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).