Supplemental Table 1. Primer sequences of rat genes probed in RT-PCR assays. Reverse primer. (5 to 3 ) CGA AAG TGT CA TTG T AGG CA CAC ATG T ACA TTT

Transcription

1 Supplemental Table 1. Primer sequences of rat genes probed in RT-PCR assays. Gene Forward primer Reverse primer Ref (5 to 3 ) (5 to 3 ) Ppia CAC CGT GTT CTT CGA CCA GTG CTC AGA GCA (1) CAT CAC CGA AAG Lpin1 TAA GGG GCT GGA GTC AGC AGC CTG GTA GAT TTT CAT TGT CA Lpin2 TAG ATG CAG ACC CTG CTG GTG CTG GCT TCT TTC CC TTG T Lpin3 AAA GAC TGG ACA CAC TGC TGG ATA TCA CTC CAG GG AGG CA Ppargc1a-a GCT TGA CTG GCG TCA ACA GAG TCT TGG CTG re-designed TTC A CAC ATG T based on (3) Ppargc1a-b GAT ATG GAT GTC GGG ACC AAC CAG GGC AGC re-designed TTT GTC A ACA TTT based on (3) Ppara TGG AGT CCA CGC ATG CGC CAG CTT TAG CCG TGA AG AAT AG Cpt1b GTC GCT TCT TCA AGG AAG AAA GCA GCA CGT TTT GG TCG AT Abbreviations used: Ppia, cyclophilin A; Ppargc1a, peroxisome proliferator-activated receptor γ co-activator-1α; Ppara, peroxisome proliferator-activated receptor α; Cpt1b, carnitine palmitoyl transferase-1b.

2 Supplemental Table 2. Primer sequences of mouse genes probed in RT-PCR assays. Gene Forward primer (5 to 3 ) Reverse primer (5 to 3 ) Ref Tbp ACC CTT CAC CAA TGA CTC ATG ATG ACT GCA GCA AAT (4) CTA TG CGC Lpin1 CAG CCT GGT AGA TTG CCA GA GCA GCC TGT GGC AAT TCA (5) Lpin2 TAG ATG CAG ACC CTG TTC CTG GTG CTG GCT TCT TTG CC T Lpin3 AAA GAC TGG ACA CAC CAG TGC TGG ATA TCA CTC AGG GG CA Ppargc1a-a GCT TGA CTG GCG TCA TTC ACA GAG TCT TGG CTG CAC (3) G ATG T Ppargc1a-b GAC ATG GAT GTT GGG ATT ACC AAC CAG AGC AGC ACA (3) GTC A TTT Ppargc1a-c AGT GAC ATG GAT GTT GGG GAA TGC CTC CGG TTA CTC (3) ATT G ACT T Ppara ACT ACG GAG TTC ACG CAT TTG TCG TAC ACC AGC TTC (6) GTG AGC Cpt1a CTC AGT GGG AGC GAC TCT GGC CTC TGT GGT ACA CGA (7) TCA CAA Cpt1b GTC GCT TCT TCA AGG TCT AAG AAA GCA GCA CGT TCG (4) GG AT

3 Abbreviations used: Tbp, TATA-binding protein; Ppargc1a, peroxisome proliferator-activated receptor γ co-activator-1α; Ppara, peroxisome proliferator-activated receptor α; Cpt1, carnitine palmitoyl transferase-1. Where not referenced, primers were designed using Primer Express version 2.0 software or with Primer-BLAST web software (National Centre for Biotechnology Information, Bethesda, MD) with default parameters. Primer specificity was also confirmed using Primer-BLAST.

4 Figure legends Supplemental Figure 1. Nucleotide sequences from the Ppargc1a genes in Rattus norgevicus and Mus musculus responsible for encoding the portions of the PGC-1α-b and PGC-1α-c splice variants different from the PGC-1α-a variant. The PGC-1α-b and PGC-1α-c splice variants only differ from PGC-1α-a in the N-terminus. The first 16 amino acids in PGC-1α-a are exchanged with the 12 and 3 amino acids of the PGC-1α-b and PGC-1α-c splice variants as denoted in the figure, respectively (3). The 5 splice site is highlighted in red. The PGC-1α-c splice variant in Rattus norvegicus does not have a functional 5 splice site and this is highlighted in green. Supplemental Figure 2. Rates of glycerolipid synthesis from glycerol and oleate in neonatal rat ventricular myocytes (NRVMs) expressing recombinant lipin-1 and -2 and pre-treated with Torin1 and/or insulin. NRVMs were infected for 38 h with recombinant adenoviral (Ad) constructs expressing green fluorescent protein (GFP), lipin-1b or -2 at a multiplicity of infection (MOI) of 7. These NRVMs were then pre-treated with vehicle, 250 nm Torin1 and/or 100 nm insulin before incubating with (A) 1 mm [ 3 H]glycerol and (B) 1 mm [ 14 C]oleate in DME/F12 medium containing 0.3 mm BSA for 3 h and the rate of glycerol and oleate incorporation into triacylglycerol (TG) was measured and expressed as pmol oleate or glycerol per pmol phospholipid phosphate. (C) NRVMs infected with recombinant lipin-1b were pre-treated with vehicle, 250 nm Torin1 and/or 100 nm insulin before incubating with 1 mm glycerol and 1 mm oleate in DME/F12 medium containing 0.3 mm BSA for 3 h. The representative Western blot shows the phosphorylation state of serine 106 on the recombinant HA-tagged lipin-1b after these treatments.

5 REFERENCES 1. Morris, K. E., L. M. Schang, and D. N. Brindley Lipid phosphate phosphatase-2 activity regulates S-phase entry of the cell cycle in Rat2 fibroblasts. J. Biol. Chem. 281: Manmontri, B., M. Sariahmetoglu, J. Donkor, M. B. Khalil, M. Sundaram, Z. Yao, K. Reue, R. Lehner, and D. N. Brindley Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically. J. Lipid Res. 49: Miura, S., Y. Kai, Y. Kamei, and O. Ezaki Isoform-specific increases in murine skeletal muscle peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC- 1alpha) mrna in response to beta2-adrenergic receptor activation and exercise. Endocrinology 149: Phan, J., and K. Reue Lipin, a lipodystrophy and obesity gene. Cell Metab. 1: Peterfy, M., J. Phan, and K. Reue Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis. J. Biol. Chem. 280: Yang, J., N. Sambandam, X. Han, R. W. Gross, M. Courtois, A. Kovacs, M. Febbraio, B. N. Finck, and D. P. Kelly CD36 deficiency rescues lipotoxic cardiomyopathy. Circ. Res. 100: Werner, A., R. Havinga, T. Bos, V. W. Bloks, F. Kuipers, and H. J. Verkade Essential fatty acid deficiency in mice is associated with hepatic steatosis and secretion of large VLDL particles. Am. J. Physiol. Gastrointest. Liver Physiol. 288: G

6 Suppl. Fig. 1

7 Suppl. Fig. 2 A B C kda 150 p-s106 Lipin HA-tagged Lipin merged image