PID1 alters antilipolytic action of insulin and increases lipolysis via Inhibited the activation of AKT/PKA Pathway

Purpose The aim was to investigate the mechanism for impaired control of lipolysis in obesity by investigating the effect of PID1 on insulin-induced activation of AKT/PKA/HSL pathway and lipolysis. Methods First, PID1 expression was detected in adipose tissue and blood insulin and glycerol levels were measured in high-fat diet-induced obese rats. Next, we examined the effect of different concentrations of insulin on lipolysis and AKT/PKA/HSL pathway in 3T3-L1cells. We also investigated the role of PID1 in regulating AKT/PKA/HSL cascade and lipolysis after insulin treatment and lipofectamine over-expression. Results PID1 expression is increased in adipose tissue from HFD rat and positive correlation with insulin levels and lipolysis. In 3T3-L1 adipocytes, we found that antilipolytic effect of insulin is mediated by AKT and AKT activated by insulin can results in phosphorylation of PKA and HSL and suppresses glycerol release. However, over-expression of PID1 counteracts insulin action as indicated by glycerol releaseand reduced level of Akt phosphorylation in accordance with a decrease in the activity of insulin-dependent PKA/HSLsignaling cascade. Conclusions All together, these data showed that activation of PID1 in adipose tissue increases lipolysis by altering the antilipolytic action of insulin. This suggests that PID1 may constitute a new strategy to ameliorate adipocyte lipolysis and hence to improve insulin sensitivity.


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Obesity is an increasing global health problem that is usuallyaccompanied byinsulin resistance and type 30 2 diabetes.Elevated serum FFA levels are frequently present in obesityand there is substantial evidence 31 implicating elevated freefatty acid levels was a consequence of inappropriate lipolysis asa major 32 etiological factor for insulin resistance and type 2diabetes mellitus (T2DM) [1-2]. Thus, 33 understandingin detail the mechanism by which the impaired insulin suppresses fat cell lipolysis is 34 critical to identifying the underlying defect in resistantadipose tissue and ultimately developing 35 effective therapeutics.

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As it is well known, insulin, an important hormone for regulating glucose metabolism, is also a key obese patients were significantly reduced in adipose tissue [7]. Thus, decreased AKT/PDE3B pathway 44 activity may be a contributing factor to the diminished antilipolytic effect of insulin in obese patients.

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PID1 were subtracted from normal-weight subjects using suppression subtractive hybridization insulin regulates lipid metabolism still needs to be confirmed by further investigations.

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In this study, we examined the effects of PID1 on lipolysis in high-fat diet (HFD)-induced obese rats, 52 and further investigated the potential molecular mechanism underlying these effects using 3T3-L1 53 cells. We present evidence that PID1 alters antilipolytic action of insulin by inhibiting the AKT/PKA 54 pathway which was activated by insulin and lead to lipolysis in obese.

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Animal care and treatments 57 Ninety-six and 3-wk-old male SD rats were individually housed in a humidity controlled room with 12 58 h light/dark cycle. All the rats consumed a commercial diet for 1 week. After that, animals were 59 randomized into two dietary groups according to ratio of 1:2: chow (36, CH, 12% kcal fat) or high fat 60 (72, HF, 60% kcal fat). Eight normal diet rats and sixteen fat diet rats were randomly selected and body 61 weights were measured at the following time points: 8, 16, 20, and 24w. The experimental protocols 62 were approved by the Animal Care and Protection Committee of Xi'an Jiao tong University.

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Blood chemistries 64 All rats were killed and samples of blood were collected to measure insulin and glycerol by ELISA

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(Sigma) at 8, 16, 20 and 24w. Enzymatic assay kits (Applygen)were used for the determination of 66 serum glucose. Samples of adipose tissue were collected to detect PID1 immunohistochemistry.

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Rat adipose tissue were fixed with 4% paraformaldehyde and embedded in paraffin, and 5μm-sections 69 were prepared. After paraffin removal, tissue sections were stained with insulin and PID1 70 antibody(Abcam).

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Differentiation was induced using described protocols [10]. When more than 90% cells were fully 74 differentiated, cells were treated with varying insulin doses (1-100nmol/L).To block PKA pathway, the

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Individual colonies were isolated, propagated and PID1 was identified by RT-PCR.  Bonferroni corrections as appropriate. Differences were considered significant at P ＜0.05.

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The body weights (BWs) for high fat diet (HFD) rats and normal diet (ND) rats were measured weekly.

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Initial mean BWs of the 2groups did not differ significantly. At week 8, HFD groups had significantly   We also examined whether the expression of PID1 were altered in WAT of HFD groups compared 139 with ND groups. After 20 weeks of the diet, protein expression of PID1 exhibited a significantly 140 increased in HFD groups compared with ND groups. After 24 weeks of the HFD feeding,

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PID1expression in HFD groups showed a further increased compared with those on the 20-week diet 142 and was significantly higher than that for ND groups (Figure1g, h), Furthermore, there were positive 143 correlations between PID1 and glycerol levels in2 groups (r=0.42, P＜0.05), suggesting that PID1 may 144 play a role in lipolysis.

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To study the effect of insulin on lipid metabolism using 3T3-L1 cell lines. After 3T3-L1cells were fully 147 differentiated, we started off examining dose-dependent effects of insulin in adipocyte lipolysis.

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Inaccordance with previous studies [11], we found that insulin produced a concentration dependent 149 decrease in glycerol release with significant elevation detectable at 100 nM insulin (Figure 2a, d), and 150 the intracellular lipids increased as insulin dose increased.

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We also detected whether Akt were required for insulin's suppression of lipolysis. when adipocytes 152 were fully differentiated, cells were treat with 1, 10 and 100nM insulin for 24 h. As expected, insulin   pathway. We found that PKA and HSL phosphorylation levels were significantly increased in PID1 10 182 over-expression cells (Figure 4c-d). This indicates that PID1 inhibits insulin antlipolytic signaling 183 pathway, which involves the inhibition of AKT phosphorylation.

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Our animal experiments show that PID1 expression is increased in adipose tissue from HFD rat and  are consistent with previous studies, which showed that the inhibitory effect of insulin on lipolysis is 202 attributed primarily to the inhibition of cAMP-mediated signaling to HSL via Akt-dependent [16,17].

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There are considerable data implicating a defect in antilipolysis as a critical etiological abnormality 11 204 initiating the positive amplifying circuit that characterizes insulin resistance [18,19]. However, the 205 molecular mechanism by which impaired control of lipolysisin obesity is still unknown at this time.