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

Ceperuelo-Mallafré V, Ejarque M, Duran X, Pachón G, Vázquez-Carballo A, Roche K, Núñez-Roa C, Garrido-Sánchez L, Tinahones FJ, Vendrell J, Fernández-Veledo S - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Hospital Universitari de Tarragona Joan XXIII, Institut d´Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, Tarragona, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas, Instituto de Salud Carlos III, Madrid, Spain.

ABSTRACT

Objective: 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.

Methods: 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.

Results: ZAG treatment increased basal glucose uptake, correlating with an increase in GLUT 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

ZAG inhibits insulin action via β1/β2-AR signaling.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. (A) 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) 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. (C) 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 vs total proteins (3 independent experiments). *, P < 0.01.
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pone.0129644.g003: ZAG inhibits insulin action via β1/β2-AR signaling.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. (A) 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) 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. (C) 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 vs total proteins (3 independent experiments). *, P < 0.01.

Mentions: The metabolic effects of ZAG in murine models have been primarily associated with β3-AR activation [7, 29]. It is widely accepted, however, that β3-AR expression in human adipose tissues is extremely low [30]. Indeed, a survey of β-AR mRNA expression in mature SGBS adipocytes demonstrated that β1-AR and β2-AR account for 65% of total β-AR expression (S3 Fig). To further explore the molecular mechanisms by which ZAG modulates glucose uptake and insulin sensitivity, mature SGBS adipocytes were pre-treated with the mixed β1/β2-AR antagonist, propranolol, prior to ZAG treatment and insulin stimulation. Propranolol treatment decreased ZAG-induced basal glucose uptake (Fig 3A, left panel), which correlated with the abolishment of ZAG-mediated effects on GLUT4 but not GLUT1 mRNA expression (Fig 3B). Furthermore, compared with control cells that did not receive propranolol, ZAG-mediated resistance to insulin-stimulated glucose uptake was abrogated in cells pretreated with the β1/ β2-AR antagonist (Fig 3A, right panel). Consequently, whereas ZAG treatment reduced the levels of phosphorylated AKT, pretreatment with propranolol restored insulin-induced activation of AKT in human adipose cells (Fig 3C). To question the potential role of β1-AR in regulating ZAG-mediated effects, SGBS mature adipocytes were treated with the specific β1-AR antagonist CGP20712A [31] prior to ZAG and/or insulin treatment. Pre-treatment with the β1-AR antagonist inhibited ZAG-mediated induction of GLUT4 mRNA expression (Fig 3B); however, insulin signaling was not reestablished (Fig 3C). Collectively, these results indicate that the effects of ZAG in this cell type might be specifically dependent on β1/β2-AR signaling, and suggest that the β3-AR subtype does not play a significant role in the insulin resistant state in adipose cells. Moreover, whereas the effects of ZAG on basal glucose uptake and GLUT4 expression are mediated via β1-AR, inhibition of insulin signaling seems to be dependent on β2-AR activation.


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

Ceperuelo-Mallafré V, Ejarque M, Duran X, Pachón G, Vázquez-Carballo A, Roche K, Núñez-Roa C, Garrido-Sánchez L, Tinahones FJ, Vendrell J, Fernández-Veledo S - PLoS ONE (2015)

ZAG inhibits insulin action via β1/β2-AR signaling.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. (A) 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) 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. (C) 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 vs total proteins (3 independent experiments). *, P < 0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465909&req=5

pone.0129644.g003: ZAG inhibits insulin action via β1/β2-AR signaling.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. (A) 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) 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. (C) 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 vs total proteins (3 independent experiments). *, P < 0.01.
Mentions: The metabolic effects of ZAG in murine models have been primarily associated with β3-AR activation [7, 29]. It is widely accepted, however, that β3-AR expression in human adipose tissues is extremely low [30]. Indeed, a survey of β-AR mRNA expression in mature SGBS adipocytes demonstrated that β1-AR and β2-AR account for 65% of total β-AR expression (S3 Fig). To further explore the molecular mechanisms by which ZAG modulates glucose uptake and insulin sensitivity, mature SGBS adipocytes were pre-treated with the mixed β1/β2-AR antagonist, propranolol, prior to ZAG treatment and insulin stimulation. Propranolol treatment decreased ZAG-induced basal glucose uptake (Fig 3A, left panel), which correlated with the abolishment of ZAG-mediated effects on GLUT4 but not GLUT1 mRNA expression (Fig 3B). Furthermore, compared with control cells that did not receive propranolol, ZAG-mediated resistance to insulin-stimulated glucose uptake was abrogated in cells pretreated with the β1/ β2-AR antagonist (Fig 3A, right panel). Consequently, whereas ZAG treatment reduced the levels of phosphorylated AKT, pretreatment with propranolol restored insulin-induced activation of AKT in human adipose cells (Fig 3C). To question the potential role of β1-AR in regulating ZAG-mediated effects, SGBS mature adipocytes were treated with the specific β1-AR antagonist CGP20712A [31] prior to ZAG and/or insulin treatment. Pre-treatment with the β1-AR antagonist inhibited ZAG-mediated induction of GLUT4 mRNA expression (Fig 3B); however, insulin signaling was not reestablished (Fig 3C). Collectively, these results indicate that the effects of ZAG in this cell type might be specifically dependent on β1/β2-AR signaling, and suggest that the β3-AR subtype does not play a significant role in the insulin resistant state in adipose cells. Moreover, whereas the effects of ZAG on basal glucose uptake and GLUT4 expression are mediated via β1-AR, inhibition of insulin signaling seems to be dependent on β2-AR activation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Hospital Universitari de Tarragona Joan XXIII, Institut d´Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, Tarragona, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas, Instituto de Salud Carlos III, Madrid, Spain.

ABSTRACT

Objective: 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.

Methods: 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.

Results: ZAG treatment increased basal glucose uptake, correlating with an increase in GLUT 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus