Hmgcoar AGCTTGCCCGAATTGTATGTG TCTGTTGTAACCATGTGACTTC. Cyp7α GGGATTGCTGTGGTAGTGAGC GGTATGGAATCAACCCGTTGTC

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Percival Dawson
6 years ago
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1 Supplement Table I: primers for Real Time RT-PCR Gene Foward Reverse Hmgcoar AGCTTGCCCGAATTGTATGTG TCTGTTGTAACCATGTGACTTC Cyp7α GGGATTGCTGTGGTAGTGAGC GGTATGGAATCAACCCGTTGTC Cyp27a1 GTGGTCTTATTGGGTACTTGC GGTATGGAATCAACCCGTTGTC Aco GCCCAACTGTGACTTCCATT GGCATGTAACCCGTAGCACT Agpat1 CACCCAGGATGTGAGAGTCTG CTGACAACGTCCAGGCGAGG Sqle CGCGCAGCGGTTACTCTGGT AGTCACGGCCGGGAACCTGT Mtp TGCGGGTCAACAGAGAGGCG CCTTGACCTTCCCCCGGACCA Gpat4 GGCAGAGGAGCTGGAGTC TGTTGTGGTACGTAATGATGG Dgat2 AGTGGCAATGCTATCATCATCGT AAGGAATAAGTGGGAACCAGATCA Fas GCTGCGGAAACTTCAGGAAAT AGAGACGTGTCACTCCTGGACTT Hmgcs GCCGTGAACTGGGTCGAA GCATATATAGCAATGTCTCCTGCAA Scd1 CCGGAGACCCCTTAGATCGA TAGCCTGTAAAAGATTTCTGCAACC Lipin1 GGTCCCCCAGCCCCAGTCCTT GCAGCCTGTGGCAATTCA Lipin2 AGTTGACCCCATCACCGTAG CCCAAAGCATCAGACTTGGT Srebp1 AGCAGCCCCTAGAACAAACAC CAGCAGTGAGTCTGCCTTGAT Srebp2 TGAAGGACTTAGTCATGGGGAC CGCAGCTTGTGATTGACCT ApoCIII AGGTACGTAGGTGCCATGC CGTCTTGGAGGCTTGTTCCA Pcsk9 CAGAGGTCATCACAGTCGGG GGGGCAAAGAGATCCACACA Hrpt TGACACTGGTAAAACAATGC AACACTTCGAGAGGTCCTTT Hmgcoar, 3-hydroxy-3-methylglutaryl coenzyme A reductase; Cyp7α, cholesterol 7alpha-hydroxylase; Cyp27a1, sterol 27-hydroxylase; Aco, Acyl coa carboxylase; Agpat1, 1-acylglycerol-3-phosphate O-acyltransferase; Sqle, Squalene monooxygenase; Mtp, Microsomal Triglyceride Transfer Protein; Gpat, Glycerol-3- phosphate acyltransferase; Dgat, Diacylglycerol acyltransferase; Fas, Fatty acid synthase; Hmgcs, HMGcoA synthase; Scd1, Stearoyl-coA desaturase-1; Srebp, Sterol regulatory element binding protein; Apo, apolipoprotein; Pcsk9, Proprotein Convertase Subtilisin/kexin type 9 ;Hrpt, Hypoxanthine guanine phosphoribosyl transferase.

2 Supplement Table II: Effect of on major plasma lipid species in Ldlr -/- mice Plasma (nmol/ml) VLDL (nmol/apob) IDL/LDL (nmol/apob) Chol * CE ** DG # TG # PC *** Chol, cholesterol; CE, cholesteryl esters; DG, diacylglycerol; TG, triglyceride; PC, phosphatidyl choline. Lipid levels were determined by LC/MSMS. Data are presented as nmol/ml for plasma and expressed nmol lipid per apob content for VLDL and IDL/LDL fractions. ApoB content of VLDL and IDL/LDL particles was determined by Western blotting. Data is mean sem from 7-8 mice per group. # p<.5, *p<, **p<.1, ***p<.1 v ctrl.

3 Supplemental Table III: Lipidomics of VLDL and LDL in v -treated Ldlr -/- mice VLDL (nmol/apob) IDL/LDL (nmol/apob) Sphingomyelin Ceramide Dihydroceramide Monohexosylceramide Dihydrohexoceramide Trihexosylceramide G M3 ganglioside Lysophosphatidylcholine Lysoalkylphosphatyidylcholine Phosphatidylcholine Alkylphosphatidylcholine Alkenylphosphatidylcholine Lysophosphatidylethanolamine Phosphatidylethanolamine Alkylphosphatidylethanolamine Alkenylphosphatidylethanolamine Phosphatidylglycerol Lysophosphatidylinositol Phosphatidylinositol Phosphatidylserine Lipid levels were determined by LC/MSMS. Data are presented relative to apob content and represent mean sem from 7-8 mice per group.

4 Supplement Table IV: Lipidomics of SM species in VLDL and LDL in v -treated Ldlr -/- mice VLDL (nmol/pc) IDL/LDL (nmol/pc) SM 32:1 SM 33:1 SM 34:1 SM 34:2 SM 34:3 SM 36:1 SM 36:2 SM 36:3 SM 38:1 SM 38:2 SM 42:1 SM 31:1 SM 33:1 SM 35:1 SM 35:2 SM 37:2 SM 39:1 SM 41:1 SM 41: ** # * # * * * ** * *** * # ** * * * * * Levels of SM species were determined by LC/MSMS. Data is presented nmol per PC content and is mean sem from 7-8 mice per group. # p<.5, *p<, ** p<.1, ***p<.1.

5 Figure I A wt Ldlr-/- B wt Ldlr-/- C wt Ldlr-/- Figure I: does not induce liver or kidney toxicity. Wt and Ldlr -/- mice were treated with 2mg/kg/day or vehicle () for 4 weeks via subcutaneous implanted Alzet Osmotic minipumps. Formalin-perfused livers (A) and kidneys (B) were paraffin embedded and stained with hematoxylin/eosin. Kidney sections were also stained with Periodic acid-schiff stain (C). Tissue sections shown were viewed using a 2x objective. Scale bar = 2µm. No significant difference was

6 Figure II A B Blood glucose (mmol/l) AUC glucose (mmol/l x 12min) ** Time (min) Figure II: does not induce insulin resistance in LDLr -/- mice. LDLr -/- mice were treated with 2mg/kg/day or vehicle () for 4 weeks. Non-fasted insulin tolerance tests were performed as described in material and methods. (A) Blood glucose levels in response to i.p. injection of insulin and (B) Glucose area under the curve (AUC). Values are mean S.E.M. (n=1 per group). ** p<.

7 Figure III Hepatic mrna level (fold induction) Hmgcoar Fas Scd1 Srebp1 Srebp2 Figure III: does not affect hepatic mrna levels of genes involved in lipid synthesis. Wt mice were treated with 2mg/kg/day or vehicle () for 4 weeks. Hepatic mrna levels were determined by qpcr as described in Materials and Methods.

8 Figure IV VLDL particle size (nm) Figure IV: does not affect VLDL particle size in LDLr -/- mice. LDLr -/- mice were treated with 2mg/kg/day or vehicle () for 4 weeks. VLDL was isolated by density ultracentrifugation and VLDL particle size was determined using dynamic light scattering as described in Material and Methods.

9 Figure V wt Ldlr -/- [ 3 H] activity (% of injected dose) Liver Heart Spleen Kidney GonWAT ScWAT Muscle SolMuscle [ 3 H] activity (% of injected dose) Liver * Heart Spleen Kidney GonWAT ScWAT Muscle SolMuscle wt Ldlr -/- [ 14 C] activity (% of injected dose) Liver Heart Spleen Kidney GonWAT ScWAT Muscle SolMuscle Figure V: inhibits hepatic uptake of lipoprotein derived cholesteryl oleate in LDLr -/- mice. Wt and LDLr -/- mice were treated with 2 mg/kg/day or cremaphore vehicle () for 4 weeks. Mice were injected with VLDL-like particles, double-labeled with [ 3 H]TO and [ 14 C]CO. 3 H- and 14 C- uptake by organs and tissues were determined at 15 min for wt and 3 min for LDLr -/- after injection. Data are expressed as percentage 3 H- or 14 C-activity of the injected dose per total tissue, with the exception of kidney, white adipose tissue pads and muscle which were determined as uptake in 1 representative tissue. GonWAT, gonadal white adipose tissue, scwat, subcutaneous white adipose tissue. Values are mean S.E.M. (n=5-7 per group). * p<.5. [ 14 C] activity (% of injected dose) Liver * Heart Spleen Kidney GonWAT ScWAT Muscle SolMuscle

10 Figure VI Figure VI: Plasma PCSK9 levels are not increased by in wt mice Wt were treated with 2 mg/kg/day or vehicle () for 4 weeks. Plasma PSCK9 levels were determined by Western Blotting. A representative sample from Ldlr -/- mice is shown. No increase in PCSK9 was observed after treatment of wt mice.

Supplemental Table 1. List of primers used for real time PCR.

Supplemental Table 1. List of primers used for real time PCR. Primer Sequence Primer Sequence Mouse Pcsk9-F TTGCAGCAGCTGGGAACTT Mouse Scd1-F CATCATTCTCATGGTCCTGCT Mouse Pcsk9-R CCGACTGTGATGACCTCTGGA Mouse