The Role of PDE3B Phosphorylation in the Inhibition of Lipolysis by Insulin - PubMed (original) (raw)

The Role of PDE3B Phosphorylation in the Inhibition of Lipolysis by Insulin

Lisa M DiPilato et al. Mol Cell Biol. 2015 Aug.

Abstract

Inhibition of adipocyte lipolysis by insulin is important for whole-body energy homeostasis; its disruption has been implicated as contributing to the development of insulin resistance and type 2 diabetes mellitus. The main target of the antilipolytic action of insulin is believed to be phosphodiesterase 3B (PDE3B), whose phosphorylation by Akt leads to accelerated degradation of the prolipolytic second messenger cyclic AMP (cAMP). To test this hypothesis genetically, brown adipocytes lacking PDE3B were examined for their regulation of lipolysis. In Pde3b knockout (KO) adipocytes, insulin was unable to suppress β-adrenergic receptor-stimulated glycerol release. Reexpressing wild-type PDE3B in KO adipocytes fully rescued the action of insulin against lipolysis. Surprisingly, a mutant form of PDE3B that ablates the major Akt phosphorylation site, murine S273, also restored the ability of insulin to suppress lipolysis. Taken together, these data suggest that phosphorylation of PDE3B by Akt is not required for insulin to suppress adipocyte lipolysis.

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Figures

FIG 1

FIG 1

Insulin suppresses glycerol release in both 3T3-L1 and brown adipocytes independently of Akt activity. (A) Glycerol release was measured at the basal state and upon stimulation with 0.3 nM isoproterenol (Iso) in the presence or absence of 25 nM insulin (Ins), 10 μM AktiVIII, or 10 μM MK-2206 for 1 h in both 3T3-L1 and brown adipocytes. Data from 3T3-L1 (n = 3) and brown adipocytes (n = 6) were normalized to those with Iso alone. Symbols indicate significant differences (P < 0.05) for insulin suppression (*) and for Iso alone versus Iso plus MK-2206 (#). (B) Cell lysates from the experiment for which results are shown in panel A were assessed for Akt phosphorylation by Western blotting. (C) The uptake of 2-deoxyglucose into brown adipocytes was measured upon stimulation with 100 nM insulin in the presence or absence of AktiVIII or MK-2206 at the same concentrations used in the experiment for which results are shown in panel A. Values represent the results of 3 experiments. (D) Glycerol release from WT brown adipocytes upon stimulation with 0.3 nM Iso in the presence or absence of 25 nM insulin, 10 μM rolipram (PDE4i), 10 μM cilostamide (PDE3i), or a combination of PDE3i and PDE4i. Data were normalized to those for Iso with no inhibitor. Values represent the results of 3 to 7 experiments. Symbols indicate significant differences (P < 0.05) for insulin suppression (*) and between Iso with a PDE inhibitor and Iso alone (#). All data are presented as means ± standard errors of the means. Statistical analysis was performed using one-way ANOVA and Tukey's posttest.

FIG 2

FIG 2

Characterization of WT and _Pde3b_-KO brown adipocytes. (A) Immunoblot for PDE3B protein in crude membrane lysates harvested from WT and _Pde3b_-KO brown adipocytes. Na/K ATPase, a membrane-bound protein, was used as a loading control. (B) A radioactive assay for PDE activity was performed on membrane lysates. For the PDE3-specific activity, 10 μM cilostamide was added to the reaction mixture for both WT and _Pde3b_-KO adipocytes, and the activity was subtracted from total activity. Other cAMP-PDE activity was calculated based on inhibition after the addition of 100 μM IBMX. (C) The protein expression of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid binding protein 4 (FABP4), adipose triglyceride lipase (ATGL), and hormone-sensitive lipase (HSL) for the two genotypes was compared via Western blot analysis. (D and E) Triglyceride (TG) content (n = 3) (D) and glucose transport (n = 3) (E) were measured in WT and _Pde3b_-KO adipocytes. All data are presented as means ± standard errors of the means.

FIG 3

FIG 3

_Pde3b_-KO brown adipocytes have impaired insulin-mediated suppression of lipolysis. (A and B) Glycerol release from WT (solid lines) and _Pde3b_-KO (dashed lines) brown adipocytes without stimulation or upon stimulation with 0.3 nM or 3 nM isoproterenol (Iso) (A) or CL 316,243 (B) in the presence (×) or absence (○) of 25 nM insulin. Data were normalized to a 0.3 nM dose in _Pde3b_-KO adipocytes. Values represent results for 3 to 7 experiments. Asterisks indicate significant differences (P < 0.05) for insulin suppression in WT cells. (C) Representative immunoblots for tubulin and phosphorylated and total perilipin 1 (PLIN1), HSL, and Akt in whole-cell lysates harvested at the termination of glycerol release assays. (D) Intracellular cAMP measurement in WT and _Pde3b_-KO adipocytes upon stimulation with 1 μM forskolin (Fsk) with or without the addition of 25 nM insulin. Data were normalized to basal values of WT cells. Values represent results for 5 experiments. All data are presented as means ± standard errors of the means. Statistical analysis was performed using one-way ANOVA with Tukey's posttest.

FIG 4

FIG 4

Phosphorylation of PDE3B at putative Akt and PKA sites is not required for the antilipolytic effect of insulin in brown adipocytes. (A) Immunoblot for PDE3B protein in crude membrane lysates harvested from WT and _Pde3b_-KO brown adipocytes and from KO adipocytes overexpressing the indicated human PDE3B proteins. Na/K ATPase was used as a membrane loading control. (B) PDE3 activity in membrane fractions. Values represent results for 2 to 3 independent experiments. *, P < 0.001. (C) Representative immunoblot of the lipid droplet fraction and whole-cell lysate (WCL) for PDE3B using perilipin 1 and HSP90 as lipid droplet and cytosolic markers, respectively. For “+WT+Ins” samples, KO plus WT adipocytes were treated with 100 nM insulin for 15 minutes prior to fractionation. (D and E) Glycerol release was measured without Iso or upon the addition of 0.3 or 3 nM Iso (D) or 3 nM Iso (E) in the presence or absence of 25 nM Ins. Data are expressed as percentages of the response of WT (D) or _Pde3b_-KO (E) adipocytes upon stimulation with 3 nM Iso. Vec, vector; NT, no treatment (basal). Values represent results for 3 experiments. Symbols indicate significant differences (P < 0.05) for insulin suppression (*), for comparison with the WT group with 3 nM Iso (#), for comparison with the KO-plus-S318A group with 3 nM Iso (^), or for comparison with the KO-plus-2SA group with 3 nM Iso (+). (F) Representative immunoblots for phosphorylated perilipin 1 (PLIN1) and HSL in whole-cell lysates harvested at the end of the glycerol release assays. All data are presented as means ± standard errors of the means. Statistical analysis was performed using one-way ANOVA and Tukey's posttest.

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