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. 2019 Apr 16;14(4):e0214606.
doi: 10.1371/journal.pone.0214606. eCollection 2019.

PID1 alters the antilipolytic action of insulin and increases lipolysis via inhibition of AKT/PKA pathway activation

Chunyan Yin  1 , Wei Hua Liu  1 , Yuesheng Liu  1 , Li Wang  1 , Yanfeng Xiao  1
Affiliations

PID1 alters the antilipolytic action of insulin and increases lipolysis via inhibition of AKT/PKA pathway activation

Chunyan Yin et al. PLoS One. .

Erratum in

Abstract

Purpose: The aim of this study was to investigate the effect of phosphotyrosine interaction domain containing 1 (PID1) on the insulin-induced activation of the AKT (protein kinase B)/protein kinase A (PKA)/hormone-sensitive lipase (HSL) pathway and lipolysis.

Methods: Sprague-Dawley rats were fed either chow or a high-fat diet (HFD). The levels of insulin, glycerol, free fatty acids (FFAs) and PID1 mRNA expression were measured in the 2 groups. Furthermore, we examined the role of PID1 in the regulation of the AKT/PKA/HSL cascade and lipolysis in the 3T3-L1 cell line.

Results: Adipose tissue from HFD rats exhibited elevated PID1 expression, which showed a positive correlation with insulin levels and lipolysis. In 3T3-L1 adipocytes, we found that the antilipolytic effect of insulin is mediated by AKT and that phosphorylated AKT results in the promotion of PDE3B expression, the dephosphorylation of PKA and HSL and the suppression of glycerol release. However, overexpression of PID1 and treatment with 1 μM isoproterenol and 100 nM insulin for 24 h resulted in an increased release of glycerol and a noticeable inhibition of AKT phosphorylation, PDE3B expression and the phosphorylation of PKA/HSL in 3T3-L1 cells. In contrast, knockdown of PID1 and treatment with the above reagents inhibited lipolysis and activated the phosphorylation of AKT, which resulted in the dephosphorylation of PKA and HSL.

Conclusions: Our findings indicate that PID1 in adipose tissue increases lipolysis by altering the antilipolytic action of insulin. This suggests that PID1 may represent a new therapeutic target to ameliorate adipocyte lipolysis and hence improve insulin sensitivity.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Body weight, total fat, insulin, glycerol, blood glucose, triglyceride, cholesterol and free fatty acid (FFA) levels in ninety-six Sprague-Dawley rats fed either chow (normal diet, ND) or a high-fat diet(HFD) at 8, 16, 20, and 24 weeks (eight rats from the ND group and sixteen rats from the HFD group were randomly selected at each time point).
Insulin bound to the receptors of fat cells, and the expression of PID1 in white adipose tissue (WAT) was determined. (a)Body weights of rats in the two groups. (b)Total fat (perirenal and epididymal fat pads) of rats in the two groups. (c)Plasma levels of insulin in the two groups. (d) Plasma levels of glycerol in the two groups. (e) Plasma levels of glucose in the two groups. (f) Plasma levels of FFAs in the two groups. (g) Plasma levels of triglycerides in the two groups. (h) Plasma levels of cholesterol in the two groups.(I)Relative mRNA expression of PID1 in WAT of the HFD and ND groups; the control ratio was normalized to 1. (j)Insulin binding to perirenal adipocytes obtained from HFD and ND rats. Isolated fat cells were incubated with mono-125I-(Tyr A14) insulin with or without various concentrations of unlabeled insulin. Specific insulin binding was determined. Data are presented as the mean±SEM.
Fig 2
Fig 2. Insulin inhibits isoproterenol-stimulated lipolysis in 3T3-L1 adipocytes via the AKT/PKA/HSL pathway.
We treated differentiated adipocytes with 100 nM insulin and 1 μM isoproterenoland examined the effects on lipolysis(in triplicate). (a)Western blot analyses ofAKT, PDE3B, PKA, HSL and ATGL protein levels in the insulin, isoproterenol and control groups. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (b)The phosphorylated protein/total protein ratios of AKT, PKA and HSL were calculated, and the control ratio was normalized to 1. (c)Western blot analyses of PDE3B protein levels in the insulin, isoproterenol and control groups. Differentiated 3T3-L1 adipocytes were treated with different doses of insulin (1, 10,100 nM) and 1 μM isoproterenol for 24 h(in triplicate). (d) Cellular triglycerides were stained with Oil red O. Bar, 50 μm. (e) The amount of lipids was quantified by the Oil red O staining technique. (f)The concentration of glycerol in the medium was detected in different groups(g, i)Western blot analyses of AKT, PDE3B, PKA, HSL and ATGL protein levels in different groups. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (h) The phosphorylated protein/total protein ratios were calculated, and the control ratio was normalized to 1.(j)cAMP levels in different groups. (k) The expression of PDE3Bwas determined in different groups by Western blot.
Fig 3
Fig 3. The effects of the depletion of AKT or PKA on the regulation of lipolysis by insulin.
Differentiated adipocytes were transfected with AKT siRNAortreated with the PKA inhibitor and incubated in the presence of 100 nM insulin and 1 μM isoproterenol for 24 h (in triplicate). (a-b)Protein expression of PDE3B, PKA, HSL and ATGL in 3T3-L1 adipocytes transfected with AKT siRNA. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (c-d)Relative protein expression of PDE3B and the phosphorylated protein/total protein ratios for PKA, HSL, and ATGL; the control ratio was normalized to 1. (e)Determination of cAMP levels. (f) Glycerol released into the medium after transfection with an siRNA targeting AKT. (g)Western blot analyses of HSL and phosphorylated HSL (p-HSL) protein expression in 3T3-L1 adipocytes treated with different doses of the PKA inhibitor. (h) The phosphorylated protein/total protein ratio for HSL in 3T3-L1 adipocytes treated with different doses of the PKA inhibitor. (i) Glycerol released into the medium after treatment with different doses of the PKA inhibitor. *P<0.05; **P<0.01.
Fig 4
Fig 4. Effects of PID1 expression on lipolysis and the phosphorylation of AKT/PDE3B/PKA/HSL signaling molecules and ATGL.
Preadipocytes were subjected to PID1knockout or upregulation and allowed to differentiate into 3T3-L1 adipocytes; these cells were treated with 1 μM isoproterenol and 100 nM insulin for 24 h(in triplicate). (a-b)Immunofluorescence analysis was performed to assess the expression of the PID1 gene in empty vector cells, PID1-overexpressing cells, and control cells.(c)RT-PCR analyses of the mRNA expression of PID1 in empty vector cells, PID1-overexpressing cells, and control cells. (d)Glycerol released into the medium after the upregulation of PID1.(e-f)Protein expression of AKT, PDE3B, PKA, HSL and ATGL in empty vector cells, PID1-overexpressing cells, and control cells. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control.(g)The phosphorylated protein/total protein ratios for AKT, PKA, HSL, and ATGL in 3T3-L1 adipocytes after transfection with the PID1 overexpression plasmid. (h)The expression of PDE3B was determined by Western blot after the PID1 overexpression plasmid. (i-j)Protein expression of AKT, PDE3B, PKA, HSL and ATGL after transfection with PID1 shRNA. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (k)RT-PCR analyses of mRNA after transfection with PID1 shRNA. (l) Glycerol was released into the medium after knockdown of PID1. (m)Phosphorylated protein/total proteinratios for AKT, PKA, HSL, and ATGL after PID1 knockdown. (n)The expression of PDE3B was determined by Western blot after transfection with PID1 shRNA.
Fig 5
Fig 5. A hypothetical model showing how PID1 promotes lipolysis by regulating the AKT/PDE3B/PKA/HSL signaling pathway, which is supported by the results of this study.
(a)Insulin inhibits isoproterenol-induced lipolysis by increasing PDE3B expression via AKT. The elevation of PDE3B catalyzes the hydrolysis of cAMP, which reduces the cellular level of cAMP. The lowering of cAMP further dephosphorylates PKA and thereby results in a decrease in hormone-sensitive lipase (HSL) and lipolysis. (b)PID1 promotes lipolysis and induces a noticeable inhibition of the phosphorylationof AKT and PDE3B expression, which further increases cAMP levels and the phosphorylation of PKA and HSL, leading to increased lipolysis.
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This work was supported by the Nature Science Foundation of China No. 81172689 to YFX and No. 81803262 to CY.