Fig 2: Loss of skeletal muscle OGT alters global muscle metabolism. (A) Metabolome heat map of gastrocnemius muscle from clamped mice. Metabolites were measured using LC-MS. (B) Glycolytic metabolites from metabolome. Fructose 1 6-bisphosphate (Fru1,6P) and phosphoenolpyruvate (PEP). (C) Pyruvate dehydrogenase (PDH) activity in the gastrocnemius muscle was assayed with the substrate [1–14C] pyruvate by measuring enzyme-catalyzed release of 14CO2. (D) Immunoblot of PDH phosphorylation at pS293 and pS300 in gastrocnemius from clamped mice. (E) Illustration of metabolites and regulation of glycolysis and hexosamine biosynthetic pathway. (F) UDP-GlcNAc levels from the metabolomics analysis. (G) Immunoblot of GFAT1 in gastrocnemius from clamped mice. (H) Acetylcarnitine levels from the metabolomics analysis. (I) Pantothenic acid levels from the metabolomics analysis. (J) Immunoblot of HSL and HSL pS563 in gastrocnemius from clamped mice. (K) Immunoblot of ATGL in gastrocnemius from clamped mice. (L) Complete oxidation of [1–14C]-palmitic acid to CO2 in gastrocnemius muscle of WT and mKO mice. (M) Incomplete oxidation of [1–14C]-palmitic acid to acid-soluble metabolites (ASM) in gastrocnemius muscle. Data represent means ± SEM from n = 8–10, 18-week-old (Panel A, B, C, F, G, H, I, J, and K) and n = 10, 16-week-old (Panel D, L, and M) male mice in each genotype. *p < 0.05; **p < 0.01; ***p < 0.001 compared with WT mice.

Fig 3: Increased phosphorylation of HSL and perilipin in PRIP-DKO adipocytes, and altered PRIP distribution after starvation.(A) Comparison of lipid metabolism-related proteins. Whole lysates obtained from WT and PRIP-DKO (DKO) epididymal fat pads were analyzed for western blotting using the indicated protein-specific antibodies. ß3AR; ß3-adrenergic receptor. The differences are not statistically significant. (B–F) Altered subcellular distribution of HSL, PRIP1, and PRIP2, and altered phosphorylation of HSL and perilipin in epididymal white adipose tissues prepared from WT and PRIP-DKO mice maintained under fed (B) and fasted (D) conditions. Whole lysates were fractionated by centrifugation into a floating fat-cake fraction, a supernatant fraction, and a pelleted membrane fraction. Western blotting was performed using the fat-cake (fat) and supernatant (sup) fractions. Indicated molecules were detected using specific antibody. Each image is a typical example from three experiments. Perilipin and ß-actin are lipid droplet and cytosol marker proteins, respectively. Subcellular distribution of HSL under fed and fasted conditions is shown in (C) and (E), respectively. Subcellular distribution of PRIP1 and PRIP2 in fed (upper panel) and fasted (lower panel) conditions is shown in (F). The black and white bars represent the amount of HSL (C, E) and PRIP (F) in the fat and sup fractions, respectively. The amount of total HSL (C, E) and PRIP (F) in the fat and sup fractions is expressed as 100%. The data represent mean ±SEM. **P<0.01 and n.s. (not significant) versus the corresponding WT value.

Fig 4: Western blot analyses of thermogenic and lipolytic proteinWestern blot analysis of beige adipocyte treated with PBS- or LPS-treated conditioned medium (CM) of BMDM, followed by a stimulation of 50 nM NE for 8 h. anti-UCP1 (1:1,000), anti-HSP90a/ß (1:10,000), anti-phospho-perilipin 1 (1:1,000), anti-perilipin 1 (1:10,000), anti-phospho-HSL (1:1,000), anti-HSL (1:1,000), goat anti-mouse IgG:HRP (1:10,000), goat anti-rabbit IgG:HRP (1:10,000).

Fig 5: Pharmacological inhibition of hKIAA1363 impairs RE degradation in LX-2 cells and primary human HSCs upon serum starvation. A: For in vitro REH activity assay, cell lysates (1,000 g supernatant) from HepG2 and LX-2 cells were prepared, and protein expression was determined by Western blotting. Cell lysates (1,000 g supernatant) of (B) HSC LX-2 and (C) primary human HSCs were incubated with RP (300 μM) as substrate. RP was emulsified with PC (300 μM) in potassium phosphate buffer (100 mM, pH 7.5) containing 2% FA-free BSA. Small-molecule inhibitors or DMSO as solvent control were added to cell lysates as indicated: Orlistat (20 μM) and JW480 (10 μM). Lipids were n-hexane extracted, and ROH content was analyzed by HPLC-FD. D: LX-2 and (E) primary human HSCs were seeded in 12-well plates and cultured in DMEM supplemented with 1% or 10% FCS and antibiotics (=basal), respectively. Then, LX-2 and primary human HSCs were incubated with DMEM containing 1% or 10% FCS, 40 μM or 20 μM ROH, and 300 μM or 100 μM oleic acid for 24 h (=loaded/pulse), respectively. Cells were incubated with DMEM low glucose (1 g/l) containing 2% FA-free BSA for 8 h (=serum starvation/chase). Serum-starvation media contained small-molecule inhibitors or DMSO as solvent control as indicated: Orlistat (20 μM) and JW480 (10 μM). Cellular neutral lipids were extracted with n-hexane:2-propanol (3:2, v/v). RP content was determined by HPLC-FD and normalized to milligram cell protein. Data are mean + SD and representative for two to three independent experiments (n = 3). Statistically significant differences were determined by Student's unpaired t-test (two-tailed: ∗∗P < 0.01; ∗∗∗P < 0.001, between control and inhibitors; ###P < 0.001 between “loaded” and “serum-starvation DMSO”). n.d., nondetectable.