Fig 2: Effect of TgHsp in 3T3−L1 cells on lipid accumulation and lipolysis. (A) ORO staining on Days 0 and 8 of differentiation in 3T3-L1 and TgHsp cells. Absorbance of extracted ORO solution was measured at 492 nm. (B) Expression of adipogenesis biomarkers in 3T3-L1 and TgHsp cells. (C) Cell culture media were assayed for free glycerol. HSL activation was assessed on Days 0 and 8 of differentiation. The data are presented as means ± SDs of at least three replicates. Means with different superscripts are significantly different at p < 0.05 by Duncan’s multiple range test.

Fig 3: PRIP-mediated protein phosphatase dephosphorylates HSL Ser660 and Ser563 and regulates the release of fatty acids and glycerol in adipose tissue.(A, B) Effect of protein phosphatase activity on HSL Ser660 phosphorylation after combination stimulation by adrenaline (Adrn) and OA. A set of typical images from three independent experiments is shown (A). (C–E) Analysis of the dephosphorylation process of HSL. White adipose explants were stimulated with 5 µM adrenaline for 30 min and then cultured with a ß3-adrenergic receptor antagonist (SR59230A, 20 µM) and a PKA inhibitor (PKI 14–22, 10 µM) for the indicated times to monitor the HSL dephosphorylation state. The dephosphorylation assay was also performed in the presence of 1 µM OA for 30 min (lane: OA30). Samples were homogenized, and western blot analysis was conducted using anti-p-Ser660 and anti-p-Ser535 HSL antibodies. A typical image from three independent experiments is shown. WT, white bar; DKO, black bar in (D, E). (F, G) Measurement of NEFA (F) and glycerol (G) released from cultured adipose tissues after combination stimulation by Adrn and OA (n = 3 experiments). OA: 1 µM okadaic acid, Adrn: 1 µM adrenaline. The data represent mean ±SEM. *P<0.05, **P<0.01, and n.s. (not significant).

Fig 4: 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 5: Two pathways of lipolysis. (A). The canonical pathway of adipocyte lipolysis. The canonical lipolytic pathway is shown on top and is diagrammed in red below. The first step is TG hydrolysis by ATGL. Next, active phosphorylated HSL on the lipid droplet surface cleaves DGs, maintaining a low level of DGs at the lipid droplet surface. ATGL has transacylation capacity but, under conditions of low DG concentrations, this accounts for a negligible fraction of acylglycerol flux. Monoglycerols are cleaved by MGL. (B) The transacylation pathway of adipocyte lipolysis in fasting HSL-deficient WAT. In this pathway, ATGL catalyzes both TG hydrolysis and transacylation of DGs. ATGL-mediated TG hydrolysis in HSL-deficient adipocytes results in high levels of DGs. DGs are less hydrophobic than TGs and hence concentrate at the lipid droplet surface, near ATGL. From two DG molecules, transacylation produces one MG (a substrate for MGL) and one TG that can be cleaved by the lipase activity of ATGL. In HSL deficiency, the high local concentration of DGs is predicted to enhance transacylation by ATGL, which mediates a major fraction of TG turnover and acylglycerol flux under these conditions. DGs are shown in blue; TGs, in dark grey.