Zinc-α2-Glycoprotein Modulates AKT-Dependent Insulin Signaling in Human Adipocytes by Activation of the PP2A Phosphatase

<div><p>Objective</p><p>Evidence from mouse models suggests that zinc-α2-glycoprotein (ZAG) is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT) remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes.</p><p>Methods</p><p>ZAG action on glucose uptake and insulin action was analyzed. β1 and β2-adrenoreceptor (AR) antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR.</p><p>Results</p><p>ZAG treatment increased basal glucose uptake, correlating with an increase in <i>GLUT</i> expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific β1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via β1-AR, whereas inhibition of insulin action is dependent on β2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG.</p><p>Conclusions</p><p>ZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a β2-AR- and PP2A-dependent manner.</p></div

ZAG increases basal glucose uptake but impairs insulin-induced glucose uptake in human subcutaneous adipocytes by acting as a β1/2-AR agonist.

<p>ZAG enhanced GLUT4 gene exression and basal glucose uptake via β1-AR. In addition, ZAG might also activate PPA2 via β2-AR, inhibiting insulin-induced AKT phosphorylation and, in consequence, insulin-induced glucose uptake. Although some prospective cohort studies point to ZAG expression in AT as a useful biomarker to predict insulin sensitivity, our data establish ZAG as a negative modulator of insulin action.</p

Administration of ZAG increases basal glucose uptake.

<p>Differentiated SGBS adipocytes <b>(A)</b> and LiSa-2 adipocytes <b>(B)</b> were cultured for 24 hours with or without 25 μg/ml ZAG. Glucose uptake was measured during the final 10 min by incorporation of labelled 2-deoxyglucose into the cells. Results are the mean±SEM of 3–4 independent experiments performed in triplicate. <b>(C)</b> Gene expression of GLUT1, GLUT3 and GLUT4 in SGBS and <b>(D)</b> LiSa-2 adipocytes treated or not with ZAG was analyzed by quantitative real time PCR (qPCR). Data are presented as mean±SEM (n = 3). *, P < 0.01 vs control.</p

ZAG activation of PP2A phosphatase impairs insulin-stimulated glucose uptake and AKT phosphorylation in SGBS adipocytes.

<p><b>(A)</b> Differentiated human adipose cells were cultured with 25 μg/ml ZAG for 24 hours and PP2A activity was measured. <b>(B)</b> PP2A activity was measured in mature adipocytes pre-treated with 1 μM propranolol or 300 nM CPG20712A (CPG) prior to culture with ZAG. Data are presented as mean±SEM (n = 3). *, P < 0.01 vs control. (<b>C</b>) Adipose cells were transfected with 100 nM siRNA against the α-catalytic subunit of PP2A (PP2A-Cα) or RISC-free (control cells) and cultured with or without 25 μg/ml ZAG for 24 hours prior to stimulation with 100 nM insulin (Ins) for 30 minutes. Glucose uptake was measured by incorporation of labelled 2-deoxyglucose into the cells during the last 10 minutes of culture. Results from 3 independent experiments performed in triplicate are expressed as pmol glc/mg prot/10 min. Right panels represent percentage of stimulation produced by insulin over control cells (no insulin, without or with ZAG, respectively). *, P < 0.01. <b>(D)</b> PP2A-Cα protein expression was analyzed by western blot in control and transfected cells. A representative experiment is shown together with densitometric analysis (3 independent experiments). *, P < 0.01. <b>(E)</b> GLUT4 mRNA levels were analyzed by qPCR in siRNA control and PP2A-Cα-silenced adipocytes in the absence or presence of ZAG. Data are presented as mean±SEM (n = 3). *, P < 0.01 vs control. <b>(F)</b> Cells transfected as in D were cultured or not with 25 μg/ml ZAG for 24 hours prior to stimulation with 100 nM insulin (Ins) for 15 minutes. Cell lysates were analyzed by western blotting using antibodies against phosphorylated and total Akt (Ser473). A representative experiment is shown together with densitometric analysis of phosphorylated <i>vs</i> total proteins (3 independent experiments). *, P < 0.01.</p

ZAG inhibits insulin action <i>via</i> β1/β2-AR signaling.

<p>Differentiated SGBS adipocytes were pre-treated for 30 min with or without 1 μM propranolol prior to culture with 25 μg/ml ZAG for 24 hours. <b>(A)</b> Glucose uptake was measured after stimulation with 100 nM insulin (Ins) for 30 minutes by incorporation of labelled 2-deoxyglucose into the cells for the final 10 minutes of culture. Left panels represent mean±SEM of 3 independent experiments performed in triplicate and are expressed as pmol glc/mg prot/10 min. Right panels represent percentage of stimulation produced by insulin over control cells (no insulin, without or with ZAG respectively). *, P < 0.01. <b>(B)</b> GLUT1 and GLUT4 mRNA expression were analyzed by qPCR in adipocytes pre-treated with 1 μM propranolol or 300 nM CPG20712A (CPG) prior to culture with ZAG. Data are presented as mean±SEM (n = 3). *, P < 0.01 vs control. <b>(C)</b> Lysates from differentiated SGBS cells pre-treated with propranolol or CPG prior to culture with ZAG and 100 nM insulin (Ins) for 15 minutes were analyzed by western blotting using antibodies against phosphorylated and total Akt (Ser473). A representative experiment is shown together with densitometric analysis of phosphorylated <i>vs</i> total proteins (3 independent experiments). *, P < 0.01.</p

Clinical, anthropometric, and analytical characteristics (units) according to insulin resistance classification.

<p>The results are given as the mean±SD. BMI: body mass index; HOMA-IR: homeostasis model assessment of insulin resistance index; DBP: Diastolic Blood Pressure; SBP: Systolic Blood Pressure. ^a and ^b indicate significant differences between the means of the different groups:</p><p>^a: P<0.05</p><p>^b: P<0.01(ANOVA or Student´s t where appropriate).</p><p>Clinical, anthropometric, and analytical characteristics (units) according to insulin resistance classification.</p

ZAG treatment impairs insulin action on glucose uptake and insulin signaling.

<p>Differentiated human subcutaneous SGBS adipocytes <b>(A)</b> and visceral LiSa-2 adipocytes <b>(B)</b> were cultured for 24 hours with or without 25 μg/ml ZAG before stimulation with 100 nM insulin (Ins) for 30 minutes. Glucose uptake was measured during the final 10 min by incorporation of labelled 2-deoxyglucose into the cells. Left panels represent mean±SEM of 3–4 independent experiments performed in triplicate and are expressed as pmol glc/mg prot/10 min. Right panels represent percentage of stimulation produced by insulin over control cells (no insulin, without or with ZAG respectively). *, P < 0.01. <b>(C)</b> Lysates from differentiated SGBS cells and <b>(D)</b> LiSa-2 cells cultured with or without 25 μg/ml ZAG for 24 hours before stimulation with 100 nM insulin (Ins) for 15 minutes, were analyzed by western blotting using antibodies against phosphorylated and total IRβ (Tyr1150/1151), IRS1 (Tyr612), Akt (Ser473) and AS160 (Thr642). A representative experiment is shown together with densitometric analysis of phosphorylated <i>vs</i> total proteins (3 independent experiments). *, P < 0.01.</p