Nfected with Ergocalciferol site adenovirus to overexpress PGC-1a or GFP (control) and transfected with siRNA to knockdown HNF4a or scramble control siRNA (n = 6). *p,0.05 versus scramble control infected with the same adenovirus. **p,0.05 versus all other groups. doi:10.1371/journal.pone.0051320.gand siHNF4a were transfected onto HepG2 cells using a Lipofectamine-2000 reagent (Invitrogen). At 14 hr after siRNA transfection, the cells were infected with Ad-GFP or Ad-PGC-1aand cultured for additional 34 hr and thereafter they were harvested for RNA isolation or subjected to assays measuring rates of palmitate oxidation 22948146 as described above.Figure 2. Lipin 1 enhances HNF4a-mediated increases in fatty acid oxidation. [A] The images depict the results of co-immunoprecipitation studies using lysates from HepG2 cells infected with adenovirus driving expression of lipin 1b or lipin 1(LXXFF). HNF4a-containing complexes were immunoprecipitated with an antibody directed against HNF4a or IgG control. Immunoprecipitated proteins were then subjected to immunoblotting with antibody directed against the HA tag of overexpressed lipin 1. Input represents 5 of the total protein used in immunoprecipitation reactions. [B] Graphs depict results of luciferase assays 
using lysates from HepG2 cells transfected with Acadm.TKLuc or Ppara.Luc and cotransfected with lipin 1 and/or HNF4a expression constructs as indicated. The results are the mean of 3 independent experiments done in triplicate. *p,0.05 versus pCDNA control. **p,0.05 versus pcDNA or lipin 1 alone. ***p,0.05 versus all other groups. [C and D] Primary hepatocytes were isolated from 6 week old C57BL/6 mice and infected with adenovirus driving expression of GFP or HNF4a in the presence or absence of overexpressed lipin 1b (wild-type or LXXFF). The graphs depict [C] the expression of Ppara and Acadm (n = 5) or [D] mean rates of palmitate oxidation (mean of 3 independent experiments done in triplicate) or *p,0.05 versus GFP control. **p,0.05 versus HNF4a overexpression alone. ***p,0.05 versus all other groups. doi:10.1371/journal.pone.0051320.gLipin 1 and HNFStatistical AnalysesStatistical comparisons were made using analysis of variance (ANOVA) coupled to Scheffe’s test. All data are presented as means 6 SEM, with a statistically significant difference defined as a P value ,0.05.Results PGC-1a Induces Lipin 1 Expression through HNF4a in HepG2 CellsWe have ML 281 previously demonstrated that PGC-1a is an important regulator of lipin 1 gene expression in liver [10], but the transcription factor 
partners of PGC-1a that mediate this effect remain unclear. To further dissect the transcriptional mechanisms at play, we transfected HepG2 cells with expression constructs for PGC-1a or PGC-1b and Lpin1 promoter-luciferase reporter constructs. The PGC-1a responsive region was not contained in the 2 kb 59 flanking sequence (22045.Lpin1.Luc) 15755315 that is sufficient to confer responsiveness to several other transcription factors [22,23,24,25]. However, PGC-1a overexpression activated a promoter driven by 393 nucleotides of the 59 flanking sequence, the first Lpin1 exon (53 nucleotides), and 2,240 bp of the first intron of the mouse lipin 1 gene (+2293-Lpin1.luc). This suggests that the PGC-1a-responsive element in HepG2 cells is contained in the first intron of the Lpin1 gene (Figure 1A). Interestingly, PGC-1a overexpression did not affect Lpin1 promoter activity. To begin to narrow the transcription factor partners of lipin 1 that might be mediatin.Nfected with adenovirus to overexpress PGC-1a or GFP (control) and transfected with siRNA to knockdown HNF4a or scramble control siRNA (n = 6). *p,0.05 versus scramble control infected with the same adenovirus. **p,0.05 versus all other groups. doi:10.1371/journal.pone.0051320.gand siHNF4a were transfected onto HepG2 cells using a Lipofectamine-2000 reagent (Invitrogen). At 14 hr after siRNA transfection, the cells were infected with Ad-GFP or Ad-PGC-1aand cultured for additional 34 hr and thereafter they were harvested for RNA isolation or subjected to assays measuring rates of palmitate oxidation 22948146 as described above.Figure 2. Lipin 1 enhances HNF4a-mediated increases in fatty acid oxidation. [A] The images depict the results of co-immunoprecipitation studies using lysates from HepG2 cells infected with adenovirus driving expression of lipin 1b or lipin 1(LXXFF). HNF4a-containing complexes were immunoprecipitated with an antibody directed against HNF4a or IgG control. Immunoprecipitated proteins were then subjected to immunoblotting with antibody directed against the HA tag of overexpressed lipin 1. Input represents 5 of the total protein used in immunoprecipitation reactions. [B] Graphs depict results of luciferase assays using lysates from HepG2 cells transfected with Acadm.TKLuc or Ppara.Luc and cotransfected with lipin 1 and/or HNF4a expression constructs as indicated. The results are the mean of 3 independent experiments done in triplicate. *p,0.05 versus pCDNA control. **p,0.05 versus pcDNA or lipin 1 alone. ***p,0.05 versus all other groups. [C and D] Primary hepatocytes were isolated from 6 week old C57BL/6 mice and infected with adenovirus driving expression of GFP or HNF4a in the presence or absence of overexpressed lipin 1b (wild-type or LXXFF). The graphs depict [C] the expression of Ppara and Acadm (n = 5) or [D] mean rates of palmitate oxidation (mean of 3 independent experiments done in triplicate) or *p,0.05 versus GFP control. **p,0.05 versus HNF4a overexpression alone. ***p,0.05 versus all other groups. doi:10.1371/journal.pone.0051320.gLipin 1 and HNFStatistical AnalysesStatistical comparisons were made using analysis of variance (ANOVA) coupled to Scheffe’s test. All data are presented as means 6 SEM, with a statistically significant difference defined as a P value ,0.05.Results PGC-1a Induces Lipin 1 Expression through HNF4a in HepG2 CellsWe have previously demonstrated that PGC-1a is an important regulator of lipin 1 gene expression in liver [10], but the transcription factor partners of PGC-1a that mediate this effect remain unclear. To further dissect the transcriptional mechanisms at play, we transfected HepG2 cells with expression constructs for PGC-1a or PGC-1b and Lpin1 promoter-luciferase reporter constructs. The PGC-1a responsive region was not contained in the 2 kb 59 flanking sequence (22045.Lpin1.Luc) 15755315 that is sufficient to confer responsiveness to several other transcription factors [22,23,24,25]. However, PGC-1a overexpression activated a promoter driven by 393 nucleotides of the 59 flanking sequence, the first Lpin1 exon (53 nucleotides), and 2,240 bp of the first intron of the mouse lipin 1 gene (+2293-Lpin1.luc). This suggests that the PGC-1a-responsive element in HepG2 cells is contained in the first intron of the Lpin1 gene (Figure 1A). Interestingly, PGC-1a overexpression did not affect Lpin1 promoter activity. To begin to narrow the transcription factor partners of lipin 1 that might be mediatin.
