Adiponectin secretion is regulated by SIRT1 and the endoplasmic reticulum oxidoreductase Ero1-L alpha

Abstract

Adiponectin is secreted from adipose tissue in response to metabolic effectors in order to sensitize the liver and muscle to insulin. Reduced circulating levels of adiponectin that usually accompany obesity contribute to the associated insulin resistance. The molecular mechanisms controlling the production of adiponectin are essentially unknown. In this report, we demonstrate that the endoplasmic reticulum (ER) oxidoreductase Ero1-L alpha and effectors modulating peroxisome proliferator-activated receptor gamma (PPAR gamma) and SIRT1 activities regulate secretion of adiponectin from 3T3-L1 adipocytes. Specifically, adiponectin secretion and Ero1-L alpha expression are induced during the early phase of adipogenesis but are then down-regulated during the terminal phase, coincident with an increased expression of SIRT1. Suppression of SIRT1 or activation of PPAR gamma enhances Ero1-L alpha expression and stimulates secretion of high-molecular-weight complexes of adiponectin in mature adipocytes. Suppression of Ero1-L alpha through expression of a corresponding small interfering RNA reduces adiponectin secretion during the differentiation of 3T3-L1 preadipocytes. Moreover, ectopic expression of Ero1-L alpha in Ero1-L alpha-deficient 3T3 fibroblasts stimulates the secretion of adiponectin following their conversion into adipocytes and prevents the suppression of adiponectin secretion in response to activation of SIRT1 by exposure to resveratrol. These findings provide a framework to understand the mechanisms by which adipocytes regulate secretion of adiponectin in response to various metabolic states.

FIG. 1.

Nutrients selectively increase secretion of HMW complexes of adiponectin. 3T3-L1 cells were cultured normally until day 4 of differentiation, at which stage the medium was changed to either standard DMEM containing 5 mM glucose (C and D) or glucose-free DMEM (A and B), and in each case the DMEM was supplemented with 10% FBS. Cultures in panels A and B were also exposed to the indicated concentration of d-glucose, while those in panels C and D were exposed to 20 mM lactate. On day 7, the medium and cell layer were harvested and subjected to either reducing (A and C) or nonreducing (B and D) SDS-PAGE as outlined in Materials and Methods. Int, intracellular; Ext, extracellular (secreted into the medium).

FIG. 2.

Perturbation of SIRT1 activity affects secretion of HMW forms of adiponectin. 3T3-L1 adipocytes (4 days) were exposed to either resveratrol (50 μM) or nicotinamide (5 mM) in standard DMEM containing 10% FBS for 2 days. The cells were then cultured in fresh DMEM overnight, at which time the medium and total cell layer were harvested for Western blot analysis of intra- and extracellular proteins on reducing (A) and nonreducing (B) SDS-PAGE as outlined in Materials and Methods.

FIG. 3.

Perturbation of PPARγ activity affects secretion of adiponectin. 3T3-L1 adipocytes were exposed to troglitazone (5 μM) or a PPARγ antagonist (10 μM T0070907) in standard DMEM containing 10% FBS for 3 days, at which time the medium and total cell layer were harvested for Western blot analysis of intra- and extracellular proteins on reducing (A) and nonreducing (B) SDS-PAGE as outlined in Materials and Methods.

FIG. 4.

Inhibition of SIRT1 expression enhances secretion of HMW adiponectin. 3T3-L1 preadipocytes expressing a control vector or SIRT1 siRNA were differentiated for the indicated number of days, and medium (extracellular) as well as the total cell layer (intracellular) was harvested for Western blot analysis of proteins on reducing (A) or nonreducing (B) SDS-PAGE employing antibodies to the following proteins: SIRT1, PPARγ, C/EBPα, adiponectin, adipsin, and aP2/FABP4.

FIG. 5.

Activation of PPARγ in Swiss 3T3 fibroblasts induces Ero1-Lα expression as well as adipogenesis. Swiss 3T3 fibroblasts expressing wild-type PPARγ or a control vector were induced to differentiate by exposure to a differentiation cocktail of dexamethasone, isobutylmethylxanthine, and insulin in 10% FBS as outlined in Materials and Methods (Con). Some cultures were also exposed to 5 μM troglitazone (Trog) or 10 μM PPARγ antagonist T0070907 (Antag) along with the differentiation cocktail. At day 6, cells were harvested for analysis of individual mRNAs as indicated using RT-PCR.

FIG. 6.

SIRT1 regulates expression of the ER oxidoreductase Ero1-Lα. (A) 3T3-L1 preadipocytes were differentiated for the indicated days, and total cell RNA was extracted for RT-PCR analysis of mRNAs corresponding to PPARγ, C/EBPα, adiponectin, Ero1-Lα, FABP4/aP2, and GAPDH. (B and C) 3T3-L1 preadipocytes expressing a control vector (-) or a SIRT1 siRNA (+) were differentiated for 8 days for RT-PCR analysis of mRNAs (B) or for the indicated days for Western blot analysis of intracellular proteins (C). (D) Control and SIRT1 siRNA-expressing 3T3-L1 preadipocytes were differentiated in the absence (Con) or presence of either a PPARγ antagonist (Antag; 10 μM T0070907) or PPARγ agonist (Trog; 5 μM troglitazone), and the medium (extracellular) and total cell layer (intracellular) were harvested for Western blot analysis of the indicated proteins.

FIG. 7.

Knockdown of Ero1-Lα expression in 3T3-L1 adipocytes inhibits secretion of adiponectin. 3T3-L1 adipocytes were transiently transfected with siRNAs corresponding to Ero1-Lα (1, 2, and 3) or a control siRNA as described in Materials and Methods. On day 6, total cell extracts (intracellular) and medium (extracellular) were harvested and subjected to Western blot analysis of Ero1-Lα, adiponectin, actin, and adipsin.

FIG. 8.

Ectopic expression of Ero1-Lα enhances secretion of adiponectin in 3T3 adipocytes. (A) Swiss 3T3 fibroblasts expressing either WT-PPARγ (WT) or the mutant F372APPARγ (F) were induced to differentiate as described in Materials and Methods, and total cell extracts (Int) as well as medium (Ext) were harvested at day 5 for Western blot analysis of the indicated proteins. (B and C) Swiss-F372APPARγ cells expressing either a pREV-TET Ero1-Lα retrovirus (F-Ero) or a control vector (F-Con) were induced to differentiate in the presence (+) or absence (-) of tetracycline for 5 days as described in Materials and Methods. At this stage, medium (Ext) and total cell protein (Int) were harvested and subjected to reducing (B) or nonreducing without heat (C) SDS-PAGE followed by Western blot analysis using antibodies against the indicated proteins. In panel C, WT proteins from panel A were also analyzed as a control.

FIG. 9.

Ectopic expression of Ero1-Lα attenuates the inhibitory effects of resveratrol on adiponectin secretion. Swiss-F372APPARγ cells expressing either a pREV-TET Ero1-Lα retrovirus (F-Ero) or a control vector (F-Con) were induced to differentiate by exposure to differentiation medium and 5 μM troglitazone in the presence (+T) or absence (-T) of tetracycline for 5 days as described in Materials and Methods. At day 4, cells were treated with or without 50 μM resveratrol for 2 days, and medium (Ext) and total cell protein (Int) were harvested for Western blot analysis of the indicated proteins.