Glutamine stimulates the gene expression and processing of sterol regulatory element binding proteins, thereby increasing the expression of their target genes Jun Inoue, Yuka Ito, Satoko Shimada, Shin-ich Satoh, Takashi Sasaki, Tsutomu Hashidume, Yuki Kamoshida, Makoto Shimizu and Ryuichiro Sato Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan Keywords glutamine; processing; Sp1; SREBP; transcriptional regulation Correspondence R Sato, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Fax: 81 5841 8029 Tel: 81 5841 5136 E-mail: aroysato@mail.ecc.u-tokyo.ac.jp (Received 17 March 2011, revised 23 May 2011, accepted June 2011) doi:10.1111/j.1742-4658.2011.08204.x Here we show that the larger the amount of glutamine added to the medium, the more the expression of genes related to lipid homeostasis is promoted by the activation of sterol regulatory element binding proteins (SREBPs) at the transcriptional and post-translational levels in human hepatoma HepG2 cells Glutamine increases the mRNA levels of several SREBP targets, including SREBP-2 The gene expression of SREBP-1a, a predominant form of SREBP-1 in most cultured cells and a target of the general transcription factor Sp1, is significantly augmented by glutamine via an increased binding of Sp1 to the SREBP-1a promoter In contrast, the increased expression of SREBP targets including SREBP-2 is due to stimulation of the processing of SREBP proteins by glutamine It is also shown that glutamine accelerates SREBP processing through increased transport of the SREBP ⁄ SREBP cleavage-activating protein complex from the endoplasmic reticulum to the Golgi apparatus The processing of activating transcription factor is activated by the same proteases as SREBPs in the Golgi in response to endoplasmic reticulum stress and is not induced by glutamine Taken together, these results clearly demonstrate that glutamine brings about not only the induction of SREBP-1a transcription but also the stimulation of SREBP processing, thereby facilitating the gene expression of SREBP targets in cultured cells Introduction Because amino acids are indispensable nutrients for cell growth, cell culture media usually contain large amounts of them In addition to their role as substrates for protein synthesis, glucogenic substrates and nitrogen carriers, amino acids often act as critical regulators of the transcription of certain genes For example, amino acid supplementation with tryptophan and glutamine induce the gene expression of collagenase and argininosuccinate synthetase, respectively [1,2] Amino acid starvation also regulates the transcription of several genes, including fatty acid synthase, asparagine synthetase and C ⁄ EBP homologous protein [3–5] Amino acid metabolism is strongly linked to both glucose and fatty acid metabolism Under certain Abbreviations ATF6, activating transcription factor 6; CPT1A, carnitine palmitoyltransferase 1A; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3phosphate dehydrogenase; GFAT, glutamine:fructose-6-phosphate amidotransferase; HMG, 3-hydroxy-3-methylglutaryl; Insig, insulin-inducing gene; LDL, low density lipoprotein; MTP, microsomal triglyceride transfer protein; PI3K, phosphatidylinositol 3-kinase; PLAP, placental alkaline phosphatase; p70S6K, p70 ribosomal protein S6 kinase; SCAP, SREBP cleavage-activating protein; SQS, squalene synthase; SREBP, sterol regulatory element-binding protein; S1P, site-1 protease; S2P, site-2 protease FEBS Journal 278 (2011) 2739–2750 ª 2011 The Authors Journal compilation ª 2011 FEBS 2739 Glutamine stimulates SREBP activities J Inoue et al physiological conditions amino acids are metabolized to either glucose precursors or acetoacetyl-CoA Acetoacetyl-CoA is then converted to acetyl-CoA and it subsequently enters into the tricarboxylic acid cycle, or is used as a precursor of fatty acids in response to their demands Under fasting conditions, the acetylCoA provided by the oxidation of free fatty acids increases the consumption of amino acids as precursors of the oxaloacetate required for condensation with acetyl-CoA [6] Although the acetyl-CoA provided as a metabolite of amino acids can be a substrate for cholesterol synthesis, the interplay between amino acid and cholesterol metabolism remains largely unknown Cholesterol and fatty acid synthesis are strictly regulated at the transcriptional level SREBPs are a family of transcription factors which consists of the SREBP-1a, SREBP-1c and SREBP-2 proteins that control the transcription of genes related to cholesterol and fatty acid metabolism [7] SREBPs are synthesized as membrane proteins located on the endoplasmic reticulum (ER) and are processed to liberate the N-terminal halves that function as transcription factors in the nucleus The proteolytic processing of SREBPs is tightly regulated by the interaction between two ER membrane proteins, SREBP cleavage-activating protein (SCAP) and the insulin-inducing gene (Insig) When cells are depleted of sterols, SCAP escorts SREBPs from the ER to the Golgi Thereafter, SREBPs are processed by two proteases, site-1 protease (S1P) and site-2 protease (S2P) within the Golgi Once the ER membrane cholesterol content increases, SCAP binds to cholesterol, induces conformational change and becomes attached to Insig, thereby remaining on the ER membrane Therefore, cholesterol is a critical determinant of SREBP activation In the present study, we report that glutamine treatment results in an increase in the promoter activities of a number of SREBP targets, such as the low density lipoprotein (LDL) receptor, 3-hydroxy-3-methylglutaryl (HMG) CoA synthase and squalene synthase (SQS) genes in human hepatoma HepG2 cells We further investigated the molecular mechanism by which glutamine affects the expression of the genes involved in cholesterol homeostasis The results clearly demonstrate that glutamine stimulates SREBP processing and the gene expression of SREBP targets The same concentrations of alanine, proline and glutamate treatment did not influence SREBP processing Moreover, glutamine treatment also causes an increase in hexosamine biosynthesis as a substrate, thereby enhancing the SREBP-1a mRNA levels To our knowledge, this is 2740 the first report showing that glutamine promotes SREBP activities and stimulates the gene expression of SREBP targets Results and Discussion Glutamine stimulates the promoter activities of SREBP targets More than 50 years ago, Eagle et al [8] reported the importance of glutamine in a culture medium for cell proliferation Because cholesterol is essential for the constituent of membrane, we examined whether glutamine regulates the expression of genes involved in cholesterol homeostasis To investigate the effect of glutamine on the transcription of genes related to cholesterol metabolism, a variety of reporter assays were preformed in HepG2 cells The promoter activities of the LDL receptor, HMG CoA synthase and SQS were increased by the addition of a 4- or 10-fold excess of glutamine in DMEM, while the promoter activity of microsomal triglyceride transfer protein (MTP) was attenuated in a dose-dependent manner (Fig 1A) In contrast, carnitine palmitoyltransferase 1A (CPT1A) promoter activity was not affected by glutamine (Fig 1A) Since these observed glutamine effects mimicked SREBP actions, we next determined whether treatment with sterols, which almost completely inhibit SREBP processing, affects the glutamine-induced promoter activities The glutamine-induced promoter activities of both HMG CoA synthase and SQS were abolished by sterols (Fig 1B) When one or two of the sterol regulatory elements (SREs) in the HMG CoA synthase or SQS promoter, respectively, was mutated (SREKO), the glutamine stimulatory effects were greatly reduced (Fig 1B) Moreover, the glutamine effect on the HMG CoA synthase promoter activity was also observed in human intestinal epithelial Caco2 cells (Fig 1C) From these results, it seems likely that the glutamine actions are mediated by the activation of SREBP Next, we sought to confirm whether glutamine causes an elevation of the endogenous mRNA levels of SREBP targets HepG2 cells were treated with a 10-fold excess of glutamine for 12 h Quantitative realtime PCR analyses revealed that glutamine treatment caused a significant increase in the mRNA levels of both HMG CoA synthase and the LDL receptor, and the glutamine effects were completely abolished by the addition of sterols (Fig 2) Taken together, glutamine is evidently capable of stimulating the SREBPmediated expression of genes related to cholesterol metabolism FEBS Journal 278 (2011) 2739–2750 ª 2011 The Authors Journal compilation ª 2011 FEBS J Inoue et al 3.0 Relative luciferase activity 4.0 No addition × Gln 10 × Gln ** ** 2.5 ** 2.0 * 1.5 1.0 0.5 0.0 B HepG2 ** ** Relative mRNA level Relative luciferase activity A 3.5 Glutamine stimulates SREBP activities pLDLR pHMG S pSQS pMTP 3.5 ** ** 3.0 2.5 No addition ** ** 10 × Gln 10 × Gln + Sterols 2.0 1.5 1.0 0.5 0.0 SREKO pHMG S WT 3.0 ** 2.0 2.5 2.0 10 × Gln 10 × Gln + Sterols 1.5 1.5 1.0 1.0 0.5 0.5 0.0 HMG S LDLR Fig Glutamine enhances the gene expression of endogenous SREBP targets in HepG2 cells HepG2 cells were cultured with the low amino acid medium containing a 10-fold excess of glutamine (10 · Gln, 40 mM) for 12 h in the presence or absence of sterols (10 lgỈmL)1 of cholesterol plus lgỈmL)1 of 25-hydroxycholesterol) and total RNA was isolated Real-time PCR analysis was performed, and relative mRNA levels were obtained after normalization to GAPDH mRNA The mRNA levels without glutamine addition are represented as All data are presented as means ± SD of three independent experiments performed in triplicate **P < 0.01 SREKO pSQS Caco2 C Relative luciferase activity 2.5 WT 2.5 2.0 ** 0.0 pCPT1A HepG2 4.0 3.5 No addition 3.0 * No addition × Gln 10 × Gln ** 1.5 1.0 0.5 0.0 pHMG S pCPT1A Fig Glutamine stimulates the promoter activities of SREBP targets HepG2 cells (A and B) and Caco-2 cells (C) were transfected with 200 ng of the reporter constructs consisting of the indicated gene promoters and 200 ng of pEF-b-Gal The cells were cultured with medium A (A and B) or medium B (C) for 36 h and then re-fed with the low amino acid medium supplemented with the indicated concentration of glutamine (4 · Gln, 16 mM; 10 · Gln, 40 mM) for 12 h in the presence or absence of sterols (10 lgỈmL)1 of cholesterol plus lgỈmL)1 of 25-hydroxycholesterol) Luciferase assays were performed as described in Materials and methods The promoter activities without glutamine addition are represented as All data are presented as means ± SD of three independent experiments performed in triplicate *P < 0.05; **P < 0.01 Glutamine enhances the mRNA levels of SREBP family members There are a couple of possible explanations for the glutamine-mediated promotion of SREBP functions The most likely are, first, an increase in the gene expression of SREBPs, and second, the enhancement of SREBP processing by glutamine We therefore investigated the effect of glutamine on the gene expression of SREBP family members, SREBP-1 and SREBP-2 SREBP-1 exists in two forms, designated 1a and 1c SREBP-1a is the predominant isoform in most cultured cells, including HepG2 cells [9], and is a more potent transcription factor than SREBP-1c [10] Accordingly, we examined the effect of glutamine on the gene expression of SREBP-1a and SREBP-2 in HepG2 cells in the following experiments The mRNA levels of both SREBP-1a and SREBP-2 were significantly elevated by treatment with a 10-fold excess of glutamine for 12 h in HepG2 cells (Fig 3A) It has been demonstrated that SREBP-2 is an SREBP target gene, and that the transcription of SREBP-1a gene is predominantly regulated by the general transcription factor Sp1 [11,12] We next compared the glutamineinduced gene expression of SREBPs under various conditions When HepG2 cells were cultured with both glutamine and sterols, the increased SREBP-1a mRNA levels were not reduced by sterols, which suppressed SREBP-2 expression robustly (Fig 3A), implying that the gene expression of SREBP-2 is induced by the activation of the SREBP in response to the higher glutamine concentration In contrast, the elevation of the SREBP-1a levels by glutamine was completely abolished by azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (GFAT), whereas the SREBP-2 mRNA level was not affected These FEBS Journal 278 (2011) 2739–2750 ª 2011 The Authors Journal compilation ª 2011 FEBS 2741 Glutamine stimulates SREBP activities ** 2.5 * 2.0 10 × Gln + Sterols 10 × Gln + Azaserine 2.5 ** ** 2.0 B C Azaserine 10 × Gln 1.5 IP: anti-Sp1 1.5 1.0 – – + – + + Time after Gln (h) IB: anti-Sp1 1.0 1.7 1.9 Nuclear 1.0 1.2 0.7 1.0 0.7 0.8 IB: anti-O-GlcNAc 1.0 Cytosol IB: anti-Sp1 0.5 0.5 0.0 0.0 SREBP-1a D SREBP-2 SREBP-1a promoter SREBP-1a-distal 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 IP: IgG anti-Sp1 % of input 0.4 0.0 No addition E Relative mRNA level Relative mRNA level A 3.0 No addition 10 × Gln J Inoue et al Sp1 1.2 1.0 ** 1.5 0.6 1.0 0.4 0.5 0.2 0.0 siLuc siSp1 No addition 10 × Gln SREBP-1a 2.0 0.8 0.0 IgG anti-Sp1 2.5 siLuc siSp1 10 × Gln Fig Glutamine increases the mRNA levels of SREBPs and O-glycosylated Sp1 (A) HepG2 cells were cultured with the low amino acid medium supplemented with a 10-fold excess of glutamine (10 · Gln, 40 mM) for 12 h in the presence or absence of sterols (10 lgỈmL)1 of cholesterol plus lgỈmL)1 of 25-hydroxycholesterol) or lM azaserine Real-time PCR analysis was performed, and relative mRNA levels were obtained after normalization to GAPDH mRNA The mRNA levels without the glutamine addition are represented as All data are presented as means ± SD of three independent experiments performed in triplicate *P < 0.05; **P < 0.01 (B) HepG2 cells were cultured with the low amino acid medium supplemented with or without a 10-fold excess of glutamine (10 · Gln, 40 mM) in the presence or absence of lM azaserine for 12 h The whole cell extracts were subjected to immunoprecipitation (IP) with anti-Sp1 antibody Aliquots of immunoprecipitates were subjected to SDS ⁄ PAGE and immunoblot (IB) analysis with anti-O-GlcNAc or anti-Sp1 antibodies, and the signals were quantified with a Fujifilm LAS-3000 Luminoimager Fold change was calculated by the ratio of the intensity between the O-glycosylated Sp1 and the whole Sp1 signals The ratio in the absence of both glutamine and azaserine was set as The same results were obtained in more than three separate experiments (C) HepG2 cells were cultured with the low amino acid medium for h and then re-fed with the medium supplemented with a 10-fold excess of glutamine (10 · Gln, 40 mM) for the indicated periods The nuclear and cytosol fractions were prepared as described previously [29] and the extracts were subjected to IB with Sp1 antibody; the signals were quantified with a Fujifilm LAS-3000 Luminoimager The intensity at time was set as The same results were obtained in more than three separate experiments (D) HepG2 cells were cultured with the low amino acid medium supplemented with a 10-fold excess of glutamine (10 · Gln, 40 mM) for h and processed for chromatin immunoprecipitation analyses as described in Materials and methods After IP with anti-Sp1 IgG, real-time PCR analysis was performed with a primer set covering the Sp1-binding region or distal region in the human SREBP-1a promoter The same results were obtained in two separate experiments (E) HepG2 cells were transfected with either control (siLuc) or Sp1 siRNA oligonucleotides (siSp1), cultured with medium A for 96 h and re-fed with the medium containing a 10-fold excess of glutamine (10 · Gln, 40 mM) for 24 h before harvest Real-time PCR analysis was performed, and the relative mRNA levels were obtained after normalization to GAPDH mRNA The mRNA levels transfected with siLuc without any glutamine addition are represented as All data are presented as means ± SD of three independent experiments performed in triplicate **P < 0.01 results imply the involvement of the O-glycosylation of Sp1 in the induction of SREBP-1a transcription by glutamine The gene expression of SREBP-1c, which represented less than 20% of the SREBP-1a gene expression in our experiments, was regulated in a similar manner to SREBP-2 (data not shown) It has been reported that glutamine treatment stimulated O-glycosylation of Sp1 in Caco2 cells, in turn causing an increase in Sp1 activity through induced nuclear localization [13] To examine whether glutamine treat2742 ment promotes O-glycosylation of Sp1 in HepG2 cells, we performed immunoblotting analyses using the RL2 antibody, which recognizes N-acetylglucosamine attached to a serine or threonine residue Glutamine elevated the O-glycosylated Sp1 level, whereas azaserine completely abolished this effect and further reduced the basal O-glycosylated Sp1 (Fig 3B) Moreover, we examined whether glutamine induces the translocation of Sp1 from the cytosol to the nucleus As shown in Fig 3C, the amount of nuclear Sp1 was FEBS Journal 278 (2011) 2739–2750 ª 2011 The Authors Journal compilation ª 2011 FEBS J Inoue et al Induction of SREBP gene expression is not the initial trigger for the glutamine effects If induction of the SREBP gene expression serves as the initial trigger for the glutamine effects, the gene expression of SREBPs should be induced prior to the SREBP target gene To test this prediction, timecourse experiments in the presence of glutamine were performed in HepG2 cells At h after the addition of glutamine, the gene expression of the SREBP targets, such as HMG CoA synthase and the LDL receptor, was slightly increased, but then the mRNA levels of both SREBPs and their target genes became significantly elevated at and 12 h (Fig 4) Based on the fact that the increase in the mRNA of these genes was nearly simultaneously observed at h or later, it is unlikely that the increased SREBP gene expression served as the initial step for the glutamine effects Glutamine stimulates SREBP processing In the above experiments, HepG2 cells were cultured with a low amino acid medium in order to detect the glutamine effects with a high sensitivity Later it turned out that the glutamine effects were reproduced in cells incubated with DMEM which contained mm glutamine Indeed, when HepG2 cells were cultured in DMEM supplemented with a 10-fold excess of gluta- 8.0 8.0 HMG S LDLR SREBP-1a SREBP-2 6.0 Relative mRNA level increased by h after glutamine treatment, accompanied by a reduction in the amount of cytosolic Sp1 In order to determine whether glutamine increases the binding of Sp1 to the SREBP-1a promoter region containing the Sp1-binding elements, we performed a chromatin immunoprecipitation assay As shown in Fig 3D, glutamine increased the Sp1 binding to the SREBP-1a promoter region but not the distal region of the SREBP-1a gene, indicating a glutamine-dependent recruitment of Sp1 to the SREBP-1a promoter We next examined whether the activation of Sp1 by glutamine treatment is involved in the induction of SREBP-1a gene expression When endogenous Sp1 expression was reduced to  20% of normal with gene-specific small interfering RNA (siRNA), the gene expression of SREBP-1a was significantly decreased in HepG2 cells (Fig 3E), indicating that the basal gene expression of SREBP-1a is under the control of Sp1 Moreover, the elevation in the mRNA level of SREBP-1a by glutamine was abolished by the knockdown of Sp1 expression (Fig 3E) These results suggest that glutamine treatment facilitates Sp1 function in HepG2 cells via its increased nuclear localization, thereby stimulating SREBP-1a transcription Glutamine stimulates SREBP activities 4.0 6.0 4.0 3.0 3.0 2.0 2.0 1.0 1.0 0.0 0.0 Time (h) 12 Fig The gene expression of SREBPs is not induced prior to the SREBP target gene by glutamine HepG2 cells were cultured with the low amino acid medium for h and then re-fed with the medium supplemented with a 10-fold excess of glutamine (40 mM) for 2, 4, or 12 h, and then total RNA was isolated Real-time PCR analysis was performed, and the relative mRNA levels were obtained after normalization to GAPDH mRNA The mRNA levels at time are represented as All data are presented as means ± SD of three independent experiments performed in triplicate mine (40 mm), the mRNA levels of SREBP family members were significantly increased, as had occurred in the cells cultured in the low amino acid medium (Fig S1) To investigate the effect of glutamine on SREBP processing, HepG2 cells cultured with DMEM were re-fed with a glutamine-supplemented medium and incubated for the indicated time (Fig 5A) The whole cell extracts were subjected to immunoblotting using anti-SREBP-1 and anti-SREBP-2 antibodies SREBP-1 was detected by an antibody recognizing its N-terminus [SREBP-1(N)], and SREBP-2 was detected by antibodies recognizing its N-terminus [SREBP2(N)] and C-terminus [SREBP-2(C)] Two antibodies recognizing the N-terminus of SREBP-1 and SREBP-2 detect their precursor and mature (nuclear) forms In contrast, SREBP-2(C) detects the precursor and the cleaved form that remains in the Golgi after release of the N-terminal mature form As shown in Fig 5A, SREBP-1 and SREBP-2 processing was induced by glutamine, as judged by the increase in the mature and cleaved forms (‘mature’ or ‘cleaved’ in Fig 5A) The fact that the glutamine-induced SREBP processing started as early as 0.5 h after glutamine treatment indicates that the glutamine-induced post-translational activation of SREBPs occurs prior to the stimulation of the gene expression of SREBPs, which required h or longer (Fig 4) Similar glutamine-stimulated SREBP processing was observed when HepG2 cells FEBS Journal 278 (2011) 2739–2750 ª 2011 The Authors Journal compilation ª 2011 FEBS 2743 Glutamine stimulates SREBP activities A Time after Gln (h) 0.5 J Inoue et al Precursor IB: SREBP-1 (N) Mature Precursor IB: SREBP-2 (N) Mature Precursor IB: SREBP-2 (C) Cleaved IB: actin B Gln (mM) 10 20 30 40 Precursor IB: SREBP-1 (N) Mature Precursor IB: SREBP-2 (N) Mature Precursor IB: SREBP-2 (C) Cleaved IB: actin Fig Glutamine stimulates the processing of both SREBP-1 and SREBP-2 (A) HepG2 cells were cultured with medium A containing mM glutamine for 48 h and re-fed with the medium containing a 10-fold excess of glutamine (40 mM) for the indicated period of time before harvest The whole cell extracts were subjected to SDS ⁄ PAGE and immunoblotting (IB) with anti-SREBP-1(N) (2A4), anti-SREBP-2(N) (Rs004), anti-SREBP-2(C) (1C6) or anti-b-actin antibodies (B) HepG2 cells were cultured with medium A containing mM glutamine for 40 h and then re-fed with the medium containing the indicated concentration of glutamine for h The whole cell extracts were subjected to SDS ⁄ PAGE and IB with the antibodies as described in (A) The same results were obtained in more than three separate experiments were cultured with the low amino acid medium, which contained 0.25 mm glutamine (Fig S2A,B) In order to examine how high the glutamine concentration must be to induce SREBP processing, HepG2 cells were incubated with various concentrations of glutamine (4, 10, 20, 30 or 40 mm) for h Figure 5B demonstrates that SREBP processing is upregulated by glutamine in a dose-dependent manner in HepG2 cells 2744 Glutamine accelerates the ER-to-Golgi transport of the SREBP ⁄ SCAP complex When SREBP precursors are processed to liberate N-terminal active forms, the SREBP ⁄ SCAP complex must be translocated to the Golgi, where two proteases responsible for SREBP cleavage reside Therefore, the glutamine-induced SREBP processing is assumed to be caused by an acceleration of the ER-to-Golgi translocation of the SREBP ⁄ SCAP To determine whether the SREBP translocation is promoted by glutamine, we adopted two in vitro assays First, we performed the reporter assay devised by Sakai et al [14], which monitors SREBP processing by determining the secreted alkaline phosphatase activity HEK293 cells were cotransfected with the expression plasmids for SCAP and the C-terminal half of SREBP-2 that was fused to the secreted form of placental alkaline phosphatase (PLAP-BP2) In agreement with previous studies [14,15], the secreted PLAP activity was increased when the cells were cultured in cholesteroldepleted conditions only in the presence of SCAP (data not shown) As shown in Fig 6A, the PLAP secretion was remarkably induced in the presence of SCAP, and a 10-fold excess of glutamine significantly enhanced the secretion Since the PLAP cleavage is mediated by S1P in this assay, it is possible that glutamine stimulated the S1P activity Alternatively, glutamine might simply accelerate the ER-to-Golgi transport of the PLAP-BP2 ⁄ SCAP complex Second, we used the SCAP-null CHO cells expressing GFPSCAP established by Nohturfft et al [16] to determine whether glutamine influences the movement of SCAP When the cells were cultured in medium C containing 2.5 mm glutamine, GFP-SCAP was diffusively distributed within the cells (Fig 6B), which partially overlapped with the Golgi marker GM130 (Fig 6C, D, H) Treatment with a 10-fold excess of glutamine for h brought about a remarkable degree of overlap between GFP-SCAP and GM130 (Fig 6E–H), indicating that GFP-SCAP had moved to the Golgi in response to glutamine treatment These data indicate that glutamine treatment promotes the ER-to-Golgi transport of the SREBP ⁄ SCAP complex, thereby stimulating SREBP processing Next, we examined whether activating transcription factor (ATF6) processing is stimulated by glutamine, because ATF6 is also processed by S1P and S2P after its translocation from ER to the Golgi in response to ER stress Unexpectedly, glutamine suppressed the gene expression of ATF6 (Fig 7A) However, the gene expression of BiP, which is known to be an ATF6 target gene, was not altered by glutamine treatment (Fig 7A) Therefore, we next FEBS Journal 278 (2011) 2739–2750 ª 2011 The Authors Journal compilation ª 2011 FEBS J Inoue et al Glutamine stimulates SREBP activities PLAP-BP2 cleavage (relative light units) 25 20 × Gln 10 × Gln Golgi ** PLAP-BP2 15 SCAP PLAP 10 S1P Secretion into medium SCAP – ** ATF6 B 10 × Gln GM130 C 1.4 1.2 1.0 0.8 0.6 0.4 0.2 × Gln 10 × Gln BiP B + Flag-ATF6 Merged D – + – – – – – – – + + + – + + Flag-ATF6α (P) Flag-ATF6α (P*) IB: anti-Flag Flag-ATF6α (N) 10 μm 40 mM Gln 1.4 1.2 1.0 0.8 0.6 0.4 0.2 Tunicamycin GFP - SCAP 2.5 mM Gln A ER Relative mRNA level A E IB: anti-β-actin F % of golgi localized GFP-SCAP H G Fig Glutamine does not stimulate ATF6 processing (A) HepG2 cells were cultured with medium A containing mM glutamine for 48 h and then re-fed with the medium containing either mM (1 · Gln) or 40 mM (10 · Gln) glutamine for 24 h Real-time PCR analysis was performed, and the relative mRNA levels were obtained after normalization to GAPDH mRNA All data are presented as means ± SD of three independent experiments performed in triplicate **P < 0.01 (B) HepG2 cells transfected with pCMV-Flag-ATF6 were treated with lgỈmL)1 tunicamycin for h or 40 mM glutamine for h The whole cell extracts were subjected to SDS ⁄ PAGE and immunoblotting (IB) with anti-Flag or antib-actin antibodies Flag-ATF6a(P*) denotes the non-glycosylated form of pATF6a(P) ** 40 30 20 10 Gln (mM) 2.5 40 Fig Glutamine stimulates the ER-to-Golgi transport of the SREBP ⁄ SCAP complex (A) HEK293 cells were transfected with pCMV-PLAP-BP2(513-1141), pCMV-b-gal and either pCMV-SCAP or its empty vector After transfection, the cells were cultured with the medium containing either mM (1 · Gln) or 40 mM (10 · Gln) glutamine for 16 h Then, aliquots of the medium were removed and assayed for the PLAP activity The data were normalized to the cellular b-galactosidase activity as described in Materials and methods All data are presented as means ± SD of three independent experiments performed in triplicate **P < 0.01 (B–G) Stably transfected CHO ⁄ pGFP-SCAP cells were set up as described in Materials and methods On day 1, the cells were cultured with medium C, which contained 2.5 mM (B, C and D) or 40 mM (E, F and G) glutamine, for h Then, the cells were fixed and incubated with the primary antibody against GM130, and subsequently incubated with the Cy3-conjugated secondary antibody The cells were imaged for GFP-SCAP (B and E) or GM130 (C and F) Panels D and G are merged images of GFP-SCAP and the Golgi marker GM130 Scale bar, 10 lm (H) Quantification of the percentage of Golgi-localized GFP-SCAP in (B)–(G) The signals were quantified with an ImageJ All data are presented as means ± SD of three independent experiments determined whether glutamine stimulates ATF6 processing using exogenously expressed flag-tagged ATF6a HepG2 cells were transfected with the expression plasmid for Flag-ATF6a, and then treated with either tunicamycin, an inhibitor of protein N-glycosylation, or glutamine While treatment with tunicamycin, which causes ER stress, increased the amount of the processed form of Flag-ATF6a (denoted as N in Fig 7B, lanes and 2), glutamine treatment had no effect (Fig 7B, lanes and 5) These results indicate that glutamine stimulates the ER-to-Golgi transport of the SREBP ⁄ SCAP complex without any significant influence on the S1P and S2P protease activities Cell swelling is not involved in the glutamine-stimulated SREBP processing The uptake of glutamine into HepG2 cells is mediated by a sodium-dependent transporter [17] When a large amount of glutamine is taken up by cells, osmotic cell FEBS Journal 278 (2011) 2739–2750 ª 2011 The Authors Journal compilation ª 2011 FEBS 2745 Glutamine stimulates SREBP activities J Inoue et al swelling often occurs A previous report has demonstrated that culture of CHO-7 cells with a hypotonic medium caused osmotic cell swelling and the ER stress that inhibits general protein synthesis, thereby stimulating SREBP processing though a reduction in the Insig-1 protein due to its rapid rate of turnover [18] This raises the possibility that glutamine-mediated cell swelling, if it occurs, could result in the stimulation of SREBP processing though various ER stress responses Since both alanine and proline also induce cell swelling [19], we examined the effect of these amino acids on SREBP processing While glutamine stimulated SREBP processing, the same concentration of alanine or proline did not influence SREBP processing (Fig 8), implying that an osmotic change, if it did occur in the cells, was not involved in the stimulation of SREBP processing by glutamine In addition, the effect of glutamate on SREBP processing was also examined because glutamine is capable of being converted to glutamate [20] As shown in Fig 8, treatment with glutamate did not influence SREBP processing, indicating that the SREBP processing induced by glutamine is not mediated by glutamate function Furthermore, it has been reported that the accelerated SREBP processing driven by osmotic cell swelling is not inhibited by treatment with sterols because of the reduction of Insig-1 protein level by cell swelling [18] In order to examine the effect of sterols on the glutamine-induced SREBP processing, HepG2 cells were treated with glutamine in the presence or absence of sterols for h, and then immunoblotting analyses were performed The stimulation of SREBP processing by glutamine was completely attenuated by sterols (Fig 9) These results suggest that the stimulatory effect of glutamine on SREBP processing is not mediated by the reduction of Insig-1 protein level caused by osmotic cell swelling in HepG2 cells PI3K-Akt pathway is involved in SREBP-1 processing but not in SREBP-2 processing Next, we examined how glutamine stimulates SREBP processing in HepG2 cells One possible mechanism could be that glutamine modulates certain protein kinase signaling pathways, which in turn activates SREBP processing It has been reported that glutamine stimulates Akt phosphorylation in HepG2 cells [21] and both phosphatidylinositol 3-kinase (PI3K) and p70 ribosomal protein S6 kinase (p70S6K) in rat primary hepatocytes [22] When HepG2 cells were treated with 40 mm glutamine for h, the levels of active phosphorylated Akt, which is a substrate of PI3K, were increased (Fig 10A) We were unable to detect 2746 Treated amino acid (20 mM) – Gln Ala Pro Glu Precursor IB: anti-SREBP-1 (N) Mature Precursor IB: anti-SREBP-2( C) Cleaved IB: anti-β-actin Fig Treatment with alanine, proline or glutamate does not stimulate SREBP processing HepG2 cells were cultured with medium A containing mM glutamine for 48 h and re-fed with the medium containing the indicated amino acid at a concentration of 20 mM for h before harvest The whole cell extracts were subjected to SDS ⁄ PAGE and immunoblotting (IB) with anti-SREBP-1(N), antiSREBP-2(C) or anti-b-actin antibodies The same results were obtained in more than three separate experiments phosphorylated p70S6K despite the presence of glutamine (data not shown) It has been shown that SREBP processing is stimulated by the activation of the PI3KAkt [15,23,24] and mTORC-p70S6K1 pathways [25] Therefore, we next treated cells with the PI3K inhibitor LY294002 and the mTORC1 inhibitor rapamycin to assess whether these kinase pathways are involved in glutamine-stimulated SREBP processing Interestingly, LY294002 inhibited glutamine-stimulated SREBP-1 processing, whereas it did not affect the stimulation of SREBP-2 processing by glutamine (Fig 10B) In contrast, rapamycin did not influence glutamineinduced SREBP processing (Fig 10B) Taken together, these results suggest that the PI3K signaling pathway plays a key role in SREBP-1 processing in the presence of glutamine but not in glutamine-stimulated SREBP-2 processing Recent reports indicate that insulin enhances SREBP-1 processing because of the phosphorylation of precursor SREBP-1 that is induced by Akt, which in turn enhances the affinity of the SREBP-1 ⁄ SCAP complex for the Sec23 ⁄ 24 proteins of the COPII vesicles and its transport to the Golgi apparatus [26] Thus, it is conceivable that the glutamine-stimulated SREBP-1 processing is mediated by the direct phosphorylation of the precursor SREBP-1 induced by Akt Further studies are required to determine how the glutamine-activated PI3K pathway is involved in SREBP-1 processing In conclusion, the present study shows that treatment with glutamine causes the stimulation of FEBS Journal 278 (2011) 2739–2750 ª 2011 The Authors Journal compilation ª 2011 FEBS J Inoue et al Glutamine stimulates SREBP activities 10 × Gln Sterols A – + – + – – + + Time after Gln (h) IB: p-Akt Precursor IB: Akt IB: SREBP-1 (N) Mature B Time after Gln (h) Precursor LY294002 Rapamycin IB: SREBP-2 (N) Mature – – + – – + Precursor IB: SREBP-1 (N) Precursor Mature IB: SREBP-2 (C) Precursor Cleaved IB: SREBP-2 (C) IB: actin Fig Stimulation of SREBP processing by glutamine is attenuated by sterols HepG2 cells were cultured with medium A containing mM glutamine for 48 h and re-fed with a 10-fold excess of glutamine (10 · Gln, 40 mM) containing medium in the presence or absence of sterols (10 lgỈmL)1 of cholesterol plus lgỈmL)1 of 25-hydroxycholesterol) for h before harvest The whole cell extracts were subjected to SDS ⁄ PAGE and immunoblotting (IB) with anti-SREBP-1(N), anti-SREBP-2(N), anti-SREBP-2(C) or anti-bactin antibodies The same results were obtained in more than three separate experiments SREBP-1a gene expression in HepG2 cells due to the activation of hexosamine biosynthesis pathway Furthermore, the post-translational processing of SREBPs is stimulated by treatment with glutamine Since it is well known that the activation of SREBPs stimulates the synthesis of lipids such as fatty acids and cholesterol, the new concept presented here is that glutamine acts as a stimulator of SREBP activities in addition to serving as a nutrient and ⁄ or signaling molecule These multiple and combined effects of glutamine may fulfill the cellular demand for lipids that is physiologically necessary for rapid cellular growth Given that serum glutamine concentration is approximately mm and rarely reaches 10 mm, glutamine has a low potential for affecting SREBP processing in the liver In contrast, because it is assumed that the glutamine concentration in the cells of the small intestine could transiently and sufficiently increase just after the intake of glutamine-rich foods, amino acids may strongly activate SREBPs in vivo, as was observed with the cultured cells in this study Further studies are required to determine whether glutamine-stimulated SREBP activities contribute to the rapid growth of cultured cells and whether dietary glutamine activates SREBP in the liver and intestine in vivo Cleaved IB: actin Fig 10 Glutamine activates the PI3K-Akt pathway, and the inhibitor of this pathway differentially affects the processing of SREBP-1 and SREBP-2 (A) HepG2 cells were cultured with medium A containing mM glutamine for 48 h and re-fed with a 10-fold excess of glutamine (40 mM) containing medium for the indicated period of time before harvest Whole cell extracts were subjected to SDS ⁄ PAGE and immunoblotting (IB) with anti-phospho-Akt (Ser473) and anti-Akt antibodies (B) HepG2 cells were cultured with medium A containing mM glutamine for 48 h After pre-incubation with or without 10 lM LY294002 or 25 nM rapamycin for 30 min, the cells were re-fed with a 10-fold excess of glutamine (40 mM) containing medium in the presence or absence of either 10 lM LY294002 or 25 nM rapamycin for h before harvest The whole cell extracts were subjected to SDS ⁄ PAGE and IB with antiSREBP-1(N), anti-SREBP-2(C) or anti-b-actin antibodies The same results were obtained in triplicate experiments Materials and methods Materials Cholesterol, 25-hydroxycholesterol, lipoprotein-deficient serum, dialyzed fetal bovine serum, azaserine, tunicamycin, LY294002 and rapamycin were purchased from Sigma (St Louis, MO, USA) DMEM and DMEM ⁄ Ham’s F-12 medium were from Wako (Osaka, Japan) Low amino acid medium The low amino acid medium [8] was kindly provided by Ajinomoto (Kawasaki, Japan) It contains comparatively low concentrations of 20 amino acids compared with DMEM The amino acid concentrations are as follows: lm Trp; 12.5 lm Cys, Met, His; 25 lm Gly, Ala, Ser, Asn, Glu, Asp, Phe, Typ, Arg, Pro; 50 lm Thr, Val, Leu, Ile, Lys; 250 lm Gln FEBS Journal 278 (2011) 2739–2750 ª 2011 The Authors Journal compilation ª 2011 FEBS 2747 Glutamine stimulates SREBP activities J Inoue et al Cell culture HEK293T and HepG2 cells were maintained in medium A (DMEM supplemented with 100 unitsỈmL)1 penicillin, 100 lgỈmL)1 streptomycin and 10% fetal bovine serum) Caco-2 cells were maintained in medium B (DMEM supplemented with 100 unitsỈmL)1 penicillin, 100 lgỈmL)1 streptomycin, nonessential amino acids and 10% fetal bovine serum) CHO ⁄ pGFP-SCAP cells were maintained in medium C (DMEM ⁄ Ham’s F-12 supplemented with 100 unitsỈmL)1 penicillin, 100 lgỈmL)1 streptomycin and 5% lipoprotein-deficient serum) Cells were incubated at 37 °C under 5% CO2 atmosphere Plasmid constructs Reporter plasmids, pHMG S containing the human HMG CoA synthase promoter, pHMG S SREKO containing a mutated sequence of the SRE, pSQS containing the human SQS promoter, pSQS SREKO containing two mutated SREs, pLDLR containing the human LDL receptor promoter, and pMTP containing the human MTP promoter were described previously [27,28] A reporter plasmid, pCPT1A, was constructed by inserting a 2.8-kb PCR fragment coding the 5¢-promoter region and intron (–286 ⁄ +2540) of the human CPT1A gene into a pGL4 basic vector (Promega, Madison, WI, USA) An expression plasmid pCMV-PLAP-BP2(513-1141) encodes an 1136 amino acid fusion protein consisting of an initiator methionine followed by the secreted form of human PLAP (amino acids 2–506), one novel amino acid (Y) generated by blunt ligation, and the COOH-terminal half of human SREBP-2 (amino acids 513–1141) pCMV-PLAP-BP2(513-1141) was constructed according to the method reported in a previous paper [14] An expression plasmid for pCMV-SCAP was constructed by inserting a 4.3-kb BglII-ClaI PCR fragment coding full length human SCAP into the same restriction sites of the pCMV vector (Stratagene, Santa Clara, CA, USA) An expression plasmid for Flag-ATF6 was constructed by inserting a 1.7-kb BglII-XbaI PCR fragment coding full length human ATF6 into the same restriction sites of the p3 · FLAG-CMV vector (Sigma) Antibodies Monoclonal anti-SREBP-1 (2A4), anti-SREBP-2 (1C6) and polyclonal anti-Sp1 (PEP2) were obtained from Santa Cruz Monoclonal anti-b-actin (AC-15) was from Sigma Monoclonal anti-GM130 (35) was from BD Biosciences (Franklin Lakes, NJ, USA) Monoclonal anti-O-GlcNAC (RL2) was from Thermo Scientific (Waltham, MA, USA) Polyclonal anti-Sp1 used for chromatin immunoprecipitation assays was from Millipore (Billerica, MA, USA) Polyclonal antiAkt and anti-phospho-Akt were from Cell Signaling Technology (Beverly, MA, USA) Peroxidase-conjugated affinity 2748 purified goat anti-rabbit IgG, peroxidase-conjugated affinity purified goat anti-mouse IgG and Cy3-conjugated affinity purified donkey anti-mouse IgG were from Jackson Immunoresearch Laboratories (West Grove, PA, USA) The anti-SREBP-2 polyclonal serum (Rs004) has been described previously [11] Luciferase assays HepG2 cells and Caco-2 cells were plated in 12-well plates at a density of 1.0 · 105 cellsỈwell)1, cultured with DMEM for 20 h, and then transfected with 200 ng of one of the reporter plasmids and 200 ng pEF-b-Gal, an expression plasmid for b-galactosidase, by the calcium phosphate method Twentyfour hours later, the medium was replaced with the low amino acid medium supplemented with 5% dialyzed fetal bovine serum and the indicated concentration of glutamine After incubation for another 12 h, the luciferase and b-galactosidase activities were measured as described previously [28] Normalized luciferase values were determined by dividing the luciferase activity by the b-galactosidase activity Real-time PCR Total RNA was extracted from HepG2 cells using an RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions cDNA was synthesized and amplified from lg total RNA using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) Quantitative real-time PCR (Taqman probe and SYBR green) analysis was performed on an Applied Biosystems 7000 sequence detection system Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control The TaqMan ID number for genes analyzed are as follows: SREBP-2, Hs00231882_m1; LDL receptor, Hs00181192_m1; HMGCoA synthase, Hs00266810_m1; CPT1A, Hs00157079_m1; Sp1, Hs00916521_m1; GAPDH, 4352934 The sequences of the primer sets used were as follows: SREBP-1a, 5¢-TCAG CGAGGCGGCTTTGGAGCAG-3¢ and 5¢-CATGTCTTC GATGTCGGTCAG-3¢ [9]; ATF6, 5¢-ATGTCTCCCCTTT CCTTATATGGT-3¢ and 5¢-AAGGCTTGGGCTGAATT GAA-3¢; BiP, 5¢-GACCTGGGGACCACCTACTC-3¢ and 5¢-TTCAGGAGTGAAGGCGACAT-3¢ Chromatin immunoprecipitation assays Chromatin immunoprecipitation assays were performed as described previously [14] Real-time PCR was performed with the following primers: SREBP-1a promoter region forward (5¢-CGAGGCTGGATAAAATGAATGA-3¢), SRE BP-1a promoter region reverse (5¢-GGTCTGCGCCACAA ATCTC-3¢), SREBP-1a distal region forward (5¢-AAAGTA CATAAAAGACAATGACCATCAC-3¢) and SREBP-1a distal region reverse (5Â-CTTGAGTTGTTTCTCTGCAGCTT-3Â) FEBS Journal 278 (2011) 27392750 ê 2011 The Authors Journal compilation ª 2011 FEBS J Inoue et al siRNA experiments siRNA (150 pmol per six-well plate) for human Sp1 (Stealth RNAi, VHS40867; Invitrogen, Carlsbad, CA, USA), with luciferase as a control (GL2 Luciferase, Bonac Co., Fukuoka, Japan), were transfected using lipofectamine RNAiMAX (Invitrogen) into HepG2 cells according to the manufacturer’s instructions Immunoblotting Cells were treated as described in the figure captions The cells were lysed in RIPA buffer (50 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl, 0.1% SDS, 0.5% deoxycholate and 1% Triton X-100) supplemented with protease inhibitors The lysates were subjected to SDS ⁄ PAGE, transferred onto a poly(vinylidene difluoride) membrane and probed with the antibodies indicated in the figure captions The immunoreactive proteins were visualized using ECL (GE Healthcare, Milwaukee, WI, USA) or Immobilon (Millipore) western blotting detection reagents The signals on the membrane were quantified with a LAS-3000 Luminoimager (Fujifilm, Tokyo, Japan) Assays for secreted alkaline phosphatase HEK293T cells were plated in 12-well plates at a density of 2.0 · 105 cellsỈwell)1, cultured with medium A for 20 h, and then transfected with 500 ng of pCMV-PLAP-BP2 (5131141), 50 ng of pCMV-b-gal and 300 ng of either pCMVSCAP or pcDNA empty vector by the calcium phosphate method After h of incubation, the cells were re-fed with fresh medium A containing the indicated concentration of glutamine After incubation for another 24 h, the medium was collected for determination of secreted PLAP using a Phospha-Light system (Applied Biosystems), as described previously [14] After removal of the medium, the cells were lysed and then measured for b-galactosidase activity To normalize differences in transfection efficiency, the PLAP activity was divided by the b-galactosidase activity Immunofluorescence staining and fluorescence microscopy CHO ⁄ pGFP-SCAP cells, a stable cell line expressing GFPSCAP [16], were plated on a human fibronectin cellware four-well culture slide (BD Biocoat, Franklin Lakes, NJ, USA), cultured with medium C for 20 h, and then re-fed with fresh medium C containing the indicated concentration of glutamine After incubation for another h, the cells were fixed with 4% paraformaldehyde ⁄ phosphate buffered saline (NaCl ⁄ Pi), penetrated with 0.2% Triton X-100 ⁄ NaCl ⁄ Pi, blocked with 2% bovine serum albumin ⁄ NaCl ⁄ Pi, treated with anti-GM130 serum and subjected to reaction with Cy3conjugated secondary antibody Then the cells were mounted Glutamine stimulates SREBP activities on glass slides with Mount aqueous mounting medium (Sigma) and visualized with a laser-scanning IX70 microscope (Olympus, Tokyo, Japan) equipped with a DP71 digital camera (Olympus) Subcellular localization of GFP-fused SCAP was also determined Acknowledgements We thank Drs Joseph L Goldstein, Michael S Brown and Andrew J Brown for generously sharing their valuable tools We also thank Dr Ken-ichi Nishida (Daiichi-Sankyo) for expression plasmids We are grateful to Dr Kevin Boru of Pacific Edit for review of the manuscript This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (R.S.), Nagase Science and Technology Foundation (R.S.), the Salt Science Research Foundation, no 0916 (J.I.) and Nestle Nutrition Council, Japan (J.I.) 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supplementary material is available: Fig S1 DMEM containing 10 · Gln stimulates the mRNA levels of SREBP family members Fig S2 Glutamine stimulates SREBP processing in HepG2 cells cultured with the low amino acid medium This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 2739–2750 ª 2011 The Authors Journal compilation ª 2011 FEBS ... an increase in the gene expression of SREBPs, and second, the enhancement of SREBP processing by glutamine We therefore investigated the effect of glutamine on the gene expression of SREBP family... increased, but then the mRNA levels of both SREBPs and their target genes became significantly elevated at and 12 h (Fig 4) Based on the fact that the increase in the mRNA of these genes was nearly... imply the involvement of the O-glycosylation of Sp1 in the induction of SREBP-1a transcription by glutamine The gene expression of SREBP-1c, which represented less than 20% of the SREBP-1a gene expression