SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth

Abstract

Cell growth (accumulation of mass) needs to be coordinated with metabolic processes that are required for the synthesis of macromolecules. The PI3-kinase/Akt signaling pathway induces cell growth via activation of complex 1 of the target of rapamycin (TORC1). Here we show that Akt-dependent lipogenesis requires mTORC1 activity. Furthermore, nuclear accumulation of the mature form of the sterol responsive element binding protein (SREBP1) and expression of SREBP target genes was blocked by the mTORC1 inhibitor rapamycin. We also show that silencing of SREBP blocks Akt-dependent lipogenesis and attenuates the increase in cell size in response to Akt activation in vitro. Silencing of dSREBP in flies caused a reduction in cell and organ size and blocked the induction of cell growth by dPI3K. Our results suggest that the PI3K/Akt/TOR pathway regulates protein and lipid biosynthesis in an orchestrated manner and that both processes are required for cell growth.

Figure 1

mTORC1 Is Required for Akt-Dependent Cell Growth and Induction of Lipid Synthesis (A) RPE myrAkt-ER cells were treated with 100 nM 4-hydroxytamoxifen (4-OHT) or solvent (ethanol) for 24 hr in medium with 1% lipoprotein deficient serum (LPDS) in the presence or absence of 50 nM rapamycin. In (A), cell size was determined by measuring electronic cell volume. (B) Changes in median cell volume (MCV) induced by Akt activation relative to solvent control. (C) RPE myrAkt-ER cells were treated with ethanol (dark gray bars), 4-OHT (black bars), rapamycin (open bars), or rapamycin and 4-OHT (light gray bars) for 48 hr in medium containing 1% LPDS. Glucose uptake and lactate production was determined by measuring metabolite concentrations in culture supernatant using NMR spectroscopy. Metabolite levels were normalized to cell number and compared to medium without cells. Values represent mean percent changes relative to ethanol-treated cells. (^∗) p ≤ 0.01 ethanol versus 4-OHT. (D) RPE myrAkt-ER cells were treated as in (A), and incorporation of D-[6-¹⁴C]glucose, [2-¹⁴C]-pyruvate, and [1-¹⁴C]-acetate into cellular lipids was measured. (E) Cellular lipids were extracted from cells treated as in (C) and analyzed by NMR spectroscopy. Metabolite concentrations were normalized to cell number and values represent mean percent changes relative to ethanol treated cells. (^∗) p < 0.01 ethanol versus 4-OHT; (^∗∗) p < 0.05 4-OHT versus 4-OHT plus rapamycin. UFA, unsaturated fatty acids; FA,saturated fatty acids; PE = phosphatidylethanolamine; PC = phosphatidylcholine; PG = phosphatidylglycerol. (F) RPE myrAkt-ER cells were stimulated with 4-OHT for 24 hr in the presence or absence of 60 μM SB20490, and cell volume was determined. (G) Changes in MCV after activation of Akt in the presence or absence of 60 μM SB20490. In (B), (D), and (G), error bars represent standard deviation (SD). In (C) and (E), error bars represent standard error of the mean (SEM).

Figure 2

Akt Induces Rapid Accumulation of mSREBP1 in the Nucleus (A) RPE myrAkt-ER cells were cultured in medium with 1% LPDS for 16 hr and treated with 100 nM 4-OHT for the indicated times. Nuclear extracts were prepared and analyzed for expression of mature SREBP1 (mSREBP1). Full-length SREBP1 (flSREBP) was detected in the supernatant. (B) Quantitative representation of levels of mature and flSREBP1 in response to Akt activation in RPE cells. (C and D) Cells were treated with 100 nM 4-OHT for the indicated times. Relative mRNA abundance of FASN was determined by qPCR. Values are normalized to GAPDH and are shown relative to solvent treated control. A representative of two independent experiments performed in duplicate is shown (D). Whole cell lysates prepared from cells treated in parallel to (C) were used to determine abundance of FASN, flSREBP1, and phospho-Akt-ER. (E) Cells were cultured in medium with 1% LPDS and treated with 100 nM 4-OHT or solvent in the presence or absence of a mixture of 10 μg/ml cholesterol and 1 μg/ml 25-hydroxycholesterol (sterols) for 2 hr, and nuclear extracts were analyzed for expression of mSREBP1. (F) Cells were treated with 100 nM 4-OHT in the presence or absence of sterols for 24 hr. Whole cell lysates were analyzed for expression of FASN, flSREBP1, and phospho-Akt-ER. (G) Cells were cultured in medium with 1% LPDS for 16 hr and then placed in glucose-free medium or medium containing 2.5 mM glucose. Cells were treated with 2-deoxy-glucose (2-DG) and 4-OHT as indicated. Nuclear extracts were prepared and analyzed for the presence of mSREBP1. (H) Cells were placed in glucose-free medium containing 1% LPDS or medium supplemented with 2.5 mM glucose or glucose and 2-DG for 30 min prior to stimulation with 100 nM 4-OHT for 24 hr. Whole cell lysates were analyzed for expression of FASN and full-length SREBP1. (I) Cells were treated with 4-OHT for 2 hr with or without pretreatment with AICAR for 30 min. Nuclear extracts were analyzed for the presence of mSREBP1. Error bars represent SD.

Figure 3

Akt-Dependent Activation of SREBP1 and Lipogenesis Requires mTORC1 Activity (A) RPE-myrAkt-ER cells were cultured in medium with 1% LPDS for 16 hr and treated with 100 nM 4-OHT for 2 hr with or without pretreatment with 50 nM rapamycin for 30 min. Nuclear extracts were prepared and analyzed for expression of of mSREBP1. (B) Cells were cultured in medium with 1% LPDS and treated with 100 nM 4-OHT or solvent for 24 hr in the presence or absence of 50 nM rapamycin. Total RNA was extracted and analyzed by qPCR for expression of ACLY and FASN. Values are normalized to GAPDH and are representative of one of three independent experiments. (C) Whole cell lysates of cells treated as in (B) were used to determine the abundance of FASN, ACLY, and flSREBP1 as well as phosphorylation of ACLY and GSK3α/β. (D) Cells were transfected with 1 μg of expression vector for myc-SREBP1 (aa 1-490) wild-type or the T426A/S430A mutant. 24 hr post-transfection, cells were placed in medium containing 1% LPDS and treated with 100 nM 4-OHT or solvent in the presence or absence of 50 nM rapamycin for 24 hr. Abundance of myc-mSREBP1 was determined using the 9E10 antibody. (E) Cells were transfected with 100 nM siRNA oligonucleotides specific for SREBP1 and SREBP2 (BP1+BP2), mTOR, raptor, rictor or an unspecific control. 72 hr post-transfection, cells were placed in medium containing 1% LPDS and treated with 4-OHT for 24 hr. Lysates were used to confirm silencing efficiency and to determine FASN and ACLY expression. (F) Cells treated in parallel to (E) were used to determine incorporation of D-[6-¹⁴C]glucose, [2-¹⁴C]-pyruvate or [1-¹⁴C]-acetate into lipid fractions. Error bars represent SD.

Figure 4

Silencing of SREBP1 Restricts Akt-Induced Cell Growth in Mammalian Cells (A) RPE-myrAkt-ER cells were transfected with either 50 or 100 nM siRNA oligonucleotides specific for SREBP1 and SREBP2 (BP1 + BP2) or an unspecific control. Seventy-two hours post transfection, cells were placed in medium containing 1% LPDS and treated with 100 nM 4-OHT or solvent for 24 hr, and cell size was determined. (B) Changes in median cell volume (MCV) following Akt activation relative to solvent control. (C) Whole cell lysates of cells treated in parallel to (A) were used to determine levels of FASN, SREBP1, SREBP2 and phospho-Akt-ER. Error bars represent SD.

Figure 5

Silencing of dSREBP Reduces Cell and Organ Size in Drosophila (A) Kc167 cells were treated with dsRNAs corresponding to GFP, dSREBP, or dFAS in the absence or presence of dTSC2 dsRNA for 5 days, and electronic cell volume was determined. (B) Cells treated as in (A) were used to determine FSC-A of the G1 population by FACS. (C) dSREBP RNAi was expressed ubiquitously during embryogenesis using the daughterless-GAL4 (da-GAL4) driver. Expression of dSREBP (white bars) and dFAS (gray bars) in second instar larvae was determined by qPCR. Transcript levels are normalized to actin mRNA levels and represent three independent experiments. (RNAi-S1, single insertion, RNAi-S2 and RNAi-S3, double insertions). (D) Representative phenotype of female flies expressing dSREBP RNAi using the da-GAL4 driver compared to control (yw). (E) Wings of flies expressing dSREBP^RNAi-S3 in the dorsal cell layer of the wing. (F) Wing area analysis of control (yw), dSREBP^RNAi-S1, dSREBP^RNAi-S2, and dSREBP^RNAi-S3. (^∗) p ≤ 10⁻⁵. (G) Wings of flies expressing dSREBP^RNAi-S3 in the posterior compartment of the developing wing. (H) Changes in wing area of control flies (white bars) or flies expressing SREBP RNAi-2 (gray bars) or SREBP RNAi-3 (black bars) in the posterior compartment of the wing. (^∗) p ≤ 10⁻⁸. (I) High-magnification images of the anterior and posterior compartments of wings shown in (G). (J) Changes in wing epithelial cell number of flies expressing GFP (white bars) or dSREBP^RNAi-S3 (black bars) in posterior compartment of the wing. Error bars represent SD.

Figure 6

dSREBP Is Regulated by the PI3K Pathway and Required for dp110-Dependent Cell Growth (A) Kc167 cells were treated with dsRNAs corresponding to GFP, dSREBP, dFAS, dp110, dAKT, and dPTEN for 5 days. Expression of dSREBP (white bars) and dFAS (gray bars) was determined by qPCR. Values are normalized to actin mRNA levels and show a representative of three experiments performed in duplicate. (B) Second instar larvae expressing dp110 ubiquitously throughout development were analyzed for dSREBP (white bars) and dFAS (gray bars) by qPCR. Values are normalized to actin mRNA levels. (C) Flies expressing fl-dSREBP or m-dSREBP under the control of the UAS promoter (UAS-fl-dSREBP1) were crossed with flies expressing GAL4 (MS1096-G4) or dp110 and GAL4 in the dorsal cell layer of the wing (MS1096-G4-UAS-dp110). Representative individual flies are shown. Arrows indicate severe malformation of the wing. (D) Wing size analysis of flies expressing dSREBP^RNAi-S2 or dSREBP^RNAi-S3 with (MS1096-G4-UAS-dp110) or without (MS1096-G4) coexpression of dp110. n ≥ 7 wings; (^∗) p ≤ 5 × 10⁻⁸. (E) Quantification of wing area of flies expressing dSREBP^RNAi-S3 with (dpp-G4-UAS-dp110[KD]) or without (dpp-G4) coexpression of kinase-dead dp110. Error bars represent SD.

Figure 7

Model of Regulation of Lipid Biosynthesis by Akt and mTORC1 Akt and mTORC1 regulate lipid biosynthesis on several levels. Activation of glucose uptake and induction of glycolysis is required for the generation of mitochondrial citrate. Activation of SREBP1 by Akt induces the expression of enzymes involved in lipid biosynthesis, including ACLY, FASN, and ACC. Akt stabilizes mSREBP1 by inhibition of GSK3. Inhibition of mTORC1 blocks accumulation of mSREBP1 in response to Akt activation through an unknown mechanism.