EFFECTS OF EXENDIN-4 ON ENDOPLASMIC RETICULUM STRESS-MEDIATED INSULIN RESISTANCE IN 3T3-L1 ADIPOCYTES

Abstract

Objective: Endoplasmic reticulum stress (ERS) is suspected as an important factor in the initiation of insulin resistance.

Aim: To explore the effects of exendin-4 (Ex-4) on the endoplasmic reticulum stress (ERS)-mediated insulin resistance in 3T3-L1 adipocytes. In our study, 3T3-L1 adipocytes were pre-treated with ERS inhibitors tauroursodeoxycholic acid (TUDCA), Ex-4 and an ERS inducer tunicamycin (TM) then induced insulin resistance. Glucose consumption of the adipocytes was measured. Western blots determined the protein levels of ERS markers and insulin signaling pathway.

Results: TM treatment reduced insulin-stimulated glucose consumption by 19.7% in 3T3-L1 adipocytes. This repression was blunted by 24h pre-treatment with TUDCA or Ex-4. Ex-4 augmented insulin-stimulated glucose consumption in adipocytes by 14.9%. Western blotting showed that TM treatment significantly increased the ER stress markers including p-IRE, p-JNK, p-PERK, p-eIF2a and ATF6 expression, whereas 24h pre-treatment of adipocytes with TUDCA or Ex-4 alleviated the ER stress. Ex-4 alleviates ERS-induced insulin resistance by upregulating the expression of phosphorylated Akt.

Conclusion: ERs mediates insulin resistance in 3T3-L1 adipocytes, and exendin-4 significantly improves this insulin resistance.

Keywords: 3T3-L1 Adipocytes; ER stress; Exendin-4; insulin resistance.

Figure 1.

Staining 3T3-L1 adipocytes with oil red (*400).

Figure 2.

Glucose consumption in 3T3-L1 adipocytes. 3T3-L1 adipocytes were pre-treated with TUDCA (1 mmol/L) or exendin-4 (100 nmol/L) for 24 h, followed with or without TM at 5 μg/mL for 5 h. Insulin (10 nM) was added for 30 min in the last step. Glucose consumption was measured as described in “Material and methods” section. The results are shown as mean ± S.E.M from four independent experiments and two replicates each.***p<0.001. Con=Control; INS=Insulin; TM=Tunicamycin; TU=Tauroursodeoxycholic acid; EX-4=Exendin-4.

Figure 3.

Exendin-4 alleviates ER stress in 3T3-L1 adipocytes. 3T3-L1 adipocytes were pre-treated with TUDCA (1 mmol/L) or exendin-4 (100 nmol/L) for 24 h, followed with or without TM at 5 ug/ml for 5 h. Insulin (10 nM) was added for 30 min in the last step. The whole cell lysates were resolved by SDS-polyacrylamide gel electrophoresis and the ER stress markers including the phosphorylation and/or expression of IRE-1, JNK, PERK, eIF-2a, ATF-6 were determined by Western blot analysis with specific antibodies as indicated. β-actin was shown as a loading control. The representative immunoblots are shown from one of three independent experiments, with a quantification analysis presented on the right. All results are presented as the means ± S.E.M from three independent experiments.**P<0.01,***P<0.001. Con=Control; INS=Insulin; TM=Tunicamycin; TU=Tauroursodeoxycholic acid; EX-4=Exendin-4.

Figure 4.

Exendin-4 increases insulin sensitivity in 3T3-L1 adipocytes. Immunoblotting for phospho-Akt (Ser473) and Akt from whole cell lysates. β-actin was shown as a loading control. The representative immunoblots are shown from one of three independent experiments, with a quantification analysis presented below. All results are presented as the means ± S.E.M from three independent experiments. *P< 0.05. Con=Control; INS=Insulin; TM=Tunicamycin; TU=Tauroursodeoxycholic acid; EX-4=Exendin-4.