Fig 2: Losartan attenuated HIF-1a expression and LDs formation in EWAT. (A) Representative HIF-1a staining of EWAT from losartan-treated ob/ob mice and normal mice. Red arrow highlights the positive staining. Scale bar: 100 µm. Quantification of (B) HIF-1a protein level by Western blot. Below graphs indicate quantification relative to Histone. (C) Representative HE, PLIN1, PLIN2, CIDEA and CIDEC staining of EWAT from losartan-treated ob/ob mice and normal mice. Scale bar: 100 µm. Quantification of (D) PLIN1, PLIN2, CIDEA, and CIDEC protein levels by Western blot of EWAT after losartan treatment. Below graphs indicate quantification relative to ß-actin. For each animal group, n = 5. All values represent the mean ± SEM. Data were analyzed by Student’s t test. * p = 0.05; normal vs. ob/ob. # p = 0.05; ob/ob vs. ob/ob + Losartan. HIF-1a, hypoxia-inducible factor-1a; LDs, lipid droplets; EWAT, epididymis white adipose tissue; HE, hematoxylin and eosin; PLIN, perilipin; CIDE, cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector.

Fig 3: CMA activation reduces diclofenac-induced lipid accumulation in hepatocytes. (A) MPH were incubated with diclofenac (300 µM) or AR7 (10 µM) for 24 h. Confocal microscopy images was analyzed to detect LAMP2A. Representative fluorescent images and quantitative data of the cells are shown (Scale bars: 20 µm). (B) The protein levels of LAMP2A, PLIN2, and GAPDH were measured in MPH by western blot analysis. Representative western blot images and the densitometric quantification of proteins are shown. (C) MPH were incubated with diclofenac (300 µM) and AR7 (10 µM) for 24 h. Intracellular lipid droplets (LDs) were determined by BODIPY 493/503 fluorescence (green). Nuclei were stained with DAPI (blue) (Scale bars: 20 µm). (D) TG concentration in MPH was quantified using an enzymatic kit. (E) MPH were transfected with pCMV-LAMP2A plasmid and were incubated with diclofenac for 24 h. Western blot analysis with antibodies against LAMP2A, PLIN2 and GAPDH and the densitometric quantification of the PLIN2 and GAPDH level are shown. (F) Intracellular LDs were determined by BODIPY 493/503 fluorescence (green). Nuclei were stained with DAPI (blue) (Scale bars: 20 µm). (G) TG concentration in MPH was quantified using the enzymatic kit. Data are presented as mean ± SD of at least 3 independent experiments, as analyzed by one-way ANOVA followed by Tukey's test. *p < 0.05, **p < 0.01, and ***p < 0.001, relative to the control group. #p < 0.05, ##p < 0.01, ###p < 0.001, relative to the indicated group.

Fig 4: Mboat7 knockdown promotes hepatic steatosis by altering the lipidome and proteome of the cytosolic lipid droplets.Male C57BL/6 mice were fed either chow or a HFD for 12–20 weeks along with receiving a control non-targeting ASO or an ASO targeting the knockdown of Mboat7. (A) Mice were necropsied either in the fed or fasted (12 hr fast) state, and the hepatic expression of genes involved in lipogenesis, fatty acid oxidation, or sterol sensing analyzed using qPCR. (B) Plasma triglyceride (TG) secretion rate in mice administered Triton WR-1339 (tyloxapol). (C) Fed mice were treated with either saline vehicle or the ß3-adrenergic receptor agonist CL316243 (0.1 mg/g body weight) to stimulate adipocyte lipolysis, and plasma glycerol and NEFA were measured after 15 min. Panels D–F), ASO-treated mice were fasted for 4 hr (from 9:00 a.m. to 1:00 p.m.) prior to necropsy, and hepatic lipid droplets were isolated using sucrose gradient fractionation to analyze both the lipidome and proteome of cytosolic lipid droplets. Lipid droplet fractions were extracted and analyzed by LC-MS/MS to quantify either lysophosphatidylinositol (D) or phosphatidylinositol (E) species on isolated lipid droplets. (F) Lipid droplet fractions were analyzed using Western blotting for MBOAT7, glycerol 3-phosphate dehydrogenase (as a non-lipid droplet fraction control), perilipin 2 (PLIN2), perilipin 3 (PLIN3), CTP:phosphocholine cytidylyltransferase a (CCTa), or glycerol-3-phosphate 4 (GPAT4); four individual mice per group are shown. Data are presented as mean ± S.E.M, (n = 4–6 per group; *p=0.05, **p=0.01, ***p=0.001, ****p=0.0001; Two-way ANOVA with Tukey’s post-hoc test).

Fig 5: HMGCS2 overexpression ameliorates lipid accumulation in HepG2 cells. (A) Schematic representing timeline of in vitro experiment, starting with oleic acid treatment at 0-hours, adenovirus-mediated overexpression of GFP (Ad-GFP) or HMGCS2 (Ad-HMGCS2) at 24-hours and cell collection at 48-hours. (B) RT-qPCR (n = 4/group) and (C) western blot quantification of HMGCS2 expression (n = 3/group) in HepG2 cells after Ad-HMGCS2 infection, compared to Ad-GFP. (D) Representative images of Oil-red-O staining and (E) its quantification of Ad-GFP or Ad-HMGCS2 treated HepG2 cells in the absence (-) and presence (+) of oleic acid (n = 3/group). (F) RT-qPCR of lipid synthesis (SREBP1C, ACC1, FASN) and lipid accumulation (FSP27, PLIN2) genes in Ad-GFP and Ad-HMGCS2 treated HepG2 cells (n = 4/group). Data are represented as mean ± SEM. Statistical analysis was performed by student's t-test or two-way ANOVA. *P = 0.05; **P = 0.01, ***P = 0.001; ****P = 0.0001. (Created with BioRender.com).