Fig.4.

Endogenous MBOAT7 is attached to the ER, MAM, LD and MBOAT7/TMC4 rs641738 variant decreases MBOAT7 mRNA expression and synthesis level in liver biopsies of obese patients.

(A) Post-nuclear supernatant (PNS), cytosol and endoplasmic reticulum (ER) were obtained by a differential centrifugation. (B) Crude mitochondria were used to isolate purified mitochondria and mitochondria-associated membranes (MAM) by Percoll density gradient. (C) Post-nuclear supernatant was used to isolate lipid droplets (LD) by sucrose gradient. (D) Hepatic mRNA levels of MBOAT7 and TMC4 in 98 severely obese stratified by the rs643718 C>T genotype (CC n=29; CT n=51; TT n=18). Data are expressed as fold increase as compared to the protective CC genotype. p=0.03 for the effect of the rs641738 T allele on MBOAT7 expression (additive model). Demographic, anthropometric and clinical characteristics of these patients are shown in supplementary Table 7. (E) Western blotting analysis and quantification of MBOAT7 protein levels in obese patients carrying the three MBOAT7/TMC4 different genotypes, of whom hepatic gene expression was available (Supplemental Table 7). Seven samples for each rs641738 genotype (CC/CT/TT) were collected from liver biopsies and pooled according to genotype. All reactions (25 μg of lysates) were performed in triplicate in the same gel. Data are expressed as fold increase of the three MBOAT7 isoforms 1-3 (molecular weight: 52 KDa, 44 KDa and 38 KDa), as compared to the protective CC genotype. p<0.005 (additive model; it represents the difference in the trait examined across MBOAT7 genotypes).

Abbreviations: CNX: calnexin; COX IV, cytochrome c oxidase, Complex IV; FACL4, long-chain acyl-CoA synthetase; ADFP, Adipose differentiation-related protein; IN, Infranatant, MBOAT7, Membrane Bound O-Acyltransferase domain containing 7; TMC4, Transmembrane Channel-like 4.