Although we could demonstrate a tendential increase in phosphor-AMPK in the livers of CB1?/?, AMPK was not activated in HBs/CB1?/? mice (Supplementary Fig.?5C). expression was associated with suppressed lipogenesis and triglyceride (TG) synthesis and enhanced autophagy as shown by increased colocalization of LC3B with lysosomal-associated membrane protein 1 (LAMP1) in HBs/CB1?/? mice. The induction of autophagy was further supported by the increased expression of LAMP1 in CB1?/? and HBs/CB1?/??mice. LAMP1 and PLIN2 were co-localized in HBs/CB1?/? indicating autophagy of cytoplasmic lipid droplets (LDs) i.e., lipophagy. Lipolysis of lipid droplets was additionally indicated by elevated expression of lysosomal acid lipase. In conclusion, these results suggest that loss of CB1 signaling leads to reduced PLIN2 abundance, which triggers lipophagy. Our new findings about the association between CB1 signaling and PLIN2 may stimulate translational studies analyzing new diagnostic and therapeutic options for NAFLD. for 10?min. The supernatant was transferred to a new vial and dried under a nitrogen gas, resuspended in an appropriate volume of chloroform/methanol 2:1(v/v) and applied in equal amount onto pretreated, prewashed TLC plates. The standard (Nonpolar TLC standard, Bio trend Chemikalien, cat no.1130) and samples were applied onto the TLC plates and chromatographically separated with the solvent system containing hexane/diethylether/acetic acid BN82002 (90:10:1(v/v/v) to the top of the plate. Detection and quantification was performed with charring method using N/10 sulfuric acids. Image was captured by gel doc analyzer (Biometra, G?ttingen, Germany). Quantitative triglyceride estimation Quantitative estimation of triglycerides from the liver samples was performed as per the protocol provided in Abcam Triglycerides estimation kit (ab65336). Real-time PCR RNA isolation from the liver samples were performed using Direct-zol RNA extraction kit (Zymo research, Cat no. R2072). cDNA synthesis was done using iScript cDNA synthesis kit (BIO-RAD Cat no. 1708891) qRT-PCR was performed as described previously [31]. Primers were ordered from Microsynth (Switzerland). qPCR data were analyzed using Ct method [32]. Primers used: PLIN2 forward. 5-GACCTTGTGTCCTCCGCTTAT-3, reverse-5-CAACCGCAATTTGTGGCTC-3; -actin forward: 5-CAG CTT CTT TGC AGC TCC TT-3, reverse: 5-AGT CCT TCT GAC CCA TTC CC-3. Cell culture and treatment with rimonabant The AML12 liver cell line (kind gift from Prof. Ralf Weiskirchen, Aachen) was cultured up to 90% confluency for all the assay inside a 1:1 mixture of Dulbeccos revised Eagles medium and Hams F12 medium (Gibco, Grand Island, USA) with 0.005?mg/ml insulin, 0.005?mg/ml transferrin, 5?ng/ml selenium (ITS combination, Gibco), 40?ng/ml dexamethasone (Sigma-Aldrich, St. Louis, MO), and supplemented with 10% fetal bovine serum (Gibco) at 37?C inside a humidified atmosphere with 5% CO2. For in vitro assay, rimonabant and methanandamide were dissolved in DMSO and applicated in presence of 2% FBS for 48?h in presence and absence of oleic acid (OA) as per the indicated doses for each assay. For induction of steatosis in vitro, cells were treated with OA (Sigma-Aldrich, St. Louis, MO) at FZD3 a final concentration of?200?M. Statistics All measurements were performed in technical triplicates. All results are indicated as means??standard error of the mean. Significant variations between four organizations were determined by one-way ANOVA with Tukeys multiple BN82002 comparisons test using to compare test groups. Variations between two organizations were determined by MannCWhitneys test. All statistical analyses were performed with GraphPad Prism software (v5.0; GraphPad Software Inc., San Diego, CA) and * em p /em ? ?0.05, was considered statistically significant. Results CB1 knockout (CB1KO) reduced hepatic triglycerides and lipid droplet build up in HBs transgenic mice Maintenance of body weight and energy homeostasis depends on the coordinated rules of appetitive behavior and peripheral energy rate of metabolism [33]. CB1?/? and HBs/CB1?/??mice showed decreased body and liver weight compared with WT and HBs mice (Fig.?1a, b). As leanness has been associated with reduced steatosis, the effect of CB1KO on hepatic extra fat was analyzed. Oil reddish staining visualized improved hepatic steatosis in HBs transgenic mice (Fig.?1c). The BN82002 CB1KO diminished the size of hepatic LDs in HBs/CB1?/??mice. Quantification of liver TGs from liver samples of 52-week-old mice further shown the reduction of TGs in CB1?/? and HBs/CB1?/? compared with WT and HBs mice (Fig.?1d). Open in a separate windowpane Fig. 1 CB1 receptor knockout decreased body weight and hepatic lipid build up in mice. a Bar diagram showed significant reduction of body weight in 52-week-old CB1?/? and HBs/ CB1?/? mice in comparison with WT and HBs. Depicted are means??SEM, em n /em ?=?8C12. b Liver weight was reduced in CB1?/?-mice and enhanced in HBs transgenic mice. c Representative Oil reddish O staining of a 52-week-old mouse liver. d Liver triglyceride analysis shown significant reduction of TGs level in the liver of 52-week-old CB1?/? and HBS/CB1?/? mice. em n /em ?=?7C9. e Representative thin.