Figure 5
From: Phospholipase D1 deficiency in mice causes nonalcoholic fatty liver disease via an autophagy defect
PA restores autophagy.
(a) Primary hepatocytes were isolated from Pld1+/+ and Pld1−/− littermates, treated with 5 μM VU0155069 in serum-fed conditions for 24 h, and the lysates were analyzed by western blot. (b) The Pld1+/+ and Pld1−/− littermates were fed HFD for 4 weeks starting at the age of 13 weeks (n = 3 per group), livers were collected, and total phosphatidic acid levels were measured by LC-MS/MS. (c,d) The mice were fed as described in (b). (c) The lysates and (d) the lysosomal fractions of livers were isolated and a PLD assay was performed using a PLD assay kit. (e) Primary hepatocytes were isolated from Pld1+/+ and Pld1−/− littermates and treated with or without 300 μM C8-PA in serum-fed conditions for 24 h; the lysates were analyzed by western blot. (f) Pld1+/+ and Pld1−/− MEFs were infected with RFP-GFP-LC3B (60 particles per cell) for 24 h. Pld1+/+ MEFs, Pld1−/− MEFs, and Pld1−/− MEFs treated with 300 μM C8-PA were maintained in either serum-fed or serum-starved conditions for 24 h. Representative confocal microscopy images from 3–5 independent experiments are shown. The average numbers of GFP-negative/RFP-positive or GFP- and RFP-positive cells were counted using ZEN software from three independent experiments (60 cells per experiment). The data are presented as means ± SE or representative blots from 3 to 5 independent experiments. *P < 0.05 versus Pld1+/+ mice. Abbreviations: C8-PA, C8-phosphatidic acid; GFP, green fluorescence protein; HFD, high-fat diet; LC-MS/MS, liquid chromatography–mass spectrometry/mass spectrometry; MEF, mouse embryonic fibroblasts; RFP, red fluorescence protein; SE, standard error.
