PMC

Abundance of mRNA of PPAR-α and its target genes was quantified with qRT-PCR in livers of mice fed the chow diet for 7 d after adenoviral transduction. Data are presented as means ± SEM. *P<0.05 vs control shRNA group. PPAR-α, peroxisome proliferator-activated receptor alpha; CPT-1, carnitine palmitoyltransferase I; ACOT-1, acyl-CoA thioesterase I; LCAD, long-chain acyl-CoA dehydrogenase; ACSL1, acyl-CoA synthetase 1; PEPCK, phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase. *P<0.05 vs control shRNA group.

(A) Liver weights in mice treated with control or ATGL shRNA in both chow and HF diet groups (n = 8-10). (B) Liver sections from these mice were stained with hematoxylin and eosin and imaged at 20× magnification. (C) Liver TAG was quantified from mice fed chow or high fat diets (n = 8-10). (D) In order to determine composition of TAG, lipids extracted from livers of chow-fed mice were separated by TLC and TAG was methylated with 5% HCl in methanol to produce fatty acid methyl esters (FAMEs), which were analyzed by GC (n = 6). Data are presented as means ± SEM. *P<0.05 vs control shRNA group.

C57/Bl6 mice at 8-10 weeks of age were infected with control or ATGL shRNA adenovirus (n = 8-10 per group) and were fed with either chow or high fat (HF) diets. After an overnight fast, animals were sacrificed 7 d post-infection followed by determination of mRNA (A) and protein (B) expression of hepatic ATGL liver in chow fed mice. (C) Cytosolic extracts were also used to measure hepatic TAG hydrolase activity from chow-fed mice (n = 3-4). Body (D) and gonadal fat (E) weights of mice fed chow or HF diets. Data are presented as means ± SEM. *P<0.05 vs control shRNA group.

One day after adenoviral injections, mice were treated with 125 mg/kg of fenofibrate suspended in 0.5% carboxylmethylcellulose for 6 d via oral gavage. Mice were fed with the HF diet and sacrificed 7 d after infection following an overnight fast. Liver weight (A) and triglyceride (B) were measured in mice treated with fenofibrate (FF) and compared to vehicle-treated mice (n = 7-8). (C) mRNA abundance of oxidative genes was determined in liver tissues of mice treated with FF (n=6). Abbreviations are described in the legend to . Data are presented as means ± SEM. *P<0.05 versus control shRNA group. ^#P<0.05 vs pulse period.

(A) Serum β-hydroxybutyrate was measured with a colorimetric enzymatic kit in serum samples collected from chow-fed mice 7 d after infection with control or ATGL shRNA adenovirus and following an overnight fast. (B-E) Primary hepatocytes isolated from chow-fed mice were transduced with control or ATGL shRNA for 66 h and Ad-GFP or Ad-ATGL for 24 h, at which time pulse (1.5 h) and chase (6 h) experiments were performed with 500 μM [1-¹⁴C]oleate. Media from hepatocytes were harvested after pulse and chase, and CO₂ and ASM were quantified as outlined in the experimental procedures to measure fatty acid oxidation. Data are presented as means ± SEM. *P<0.05 vs control shRNA group. ^#P<0.05 vs pulse period.

Mice fed the chow diet were treated with control or ATGL shRNA adenovirus and were fasted overnight followed by harvesting of serum for analysis of (A) free fatty acid (FFA) and (B) TAG (n = 8-10). (C) For measurement of hepatic TAG production, overnight fasted mice fed the chow diet were treated with Tyloxapol and blood collections were performed at 0, 1, 2 and 3 h intervals (n = 4-6) and serum TAG was subsequently analyzed. (D) Primary hepatocytes were transduced with control or ATGL shRNA for 66 h, at which time pulse (1.5 h) and chase (6 h) experiments were performed with 500 μM [1-¹⁴C]oleate. [¹⁴C]TAG was quantified during the chase period. (E) Primary hepatocytes were transduced with control or ATGL shRNA for 66 h followed by 8 h of pulse with 500 μM [1-¹⁴C]oleate. Cells were harvested and radiolabeled oleate incorporation into different lipid fractions was determined (n = 3). (F) Primary hepatocytes were also transduced with Ad-GFP or Ad-ATGL adenoviruses and after 24 h pulse (1.5 h) and chase (6 h) experiments were performed with 500 μM [1-¹⁴C]oleate and [¹⁴C]TAG secretion during the chase period was measured. Data are presented as means ± SEM.

Primary mouse hepatocytes were isolated from 8-10 wk old C57/Bl6 chow-fed mice and cells were transduced with control or ATGL shRNA adenovirus for 66 h, at which time pulse (1.5 h) and chase (6 h) experiments were performed with 500 μM [1-¹⁴C]oleate. (A) TAG, (B) PL, (C) CE and (D) DAG were isolated from cells by lipid extraction and separation by TLC to measure incorporation of radiolabeled oleate into different lipid species (n = 3-5). (E) Cells were transduced with Ad-GFP and Ad-ATGL virus for 24 h, at which time pulse (1.5 h) and chase (6 h) experiments were performed with 500 μM [1-¹⁴C]oleate (n = 3). (F) Cells were transduced with control and ATGL shRNA for 66 h, at which time cells were pulsed with 500 μM [1-¹⁴C]oleate for 8 h. TAG, PL & CE were isolated from harvested cells by TLC to measure incorporation of radiolabeled oleate into different lipid species (n = 3). (G) Primary hepatocytes were transduced with ATGL knockdown or overexpression adenovirus as described above and then exposed to 500 μM of oleate for 30 h, at which time cells were washed, fixed and stained with Oil Red O followed by imaging with light microscopy at 20× magnification (representative of 3 experiments). Data are presented as means ± SEM. CE, cholesteryl ester; DAG, diacylglycerol; PL, phospholipid. *P< 0.05 vs control shRNA group. ^#P<0.05 vs pulse period.