Dried lipids were redissolved in 1% (v/v) Triton X-100, and TG content was measured using the reagent for quantitative TG measurement (DiaSys, Holzheim, Germany). Mouse liver was homogenized in 0.5 mL of
phosphate buffered saline and 0.5 mL of methanol. Each sample was spiked with 375 nmol of a C15:0 FA as an internal standard immediately. Lipids were then extracted according to Bligh and Dyer. Lipid extracts were taken to dryness and resuspended Selleck Tamoxifen in 1 mL of methanolic NaOH. After 10 minutes at 80°C and 5 minutes on ice, 1 mL of BF3 was added, followed by another 10 minutes at 80°C. FA methyl esters were extracted with 1 mL of saturated NaCl solution and 2 mL of hexane. The hexane phase was taken to dryness and redissolved in 1.5 Decitabine datasheet mL of hexane. Gas chromatography (GC)/electron impact ionization/mass spectrometry (MS) and GC/negative ion chemical ionization/MS was performed
as described in the Supporting Materials and Methods. Statistical analysis was performed using SPSS V.18.0 (SPSS, Inc., Chicago, IL), using the unpaired Student’s t test. Data are reported as means ± standard deviation (SD). Unless otherwise noted, animal numbers were as follows: WT control: n = 5; ATGL KO control: n = 5; WT TM treatment: n = 5; and ATGL KO TM treatment: n = 6. Cell-culture experiments were performed in triplicate. A P value ≤0.05 was considered significant. Lipotoxicity is known to induce ER stress in vitro32 and in vivo.8 Therefore, we injected
TM to induce ER stress in WT and ATGL KO fed mice, which have a defect in cellular TG catabolism.25, 26, 33 Serum ALT levels were not significantly increased in mice injected with TM; ALP levels were increased, whereas total CHOL, TG, and FA were decreased in both genotypes (Fig. 1A). Although serum parameters suggested that WT and ATGL KO mice challenged with TM have disturbed lipid metabolism, only ATGL KO mice showed hepatic lipid Thiamet G accumulation (Fig. 1B; Supporting Fig. 1A). Notably, the liver/body-weight ratio was not changed by TM treatment in both WT and ATGL KO mice (Supporting Fig. 1B). Liver histology in WT mice was not affected by TM, whereas untreated ATGL KO controls exhibited a moderate lipid infiltration, which was further pronounced by TM, as shown by H&E and Oil Red O staining (Fig. 1B; Supporting Fig. 1A). Biochemical quantification of hepatic lipid content revealed a more than 2-fold increase in hepatic TG accumulation in TM-treated ATGL KO mice, compared to TM-treated WT mice (Fig. 1C). Taken together, our data demonstrate that ATGL KO mice show elevated hepatic TG stores after induction of ER stress. To further address the role of ATGL in the hepatic ER stress response, we checked mRNA expression levels of ER stress markers in the presence and absence of ATGL in vivo.