepatic lipid synthesis on lipin 1 phosphatidate phosphatase LPIN1 LPL lipoprotein lipase its own [18]. In fact, these mice were found on two independ­ LST8 lethal with SEC13 protein 8 ent strain backgrounds to be resistant to the development of MAG monoacylglycerol both age­ and diet­induced hepatic steatosis due to decreased MEF mouse embryonic fibroblast SREBP1c activation [18,26]. These seemingly paradoxical find­ N‑CoR1 nuclear receptor co‑repressor 1 ings are the result of a strong feedback attenuation of Akt signal­ PCSK9 proprotein convertase subtilisin/kexin type 9 ling that accompanies loss of function of the TSC1–TSC2 complex PKA/C protein kinase A/C in all settings [27]. A crucial role for Akt signalling in the induc­ PPARα/γ peroxisome proliferator‑activated receptor α/γ tion of SREBP1c and lipogenesis in the liver has been established Raptor regulatory‑associated protein of mTOR through rodent models [28–30], and this has been extended by Rictor Raptor‑independent companion of mTOR using mice with liver­specific Rictor knockout, which results in S6K1/2 ribosomal S6 kinase 1/2 SCAP SREBP cleavage‑activating protein the loss of mTORC2 activity and its activating phosphorylation of SCD stearoyl‑CoA desaturase Akt [31]. Consistent with the essential nature of Akt signalling to SGK serum and glucocorticoid regulated kinase hepatic SREBP1c, a restoration of Akt activity in LTsc1KO hepato­ shRNA short hairpin RNA cytes restores SREBP1c activation and lipogenesis [18]. Whilst siRNA small interfering RNA many mTORC1­independent pathways might function in parallel SRE sterol response element downstream from Akt to help to promote the activation of hepatic SREBP sterol regulatory element‑binding protein SREBP1c, including GSK3 inhibition [32], data from the LTsc1KO TAG triacylglycerol mice suggest that one pathway involves the repression of an iso­ TBC1D7 TBC1 domain family, member 7 form of the SREBP inhibitor Insig, Insig2a, which is only expressed TCA tricarboxylic/citric acid in the liver [18]. A liver­specific mechanism is also consistent with TSC1/2 tuberous sclerosis complex 1/2 VLDL very low density lipoprotein the fact that mTORC1 activation alone is sufficient to promote SREBP activation and lipogenesis in other settings, even in the absence of Akt signalling [15]. of SREBP (SREBP1c) and its targets, and to promote de novo lipid The molecular mechanism by which S6K1 promotes SREBP pro­ synthesis in a manner that is sensitive to rapamycin  [17–19]. cessing is unknown, and it is clear from additional studies that S6K1 Insulin activates mTORC1 through a pathway involving the Akt­ is not the only direct target downstream from mTORC1 involved mediated phosphorylation and inhibition of TSC2, within a com­ in SREBP isoform regulation, which might vary by cellular context. plex with TSC1 and TBC1D7 [2–4]. Expression of constitutively For instance, siRNA knockdown of the mRNA capbinding protein ­ active Akt or loss of either TSC1 or TSC2, both of which result in eIF4E, which is normally activated by mTORC1 signalling through insulin­independent activation of mTORC1 signalling, stimulates the phosphorylation and release of its inhibitory binding partner the global expression of SREBP1 and SREBP2 targets and drives 4EBP1, decreases o ­ verall levels of SREBP1 and its canonical target lipogenesis through mTORC1 [15,16]. These latter studies found SCD in breast cancer cell lines [33]. The potential involvement of that mTORC1 signalling promotes accumulation of the processed, 4EBP1 regulation b ­ y mTORC1 in some cells might explain the resist ­ mature form of SREBP1, which resides in the nucleus to induce ance of SREBP1 or SREBP2 activation to rapamycin in specific set­ its own expression and that of genes involved in both steroid and tings [22,34]. The resistance of some mTORC1 targets to rapamycin fatty acid biosynthesis. In exploring the molecular mechanism of (Sidebar A) is an important consideration when examining the role this regulation, it was found that S6K1 is required downstream of mTORC1 signalling in any aspect of lipid metabolism. Another from mTORC1 to stimulate the increase in levels of active SREBP1, direct target of mTORC1 that, as with 4EBP1, is partly resistant to ­ expression of SREBP1 and SREBP2 targets, and de  novo lipo­ rapamycin for its regulation is the phosphatidic acid phosphatase genesis in TSC2­deficient cells [15]. SREBP1 regulation in this set ­ lipin 1, which has also been implicated in SREBP regulation [25,35]. ting is independent of the effects on the proteasomal degradation Lipin 1 seems to have a role in the remodelling of the nuclear lamina, of its active form, suggesting that S6K1 promotes the processing which is inhibited by mTORC1mediated phosphorylation of man ­ y of SREBP1. Consistent with these findings, S6K1 has been found residues on this enzyme. Lipin 1 phosphorylation also coincides 244 EMBO reports VOL 14 | NO 3 | 2013 ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION mTORC1 signalling regulates lipid metabolism review SREBF1, SREBF2 S6K SREBP mTORC1 processing lipin1 Lipogenic targets Cytosol RELEASE Cytosol + sterols – sterols Insig SCAP SREBP SCAP SREBP N N C C Endoplasmic reticulum Endoplasmic reticulum TRANSPORT Cytosol SCAP SREBP COP II vesicle CLEAVAGE Cytosol SCAP SREBP Nucleus TRANSCRIPTION DEGRADATION S1P S6K SREBP S2P Rapa- Golgi mycin mTOR kinase inhibitors SRE mTORC1 lipin1 Cytosol Cytosol Fig 2 | The complex steps leading to SREBP activation and input from mTORC1 signalling. (A) SREBP processing and activation is regulated by mTORC1 through S6K and lipin 1 leading to the transcriptional induction of the SREBF1 and SREBF2 genes, encoding SREBP1 and SREBP2, respectively, and genes encoding many lipogenic enzymes involved in both fatty acid and sterol synthesis. The mTORC1‑ mediated transcriptional activation of SREBF1 could result from either autoregulation by SREBP1 or from an unknown parallel pathway downstream from mTORC1. (B) In the presence of sterols, SREBP resides in the endoplasmic reticulum bound to SCAP and the Insig proteins. When sterols become scarce SCAP undergoes a conformational change, which releases the SCAP–SREBP complex from the Insig, allowing its transport from the endoplasmic reticulum to the Golgi apparatus through COPII vesicles. Once in the Golgi, SREBP comes into contact with two site‑ specific proteases. S1P cleaves the luminal loop of SREBP and S2P cleaves the amino‑ terminal transmembrane region of SREBP, which releases the N‑terminal region of SREBP containing the DNA ‑ binding and ‑ transactivating domains. The NLS‑ containing processed form of SREBP enters the nucleus to activate transcription of genes containing SREs in their promoters. Finally, the processed form of SREBP is unstable and subject to proteasome‑ mediated degradation. In some settings, SREBP processing has been found to require S6K1 downstream from mTORC1 and is therefore sensitive to rapamycin. However, the nuclear shuttling of SREBP has been found to require lipin 1 downstream from mTORC1, the phosphorylation of which is largely resistant to rapamycin but sensitive to mTOR kinase domain inhibitors (Sidebar A). The precise molecular mechanisms by which either of these two mTORC1 targets regulates SREBP activation are unknown. COPII, coatamer protein II; Insig, insulin‑ induced gene; lipin 1, phosphatidate phosphatase LPIN1; mTORC1, mechanistic target of rapamycin complex 1; NLS, nuclear localization signal; S1/2P, site 1/2 protease; S6K1, ribosomal S6 kinase 1; SCAP, SREBP cleavage‑ activating protein; SRE, sterol response element; SREBP1/2, sterol regulatory element‑ binding protein 1/2. ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 14 | NO 3 | 2013 245 mTORC1 signalling regulates lipid metabolism review mTORC1­ dependent transcriptional response leads to an increase Mesenchymal in full­ length SREBP isoforms that accompany the increased process­ stem cell ing and activation of SREBP. However, it remains unclear whether this transcriptional effect is simply a result of autoregulation by pro­ cessed SREBPs at the SREBF1 or SREBF2 promoter or a parallel path­ Lineage S6K way independent from the effects of mTORC1 on SREBP processing commitment (Fig 2). Both SREBF1 and SREBF2 contain a characterized SRE in their promoters [36,37]. In cell culture models, exogenous expression of Early preadipocytes processed SREBP1a stimulates the expression of endogenous SREBP1 Rapamycin and SREBP2 transcripts in a manner that is no longer sensitive to rapa­ mycin, suggesting that the transcriptional effects of mTORC1 signal­ ling on SREBP expression are upstream from processed SREBP [15]. However, elegant studies with a transgenic version of SREBP1c in rats suggest that the role of mTORC1 in SREBP1c processing and gene C/EBP-β mTORC1 Clonal expression is separable [21]. More studies are needed to understand C/EBP-δ expansion the many inputs of mTORC1 signalling, especially in vivo, into the regulation of SREBP isoforms. Preadipocytes Adipogenesis Adipocytes are specialized mesenchymal cells that either store lipids as energy reserves (white adipose tissue) or burn lipids through oxidation to generate heat (brown adipose tissue). Pharmacological and genetic studies have demonstrated that the differentiation of C/EBP-α, PPARγ Terminal mesenchymal stem cells into mature adipocytes—adipogenesis— 4E-BP differentiation requires mTOR signalling (Fig  3). Rapamycin treatment has been reported to reduce adipogenesis in a variety of cell culture models. Rapamycin seems to block the early determination step in brown Mature adipocytes adipocyte differentiation, in which a mesenchymal stem cell com­ mits to becoming a preadipocyte [38]. Similarly, rapamycin treat­ ment or shRNA­ mediated knockdown of S6K1 in embryoid bodies hinders their commitment to preadipocytes [39]. However, much of our knowledge of adipogenesis comes from cell culture models Fig 3 | mTORC1 signalling has been implicated in promoting the three of preadipocytes after lineage commitment and also from MEFs, and main steps of adipogenesis. Adipogenesis consists of the differentiation of a has therefore been focused on the later steps of white adipose dif­ mesenchymal stem cell to a mature adipocyte, which makes up a significant ferentiation. Treatment of preadipocytes with rapamycin leads to a part of adipose tissue in which energy is stored as lipids. The commitment marked decrease in adipocyte differentiation  [40–44]. mTOR has of the mesenchymal stem cells to the adipocyte lineage is the first step of been implicated in hormonal induction of clonal expansion, which adipogenesis and is facilitated by S6K1 activity. C/EBP‑ β and ‑ δ are the is an initial step of differentiation that occurs through the action primary drivers of clonal expansion, which is crucial for preadipocyte of two C/EBP family transcription factors, C/EBP­ β and ­ δ. Overall maturation, and the former has been suggested to be activated by mTORC1 levels of C/EBP­ β have been found to decrease on rapamycin treat­ signalling. The terminal differentiation of preadipocytes to mature adipocytes ment, which corresponds with a repression of clonal expansion of is mediated by PPARγ and C/EBP‑α. mTORC1 promotes this final step preadipo cytes [41]. However, rapamycin has also been shown to through both its inhibition of 4E‑ BP and its activation of PPARγ through a inhibit preadipocyte differentiation after clonal expansion, thereby poorly understood mechanism. Although the precise molecular mechanisms ruling out the anti­ proliferative effects of rapamycin as its primary have yet to be defined, rapamycin blocks adipogenesis. 4E‑ BP, eIF4E‑ mode of inhibiting adipogenesis [42–44]. binding protein; C/EBP‑α/β/δ, CCAAT/enhancer ‑ binding protein‑ α/β/δ; Several genetic models have further supported a crucial role for mTORC1, mechanistic target of rapamycin complex 1; PPARγ, peroxisome mTORC1 activation in terminal adipocyte differentiation, in which proliferator‑ activated receptor γ; S6K1, ribosomal S6 kinase 1. it seems to be both necessary and sufficient. For instance, MEFs lacking TSC1 or TSC2, which have sustained, insulin­ independent activation of mTORC1 signalling, have an mTORC1­ dependent with an increase in the levels of processed, nuclear SREBP1 and enhanced capacity to differentiate into adipocytes despite these SREBP2, and the expression of SREBP targets. Although the phospha­ cells being severely resistant to insulin, a major adipogenic fac­ tidic acid phosphatase activity of lipin 1 was shown to be important tor  [45]. Reciprocally, TSC2­ deficient MEFs that express a phos­ for its inhibitory effect on nuclear SREBP levels [35], the molecular phorylation site mutant of TSC2, which blocks the ability of mechanism and tissue specificity of this regulation, as with S6K1 and mTORC1 to be activated by insulin and Akt signalling, show 4E­ BP1, remains unknown. Finally, it is clear that mTORC1 signal­ reduced adipogenesis [45]. The enhanced adipogenesis in mesen­ ling also increases the transcript levels of SREBP1 and SREBP2  in chymal cells lacking the TSC tumour suppressors probably explains cell culture models [15], and SREBP1c in both rodent hepatocytes the common development of adipocyte­ rich renal angiomyo­ and the intact liver in response to insulin or feeding [18–21]. This lipomas in patients with TSC [46]. Consistent with an essential role 246 EMBO reports VOL 14 | NO 3 | 2013 ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION mTORC1 signalling regulates lipid metabolism review for mTORC1, RNA interference knockdown of Raptor also blocks adipocyte. Interestingly, adiposespecific ­ Atg7 knockout mice that adipogenesis in preadipocytes [47]. Downstream from mTORC1, have a defect in autophagy, show decreased adipocyte lipolysis [59], genetic evidence suggests a role for both S6K and 4E­BP in the suggesting that the inhibitory effects of mTORC1 on lipolysis could control of adipogenesis. The involvement of S6K in the commit­ be, at least in part, through its attenuation of autophagy. ment of stem cells to preadipocytes was reinforced by the reduced Although the molecular mechanisms of lipolytic regulation size of this progenitor cell population in S6K1 knockout mice and by mTOR are not fully understood, mTORC1 signalling has been a defect in the capacity of embryonic stem cells from these mice found to influence three distinct lipases: ATGL, HSL and LPL [60]. to commit to the adipocyte lineage [39]. Reciprocally, 4E-BP1/2 In adipocytes, ATGL catalyses the lipolysis of TAGs to DAGs within double­knockout MEFs show enhanced differentiation towards lipid droplets. HSL then converts the DAGs to MAGs. In 3T3­L1 adipocytes [48], suggesting that the ability of mTORC1 to both adipo cytes, mTORC1 suppression increases the transcription of activate S6K and inhibit 4E­BP contributes to its role in promoting ATGL, which parallels the enhanced lipolysis induced by rapa­ adipogenesis. Interestingly, the S6K1 knockout mice have a lean mycin or siRNA knockdown of Raptor [57]. The phosphorylation phenotype on both normal and high­fat diets [39,49], whereas of HSL at Ser 563, an established PKA site, is associated with an the 4E-BP1/2 double­knockout mice are more sensitive to diet­ increase in its lipase activity. A decrease in HSL phosphorylation induced obesity than their wild­type counterparts [48]. However, correlates with mTORC1 activation and the diminished release of the differences in adiposity in these systemic mouse models prob­ free fatty acids  [58]. However, as with ATGL transcriptional sup­ ably reflect many effects of mTORC1 signalling on lipid synthesis pression, how mTORC1 signalling negatively affects HSL phos­ and mobilization, discussed elsewhere in this review, in addition phorylation on this PKA site is unknown. Similarly to mTORC1 to its role in promoting the development of adipose deposits. inhibition, adipocyte­specific Rictor knockout also leads to the The molecular mechanisms by which mTORC1 and its down­ phosphorylation of HSL at Ser 563 [61]. In addition to adipo­ stream targets stimulate adipocyte differentiation have yet to be cyte lipolysis, mTORC1 has been implicated in the control of fully defined. The temporal activation of two transcription factors, the extracellular lipase LPL. LPL is a water­soluble lipase present C/EBP­ α and PPARγ—the master regulator of terminal adipocyte in plasma, as well as on the surface of endothelial cells, primar­ differentiation—is responsible for inducing the final stages of dif­ ily in muscle and adipose tissue. It hydrolyses TAG in circulating ferentiation [50]. mTORC1 signalling has been shown to increase VLDL to promote conversion to IDL and LDL, which facilitates the PPARγ transcript and protein levels, as well as its transactivating activ­ uptake of lipoprotein into tissues [62]. Systemic rapamycin treat­ ity [45,47,51,52], albeit through unknown mechanisms. Cell culture ment has been found to decrease LPL activity in mouse adipose experiments have suggested that regulation of the final differentiation tissue, and mouse and human plasma, albeit through an unknown steps is primarily independent of S6K and is probably dependent on mechanism  [63,64]. The collective studies in patients treated 4EBP ­ inhibition downstream from mTORC1 [40,48]. However, a with rapamycin and a variety of cell and mouse models suggest study has indicated that PPARγ activation can also be suppressed by that mTORC1 activation, which occurs in metabolic tissues after hyperactive mTORC1 signalling through its negative feedback effects feeding, promotes the synthesis and storage of lipids. By contrast, on insulin signalling [53]. These findings indicate that there are prob­ mTORC1 inhibition, such as during fasting, stimulates lipolysis and ably mTORC1dependent and ­ independent inputs into PP ­ AR γ the release of free fatty acids into the circulation. activation and adipocyte differentiation downstream from insulin signalling, with more in vivo experiments needed. β­oxidation and ketogenesis Consistent with the inhibition of mTORC1 signalling promoting Lipolysis fatty acid release and consumption, there is growing evidence that In addition to its role in stimulating lipogenesis through SREBP, mTORC1 suppresses the β­oxidation of fatty acids for energy or mTORC1 signalling is believed to promote the storage of fatty acids ketogenesis. Rapamycin has been found to increase β­oxidation in in lipid stores by inhibiting lipolysis. Neutral lipids, in the form of rat hepatocytes and this has been attributed to increased expression MAG, DAG and TAG inside the cell are subject to lipolysis to mobi­ of β­oxidation enzymes, including long­chain acyl­CoA dehydro­ lize free fatty acids for energy production or remodelling into new genase and carnitine acyltransferase [17,65]. This effect of rapa­ lipid species, including specific membrane and signalling lipids. mycin could be due to the induction of autophagy, which seems Patients treated with rapamycin frequently have dyslipidaemia, one to promote the β­oxidation of fatty acids from TAGs in hepato ­ facet of which is elevated levels of plasma free fatty acids, which cytes  [66]. However, genetic evidence suggests that autophagy could reflect an increase in lipolysis in adipose tissue [54,55]. has inhibitory effects on β­oxidation in adipose tissue  [59,67]. Mice treated with rapamycin show a reduction in adipocyte size Mice with whole­body knockout of S6K1 seem to have enhanced and overall adiposity, and rapamycin stimulates lipolysis in cul­ β­oxidation, as evidenced by increased levels of CPT1 transcript tured adipocytes [56–58]. Genetic manipulations of mTORC1 sig­ in isolated adipocytes [49]. Consistent with mTORC1 signal­ nalling in several mouse models have reinforced the link between ling attenuating β­oxidation, myoblasts isolated from S6K1/S6K2 mTORC1 activation and an inhibition of lipolysis. The adipose tissue double­knockout mice also show enhanced β­oxidation of fatty of 4E-BP1/2 doubleknoc ­ kout mice shows decreased lipo lysis [48], acids [68]. However, this phenotype was attributed to indirect and S6K1 knockout mice are leaner with elevated rates of lipo­ effects from energy stress and AMPK activation in this setting. As lysis  [49]. However, mice with adiposespecific ­ Raptor knockout, with the S6K1 knockout and the S6K1/S6K2 double­ knockout whilst also lean with reduced adiposity, do not show an obvious mice, mice with adipose­specific Raptor knockout are lean increase in lipolysis [47]. This suggests that the lipolysis phenotypes with adipocytes that show increased mitochondrial uncoupling, observed in the wholebod ­ y 4E-BP and S6K1 knockout models which could allow them to burn lipids rapidly without generat­ could be due to systemic effects rather than those intrinsic to the ing ATP [47,49,68]. Paradoxically, mTORC1 activation has also ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 14 | NO 3 | 2013 247 mTORC1 signalling regulates lipid metabolism review FOOD INTAKE LIVER ADIPOSE TISSUE Gluconeogenesis Glucose Glucose Glucose GLUT4 Lipogenesis Lipogenesis β-oxidation β-oxidation Fatty Fatty Ac-CoA Ac-CoA acids acids mTORC1 mTORC1 Insulin HSL Lipolysis DAG ATGL Cholesterol TAG Ketogenesis TAG LDLR STORAGE TRANSPORT Lipoproteins VLDL IDLLDL LPL Fig 4 | The increase in insulin levels after a meal alters hepatic and adipose lipid metabolism, at least in part, through mTORC1 signalling (a working model). In the liver, mTORC1 promotes lipid synthesis through SREBP1c activation. In addition, mTORC1 signalling blocks lipid catabolism by blocking β‑oxidation and ketogenesis in the liver. Consequently, mTORC1 activation in the liver promotes the synthesis of TAGs and perhaps cholesterol, which are incorporated into VLDL for transport to peripheral tissues. Evidence suggests that mTORC1 signalling positively influences LPL activity, which promotes lipid delivery to peripheral tissues by hydrolysing VLDL to IDL, which is then converted to LDL. Lipoprotein‑ bound TAGs are taken up by tissues, including adipocytes, through the LDLR. Both the expression and stability of LDLR, at least in the liver, are probably promoted by mTORC1 activation. In response to insulin, mTORC1 has been suggested to inhibit lipolysis in adipocytes by downregulating ATGL and HSL. Therefore, the systemic effects of postprandial mTORC1 activation are to promote the flux of carbon from glucose towards TAG storage in adipose tissue. See text for details regarding the evidence underlying this model. Ac‑ COA, acetyl‑ CoA; ATGL, adipose triglyceride lipase; DAG, diacylglycerol; GLUT4, glucose transporter type 4; HSL, hormone‑ sensitive lipase; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; LDLR, LDL receptor; LPL, lipoprotein lipase; mTORC1, mechanistic target of rapamycin complex 1; SREBP1c, sterol regulatory element‑ binding protein 1c; TAG, triacylglycerol; VLDL, very low density lipoprotein. been linked to increased mitochondrial biogenesis in some set­ transcriptional targets could also explain the negative regulation of tings [69]. This could explain the decrease in oxidative capacity fatty acid oxidation by mTORC1. The repression of β­ oxidation and of muscle [69–71] and Jurkat T  cells [72] after the inhibition or ketogenesis by mTORC1 probably acts together with its stimulation complete loss of mTORC1 signalling. However, further studies are of lipogenesis, further promoting the flux of acetyl­ CoA towards needed to determine how the observed changes in mitochondrial lipid synthesis and storage. gene expression and oxygen consumption in these settings influ­ ence the β­ oxidation of fatty acids. The collective data suggest Lipid transport that mTORC1 signalling inhibits fatty acid oxidation, whilst also Several lines of evidence suggest a role for mTORC1 signal­ promoting mitochondrial biogenesis in some settings. ling in the control of lipid mobilization and transport. As stated The acetyl­ CoA released from β­ oxidation can either enter the above, patients treated with mTORC1 inhibitors suffer frequently TCA cycle or, under fasting conditions in the liver, be converted to from a dyslipidaemia consisting of hypertriglyceridaemia and ketone bodies. Genetic evidence suggests that mTORC1 signalling hypercholesterolaemia, as well as increased levels of plasma free in the liver, which is respectively inhibited and activated by fast­ fatty acids  [55]. The source of the elevated circulating lipids in ing and feeding, suppresses ketogenesis [73]. Mice with LTsc1KO these patients is unknown. However, TAG and cholesterol trans­ that show sustained mTORC1 signalling under fasting have a defect port out of the liver involves their packaging into apolipoprotein in ketogenesis, whereas mice with liver­ specific Raptor knockout complexes, and plasma levels of both apolipoprotein B­ 100 and show an increase in fasting­ induced ketogenesis. mTORC1 seems apolipoprotein C­ III have been found to be increased in patients to suppress the expression of ketogenic enzymes through its reg­ treated with rapamycin [54]. A study in guinea pigs revealed that ulation of N­ CoR1 and PPARα [73], by a mechanism probably the increase in circulating TAGs observed in the response to rapa­ dependent on S6K2 [74]. These inhibitory effects on PPARα and its mycin correlates with an increase in VLDL, the primary mode of 248 EMBO reports VOL 14 | NO 3 | 2013 ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION mTORC1 signalling regulates lipid metabolism review TAG export from the liver [75]. In cultured hepatocytes, the abil­ Sidebar B | In need of answers ity of insulin to repress the expression of both apolipoprotein B (i) What are the molecular mechanisms by which mTORC1 regulates and apolipoprotein A­5 is sensitive to rapamycin, suggesting that SREBP1 and SREBP2? the increase in apolipoproteins observed on rapamycin treatment (ii) Which lipid species are most influenced by the activation state of in vivo might be due to direct effects on hepatocytes [76,77]. How mTORC1 signalling? mTORC1 negatively regulates the expression or protein levels of (iii) Does mTORC1 stimulate the synthesis of membrane lipids in addition specific apolipoproteins is unknown and could be secondary to to storage lipids? changes in apolipoprotein uptake or degradation. Conversely, (iv) How do lipids influence mTORC1 signalling? (v) How does mTORC1 become dysregulated under conditions of obesity? mTORC1 signalling seems to upregulate LDLR, which facili­ (vi) Does mTORC1 inhibition contribute to the effects of AMPK‑ tates the uptake of cholesterol­rich LDL from the plasma into the activating compounds on cellular and systemic metabolism? liver and peripheral tissues. LDLR gene expression is controlled (vii) What is the role of mTORC1 activation in the common lipogenic by SREBP [78] and would, therefore, be predicted to be stimu­ phenotype of cancer cells? lated by  insulin in an mTORC1­dependent manner. In addition, (viii) How is lipid metabolism differentially regulated by mTORC1 in mTORC1 signalling downstream from the insulin receptor in the different tissues? liver has been found to repress the expression of PCSK9, a known negative regulator of LDLR protein levels [79]. Consequently, rapa­ mycin treatment decreases LDLR levels in a PCSK9­dependent these feedback mechanisms to insulin resistance is well illustrated manner, thereby reducing LDL uptake and increasing its circulat­ by loss and gain ­ of ­ function mouse models of mT ­ ORC1 signalling. ing levels. Combined with the rapamycin­stimulated increase in For instance, S6K1 knockout mice have enhanced peripheral insulin lipolysis and apolipoprotein levels, these effects on the LDLR sug­ sensitivity [49], whereas mice with LTsc1KO show hepatic insulin gest a mechanistic basis for the dyslipidaemia observed in patients resistance with greatly reduced Akt signalling [18]. Therefore, under treated with mTORC1 inhibitors. conditions of obesity, mTORC1 activation in metabolic tissues prob­ ably both perpetuates obesity and promotes insulin resistance, mTORC1 in physiology, obesity and diabetes thereby expediting the progression to type II diabetes. The global effects of the mTORC1­mediated regulation of lipid The fundamental role of mTORC1 in regulating wholebod ­ y lipid metabolism detailed above are predicted to promote the systemic homeostasis, paired with its frequent upregulation in obesity and flux of carbon into lipids and their storage as TAGs within adipose type 2 diabetes, suggests that mTOR inhibitors might offer some tissue (Fig 4). The postprandial increase in both glucose and insulin thera peutic benefit in metabolic diseases. In theory, mTORC1­ stimulates the acute activation of mTORC1 within metabolic tis­ specific inhibitors should suppress lipid synthesis and promote sues, in which mTORC1 has contextual roles in controlling lipid lipolysis and lipid catabolism, in addition to blocking mTORC1­ metabolism. In the liver, and probably in adipose tissue, mTORC1 dependent feedback mechanisms to resensitize tissues to insulin. activation induces lipogenesis. At the same time, mTORC1 prob­ However, important caveats arise from the use of mTORC1 inhibitors ably blocks the β­oxidation of fatty acids in the liver, adipose, and to combat obesity and diabetes. First, prolonged treatment with rapa­ perhaps muscle, instead promoting the use and storage of glu­ mycin disrupts mTORC2 and therefore Akt activation downstream cose in these tissues. TAGs and cholesterol produced in the liver from the insulin receptor, further exacerbating the insulinresistant ­ facilitate the packaging and release of VLDL into circulation. phenotype (Sidebar A; [82]). Second, patients treated with rapa­ mTORC1 signalling might enhance uptake of lipids by peripheral mycin frequently have increased levels of circulating TAGs, choles­ tissues through the activation of LPL, which hydrolyses VLDL to terol and free fatty acids [55]. Therefore, whilst rapamycin treatment IDL, and an increase in the levels of LDLR. In adipose tissue, the might help mobilize lipids and deplete fat stores, lipid clearance insulin­stimulated activation of mTORC1 is predicted to contrib ­ offers an additional pathological challenge. Targeting mTORC1 ute to the inhibition of lipolysis, further promoting the storage of signalling indirectly might offer a more promising avenue. AMPK TAGs, either mobilized from the liver or produced de novo within is a potent negative regulator of mTORC1, blocking its function the adipocytes. through phosphorylation of both the TSC–TBC complex [2,5] and Whilst mTORC1 is activated transiently within metabolic tis­ Raptor [6]. Therefore, mTORC1 signalling is blocked on activation sues by normal feeding, conditions of nutrient overload and obesity of AMPK, which is stimulated by a large variety of natural and syn­ can lead to chronically elevated mTORC1 signalling in these tis­ thetic compounds, including metformin, resveratrol and aspirin [83]. sues  [49,80]. The mechanism by which obesity leads to hyper­ Importantly, metformin is the most widely prescribed antidiabetes ­ activation of mTORC1 is unknown but happens probably through drug in the world. Whether any of the beneficial metabolic effects a combination of hyperglycaemia and hyperinsulinaemia under of metformin are attributed to its inhibition of mTORC1 signalling is these conditions. Furthermore, evidence suggests that increased one of several important outstanding questions (Sidebar B). circulating levels of branchc ­ hain amino acids, which are known to activate mTORC1, correlates with the development of obesity ACKNOWLEDGEMENTS We apologize to our colleagues whose work we were not able to cover in and insulin resistance [81]. In addition to potentially exacerbating this review due to space constraints. Research in the Manning laboratory obesity by further promoting lipid storage in adipose depots, chronic related to the subject of this review was supported by a predoctoral training mTORC1 activation under such conditions is believed to contribute grant DGE­1144152 from the National Science Foundation (S.J.H.R.) and to the development of insulin resistance, which frequently accom­ by National Institutes of Health grants R01­CA122617 and P01­CA120964, panies obesity. Increased mTORC1 signalling can trigger several Department of Defense grants TS093033 and TS110065, a Sanofi Innovation distinct feedback mechanisms, which in a cellautonomous manner ­ , Award and grants from the American Diabetes Association and Ellison Medical Foundation. dampens the cellular response to insulin. The in vivo contribution of ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 14 | NO 3 | 2013 249 mTORC1 signalling regulates lipid metabolism review 23. 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The multifaceted role of mTORC1 in the control of lipid metabolism

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Abstract

review review The multifaceted role of mTORC1 in the control of lipid metabolism Stéphane J.H. Ricoult & Brendan D. Manning Department of Genetics & Complex Diseases, Harvard School of Public Health, Boston, Massachusetts, USA The mechanistic target of rapamycin is a protein kinase that, as part mTOR is an evolutionarily conserved serine/threonine kinase of the mechanistic target of rapamycin complex 1 (mTORC1), senses that exists within two functionally distinct protein complexes, the both local nutrients and, through insulin signalling, systemic nutri­ mechanistic target of rapamycin complexes 1 (mTORC1) and 2 ents to control a myriad of cellular processes. Although roles for (mTORC2). mTORC1 senses and integrates a diverse array of cell­ mTORC1 in promoting protein synthesis and inhibiting autophagy in ular signals, with mTOR kinase activity within the complex being response to nutrients have been well established, it is emerging as a influenced by a variety of nutrients—for example, amino acids, central regulator of lipid homeostasis. Here, we discuss the growing glucose and oxygen, cellular energy levels, such as ATP, and many genetic and pharmacological evidence demonstrating the functional secreted growth factors, cytokines and hormones, including insu­ importance of its signalling in controlling mammalian lipid meta­ lin. All of these signals require the Ras­related small G protein bolism, including lipid synthesis, oxidation, transport, storage and Rheb, which on GTP­loading is an essential upstream activator lipolysis, as well as adipocyte differentiation and function. Defining of mTORC1 [1]. Many of the signals that regulate mTORC1 do so the role of mTORC1 signalling in these metabolic processes is crucial by altering the GTP­binding status of Rheb through activation or to understanding the pathophysiology of obesity and its relationship inhibition of a GTPase­activating protein complex, comprised of to complex diseases, including diabetes and cancer. TSC1, TSC2 and TBC1D7—the TSC–TBC complex [2]. For instance, Keywords: adipocytes; Akt; insulin; liver; mTOR insulin, IGF1 and other growth factors inhibit the complex to acti­ EMBO reports (2013) 14, 242–251; published online 12 February 2013; vate Rheb and mTORC1 through Akt­mediated phosphorylation doi:10.1038/embor.2013.5 of TSC2 [3,4]. By contrast, a decrease in cellular ATP, such as the See the Glossary for abbreviations used in this article. decrease that occurs during glucose depletion, activates the com­ plex to inhibit Rheb and mTORC1, at least in part, through the Introduction action of AMPK (Fig  1; [5–7]). On activation, mTORC1 directly Of the four main classes of biological macromolecule, our under­ phosphorylates S6K1 and S6K2, 4E­BP1 and 4E­BP2, and a grow ­ standing of the molecular mechanisms by which cellular signal­ ing number of other downstream targets [8]. Whilst the overall ling pathways regulate lipid metabolism has lagged behind that of effects of mTORC1 signalling differ in cells and tissues, it has an carbohydrates, proteins and nucleic acids. However, lipids are cru­ evolutionarily conserved role in promoting anabolic cell growth cially important both structurally and functionally in all living organ­ and inhibiting the catabolic process of autophagy. On the other isms. An obvious reason for this dependence is the lipid makeup of hand, mTORC2 seems to be regulated primarily by growth factor the plasma membrane and many subcellular organelles. Moreover, signalling and phosphorylates a conserved hydrophobic motif in lipids act as signalling molecules on both a cellular, for example phos­ the protein kinases Akt, SGK and some isoforms of PKC, thereby phoinositides, and organismal, for example steroid hormones, scale. increasing their kinase activity [9]. Through these targets, and prob­ Lipids are also used for energy storage, primarily as triacylglycerides in ably through others, mTORC2 signalling is believed to promote cell adipocytes, and as an alternative to glucose for catabolic meta bolism. survival, proliferation, metabolism and changes in the actin cyto­ Despite the dependence of living organisms on lipids, we know little skeleton. The two mTOR complexes can be distinguished from one about how lipid homeostasis is controlled by the intricate network of another by their differential sensitivity to rapamycin, an allosteric cellular signalling pathways that sense cellular growth conditions. As and partial inhibitor of mTOR (Sidebar A). detailed in this review, the mechanistic target of rapamycin (mTOR) Many studies in cell and mouse models, combined with pre­ protein kinase has emerged as a crucial link between cellular and clinical and clinical data on mTOR inhibitors, have revealed a systemic growth signals and the regulation of lipid metabolism. pivotal role for mTOR—particularly within mTORC1—in con­ trolling lipid homeostasis in many settings, both physiological and pathological. We review this evidence below, with a focus Department of Genetics & Complex Diseases, Harvard School of Public Health, on the key aspects of lipid synthesis, storage and mobilization. 665 Huntington Avenue, SPH2‑117, Boston, Massachusetts 02115, USA Corresponding author. Tel: +1 617 432 5614; Fax: +1 617 432 5236; The emerging picture is that, through a variety of molecular E‑mail: [email protected] mechanisms, mTORC1 signalling promotes processes to syn­ thesize and store  lipids, whilst inhibiting those leading to lipid Received 6 November 2012; accepted 16 January 2013; published online 12 February 2013 consumption (Fig 1). 242 EMBO reports VOL 14 | NO 3 | 2013 ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION m mTORC1 signalling regulates lipid metabolism review Sidebar A | mTORC1 versus mTORC2 and the differential effects Glucose Insulin / IGF1 (ATP) of mTOR inhibitors In studying the mechanistic target of rapamycin (mTOR) signalling network, or interpreting the mTOR literature, it is crucial to understand some of the basic complexities of mTOR signalling and inhibition. The AMPK AKT mechanistic target of rapamycin complex 1 (mTORC1) is composed of the core essential components mTOR, mTOR‑ associated protein, LST8 homologue (mLST8) and the regulatory‑ associated protein of mTOR TSC1 TSC2 (Raptor), whereas mTORC2 is composed of mTOR, mLST8, SAPK‑ TBC1D7 interacting protein 1 (SIN1) and the Raptor‑ independent companion of mTOR (Rictor). Although these complexes are functionally distinct, they can have an influence on each other’s activity. For instance, as mTORC2 stimulates an increase in Akt activity [84], it might influence its downstream Rheb signalling from mTORC1. On the other hand, several negative feedback GTP mechanisms are triggered by mTORC1 activation, which influences Amino mTORC2 activity, including one leading to direct phosphorylation of acids Rictor within mTORC2 by ribosomal S6 kinase 1 (S6K1) downstream from mTORC1 [85,86]. Regarding mTOR inhibitors, the widely used rapamycin mTOR and its many analogues, which on interaction with the ubiquitous protein Lipogenesis Lipolysis FK506 binding protein of 12 kDa (FKBP12) binds to an allosteric site amino Raptor mLST8 terminal to the mTOR kinase domain—the FKBP12‑ rapamycin binding β-oxidation Adipogenesis domain—only has access to mTOR within mTORC1. However, it is evident that in both cell culture and mice, prolonged exposure to rapamycin can Lipid storage Ketogenesis block the assembly of mTORC2 by sequestering uncomplexed mTOR [82,87]. Therefore, although rapamycin is specific to mTORC1 for acute inhibition and generally leads to an increase in upstream signalling from Fig 1 | Upstream regulation from the mTORC1 and its downstream functions mTORC2 and Akt by blocking negative feedback mechanisms, one must related to lipid metabolism. The presence of amino acids is required for the consider that the observed effects of long‑ term rapamycin treatment might activation of mTORC1 by GTP‑ bound Rheb. Upstream from Rheb, the TSC–TBC be due to loss of mTORC2 in some experimental systems, which affects the many processes downstream from Akt. Also, the development of mTOR complex receives signals about systemic and local nutrient and energy availability, kinase domain inhibitors, which completely block mTOR within both in part through AMPK and Akt. These signals either activate or inhibit the complexes, has revealed that rapamycin only partly inhibits mTORC1 ability of the TSC–TBC complex to act as a GAP for Rheb, thereby inhibiting activity. Whilst the nature of this differential sensitivity is unknown, or activating mTORC1, respectively. Activated mTORC1 leads to enhanced rapamycin strongly affects the phosphorylation of some mTORC1 targets phosphorylation of IRS1, which serves as negative feedback to dampen the insulin (for example, S6K1) but only modestly inhibits other targets (for example, response. mTORC1 has many roles in regulating lipid metabolism, including eIF4E‑ binding protein 1; [88]). the promotion of lipid synthesis and storage and inhibition of lipid release and consumption, which are detailed in the text. AMPK, adenosine monophosphate‑ Lipogenesis activated protein kinase; GAP, GTPase‑ activating protein; IRS1, insulin receptor The regulation of de  novo sterol and fatty acid synthesis by sig­ substrate 1; IGF1, insulin‑ like growth factor 1; mTORC1, mechanistic target of nalling pathways, especially insulin signalling, has garnered rapamycin complex 1; Raptor, regulatory‑ associated protein of mTOR; TSC, intense interest. Unlike most terminally differentiated cells, tuberous sclerosis complex; TBC, Tre‑ 2/Bub2/Cdc16 domain‑ containing protein. hepatocytes and adipocytes synthesize significant amounts of lipid de  novo through pathways in which cytosolic acetyl­ CoA, induces transcription from SREs within target genes. SREBP1a derived from glucose or amino acid catabolism, is used to form and 1c are products of alternative splicing of the SREBF1 gene and the hydro phobic carbon backbone of lipids. Acetyl­ CoA is have been primarily implicated in the control of genes involved either committed to sterol and isoprenoid biosynthesis through in fatty acid synthesis, although SREBP1a is thought to activate the action of HMG­ CoA synthase or to fatty acid biosynthesis most SRE­ containing genes [12]. SREBP2 is encoded by SREBF2 through acetyl­ CoA carboxylase. Both the sterol and fatty acid and is believed to have a more important role in the transcription synthesis branches comprise many steps requiring many specific of steroido genic genes, including those involved in cholesterol enzymes. Importantly, the SREBPs are transcription factors that synthesis in the liver [13,14]. Although the SREBPs preferentially stimulate the expression of genes encoding nearly all of these activate transcription of different sets of genes, there is substan­ lipogenic enzymes [10]. The three SREBP isoforms, encoded by tial overlap between the targets of the SREBP isoforms and the two genes, are produced as inactive transmembrane proteins at tissue specificity of these preferences, which has not been fully the endoplasmic reticulum (Fig 2). Under conditions of abundant established. Importantly, independent studies have identified sterols, full­ length SREBP, through its sterol­ sensing binding part­ the SREBPs as major transcriptional effectors of mTORC1 signal­ ner SCAP, is retained in the endoplasmic reticulum by the INSIG ling and have demonstrated that mTORC1 activation promotes proteins [11]. Depletion of intracellular sterols results in release lipogenesis through this family of transcription factors [15,16]. of the SREBP–SCAP complex from Insig and their transport to mTORC1 signalling promotes SREBP activation and lipo genesis the Golgi apparatus, in which two proteolytic cleavage events by in response to both physiological and genetic stimuli. In primary the site­ specific proteases S1P and S2P liberate the active amino­ rodent hepatocytes and the intact liver, insulin or feeding has terminus of SREBP. This fragment then enters the nucleus and been shown to increase the expression of the major liver isoform ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 14 | NO 3 | 2013 243 1 mTORC1 signalling regulates lipid metabolism review to promote the activation of hepatic SREBP1c by having an effect Glossary on its processing [20,21], and to affect the processing of SREBP2 4E‑BP1/2 eIF4E‑binding protein 1/2 in a hepatocellular carcinoma cell line [22]. mTORC1 signal­ AMPK adenosine monophosphate‑activated protein kinase ling has also been suggested to increase SREBP1 activation in an ATG5/7 autophagy‑related 5/7 S6K1­dependent manner in cultured myotubes [23]. ATGL adipose triglyceride lipase Genetic mouse models have demonstrated that mTORC1 C/EBP CCAAT/enhancer‑binding protein CPT1 carnitine palmitoyltransferase 1 activation is essential, but not sufficient, to stimulate hepatic DAG diacylglycerol SREBP1c and its lipogenic targets in response to feeding [18,24]. eIF4E eukaryotic translation initiation factor 4E Mice lacking mTORC1 in their liver, through liver­specific Raptor GSK3 glycogen synthase kinase 3 knockout, fail to induce SREBP1c and lipogenesis [24], and have HSL hormone‑sensitive lipase reduced levels of both liver triglycerides and circulating choles­ IDL intermediate density lipoprotein terol on a ‘Western’ diet [25]. However, characterization of mice IGF1 insulin‑like growth factor 1 with a liver­specific knockout of Tsc1 (LTsc1KO) and constitu­ Insig insulin‑induced gene tive activation of mTORC1, which is independent of insulin and LDL low density lipoprotein feeding, revealed that mTORC1 signalling, although essential, is LDLR LDL receptor not capable of activating SREBP1c and hepatic lipid synthesis on lipin 1 phosphatidate phosphatase LPIN1 LPL lipoprotein lipase its own [18]. In fact, these mice were found on two independ­ LST8 lethal with SEC13 protein 8 ent strain backgrounds to be resistant to the development of MAG monoacylglycerol both age­ and diet­induced hepatic steatosis due to decreased MEF mouse embryonic fibroblast SREBP1c activation [18,26]. These seemingly paradoxical find­ N‑CoR1 nuclear receptor co‑repressor 1 ings are the result of a strong feedback attenuation of Akt signal­ PCSK9 proprotein convertase subtilisin/kexin type 9 ling that accompanies loss of function of the TSC1–TSC2 complex PKA/C protein kinase A/C in all settings [27]. A crucial role for Akt signalling in the induc­ PPARα/γ peroxisome proliferator‑activated receptor α/γ tion of SREBP1c and lipogenesis in the liver has been established Raptor regulatory‑associated protein of mTOR through rodent models [28–30], and this has been extended by Rictor Raptor‑independent companion of mTOR using mice with liver­specific Rictor knockout, which results in S6K1/2 ribosomal S6 kinase 1/2 SCAP SREBP cleavage‑activating protein the loss of mTORC2 activity and its activating phosphorylation of SCD stearoyl‑CoA desaturase Akt [31]. Consistent with the essential nature of Akt signalling to SGK serum and glucocorticoid regulated kinase hepatic SREBP1c, a restoration of Akt activity in LTsc1KO hepato­ shRNA short hairpin RNA cytes restores SREBP1c activation and lipogenesis [18]. Whilst siRNA small interfering RNA many mTORC1­independent pathways might function in parallel SRE sterol response element downstream from Akt to help to promote the activation of hepatic SREBP sterol regulatory element‑binding protein SREBP1c, including GSK3 inhibition [32], data from the LTsc1KO TAG triacylglycerol mice suggest that one pathway involves the repression of an iso­ TBC1D7 TBC1 domain family, member 7 form of the SREBP inhibitor Insig, Insig2a, which is only expressed TCA tricarboxylic/citric acid in the liver [18]. A liver­specific mechanism is also consistent with TSC1/2 tuberous sclerosis complex 1/2 VLDL very low density lipoprotein the fact that mTORC1 activation alone is sufficient to promote SREBP activation and lipogenesis in other settings, even in the absence of Akt signalling [15]. of SREBP (SREBP1c) and its targets, and to promote de novo lipid The molecular mechanism by which S6K1 promotes SREBP pro­ synthesis in a manner that is sensitive to rapamycin  [17–19]. cessing is unknown, and it is clear from additional studies that S6K1 Insulin activates mTORC1 through a pathway involving the Akt­ is not the only direct target downstream from mTORC1 involved mediated phosphorylation and inhibition of TSC2, within a com­ in SREBP isoform regulation, which might vary by cellular context. plex with TSC1 and TBC1D7 [2–4]. Expression of constitutively For instance, siRNA knockdown of the mRNA capbinding protein ­ active Akt or loss of either TSC1 or TSC2, both of which result in eIF4E, which is normally activated by mTORC1 signalling through insulin­independent activation of mTORC1 signalling, stimulates the phosphorylation and release of its inhibitory binding partner the global expression of SREBP1 and SREBP2 targets and drives 4EBP1, decreases o ­ verall levels of SREBP1 and its canonical target lipogenesis through mTORC1 [15,16]. These latter studies found SCD in breast cancer cell lines [33]. The potential involvement of that mTORC1 signalling promotes accumulation of the processed, 4EBP1 regulation b ­ y mTORC1 in some cells might explain the resist ­ mature form of SREBP1, which resides in the nucleus to induce ance of SREBP1 or SREBP2 activation to rapamycin in specific set­ its own expression and that of genes involved in both steroid and tings [22,34]. The resistance of some mTORC1 targets to rapamycin fatty acid biosynthesis. In exploring the molecular mechanism of (Sidebar A) is an important consideration when examining the role this regulation, it was found that S6K1 is required downstream of mTORC1 signalling in any aspect of lipid metabolism. Another from mTORC1 to stimulate the increase in levels of active SREBP1, direct target of mTORC1 that, as with 4EBP1, is partly resistant to ­ expression of SREBP1 and SREBP2 targets, and de  novo lipo­ rapamycin for its regulation is the phosphatidic acid phosphatase genesis in TSC2­deficient cells [15]. SREBP1 regulation in this set ­ lipin 1, which has also been implicated in SREBP regulation [25,35]. ting is independent of the effects on the proteasomal degradation Lipin 1 seems to have a role in the remodelling of the nuclear lamina, of its active form, suggesting that S6K1 promotes the processing which is inhibited by mTORC1mediated phosphorylation of man ­ y of SREBP1. Consistent with these findings, S6K1 has been found residues on this enzyme. Lipin 1 phosphorylation also coincides 244 EMBO reports VOL 14 | NO 3 | 2013 ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION mTORC1 signalling regulates lipid metabolism review SREBF1, SREBF2 S6K SREBP mTORC1 processing lipin1 Lipogenic targets Cytosol RELEASE Cytosol + sterols – sterols Insig SCAP SREBP SCAP SREBP N N C C Endoplasmic reticulum Endoplasmic reticulum TRANSPORT Cytosol SCAP SREBP COP II vesicle CLEAVAGE Cytosol SCAP SREBP Nucleus TRANSCRIPTION DEGRADATION S1P S6K SREBP S2P Rapa- Golgi mycin mTOR kinase inhibitors SRE mTORC1 lipin1 Cytosol Cytosol Fig 2 | The complex steps leading to SREBP activation and input from mTORC1 signalling. (A) SREBP processing and activation is regulated by mTORC1 through S6K and lipin 1 leading to the transcriptional induction of the SREBF1 and SREBF2 genes, encoding SREBP1 and SREBP2, respectively, and genes encoding many lipogenic enzymes involved in both fatty acid and sterol synthesis. The mTORC1‑ mediated transcriptional activation of SREBF1 could result from either autoregulation by SREBP1 or from an unknown parallel pathway downstream from mTORC1. (B) In the presence of sterols, SREBP resides in the endoplasmic reticulum bound to SCAP and the Insig proteins. When sterols become scarce SCAP undergoes a conformational change, which releases the SCAP–SREBP complex from the Insig, allowing its transport from the endoplasmic reticulum to the Golgi apparatus through COPII vesicles. Once in the Golgi, SREBP comes into contact with two site‑ specific proteases. S1P cleaves the luminal loop of SREBP and S2P cleaves the amino‑ terminal transmembrane region of SREBP, which releases the N‑terminal region of SREBP containing the DNA ‑ binding and ‑ transactivating domains. The NLS‑ containing processed form of SREBP enters the nucleus to activate transcription of genes containing SREs in their promoters. Finally, the processed form of SREBP is unstable and subject to proteasome‑ mediated degradation. In some settings, SREBP processing has been found to require S6K1 downstream from mTORC1 and is therefore sensitive to rapamycin. However, the nuclear shuttling of SREBP has been found to require lipin 1 downstream from mTORC1, the phosphorylation of which is largely resistant to rapamycin but sensitive to mTOR kinase domain inhibitors (Sidebar A). The precise molecular mechanisms by which either of these two mTORC1 targets regulates SREBP activation are unknown. COPII, coatamer protein II; Insig, insulin‑ induced gene; lipin 1, phosphatidate phosphatase LPIN1; mTORC1, mechanistic target of rapamycin complex 1; NLS, nuclear localization signal; S1/2P, site 1/2 protease; S6K1, ribosomal S6 kinase 1; SCAP, SREBP cleavage‑ activating protein; SRE, sterol response element; SREBP1/2, sterol regulatory element‑ binding protein 1/2. ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 14 | NO 3 | 2013 245 mTORC1 signalling regulates lipid metabolism review mTORC1­ dependent transcriptional response leads to an increase Mesenchymal in full­ length SREBP isoforms that accompany the increased process­ stem cell ing and activation of SREBP. However, it remains unclear whether this transcriptional effect is simply a result of autoregulation by pro­ cessed SREBPs at the SREBF1 or SREBF2 promoter or a parallel path­ Lineage S6K way independent from the effects of mTORC1 on SREBP processing commitment (Fig 2). Both SREBF1 and SREBF2 contain a characterized SRE in their promoters [36,37]. In cell culture models, exogenous expression of Early preadipocytes processed SREBP1a stimulates the expression of endogenous SREBP1 Rapamycin and SREBP2 transcripts in a manner that is no longer sensitive to rapa­ mycin, suggesting that the transcriptional effects of mTORC1 signal­ ling on SREBP expression are upstream from processed SREBP [15]. However, elegant studies with a transgenic version of SREBP1c in rats suggest that the role of mTORC1 in SREBP1c processing and gene C/EBP-β mTORC1 Clonal expression is separable [21]. More studies are needed to understand C/EBP-δ expansion the many inputs of mTORC1 signalling, especially in vivo, into the regulation of SREBP isoforms. Preadipocytes Adipogenesis Adipocytes are specialized mesenchymal cells that either store lipids as energy reserves (white adipose tissue) or burn lipids through oxidation to generate heat (brown adipose tissue). Pharmacological and genetic studies have demonstrated that the differentiation of C/EBP-α, PPARγ Terminal mesenchymal stem cells into mature adipocytes—adipogenesis— 4E-BP differentiation requires mTOR signalling (Fig  3). Rapamycin treatment has been reported to reduce adipogenesis in a variety of cell culture models. Rapamycin seems to block the early determination step in brown Mature adipocytes adipocyte differentiation, in which a mesenchymal stem cell com­ mits to becoming a preadipocyte [38]. Similarly, rapamycin treat­ ment or shRNA­ mediated knockdown of S6K1 in embryoid bodies hinders their commitment to preadipocytes [39]. However, much of our knowledge of adipogenesis comes from cell culture models Fig 3 | mTORC1 signalling has been implicated in promoting the three of preadipocytes after lineage commitment and also from MEFs, and main steps of adipogenesis. Adipogenesis consists of the differentiation of a has therefore been focused on the later steps of white adipose dif­ mesenchymal stem cell to a mature adipocyte, which makes up a significant ferentiation. Treatment of preadipocytes with rapamycin leads to a part of adipose tissue in which energy is stored as lipids. The commitment marked decrease in adipocyte differentiation  [40–44]. mTOR has of the mesenchymal stem cells to the adipocyte lineage is the first step of been implicated in hormonal induction of clonal expansion, which adipogenesis and is facilitated by S6K1 activity. C/EBP‑ β and ‑ δ are the is an initial step of differentiation that occurs through the action primary drivers of clonal expansion, which is crucial for preadipocyte of two C/EBP family transcription factors, C/EBP­ β and ­ δ. Overall maturation, and the former has been suggested to be activated by mTORC1 levels of C/EBP­ β have been found to decrease on rapamycin treat­ signalling. The terminal differentiation of preadipocytes to mature adipocytes ment, which corresponds with a repression of clonal expansion of is mediated by PPARγ and C/EBP‑α. mTORC1 promotes this final step preadipo cytes [41]. However, rapamycin has also been shown to through both its inhibition of 4E‑ BP and its activation of PPARγ through a inhibit preadipocyte differentiation after clonal expansion, thereby poorly understood mechanism. Although the precise molecular mechanisms ruling out the anti­ proliferative effects of rapamycin as its primary have yet to be defined, rapamycin blocks adipogenesis. 4E‑ BP, eIF4E‑ mode of inhibiting adipogenesis [42–44]. binding protein; C/EBP‑α/β/δ, CCAAT/enhancer ‑ binding protein‑ α/β/δ; Several genetic models have further supported a crucial role for mTORC1, mechanistic target of rapamycin complex 1; PPARγ, peroxisome mTORC1 activation in terminal adipocyte differentiation, in which proliferator‑ activated receptor γ; S6K1, ribosomal S6 kinase 1. it seems to be both necessary and sufficient. For instance, MEFs lacking TSC1 or TSC2, which have sustained, insulin­ independent activation of mTORC1 signalling, have an mTORC1­ dependent with an increase in the levels of processed, nuclear SREBP1 and enhanced capacity to differentiate into adipocytes despite these SREBP2, and the expression of SREBP targets. Although the phospha­ cells being severely resistant to insulin, a major adipogenic fac­ tidic acid phosphatase activity of lipin 1 was shown to be important tor  [45]. Reciprocally, TSC2­ deficient MEFs that express a phos­ for its inhibitory effect on nuclear SREBP levels [35], the molecular phorylation site mutant of TSC2, which blocks the ability of mechanism and tissue specificity of this regulation, as with S6K1 and mTORC1 to be activated by insulin and Akt signalling, show 4E­ BP1, remains unknown. Finally, it is clear that mTORC1 signal­ reduced adipogenesis [45]. The enhanced adipogenesis in mesen­ ling also increases the transcript levels of SREBP1 and SREBP2  in chymal cells lacking the TSC tumour suppressors probably explains cell culture models [15], and SREBP1c in both rodent hepatocytes the common development of adipocyte­ rich renal angiomyo­ and the intact liver in response to insulin or feeding [18–21]. This lipomas in patients with TSC [46]. Consistent with an essential role 246 EMBO reports VOL 14 | NO 3 | 2013 ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION mTORC1 signalling regulates lipid metabolism review for mTORC1, RNA interference knockdown of Raptor also blocks adipocyte. Interestingly, adiposespecific ­ Atg7 knockout mice that adipogenesis in preadipocytes [47]. Downstream from mTORC1, have a defect in autophagy, show decreased adipocyte lipolysis [59], genetic evidence suggests a role for both S6K and 4E­BP in the suggesting that the inhibitory effects of mTORC1 on lipolysis could control of adipogenesis. The involvement of S6K in the commit­ be, at least in part, through its attenuation of autophagy. ment of stem cells to preadipocytes was reinforced by the reduced Although the molecular mechanisms of lipolytic regulation size of this progenitor cell population in S6K1 knockout mice and by mTOR are not fully understood, mTORC1 signalling has been a defect in the capacity of embryonic stem cells from these mice found to influence three distinct lipases: ATGL, HSL and LPL [60]. to commit to the adipocyte lineage [39]. Reciprocally, 4E-BP1/2 In adipocytes, ATGL catalyses the lipolysis of TAGs to DAGs within double­knockout MEFs show enhanced differentiation towards lipid droplets. HSL then converts the DAGs to MAGs. In 3T3­L1 adipocytes [48], suggesting that the ability of mTORC1 to both adipo cytes, mTORC1 suppression increases the transcription of activate S6K and inhibit 4E­BP contributes to its role in promoting ATGL, which parallels the enhanced lipolysis induced by rapa­ adipogenesis. Interestingly, the S6K1 knockout mice have a lean mycin or siRNA knockdown of Raptor [57]. The phosphorylation phenotype on both normal and high­fat diets [39,49], whereas of HSL at Ser 563, an established PKA site, is associated with an the 4E-BP1/2 double­knockout mice are more sensitive to diet­ increase in its lipase activity. A decrease in HSL phosphorylation induced obesity than their wild­type counterparts [48]. However, correlates with mTORC1 activation and the diminished release of the differences in adiposity in these systemic mouse models prob­ free fatty acids  [58]. However, as with ATGL transcriptional sup­ ably reflect many effects of mTORC1 signalling on lipid synthesis pression, how mTORC1 signalling negatively affects HSL phos­ and mobilization, discussed elsewhere in this review, in addition phorylation on this PKA site is unknown. Similarly to mTORC1 to its role in promoting the development of adipose deposits. inhibition, adipocyte­specific Rictor knockout also leads to the The molecular mechanisms by which mTORC1 and its down­ phosphorylation of HSL at Ser 563 [61]. In addition to adipo­ stream targets stimulate adipocyte differentiation have yet to be cyte lipolysis, mTORC1 has been implicated in the control of fully defined. The temporal activation of two transcription factors, the extracellular lipase LPL. LPL is a water­soluble lipase present C/EBP­ α and PPARγ—the master regulator of terminal adipocyte in plasma, as well as on the surface of endothelial cells, primar­ differentiation—is responsible for inducing the final stages of dif­ ily in muscle and adipose tissue. It hydrolyses TAG in circulating ferentiation [50]. mTORC1 signalling has been shown to increase VLDL to promote conversion to IDL and LDL, which facilitates the PPARγ transcript and protein levels, as well as its transactivating activ­ uptake of lipoprotein into tissues [62]. Systemic rapamycin treat­ ity [45,47,51,52], albeit through unknown mechanisms. Cell culture ment has been found to decrease LPL activity in mouse adipose experiments have suggested that regulation of the final differentiation tissue, and mouse and human plasma, albeit through an unknown steps is primarily independent of S6K and is probably dependent on mechanism  [63,64]. The collective studies in patients treated 4EBP ­ inhibition downstream from mTORC1 [40,48]. However, a with rapamycin and a variety of cell and mouse models suggest study has indicated that PPARγ activation can also be suppressed by that mTORC1 activation, which occurs in metabolic tissues after hyperactive mTORC1 signalling through its negative feedback effects feeding, promotes the synthesis and storage of lipids. By contrast, on insulin signalling [53]. These findings indicate that there are prob­ mTORC1 inhibition, such as during fasting, stimulates lipolysis and ably mTORC1dependent and ­ independent inputs into PP ­ AR γ the release of free fatty acids into the circulation. activation and adipocyte differentiation downstream from insulin signalling, with more in vivo experiments needed. β­oxidation and ketogenesis Consistent with the inhibition of mTORC1 signalling promoting Lipolysis fatty acid release and consumption, there is growing evidence that In addition to its role in stimulating lipogenesis through SREBP, mTORC1 suppresses the β­oxidation of fatty acids for energy or mTORC1 signalling is believed to promote the storage of fatty acids ketogenesis. Rapamycin has been found to increase β­oxidation in in lipid stores by inhibiting lipolysis. Neutral lipids, in the form of rat hepatocytes and this has been attributed to increased expression MAG, DAG and TAG inside the cell are subject to lipolysis to mobi­ of β­oxidation enzymes, including long­chain acyl­CoA dehydro­ lize free fatty acids for energy production or remodelling into new genase and carnitine acyltransferase [17,65]. This effect of rapa­ lipid species, including specific membrane and signalling lipids. mycin could be due to the induction of autophagy, which seems Patients treated with rapamycin frequently have dyslipidaemia, one to promote the β­oxidation of fatty acids from TAGs in hepato ­ facet of which is elevated levels of plasma free fatty acids, which cytes  [66]. However, genetic evidence suggests that autophagy could reflect an increase in lipolysis in adipose tissue [54,55]. has inhibitory effects on β­oxidation in adipose tissue  [59,67]. Mice treated with rapamycin show a reduction in adipocyte size Mice with whole­body knockout of S6K1 seem to have enhanced and overall adiposity, and rapamycin stimulates lipolysis in cul­ β­oxidation, as evidenced by increased levels of CPT1 transcript tured adipocytes [56–58]. Genetic manipulations of mTORC1 sig­ in isolated adipocytes [49]. Consistent with mTORC1 signal­ nalling in several mouse models have reinforced the link between ling attenuating β­oxidation, myoblasts isolated from S6K1/S6K2 mTORC1 activation and an inhibition of lipolysis. The adipose tissue double­knockout mice also show enhanced β­oxidation of fatty of 4E-BP1/2 doubleknoc ­ kout mice shows decreased lipo lysis [48], acids [68]. However, this phenotype was attributed to indirect and S6K1 knockout mice are leaner with elevated rates of lipo­ effects from energy stress and AMPK activation in this setting. As lysis  [49]. However, mice with adiposespecific ­ Raptor knockout, with the S6K1 knockout and the S6K1/S6K2 double­ knockout whilst also lean with reduced adiposity, do not show an obvious mice, mice with adipose­specific Raptor knockout are lean increase in lipolysis [47]. This suggests that the lipolysis phenotypes with adipocytes that show increased mitochondrial uncoupling, observed in the wholebod ­ y 4E-BP and S6K1 knockout models which could allow them to burn lipids rapidly without generat­ could be due to systemic effects rather than those intrinsic to the ing ATP [47,49,68]. Paradoxically, mTORC1 activation has also ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 14 | NO 3 | 2013 247 mTORC1 signalling regulates lipid metabolism review FOOD INTAKE LIVER ADIPOSE TISSUE Gluconeogenesis Glucose Glucose Glucose GLUT4 Lipogenesis Lipogenesis β-oxidation β-oxidation Fatty Fatty Ac-CoA Ac-CoA acids acids mTORC1 mTORC1 Insulin HSL Lipolysis DAG ATGL Cholesterol TAG Ketogenesis TAG LDLR STORAGE TRANSPORT Lipoproteins VLDL IDLLDL LPL Fig 4 | The increase in insulin levels after a meal alters hepatic and adipose lipid metabolism, at least in part, through mTORC1 signalling (a working model). In the liver, mTORC1 promotes lipid synthesis through SREBP1c activation. In addition, mTORC1 signalling blocks lipid catabolism by blocking β‑oxidation and ketogenesis in the liver. Consequently, mTORC1 activation in the liver promotes the synthesis of TAGs and perhaps cholesterol, which are incorporated into VLDL for transport to peripheral tissues. Evidence suggests that mTORC1 signalling positively influences LPL activity, which promotes lipid delivery to peripheral tissues by hydrolysing VLDL to IDL, which is then converted to LDL. Lipoprotein‑ bound TAGs are taken up by tissues, including adipocytes, through the LDLR. Both the expression and stability of LDLR, at least in the liver, are probably promoted by mTORC1 activation. In response to insulin, mTORC1 has been suggested to inhibit lipolysis in adipocytes by downregulating ATGL and HSL. Therefore, the systemic effects of postprandial mTORC1 activation are to promote the flux of carbon from glucose towards TAG storage in adipose tissue. See text for details regarding the evidence underlying this model. Ac‑ COA, acetyl‑ CoA; ATGL, adipose triglyceride lipase; DAG, diacylglycerol; GLUT4, glucose transporter type 4; HSL, hormone‑ sensitive lipase; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; LDLR, LDL receptor; LPL, lipoprotein lipase; mTORC1, mechanistic target of rapamycin complex 1; SREBP1c, sterol regulatory element‑ binding protein 1c; TAG, triacylglycerol; VLDL, very low density lipoprotein. been linked to increased mitochondrial biogenesis in some set­ transcriptional targets could also explain the negative regulation of tings [69]. This could explain the decrease in oxidative capacity fatty acid oxidation by mTORC1. The repression of β­ oxidation and of muscle [69–71] and Jurkat T  cells [72] after the inhibition or ketogenesis by mTORC1 probably acts together with its stimulation complete loss of mTORC1 signalling. However, further studies are of lipogenesis, further promoting the flux of acetyl­ CoA towards needed to determine how the observed changes in mitochondrial lipid synthesis and storage. gene expression and oxygen consumption in these settings influ­ ence the β­ oxidation of fatty acids. The collective data suggest Lipid transport that mTORC1 signalling inhibits fatty acid oxidation, whilst also Several lines of evidence suggest a role for mTORC1 signal­ promoting mitochondrial biogenesis in some settings. ling in the control of lipid mobilization and transport. As stated The acetyl­ CoA released from β­ oxidation can either enter the above, patients treated with mTORC1 inhibitors suffer frequently TCA cycle or, under fasting conditions in the liver, be converted to from a dyslipidaemia consisting of hypertriglyceridaemia and ketone bodies. Genetic evidence suggests that mTORC1 signalling hypercholesterolaemia, as well as increased levels of plasma free in the liver, which is respectively inhibited and activated by fast­ fatty acids  [55]. The source of the elevated circulating lipids in ing and feeding, suppresses ketogenesis [73]. Mice with LTsc1KO these patients is unknown. However, TAG and cholesterol trans­ that show sustained mTORC1 signalling under fasting have a defect port out of the liver involves their packaging into apolipoprotein in ketogenesis, whereas mice with liver­ specific Raptor knockout complexes, and plasma levels of both apolipoprotein B­ 100 and show an increase in fasting­ induced ketogenesis. mTORC1 seems apolipoprotein C­ III have been found to be increased in patients to suppress the expression of ketogenic enzymes through its reg­ treated with rapamycin [54]. A study in guinea pigs revealed that ulation of N­ CoR1 and PPARα [73], by a mechanism probably the increase in circulating TAGs observed in the response to rapa­ dependent on S6K2 [74]. These inhibitory effects on PPARα and its mycin correlates with an increase in VLDL, the primary mode of 248 EMBO reports VOL 14 | NO 3 | 2013 ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION mTORC1 signalling regulates lipid metabolism review TAG export from the liver [75]. In cultured hepatocytes, the abil­ Sidebar B | In need of answers ity of insulin to repress the expression of both apolipoprotein B (i) What are the molecular mechanisms by which mTORC1 regulates and apolipoprotein A­5 is sensitive to rapamycin, suggesting that SREBP1 and SREBP2? the increase in apolipoproteins observed on rapamycin treatment (ii) Which lipid species are most influenced by the activation state of in vivo might be due to direct effects on hepatocytes [76,77]. How mTORC1 signalling? mTORC1 negatively regulates the expression or protein levels of (iii) Does mTORC1 stimulate the synthesis of membrane lipids in addition specific apolipoproteins is unknown and could be secondary to to storage lipids? changes in apolipoprotein uptake or degradation. Conversely, (iv) How do lipids influence mTORC1 signalling? (v) How does mTORC1 become dysregulated under conditions of obesity? mTORC1 signalling seems to upregulate LDLR, which facili­ (vi) Does mTORC1 inhibition contribute to the effects of AMPK‑ tates the uptake of cholesterol­rich LDL from the plasma into the activating compounds on cellular and systemic metabolism? liver and peripheral tissues. LDLR gene expression is controlled (vii) What is the role of mTORC1 activation in the common lipogenic by SREBP [78] and would, therefore, be predicted to be stimu­ phenotype of cancer cells? lated by  insulin in an mTORC1­dependent manner. In addition, (viii) How is lipid metabolism differentially regulated by mTORC1 in mTORC1 signalling downstream from the insulin receptor in the different tissues? liver has been found to repress the expression of PCSK9, a known negative regulator of LDLR protein levels [79]. Consequently, rapa­ mycin treatment decreases LDLR levels in a PCSK9­dependent these feedback mechanisms to insulin resistance is well illustrated manner, thereby reducing LDL uptake and increasing its circulat­ by loss and gain ­ of ­ function mouse models of mT ­ ORC1 signalling. ing levels. Combined with the rapamycin­stimulated increase in For instance, S6K1 knockout mice have enhanced peripheral insulin lipolysis and apolipoprotein levels, these effects on the LDLR sug­ sensitivity [49], whereas mice with LTsc1KO show hepatic insulin gest a mechanistic basis for the dyslipidaemia observed in patients resistance with greatly reduced Akt signalling [18]. Therefore, under treated with mTORC1 inhibitors. conditions of obesity, mTORC1 activation in metabolic tissues prob­ ably both perpetuates obesity and promotes insulin resistance, mTORC1 in physiology, obesity and diabetes thereby expediting the progression to type II diabetes. The global effects of the mTORC1­mediated regulation of lipid The fundamental role of mTORC1 in regulating wholebod ­ y lipid metabolism detailed above are predicted to promote the systemic homeostasis, paired with its frequent upregulation in obesity and flux of carbon into lipids and their storage as TAGs within adipose type 2 diabetes, suggests that mTOR inhibitors might offer some tissue (Fig 4). The postprandial increase in both glucose and insulin thera peutic benefit in metabolic diseases. In theory, mTORC1­ stimulates the acute activation of mTORC1 within metabolic tis­ specific inhibitors should suppress lipid synthesis and promote sues, in which mTORC1 has contextual roles in controlling lipid lipolysis and lipid catabolism, in addition to blocking mTORC1­ metabolism. In the liver, and probably in adipose tissue, mTORC1 dependent feedback mechanisms to resensitize tissues to insulin. activation induces lipogenesis. At the same time, mTORC1 prob­ However, important caveats arise from the use of mTORC1 inhibitors ably blocks the β­oxidation of fatty acids in the liver, adipose, and to combat obesity and diabetes. First, prolonged treatment with rapa­ perhaps muscle, instead promoting the use and storage of glu­ mycin disrupts mTORC2 and therefore Akt activation downstream cose in these tissues. TAGs and cholesterol produced in the liver from the insulin receptor, further exacerbating the insulinresistant ­ facilitate the packaging and release of VLDL into circulation. phenotype (Sidebar A; [82]). Second, patients treated with rapa­ mTORC1 signalling might enhance uptake of lipids by peripheral mycin frequently have increased levels of circulating TAGs, choles­ tissues through the activation of LPL, which hydrolyses VLDL to terol and free fatty acids [55]. Therefore, whilst rapamycin treatment IDL, and an increase in the levels of LDLR. In adipose tissue, the might help mobilize lipids and deplete fat stores, lipid clearance insulin­stimulated activation of mTORC1 is predicted to contrib ­ offers an additional pathological challenge. Targeting mTORC1 ute to the inhibition of lipolysis, further promoting the storage of signalling indirectly might offer a more promising avenue. AMPK TAGs, either mobilized from the liver or produced de novo within is a potent negative regulator of mTORC1, blocking its function the adipocytes. through phosphorylation of both the TSC–TBC complex [2,5] and Whilst mTORC1 is activated transiently within metabolic tis­ Raptor [6]. Therefore, mTORC1 signalling is blocked on activation sues by normal feeding, conditions of nutrient overload and obesity of AMPK, which is stimulated by a large variety of natural and syn­ can lead to chronically elevated mTORC1 signalling in these tis­ thetic compounds, including metformin, resveratrol and aspirin [83]. sues  [49,80]. The mechanism by which obesity leads to hyper­ Importantly, metformin is the most widely prescribed antidiabetes ­ activation of mTORC1 is unknown but happens probably through drug in the world. Whether any of the beneficial metabolic effects a combination of hyperglycaemia and hyperinsulinaemia under of metformin are attributed to its inhibition of mTORC1 signalling is these conditions. Furthermore, evidence suggests that increased one of several important outstanding questions (Sidebar B). circulating levels of branchc ­ hain amino acids, which are known to activate mTORC1, correlates with the development of obesity ACKNOWLEDGEMENTS We apologize to our colleagues whose work we were not able to cover in and insulin resistance [81]. In addition to potentially exacerbating this review due to space constraints. Research in the Manning laboratory obesity by further promoting lipid storage in adipose depots, chronic related to the subject of this review was supported by a predoctoral training mTORC1 activation under such conditions is believed to contribute grant DGE­1144152 from the National Science Foundation (S.J.H.R.) and to the development of insulin resistance, which frequently accom­ by National Institutes of Health grants R01­CA122617 and P01­CA120964, panies obesity. Increased mTORC1 signalling can trigger several Department of Defense grants TS093033 and TS110065, a Sanofi Innovation distinct feedback mechanisms, which in a cellautonomous manner ­ , Award and grants from the American Diabetes Association and Ellison Medical Foundation. dampens the cellular response to insulin. The in vivo contribution of ©2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 14 | NO 3 | 2013 249 mTORC1 signalling regulates lipid metabolism review 23. 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Journal

The EMBO Reports – Springer Journals

Published: Mar 1, 2013

Keywords: adipocytes; Akt; insulin; liver; mTOR

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