Fig 2: PNPLA2 enhanced the production of 11-cis-retinol by RPE65(A–C) PNPLA2 enhances intracellular 11-cis-retinol production by RPE65 in a cell culture assay. (A) HPLC profiles of retinol isomers from saponified REs extracted from HEK293A-LRAT cells infected with Adv (null, control) (MOI = 50), Ad-RPE65 (RPE65) (MOI = 30) plus Adv (MOI = 20), and RPE65 (MOI = 30) plus Ad-PNPLA2 (PNPLA2) (MOI = 20). The inset shows the absorbance spectra of peak 2, 11-cis-ROL at 318.5 nm. HEK293A-LRAT cells were supplemented with 25 µM all-trans-retinol for 48 h before infection with the adenoviruses. Peak 1, REs; Peak 2, 11-cis-retinol (blue dotted line); Peak 3, 13-cis-retinol; Peak 4, all-trans-retinol. (B) Western blot analysis using the samples for retinoid profile analysis (40 µg of protein per lane). (C) Comparison of the amount of 11-cis-retinol production in HEK293A-LRAT cells expressing RPE65 alone (tan) and that coexpressing RPE65 and PNPLA2 (teal). Data are shown as mean ± SEM. Statistical analysis was performed using an unpaired two-tailed t test. ***p = 0.00023 (n = 9 per group).(D and E) PNPLA2 increased 11-cis-retinol production in vitro retinol isomerase assay. Retinoids generated after incubation of HEK293A-LRAT cell homogenates expressing RPE65 alone or together with PNPLA2 and CGI-58 (250 µg of total cellular protein) with 0.3 µM of all-trans-retinol for 2 h were analyzed by HPLC.(D) Representative retinoid profiles are shown for the samples with and without PNPLA2 and CGI-58 expression. Peak 1: REs; Peak 2: 11-cis-retinol.(E) Quantification of 11-cis-retinol produced by HEK293A-LRAT cells infected with Ad-RPE65 (MOI = 50) that were additionally infected with either Adv (null) (MOI = 100) (control), or Ad-PNPLA2 and Ad-CGI-58 (MOI = 50 each) for 24 h. An equal amount of cell homogenates was mixed with all-trans-retinol added in 2 µL of dimethyl formamide and incubated for 2 h at 37°C. Data are presented as mean ± SEM. Statistical analysis was performed using an unpaired two-tailed t test. **p < 0.0027 (n = 4 per group).

Fig 3: PNPLA2 and CGI-58 are associated with cellular lipid droplets(A–D) Immunofluorescence microscopy of HEK293A cells treated with 400 µM oleic acid for 24 h followed by infection with indicated adenoviral expression vectors. Lipid droplets were stained with LipidTox Red (red). Cells were stained with indicated antibodies for the PLIN2 (A), RPE65 (B), PNPLA2 (C), or CGI-58 (D). Scale bar, 5 µm.(E–H) RGB profile plot of IF microscopy images from (A–D). Dotted line in merged panels indicates the scan position selected for a plot. Lipid droplets (red); lipid droplet-associated proteins (green).(I) Western blot analysis of sucrose density gradient fractionation of oleate-treated HEK293A cells with transiently expressed proteins of interest and endogenous lipid droplet-associated proteins. Fractions: Input (total lysate); pellet (cell pellet); Cyto, cytosol; LD, lipid droplet.

Fig 4: REH activity of PNPLA2 is enhanced by CGI-58(A) Retinoid profiles comparing RE levels in HEK293A cells infected with adenovirus expressing CGI-58, PNPLA2 (multiplicity of infection [MOI] = 50 each) adjusted for total MOI 100 with null Adv for control (MOI = 50), and coexpressing of CGI-58 and PNPLA2 (MOI = 50 each)with null Adv alone as control (MOI = 100). The products of REH activity by PNPLA2 were detected at 320 nm and quantified by HPLC. Peak 1: REs; Peak 2: all-trans-retinol.(B) Western blot analysis using the same cell lysates used in retinoid profile assay.(C) Comparison of the amounts of REs detected in the cells. REs were quantified by HPLC (n = 4 per group). Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with a two-stage linear step-up procedure. ns: not significant, *p < 0.05, ***p < 0.001, and ****p < 0.0001.(D) Western blot analysis showing expression of endogenous PNPLA2 inhibited by the PNPLA2 siRNA. Left lane: HEK293A cells transfected with 100 nM scrambled siRNA (scr-siRNA). Right lane: HEK293A cells transfected with 100 nM PNPLA2 siRNA.(E) Comparison of the amounts of REs in scrambled and PNPLA2 siRNA-transfected cells (n = 4 per group). The amounts of REs were quantified by HPLC. Data are presented as mean ± SEM. Statistical analysis was performed using an unpaired two-tailed t test. *p < 0.05.(F and G) Measurement of REH activity of PNPLA2 using liposome assay. All-trans-retinyl palmitate (RP) containing liposomes (250 µM lipids, 3.3 µM all-trans-RP) were incubated in 200 µL of 1% BSA in 0.1 M Tris-HCl for 2 h at 37°C with 250 µg of total cellular protein. Cells were infected by either Ad-mCherry (MOI = 100) (control) or by Ad-PNPLA2 (MOI = 100) and Ad-CGI (MOI = 100). (F) Representative retinoid profile for the liposome-based assay. Peak 1: REs; Peak 2: all-trans-retinol. (G) Comparison of the amount of all-trans-retinol produced by HEK293A cell homogenates that were separately infected with Ad-mCherry (control), Ad-CGI-58 (CGI-58), or Ad-PNPLA2 (PNPLA2) (MOI = 100 each) for 24 h. An equal amount of cell homogenates (125 µg of total protein) of PNPLA2 and CGI-58 lysates were mixed with liposomes to examine the effect of the coactivator (n = 3 per group). Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. ns: not significant. ****p < 0.0001.

Fig 5: Reduced fat mass in the ob/ob/Fsp27-/- mice.Four-month-old ob/ob and ob/ob/Fsp27-/- mice were maintained on a chow diet for the analyses shown in (a–i). (a) computed tomography (CT) scan analysis of the mice; fat is shown in yellow. (b) GWAT of ob/ob and ob/ob/Fsp27-/- mice. (c) Body composition of ob/ob (n=5) and ob/ob/Fsp27-/- mice (n=6). (d) Decreased TAG content in the GWAT of the ob/ob and ob/ob/Fsp27-/- mice (n=5). (e) Abdominal magnetic resonance imaging (MRI) of ob/ob and ob/ob/Fsp27-/- mice (upper panel). Fat is shown in white in these MRI images. GWAT morphology (middle and lower panels). H&E, haematoxylin and eosin staining; EM, electron microscope. Scale bar, 64 and 2 µm for H&E staining and EM, respectively. (f) LD number per adipocyte in ob/ob and ob/ob/Fsp27-/- mice. The number of LDs in ten adipocytes was measured. (g) The average LD diameter in the GWAT of ob/ob and ob/ob/Fsp27-/- mice. The diameter of LDs in ten adipocytes was measured. (h) The distribution of fat cell size in the GWAT of ob/ob and ob/ob/Fsp27-/- mice. The fat cell area from 400 adipocytes was measured. (i) A representative western blotting showing the expression pattern of Fsp27, Perilipin1, Perilipin2, Cidea, Cyto C, Cox4, ATGL, CGI58, HSL, CEBPß and PPAR? in the GWAT of ob/ob and ob/ob/Fsp27-/- mice. Actin was used as a loading control. Quantitative data are presented as mean±s.e.m. Significance was established using a two-tailed Student’s t-test. Differences were considered significant at P<0.05. ***P<0.001.