Figure 1.
Impairment of fat metabolism in PRIP-DKO mice.
(A) Body weight changes in WT (open circle) and PRIP-DKO (DKO, closed triangle) male mice monitored 20 weeks after birth (n = 5 mice for both genotypes). (B) Comparison of the epididymal fat pad of WT and PRIP-DKO mice at the age of 10-week-old. (C) Comparison of wet tissue weight (representing major organs) obtained from WT and PRIP-DKO male mice at the age of 18-week-old. The graph shows percent of tissue weight against body weight [26.6±0.8 g (WT), 23.4±0.6 g (PRIP-DKO); n = 5 mice per genotype]. WAT, white adipose tissue; BAT, brown adipose tissue; M. (Musculus) rectus femoris; M. (Musculus) gastrocnemius. (D) Comparison of adipocyte size. Graph shows average size of cells assessed using hematoxylin and eosin-stained sections (n = 3 sections for both genotypes). Scale bar: 100 µm. (E, F) Concentration of plasma leptin (n = 5 mice for both genotypes) and adiponectin (n = 7 mice for both genotypes) at the age of 10–12 weeks. Mice were fed a standard chow ad libitum. (G, H) Plasma NEFA and glycerol concentration at the age of 10–12 weeks under conditions of (G) ad libitum feeding (n = 3 mice for both genotypes) and (H) fasting for 8 h (n = 4 mice for both genotypes). The data represent mean ±SEM. *P<0.05 and **P<0.01 versus the corresponding WT value.
Figure 2.
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. β3AR; β3-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 β-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.
Figure 3.
Alterations in subcellular PRIP distribution and HSL phosphorylation in explant adipose tissues.
(A, B) Adrenaline stimulation (Adrn) induces HSL translocation from cytosol fraction (sup) to fat-cake fraction (fat) in WT and PRIP-DKO explant adipose tissues. The bars for fat (black) and sup (white) in B represent the floating fat-cake and supernatant fractions, respectively. The amount of total HSL in fat and sup fractions is expressed as 100%. All the data represent mean ±SEM. **p<0.01 and n.s. (not significant) versus the corresponding WT value. Altered phosphorylation of HSL at Ser563 and Ser660 and perilipin at Ser492 in adipose explant lipolysis assays with or without stimulation by 1 µM adrenaline are also shown in (A). A representative image is shown; similar images were obtained from three independent experiments. (C, D) PRIP translocation to the lipid droplet fraction in response to 1 µM adrenaline (Adrn) stimulation. The bars for fat (black) and sup (white) in (D) represent the floating fat-cake and supernatant fractions, respectively. The amount of total PRIP in fat and sup fractions is expressed as 100%. Perilipin and β-actin are lipid droplet and cytosol marker proteins, respectively. A representative image is shown; similar images were obtained from three independent experiments (C). The data represent mean ±SEM. **P<0.01 and ***P<0.001 versus the corresponding values of PRIP1 and PRIP2 without adrenaline stimulation.
Figure 4.
PP2A and PP1 are translocated from the cytosol to lipid droplets in adipocytes in response to adrenaline stimulation.
(A–C) Translocation of PP2A (A, B) and PP1 (A, C) to the lipid droplet fraction in response to stimulation with 1 µM adrenaline (Adrn). The bar graph shows the amount of PP2A (B) and PP1 (C) in fat (black) and sup (white) fractions, respectively. A typical image from four independent experiments is shown (A). (D, E) Phosphatase activity in floating fat-cake fraction. Phosphatase activities of PP2A (D, n = 4) and PP1 (E, n = 5) were measured using floating fat-cake fractions from WT and PRIP-DKO explants treated with or without 1 µM adrenaline. The data represent mean ±SEM; *P<0.05, **P<0.01, ***P<0.001 and n.s. (not significant) versus the corresponding values for paired bars connected by solid and dotted lines (B–E).
Figure 5.
PRIP and PP2A are translocated to the periphery of lipid droplets in COS7 cells after adrenaline stimulation.
(A) COS7 cells cultured with oleic acid with (+) or without (–) adrenaline stimulation (5 µM). Two sets of representative images are shown. The boxed areas of the left image (scale bar: 10 µm) are enlarged in the four right images (scale bar: 5 µm). Five similar images were obtained from three independent experiments. Arrowhead in GFP-PRIP1 and PP2A images indicates an accumulation of each signal at lipid droplet periphery. (B) Intensity profiles along the white lines in the merged images of (A). Compared with unstimulated cells (–Adrn), adrenaline-stimulated cells (+Adrn) exhibit increased fluorescence intensity corresponding to GFP-PRIP1 (green) and PP2A (blue) at the edge of the lipid droplet (red).
Figure 6.
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 β3-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).
Figure 7.
Possible mechanism by which the PRIP/protein-phosphatase complex mediates lipolysis.
Lipolysis in adipocytes is mediated by the activation of a PKA-mediated pathway. The process is regulated by lipases (HSL and ATGL) and other modulatory proteins, including perilipin, CGI-58, PP2A, and PRIP (see Discussion). The disappearance of the dotted line represents postulated situations in PRIP-DKO mice. TAG, triacylglycerol; FFA, free fatty acid; P, phosphate group; PKA, protein kinase A; HSL, hormone-sensitive lipase; PRIP, phospholipase C-related catalytically inactive protein; PP2A, protein phosphatase 2A; CGI-58, comparative gene identification 58 (abhydrolase domain-containing protein 5).