Cell growth requires co-ordinated regulation of processes that provide metabolites for the synthesis of macromolecules such as proteins and membrane lipids. In recent years, a lot of emphasis has been placed on the activation of protein synthesis by mTORC1 (mammalian target of rapamycin complex 1). The contribution of anabolic pathways other than protein synthesis has only been considered recently. In the present paper, we discuss recent findings regarding the contribution of transcriptional regulation of lipogenesis genes by the SREBP (sterol-regulatory-element-binding protein) transcription factor, a central regulator of expression of lipogenic genes, to the control of cell size in vitro and cell and organ size in vivo.
- cell growth
- mammalian target of rapamycin complex 1 (mTORC1)
- sterol-regulatory-element-binding protein (SREBP)
The PI3K (phosphoinositide 3-kinase)/Akt pathway and growth control
The serine/threonine kinase Akt/PKB (protein kinase B)/c-Akt is activated in response to PI3K signalling and regulates numerous downstream effectors including components of the apoptotic machinery such as Bad, the metabolic regulators GSK3 (glycogen synthase kinase 3) and HK2 (hexokinase 2), and members of the FOXO (forkhead box O) family of transcription factors . In addition, Akt activation enhances activity of the mTORC [mTOR (mammalian target of rapamycin) complex] 1 kinase, a key signalling component in the regulation of cellular metabolism. Akt stimulates mTORC1 activity through at least two distinct mechanisms. Phosphorylation of TSC (tuberous sclerosis complex) 2 on Ser939 and Thr1462 blocks the inhibitory function of the TSC1–TSC2 complex and allows activation of mTORC1 by the Rheb GTPase . Akt has also been shown to phosphorylate PRAS40 (proline-rich Akt substrate of 40 kDa), which results in dissociation of this inhibitory component from mTORC1 [3–5]. mTORC1 phosphorylates S6K (S6 kinase) and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1), resulting in activation of translation and ribosome biosynthesis. In addition, mTORC1 has been shown to be involved in several other cellular processes, such as transcription, cell metabolism and autophagy .
Activation of the PI3K/Akt/mTORC1 pathway has been associated with cell growth in several experimental systems, including mammalian tissue culture cells, transgenic Drosophila melanogaster and knockout and transgenic mice . Overexpression of PI3K results in increased cell and organ size in D. melanogaster , whereas ablation of components of the PI3K/Akt/TOR (target of rapamycin) pathway causes reduced cell size . Akt1/Akt2 double-knockout mice exhibit severe growth deficiency  and mice deficient in S6K1 show a significant reduction in size . In contrast, expression of activated Akt in the heart caused an increase in myocyte size . These findings show that the PI3K/Akt/mTORC1 pathway is both necessary and sufficient to control the size of cells and organs, as well as entire organisms, and suggests that all anabolic processes required for growth must lie downstream of this signalling axis.
A number of metabolic targets of the Akt signalling pathway have been identified in recent years . Akt activation enhances glucose uptake through induction of GLUT (glucose transporter) 4 translocation to the plasma membrane and increased expression of GLUT1 . Akt also phosphorylates HK2 and enhances association of hexokinases with the mitochondrial membrane [15,16], resulting in an increased glycolytic rate which provides carbon units for anabolic processes. Furthermore, Akt enhances amino acid uptake by inducing translocation of amino acid transporters to the plasma membrane . Furthermore, mTORC1 can activate expression of HIF1 (hypoxia-inducible factor 1) which induced expression of glycolytic genes [17a].
We recently performed a detailed analysis of changes in intracellular metabolites following ectopic activation of Akt in human RPE (retinal pigment epithelial) cells using 1H-NMR spectroscopy. Medium metabolite concentrations were also monitored to determine changes in nutrient uptake and secretion. We observed that activation of Akt caused a significant increase in glucose and amino acid uptake, as well as enhanced secretion of lactate . We also observed increased intracellular concentrations of several amino acids, including the essential amino acids leucine, isoleucine, valine and phenylalanine. Interestingly, intracellular concentrations of glutamine were only slightly increased, although cells depleted 60% more glutamine from the medium. This observation is in keeping with a recent report showing that actively growing cells use glutamine as a carbon source for anabolic processes .
We could also show that Akt activation causes a significant increase in de novo lipid synthesis, which was determined by following the incorporation of radioactive metabolites into the lipid fraction. In addition, Akt activation caused a significant increase in the amount of cellular lipids including the membrane phosphoglycerides PE (phosphatidylethanolamine), PC (phosphatidylcholine) and PG (phosphatidylglycerol) .
Interestingly, all metabolic effects of Akt activation, including lipid synthesis and phosphoglyceride accumulation, were blocked by the mTORC1 inhibitor rapamycin. This strongly suggests that mTORC1 is the major metabolic effector downstream of Akt (Figure 1).
SREBPs (sterol-regulatory-element-binding proteins): regulators of lipid synthesis
Expression of most enzymes required for the synthesis of fatty acids, cholesterol, triacylglycerols and phosphoglycerides is regulated by the SREBPs . This family of bHLH (basic helix–loop–helix) leucine zipper transcription factors consists of three highly related members (SREBP1a, 1c and 2). SREBPs are synthesized as inactive precursors and localize to the ER (endoplasmic reticulum) membrane where they are bound by SCAP (SREBP-cleavage-activating protein) . During SREBP activation, the SREBP–SCAP complex translocates to the Golgi where a two-step proteolytic cleavage releases the 65 kDa mature form of SREBP, which can enter the nucleus and binds to SREs (sterol-regulatory elements) within the promoter regions of SREBP target genes . SREBP target genes involved in lipid synthesis include ACLY (ATP-citrate lyase), ACC (acetyl-CoA carboxylase) and FASN (fatty acid synthase) . In addition, enzymes and transporters involved in the shuttling of acetyl groups from the mitochondria to the cytosol and for the production of NADPH are regulated by SREBP. Overexpression of SREBP1a in the livers of transgenic mice is sufficient to induce increased synthesis of cholesterol and triacylglycerols , indicating that SREBP activity is sufficient to induce lipid synthesis.
SREBP processing is regulated by cellular sterol concentrations. Saturating amounts of cholesterol in the ER membrane cause a conformational shift in SCAP, which enables binding of INSIG (insulin-induced gene) and results in retention of the complex in the ER by blocking interaction with components of the COPII (coatamer protein II) vesicle coat  (Figure 2). Mature SREBP1 is regulated through phosphorylation by several kinases, including Cdk1 (cyclin-dependent kinase 1), PKA (protein kinase A) and GSK3 [26–28]. Furthermore, stability and transcriptional activity of nuclear SREBPs are regulated by ubiquitination and SUMOylation [28,29], and acetylation , as well as by association with transcriptional regulators and cofactors including SP1 (specificity protein 1), NFY (nuclear factor Y), CBP [CREB (cAMP-response-element-binding protein)-binding protein], p300 and the ARC (activator-recruited cofactor)–mediator complex . Recent studies have shown that SREBP co-operates with SP1 and NFY in regulating specific classes of target genes and suggest that SREBP could have a role in additional cellular functions apart from lipogenesis .
Regulation of SREBP by Akt: role of mTORC1
Several reports have linked SREBP and its target genes with components of the PI3K/Akt pathway. Insulin increases the expression of the SREBP1c transcript in mouse liver . Cultured hepatocytes show increased expression of SREBP1 in response to insulin treatment or Akt activation  and insulin increases SREBP processing . SREBP1 processing and expression of SREBP target genes is induced by PDGF (platelet-derived growth factor) treatment in a PI3K-dependent manner . Furthermore, ectopic activation of Akt induces nuclear accumulation of mature SREBP1, but not SREBP2, and enhances expression of SREBP target genes .
We have shown recently that accumulation of mature SREBP1 in response to Akt activation is rapid and precedes induction of SREBP target gene expression . Furthermore, activation of SREBP1 by Akt required the presence of glucose in the culture medium and was blocked by AICAR (5-amino-4-imidazolecarboxamide riboside). Energy depletion and AICAR treatment led to activation of AMPK (AMP-activated kinase), which negatively regulates the activity of mTORC1 . Subsequent experiments showed that the mTORC1-specific inhibitor rapamycin blocked accumulation of mature SREBP1 as well as expression of the SREBP target genes FASN and ACLY in response to Akt activation or following insulin treatment. Furthermore, siRNA (small interfering RNA)-mediated silencing of raptor (regulatory associated protein of mTOR), a specific component of mTORC1, blocked induction of FASN and ACLY expression by Akt and prevented Akt-dependent de novo lipogenesis. In contrast, silencing of the mTORC2 component rictor (rapamycin-insensitive companion of mTOR) had no effect confirming that this process is mediated by mTORC1 .
Akt activation was sufficient to increase the stability of ectopically expressed mature SREBP1. A mutant of SREBP in which two GSK3 phosphorylation sites, Thr426 and Ser430, were replaced with alanine showed increased stability in the absence of Akt activation, but could not be stabilized further when Akt was activated. Crucially, the observed Akt-dependent stabilization of mature SREBP was independent of mTORC1 as it could not be blocked by rapamycin treatment. However, inhibition of mTORC1 clearly blocked the formation of endogenous mature SREBP1. These results suggest that Akt contributes to accumulation of mature SREBP1 through at least two mechanisms: Akt increases formation of mature SREBP through an mTORC1-dependent mechanism and enhances the stability of mature SREBP via inhibition of GSK3. Although the exact mechanism of mTORC1-dependent regulation of SREBP1 is still unclear, our findings support a role for mTORC1 in SREBP processing. In this context, it is interesting to note that mTOR associates with the ER and Golgi membranes . It has also been found that membrane association of mTOR is required for its activity . ER and/or Golgi localization would place SREBP and mTORC1 within the same cellular compartment and could facilitate regulation of components of the SREBP-processing machinery by mTORC1 or proteins downstream of mTORC1 signalling such as S6K or 4E-BP1. We are currently investigating potential phosphorylation of SREBP1 and other components of the SREBP-processing machinery in response to mTORC1 activation. Akt activity was shown to be required for ER/Golgi translocation of the SREBP2–SCAP complex , but this process has been reported to be independent of mTORC1 . Further experiments are required to reveal the exact mechanism of regulation of SREBP1 by mTORC1 signalling.
SREBP and cell growth
The main sites of lipid synthesis in the human body are the liver and adipose tissue as well as the lactating breast. Most other adult tissues rely exclusively on lipids provided through the bloodstream for their energy requirements and structural demands. However, re-activation of de novo fatty acid synthesis is observed in many cancers, suggesting that lipogenesis could be a rate-limiting process in rapidly growing tissues . We could show that activation of SREBP is required for the induction of de novo lipid synthesis in response to Akt activation . Furthermore, SREBP silencing prevented the Akt-dependent increase in cell volume, thus demonstrating that expression of lipogenic genes is indeed required for the growth of mammalian cells.
The importance of the PI3K/Akt pathway in cell growth and the control of organ/body size has been elegantly demonstrated using D. melanogaster as a model system ( and references therein). The Drosophila genome harbours a gene encoding a single isoform of a bHLH transcription factor that has significant homology with mammalian SREBP . dSREBP (Drosophila SREBP) induces expression of genes involved in fatty acid and phospholipid synthesis, but not cholesterol biogenesis, as fruitflies are auxotrophic for this metabolite. The mechanism of regulation of dSREBP activity is similar to that of the mammalian protein; however, dSREBP processing is controlled by PE rather than by sterols . dSREBP was found to be expressed in the larval fat body, midgut and oenocytes, a specialized cell type that is functionally related to the mammalian liver . Deletion of dSREBP caused fatty acid auxotrophy, and dSREBP is essential during larval development .
We could show that silencing of dSREBP or dFAS (Drosophila fatty acid synthase) reduced the size of Drosophila tissue culture cells in vitro. Furthermore, moderate silencing of dSREBP in vivo caused a significant developmental delay and resulted in adult fruitflies with reduced body size and weight . Tissue-specific silencing of dSREBP in a single compartment of the developing wing disc led to a specific reduction in the size of the corresponding part of the adult organ. Crucially, reduced organ size was caused by a reduction in cell size rather than number, suggesting that dSREBP is required for the regulation of cell growth rather than proliferation. We also obtained results showing that dSREBP is regulated downstream of the PI3K/Akt pathway in fruitflies. Silencing of components of the PI3K pathway reduced expression of dSREBP and dFAS in vitro, whereas overexpression of dp110 (Drosophila p110), the catalytic subunit of Drosophila PI3K, enhanced expression of both transcripts in vivo. Furthermore, silencing of dSREBP prevented the increase in wing size caused by overexpression of dp110, which suggests that both factors are components of the same pathway in the regulation of cell growth in fruitflies .
Taken together, our results place SREBP activation downstream of a signalling pathway that integrates mitogenic signalling with nutrient availability and suggest that the PI3K/Akt/mTORC1 axis regulates protein and lipid biosynthesis in an orchestrated manner during cell growth (Figure 3).
The importance of cellular metabolic processes in the regulation of cell growth, proliferation and survival is increasingly being recognized. This becomes particularly important in the context of cancer. Activation of oncogenic signalling pathways as well as inactivation of tumour suppressors leads to reprogramming of cellular metabolic processes to fulfil the increased anabolic demand of a rapidly proliferating tissue . We have shown that activation of Akt is sufficient to drive lipogenesis and growth in a normal, non-transformed, cell line and that transcriptional regulation of lipogenic enzymes by SREBP is required for efficient cell growth in mammalian cells in vitro and in fruitflies in vivo. The importance of lipogenesis for growth and tumorigenic potential of transformed cells has been described previously . The simplest explanation for these observations is that lipogenesis is required for the synthesis of membrane phosphoglycerides and is thus essential for cell growth and proliferation. It is also possible that components of the lipid biosynthesis pathway are actively involved in cellular signalling processes. Some phospholipids act as second messengers in growth factor signalling, and lipid-based metabolites are required for post-transcriptional modification of many proteins, including some with signalling functions. Interestingly, a branched-chain fatty acid has recently been shown to be involved in growth control in Caenorhabditis elegans, although the exact mechanism has not been identified . Furthermore, it is possible that enhanced lipogenesis is required to balance the redox potential by utilizing reducing equivalents (NADPH). This could be particularly important under conditions of limited oxygen supply and could affect growth and survival of cancer cells.
Careful functional analysis of the metabolic processes induced by oncogenic signalling pathways will be required to fully understand their contribution to cell growth, proliferation and tumorigenesis.
C.R.S. was supported by a European Molecular Biology Organization long-term fellowship. This work was funded by Cancer Research UK.
We thank S. Leevers, N. Tapon and J. Downward for helpful discussions. We also thank Y.-L. Chung and J.R. Griffiths for collaborating on the metabolite analysis. We are very grateful to E. Hafen, R. Rawson and J. Ericsson for providing material.
mTOR Signalling, Nutrients and Disease: Biochemical Society Focused Meeting held at Medical Sciences Teaching Centre, University of Oxford, U.K., 15–16 September 2008. Organized and Edited by Richard Boyd (Oxford, U.K.), Deborah Goberdhan (Oxford, U.K.) and Richard Lamb (Cancer Research UK, London, U.K.).
Abbreviations: ACLY, ATP-citrate lyase; AICAR, 5-amino-4-imidazolecarboxamide riboside; bHLH, basic helix–loop–helix; dFAS, Drosophila fatty acid synthase; dp110, Drosophila p110; ER, endoplasmic reticulum; FASN, fatty acid synthase; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; GLUT, glucose transporter; GSK3, glycogen synthase kinase 3; HK2, hexokinase 2; INSIG, insulin-induced gene; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; NFY, nuclear factor Y; PE, phosphatidylethanolamine; PI3K, phosphoinositide 3-kinase; S6K, S6 kinase; SP1, specificity protein 1; SREBP, sterol-regulatory-element-binding protein; dSREBP, Drosophila SREBP; SCAP, SREBP-cleavage-activating protein; TSC, tuberous sclerosis complex
- © The Authors Journal compilation © 2009 Biochemical Society