Supporting Information Zhang et al. 10.1073/pnas.1110730109 SI Materials and Methods Antibodies and Reagents. Antibodies were from Cell Signaling Technology for Akt, phospho-akt (473), phospho-akt (308), mtor, phospho-mtor (2481), phospho-mtor (2448), S6K1, phospho-s6k1 (Thr389), IRS1, phospho-irs1 (636/639), mlst8, and rictor. Antibodies were from Abcam for GPAT1, AGPAT2, lipin 2, Sin1, and a-tubulin. Anti-PKC and antiphospho-pkcα (657) antibodies and protein A/G Sepharose were from Santa Cruz Biotechnology. Anti-HA antibody, Antiflag M2 antibody, BSA (fatty acid free), insulin (human recombinant), sodium-d-lactate, glucose assay kit, Avertin (2-2-2-tribromoethanol), phosphatase inhibitor mixture 1 and 2, Percoll, adenosine 5 -triphosphate (ATP) and CHAPS were from Sigma. Type I collagenase was from Worthington Biochemical Corporation. Protease inhibitor tablet was from Roche. Inactive Akt1 was from SignalChem. SuperSignal West Pico Chemiluminescent Substrate was from Thermo Scientific. Cell culture media and reagents were from Invitrogen. Animals. Animal protocols were approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee. Wild-type and Gpat1 / mice (1) on a C57BL/6J background were housed in an air-conditioned facility with ad libitum food (Prolab 5P76 Isopro 3000; 5.4% fat by weight) and water. A subset of mice was fed a high-fat safflower oil diet (#112245, Dyets; 59% calories from safflower oil and menhaden oil). Mice were studied between 8 and 12 wk of age. Cell Culture. Hepatocytes were isolated from perfused mouse livers (2, 3) and cultured in William s E medium supplemented with 10% FBS, 1% penicillin/streptomycin and 2 mm glutamine overnight. Cells were washed twice with warm PBS and placed in serum-free D/MEM with 1.0 g/l glucose and infected with adenoviruses. After 24 h, cells were treated for 1 h with or without insulin or rapamycin in glucose-free D/MEM, 2 mm sodium pyruvate, 20 mm sodium lactate. An aliquot of medium was then collected for glucose assay, or cells were lysed for Western blot analyses or for immunoprecipitation (IP). Mouse 3T3-L1 preadipocytes and HEK293T cells were cultured in D/MEM, 10% FBS, 1% penicillin/ streptomycin. Cells were washed twice with warm PBS, grown in serum-free D/MEM with 1.0 g/l glucose, and infected with adenoviruses. Twenty-four hours after infection and serum starvation, cells were harvested and lysed for Western blot analyses or for IP. Ninety-six h after adenovirusmediated rictor-shrna and LacZ-shRNA delivery, cells were harvested and lysed for Western blot or for IP. PAP Activity. PAP specific activity was measured as the release of free phosphate from Triton X-100/phosphatidic acid mixed micelles (4). Glucose Output. The effect of insulin stimulation on glucose output was measured as described (5) with minor modifications. Mouse primary hepatocytes overexpressing EGFP or GPAT1 were cultured overnight in six-well plates in DMEM without FBS. The medium was replaced with 1 ml of glucose-free DMEM, 20 mm sodium lactate, 2 mm sodium pyruvate, with or without 100 nm insulin. After a 2-h incubation, medium was collected, and the glucose concentration was measured colorimetrically (Sigma kit). Glucose concentrations were normalized to the total protein content determined from the whole-cell lysates. Recombinant Adenoviruses and Infection. The construction, generation and purification of recombinant GPAT1-FLAG adenovirus and Ad-EGFP were described (6). The AGPAT2-HA adenoviral construct was generated by cutting out an HA-tagged AGPAT2 fragment from pδu3-cmv-ha-agpat2 with XbaI, blunting the fragment with Klenow, and inserting it into EcoRVcut padtrackcmv. The HA-tagged lipin 2 adenovirus was previously described (4). The adenovirus overexpressing rictorshrna was generated by subcloning the U6 promoter-rictor shrna cassette from plko mouse rictor shrna1 (Addgene Plasmid 21341) (7) into padtrack, and purifying the adenovirus as described (8). The adenoviral titers ( 10 11 transduction particles per ml) were determined by the UNC Gene Therapy Center. MOIs of 20, 100, and 10 were used for mouse hepatocytes, 3T3-L1 preadipocytes, and HEK293T cells, respectively. Doses were based on pilot experiments to obtain equal infectivity without obvious toxic effects on the cells. Cell Lipid Extraction and LPA, PA, and DAG Profile Assays. Total lipid was extracted (9), and cellular content and species of LPA, PA, and DAG were analyzed by LC/MS on a Shimadzu Prominence UFLC (Ultra Fast Liquid Chromatography) system equipped with a C8 column (Nucleodur 5 mm, 2 125 mm, Machery- Nagel). Detection was carried out with an Applied Biosystems 4000 Q Trap triple quadrupole LC/MS/MS system equipped with an electrospray ionization system. For LPA and PA, multiple reaction monitoring protocols in negative mode were developed for each PA using commercial pure PAs; the most intense product ions were selected to analyze biological samples. DAG analyses were carried out by monitoring product ions generated by neutral loss (NL) of ammoniated acyl groups [RCOOH+NH3] from DAG ammonium adducts [M+NH4 + ] (10). The amount of each species of glycerolipid in the biological samples was calculated from the peak areas obtained using the software that controls the LC/MS system (Analyst 1.5, Applied Biosystems). Raw peak areas were corrected for recovery and sample loading as described above and then transformed into amounts of analyte using standards curves made with commercial glycerolipids. Glycerolipids were quantified using the chromatographic and spectrometric methods described above with 0.1 nmol 17:0 LPA and 17:0 ceramide as internal standards for LPA/PA and DAG, respectively, to correct for recovery and normalized to the protein concentrations of the cellular lysates. Western Blot Analysis. Cells were harvested in lysis buffer (20 mm Tris HCl, ph 7.5/0.1 mm Na 3 VO 4 /25 mm NaF/25 mm glycerophosphate/2 mm EGTA/1 mm DTT/0.5 mm phenylmethylsulfonyl fluoride, and 0.3% Triton X-100). Lysates were boiled with 2 Laemmli sample buffer (1:1, vol/vol) before SDS/PAGE. Western blotting was carried out following procedures recommended by antibody suppliers. Horseradish peroxidase-conjugated secondary antibodies were detected with SuperSignal West Pico Chemiluminescent Substrate and exposure on X-ray films. Films were scanned with an Epson scanner (Perfection 2400) and band densities were determined with Image J. Immunoprecipitation and Kinase Activity Assay. IP of rictor and the mtorc2 kinase assay were performed (11). Cells in 10-cm plates were rinsed once with PBS and lysed on ice for 10 min in 1 ml of cold IP lysis buffer (40 mm Hepes, ph 7.5/120 mm NaCl/1 mm EDTA/10 mm pyrophosphate/10 mm glycerophosphate/50 mm NaF/0.5 mm Na 3 VO 4 /EDTA-free protease 1of5
inhibitors/1% phosphatase inhibitor mixture 1 and 2, and 0.3% CHAPS). Cell lysates were centrifuged at 13,000 g for 10 min, and supernatants were transferred to new tubes. Four μg of the appropriate antibody was added to 1 mg protein from the cleared cellular lysates and incubated with rotation for 90 min. Twentyfive μl of a 50% slurry of protein A/G Sepharose was added, and lysates were incubated on a rotary mixer for 60 min. Beads were washed 4 times with IP lysis buffer and once with the rictormtor kinase buffer (25 mm Hepes, ph 7.5/100 mm potassium acetate/1 mm MgCl 2 ). For kinase activity assays, IPs were incubated in a final volume of 15 μl for 20 min at 37 C in the rictor-mtor kinase buffer containing 500 ng of inactive Akt1 and 500 μm ATP. The reaction was stopped by adding 100 ml of cold 2 Laemmli sample buffer followed by 5 min of boiling. Western blots probed S473-Akt to indicate mtorc2 activity. Lipid Vesicle Formation. LPA, PA, and DAG vesicles were prepared by water bath sonication (12). Lipid vesicles were freshly made before each experiment and added to the kinase assay system at the final concentrations indicated in the figure legends. Statistical Analysis. Values are expressed as means ± SE. Comparisons between groups (adenoviral-delivered gene overexpression or gene knockdown) of the same treatment (basal, insulin-stimulated, or NEM resistant) were determined using Student s t test. Comparisons among groups (EGFP, GPAT1, AGPAT2, and Lipin2) were determined by one-way ANOVA with Dunnett s post hoc analysis (EGFP set as the control). Data represent at least three independent experiments performed in triplicate unless otherwise indicated. P < 0.05 was considered significant. 1. Hammond LE, et al. (2002) Mitochondrial glycerol-3-phosphate acyltransferasedeficient mice have reduced weight and liver triacylglycerol content and altered glycerolipid fatty acid composition. Mol Cell Biol 22:8204 8214. 2. Klaunig JE, et al. (1981) Mouse liver cell culture. I. Hepatocyte isolation. In Vitro 17: 913 925. 3. Klaunig JE, Goldblatt PJ, Hinton DE, Lipsky MM, Trump BF (1981) Mouse liver cell culture. II. Primary culture. In Vitro 17:926 934. 4. Gropler MC, et al. (2009) Lipin 2 is a liver-enriched phosphatidate phosphohydrolase enzyme that is dynamically regulated by fasting and obesity in mice. J Biol Chem 284: 6763 6772. 5. Yoon JC, et al. (2001) Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413:131 138. 6. Lewin TM, Wang S, Nagle CA, Van Horn CG, Coleman RA (2005) Mitochondrial glycerol-3-phosphate acyltransferase-1 directs the metabolic fate of exogenous fatty acids in hepatocytes. Am J Physiol Endocrinol Metab 288:E835 E844. 7. Thoreen CC, et al. (2009) An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mtorc1. J Biol Chem 284: 8023 8032. 8. Harris TE, et al. (2007) Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1. JBiolChem282:277 286. 9. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497 509. 10. Murphy RC, et al. (2007) Detection of the abundance of diacylglycerol and triacylglycerol molecular species in cells using neutral loss mass spectrometry. Anal Biochem 366:59 70. 11. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mtor complex. Science 307:1098 1101. 12. Yoon MS, Sun Y, Arauz E, Jiang Y, Chen J (2011) Phosphatidic acid activates mammalian target of rapamycin complex 1 (mtorc1) kinase by displacing FK506 binding protein 38 (FKBP38) and exerting an allosteric effect. JBiolChem286:29568 29574. Fig. S1. Overexpressing GPAT1 in 3T3-L1 preadipocytes and HEK293T cells impairs insulin signaling. 3T3-L1 preadipocytes and HEK293T cells were infected with EGFP or Flag-GPAT1. (A) Representative images of Western blots of cell lysates confirming overexpression of Flag-GPAT1. (B) Representative Western blots of cell lysates. (C) Quantitative analysis of data from B. Asterisk indicates significant differences (P < 0.05) from EGFP basal; #, significantly different compared with EGFP stimulated with insulin. 2of5
Fig. S2. GPAT1-impaired insulin signaling is not due to altered mtorc1/s6k1 or IRS1/PI3K signaling. Mouse hepatocytes were infected for 24 h to overexpress either EGFP or Flag-GPAT1 and treated with or without insulin (100 nm) for 10 min and with or without rapamycin (100 nm) for 45 min. (A) Representative images of Western blots of cell lysates. (B) Quantitative analysis of data from A. (C) Representative images of Western blots of cell lysates and IRS1-IP product. (D) Quantitative analysis of data from C. Asterisk indicates significant differences (P < 0.05) from EGFP basal; #, significantly different compared with EGFP stimulated with insulin. Fig. S3. GPAT1-mediated impairment of insulin signaling is due to decreased mtorc2 activity. 3T3-L1 or HEK293T cells infected to overexpress either EGFP or Flag-GPAT1 were treated with or without insulin (100 nm) for 10 min. (A) Representative images of Western blots of rictor-ip products. (B) Quantitative analysis of data from A. Asterisk indicates significant differences (P < 0.05) from EGFP basal; #, significantly different compared with EGFP stimulated with insulin. 3of5
Fig. S4. Purified Flag-GPAT1 protein did not alter mtorc2 kinase activity. Overexpressed Flag-GPAT1, HA-AGPAT2, and HA-Lipin2 proteins and PAPase activity in mouse primary hepatocytes. (A) Representative images of Western blots of mtorc2 kinase assay product. Flag-GPAT1 protein was purified by Flag-IP from mouse hepatocytes overexpressing Flag-GPAT1. Cell lysates from WT mouse hepatocytes were prepared and rictor protein was pulled down by immunoprecipitation. Purified Flag-GPAT1 protein was added to the rictor-ip product and mtorc2 kinase activity was assayed. (B) Quantitative analysis of data from A. (C) Representative images of Western blots confirming respective overexpression of GPAT1, AGPAT2, and Lipin2. Primary mouse hepatocytes were infected with EGFP, Flag-GPAT1, HA-AGPAT2 or HA-lipin 2. (D) PAPase specific activity. Asterisk indicates significant differences (P < 0.05) from EGFP. Fig. S5. Glycerolipid signals alter mtorc2 to diminish insulin signaling. Fatty acids enter the cell and are esterified to glycerol-3-phosphate to form lysophosphatidic acid (LPA). A second esterification by 1-acyl-glycerol-3-phosphate acyltransferase (AGPAT) produces phosphatidic acid (PA). The phosphate is cleaved by phosphatidic acid phosphohydrolase (PAP/Lipin) and a third acylation converts the diacylglycerol (DAG) product to triacylglycerol (TAG). In liver, certain PA and/or DAG species dissociate mtor and rictor resulting in an inhibition of Akt-insulin signaling and an increase in glucose output. 4of5
Table S1. Content of lipid species in mouse primary hepatocytes that overexpress EGFP, GPAT1, AGPAT2, or Lipin2 Lipid species EGFP GPAT1 AGPAT2 Lipin 2 LPA (fmol/mg protein) 16:0 3.63 ± 0.37 30.23 ± 0.35* 4.81 ± 0.34 4.24 ± 0.19 16:1 0.21 ± 0.02 1.76 ± 0.03* 0.31 ± 0.03 0.23 ± 0.02 18:0 5.29 ± 1.55 6.22 ± 0.41 3.99 ± 0.17 4.38 ± 0.06 18:1 1.71 ± 0.34 2.63 ± 0.24* 1.87 ± 0.13 1.66 ± 0.07 18:2 0.31 ± 0.03 0.57 ± 0.12 0.82 ± 0.40 0.26 ± 0.05 20:4 0.68 ± 0.07 0.81 ± 0.06 0.45 ± 0.03* 0.72 ± 0.02 PA (pmol/mg protein) 16:0 16:0 0.22 ± 0.00 3.16 ± 0.01* 1.58 ± 0.07* 0.35 ± 0.02 16:0 18:0 0.24 ± 0.01 1.10 ± 0.02* 0.54 ± 0.01* 0.31 ± 0.01* 16:0 18:1 2.63 ± 0.07 3.23 ± 0.08* 5.24 ± 0.24* 3.28 ± 0.08* 16:0 18:2 0.59 ± 0.01 0.98 ± 0.02* 0.85 ± 0.03* 0.68 ± 0.02 18:0 18:0 0.04 ± 0.00 0.06 ± 0.00* 0.07 ± 0.00* 0.05 ± 0.00 18:0 18:1 0.74 ± 0.02 0.42 ± 0.01* 1.02 ± 0.03* 0.81 ± 0.02 18:1 18:1 1.18 ± 0.02 0.30 ± 0.00* 2.17 ± 0.05* 1.54 ± 0.09* DAG (pmol/mg protein) 16:0 16:0 51.04 ± 1.14 871.12 ± 23.96* 160.83 ± 9.04* 61.58 ± 1.08 16:0 18:1 654.56 ± 9.44 1516.34 ± 16.58* 1034.47 ± 59.95* 675.36 ± 7.08 16:0 18:2 11.42 ± 0.39 79.08 ± 0.39* 21.98 ± 1.53* 12.87 ± 0.14 18:0 20:4 38.16 ± 0.39 67.25 ± 1.14* 25.88 ± 1.44* 40.68 ± 1.10 18:1 18:1 445.01 ± 11.74 119.62 ± 2.69* 520.2 ± 20.97* 381.96 ± 10.47* 18:1 18:2 14.33 ± 0.20 13.68 ± 0.24 19.29 ± 0.82* 13.25 ± 0.50 18:2 18:2 2.58 ± 0.08 3.26 ± 0.19 2.88 ± 0.14 3.06 ± 0.23 20:4 20:5 0.11 ± 0.01 0.12 ± 0.01 0.11 ± 0.00 0.12 ± 0.01 Values represent means ± SEM of three independent experiments performed in triplicate. *P < 0.05 compared with EGFP control. 5of5