Fig 2: ETC-1002 mediates effects on lipid metabolism independently of AMPK ß1.Apoe-/- and Apoe-/-/Ampkß1-/- (DKO) mice were fed a HFHC diet for 12 weeks with or without ETC-1002 targeted to achieve a 30 mg kg-1 per day dose and (a) total and phosphorylated (T172) AMPKa and total and phosphorylated (S79) ACC measured in livers from Apoe-/- (n=4) and DKO (n=3). (b) Slc27a2 (ACSVL1) expression in liver and tibialis anterior from untreated Apoe-/- mice (n=6). (c) Representative fast performance liquid chromatography tracings from chow- and HFHC diet-fed±ETC-1002 Apoe-/- and DKO mice. Plasma (d) very-low-density lipoprotein (VLDL), (e) LDL, (f) HDL (n=4 for chow-fed mice and 6 for remaining treatment groups) and (g) total cholesterol (n=10), liver (h) cholesterol and (i) triglycerides (n=4 for chow-fed and 6 for remaining treatment groups) determined at the end of study. (j) RER was measured over 24 h following 10 weeks on diet and mean RER during (k) dark (19:00–07:00) and (l) light (07:00–19:00) cycles calculated. (m) Total lipid synthesis in primary hepatocytes isolated from WT and AMPK ß1 KO mice treated for 4 h with the indicated concentration of ETC-1002, A-769622 or vehicle (CON) in the presence of 3H-acetate (n=3–8 independent experiments performed in duplicate). (n) Srebf2, Ldlr, Pcsk9, Hmgr, Acly, Srebf1c and Slc27a2 gene expression (n=4 chow-fed and 5 for remaining treatment groups) determined at the end of study. (o) Representative images for immunohistochemistry staining of LDLR in frozen liver sections from ETC-1002-treated Apoe-/- and DKO mice (20 × ); scale bar, 100 µM. Data are expressed as mean±s.e.m. Multiple comparisons were made using an *one-way (within Apoe-/- treatment groups) or #two-way ANOVA (between Apoe-/- and DKO treatment groups); Bonferroni's multiple comparisons test; *and #P<0.05.

Fig 3: CD36 regulates ER-to-Golgi transport of SREBP1. A: The protein level of SREBP1 in Golgi and ER of organelle fractions from fl/fl and LKO mice was analyzed by immunoblotting. B: Nuclear location of SREBP1 (red-TRITC) was determined by immunofluorescence staining and confocal analysis in primary hepatocytes from fl/fl and LKO mice. Scale bars, 25 µm. C: Localization of SREBP1 (green-FITC) in the Golgi (red-TRITC) was determined by immunofluorescence staining and confocal analysis in primary hepatocytes from fl/fl and LKO mice. Scale bars, 25 µm. D: The protein levels of SREBP1 of cytoplasmic and nuclear fractions from NC and CD36OE cells were analyzed by immunoblotting. E: Nuclear location of SREBP1 (red-TRITC) was determined by immunofluorescence staining and confocal analysis in NC and CD36OE cells. Scale bars, 25 µm. F: The localization of SREBP1 (green-FITC) in the Golgi (red-TRITC) was determined by immunofluorescence staining and confocal analysis in NC and CD36OE cells. Scale bars, 25 µm. G–H: HepG2 cells were co-transfected with stably expressing HA-tagged full-length SREBP1 (FL-SREBP1) and FLAG-tagged-CD36. The precursor and active nuclear form of SREBP1 were visualized using HA antibody (G) and the SREBP1 downstream targets, including ACCa, FASN, SCD1, and ACLY, were analyzed by immunoblotting (H). Quantitation of protein levels was shown in the bottom. n = 4, *P < 0.05 vs. SREBP1OE group without CD36OE.

Fig 4: Interdependence of Gal-1 and SCD1 expression in human HCC cell lines suggests regulation of lipid synthesis. (A) siRNA-mediated Gal-1 silencing elicited a decrease in SCD1 protein levels in the human HCC cell lines PLC/PRF/5, HLF, and Snu182 as exemplified in western blot analyses (left panel). Molecular weights of observed bands are noted. Quantification of yielded bands was performed (right panel). (B) In contrast, siRNA-mediated Gal-1 silencing did not yield a regulation of other important lipogenic enzymes, such as ACLY, FASN, and ACAC. At the same time, a tendency for proliferator-activated receptor gamma (PPAR?) reduction could be observed in PLC and Snu182 cell lines. n(PLC/PRF/5) = 3 cell culture experimental repeats with 3 cell culture replicates + 1 cell culture experimental repeat with 6 cell culture replicates. n(HLF, Snu182) = 3 cell culture experimental repeats with 3 cell culture replicates. (C) 20 µm OTX008 treatment for 24 h likewise led to a decrease in SCD1 in PLC/PRF/5 and HLF cell lines as demonstrated by Western blot (left panel). Molecular weights of observed bands are noted. Quantification of yielded bands was performed (right panel). Intensities were normalized to the mean of control for all tested proteins. Tukey method box-and-whisker plots are displayed (right panels). Mann–Whitney tests were employed; ns, not significant. All pairwise comparisons in the right panel in (B) were not significant, except for the one displayed.

Fig 5: Hepatic lipogenic enzymes are significantly down-regulated as ground squirrels enter into hibernation.A) Representative western blot of lipogenic genes fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and ATP-citrate lyase (ACL) in livers from summer active (SA), fall transition (FT), interbout arousal (IBA), entering torpor (Ent), early torpor (ET), late torpor (LT), arousing from torpor (Ar), and Spring (Sp) animals. B-D) Western blot densitometry from all ground squirrels analyzed, n=6 animals per physiological stage, small letters indicate significantly different groups.