SUPPLEMENTARY INFORMATION FOR Liver X Receptor α mediates hepatic triglyceride accumulation through upregulation of G0/G1 Switch Gene 2 (G0S2) expression I: SUPPLEMENTARY METHODS II: SUPPLEMENTARY FIGURES & TABLES
SUPPLEMENTARY METHODS Reagents Sodium palmitate, sodium oleate, linoleic acid and bovine serum albumin (essentially fatty acid free) were purchased from Sigma-Aldrich. T0901349 and GW3965 were obtained from Tocris. GSK2033 was purchased from Axon MedChem and HX-531 from Tocris Bioscience. CL316243 was purchased from Sigma-Alrich. The anti-atgl antibody (#2138) was purchased from Cell Signaling Technology. Anti-G0S2 antibody was produced as previously described (Zhang et al., 2014). Antibodies for LXRα (sc- 1202) and LXRβ (sc-1001) were purchased from Santa Cruz. Horseradish peroxidaselinked secondary antibodies were purchased from Jackson Immuno-Research Laboratories. The protease inhibitor mini tablet cocktail was obtained from Roche Diagnostics. The PureLink RNA Mini Kit and High-Capacity cdna Reverse Transcription Kit were purchased from Invitrogen. Reagents for tissue culture and Bodipy 493/503 were obtained from Invitrogen. Plasma and liver metabolite analysis Plasma total triglyceride, cholesterol (Thermo Fisher Scientific), HDL/LDL (Biovision), 3-hydroxybutyrate (Wako), and glucose (Wako) were measured using enzyme colorimetric assays according to the manufacturer s protocols. Liver lipids were extracted by choloroform/methonal (2:1), dried under nitrogen gas and resuspended in 1% triton X- 100. Then total triglyceride and total cholesterol content were assayed using total TG and TC assay kits from Thermo Fisher Scientific. For hepatic glycogen measurement, liver
tissue was homogenized and glycogen extracted by acid hydrolysis as described previously (XD dibatetes paper). RNA Extraction and Real-Time PCR Total RNA was isolated from mouse liver samples and primary hepatocytes using the PureLink RNA Mini Kit (Invitrogen) according to the manufacturer's instruction. cdna was synthesized from total RNA by High-Capacity cdna Reverse Transcription Kit (Invitrogen) with Oligo d(t). The resulting cdna was subjected to real-time PCR analysis with SYBR Select Master Mix (Invitrogen) on an Applied Biosystems 7900 HT Real-Time PCR System. The sequences of qpcr primers are listed in (Supplementary Table 1). Data were analyzed using the comparative cycle threshold ( ΔΔ Ct) method. The mrna levels of genes normalized to β-actin were presented as relative to the control. PCR product specificity was verified by postamplification melting curve analysis and by running products on an agarose gel. Immunoblotting Liver samples were lysed at 4 C in a buffer containing 50 mm Tris-HCl (ph 7.4), 135 mm NaCl, 10 mm NaF, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 1.0 mm EDTA, 10% glycerol, and protease inhibitors (1 tablet per 10 ml of buffer). The lysates were clarified by centrifugation at 20,000 g, 4 C for 10 min and then mixed with equal volume of 2 SDS sample buffer. Equivalent amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Individual proteins
were blotted with primary antibodies at appropriate dilutions. Peroxide-conjugated secondary antibodies were incubated with the membrane at a dilution of 1:5000. The signals were then visualized using ECL substrate (Pierce). H&E staining, B-galactosidase staining and light microscopy Tissue samples collected for H&E analysis were immediately immersed and fixed in 10% formalin. Paraffin embedded section and subsequent H&E staining was carried out by the Mayo Clinic Microscopy Core facility. Tissues samples collected for B-galactosidase staining were immediately frozen in liquid nitrogen. The Mayo Clinic Microscopy Core, following standard procedures, conducted cryo-sectioning and staining with X-gal. All slides were visualized on a Nikon light microscope with standard digital camera system.
Supplementary Figure 1 3 (-) Glycerol (+) Glycerol Relative Expression 2 1 0 G0S2 ATGL WT primary hepatocytes were treated for 8-h in the presence or absence of 3mM glycerol. A: qpcr analysis of G0S2 and ATGL following glycerol treatment.
Supplementary Figure 2 A Relative Expression 10 9 8 7 6 5 3 2 1 *** ** ** ** GW3965 0 G0S2 LXRa ABCG5 ABCG8 SREBP1c ATGL B GW3965 G0S2 Actin C 60 GW3965 *** Hepatic TG (ug/mg) 40 20 0 GW3965 12-week old female WT mice were injected with 20mg/kg/day GW3965. A: qpcr analysis of G0S2, ATGL, LXRa and LXRa target genes in liver. B: Immunoblot analysis of G0S2 and C: total hepatic triglycerides. (n = 6 for qpcr, n = 8 for TG ** p < 0.001, *** p < 0.0001)
Supplementary Figure 3 60 50 *** Luciferase (RLU) 40 30 20 10 0 Ctrl T09 Linoleic acid Oleic acid HepG2 cells were transfected with the Gal4-LXR:MH100-Luc plasmids at a 1:8 ratio. After 24h, cells were treated for an additional 24h with 10 μm T09, 750 μm linoleic acid or 500 μm oleic acid. Luciferase activity was assayed using the Luciferase Assay kit.
Supplementary Figure 4 A Plasma 3-hydroxybutyrate (mm) 0.3 0.2 0.1 0.0 Wt ** G0S2 KO T0901317 * B 0.25 Wt G0S2 KO Plasma FFA (mm) 0.20 0.15 0.10 0.05 0.00 T0901317 C 10 Wt G0S2 KO Plasma Glucose 8 6 4 2 0 T0901317 Plasma parameters in T09 injected Wt and G0S2 KO mice, n=5.
Supplementary Figure 5 LXR agonism Fasting Reverse Cholesterol Trafficking TG FA utilization Fatty acids de novo FAs (LXR agonism) Adipose-derived FAs (Fasting) Model for the regulation of hepatic G0S2 expression and triglyceride accumulation by fasting and LXR agonism. Activation of LXRα promotes G0S2 expression and ATGL inhibition. FAs dervied from either adipose tissue (fasting) or de novo FA synthesis (LXR agonism) are packaged into intrahepatic lipid droplets as TG. Due to LXRα-mediated transcriptional activation of G0S2, inhibition of ATGL is increased and TG accumulation results. FA utilization therefore is reduced in response to decreased the availability of free FAs.
Supplementary Table 1 Gene Sequence of forward and reverse primers (5 to 3 ) G0S2 AAAGTGTGCAGGAGCTGA AGGGAGGCCGTCGTGCGGA ATGL AGACAGAGCTTTCTCCCAGTGAA CCCCGTGAAGCCCAACT LXR AGGAGTGTCGACTTCGCAAA CTCTTCTTGCCGCTTCAGTTT ABCG5 TGGATCCAACACCTCTATGCTAAA GGCAGGTTTTCTCGATGAACTG ABCG8 TGCCCACCTTCCACATGTC ATGAAGCCGGCAGTAAGGTAGA SREBP1c CGGAAGCTGTCGGGGTAG GTTGTTGATGAGCTGGAGCA FAS CCTGGATAGCATTCCGAACCT AGCACATCTCGAAGGCTACACA SCD1 GAGGCCTGTACGGGATCATA TGAGAGAAGAAGAAGCCACGG Primer sequence for qpcr. All sequences are mouse.
Supplementary Table 2 LABELED PROBES: SREBP-1C (+) LXRE 5 BIOTIN / CAGTGACCGCCAGTAACCCCAGC - 3 G0S2 LXRE 5 BIOTIN / ATCTGGCCTGTGGTTACCTTGAT - 3 NON-SPECIFIC 5 BIOTIN / AAATCAGGGGACTTTTTCAGGCG - 3 UNLABELED COMPETITORS: SREBP-1C LXRE 5 CAGTGACCGCCAGTAACCCCAGC - 3 G0S2 LXRE 5 ATCTGGCCTGTGGTTACCTTGAT - 3 NON-SPECIFIC 5 AAATCAGGGGACTTTTTCAGGCG - 3 Sequences for probes used in EMSA assays.