Roux-en-Y gastric bypass improves liver and glucose homeostasis in Zucker diabetic fatty rats by upregulating hepatic trefoil factor family 3 and activating the phosphatidylinositol 3-kinase/protein kinase B pathway

Abstract

Background: Roux-en-Y gastric bypass (RYGB) surgery is effective in ameliorating type 2 diabetes mellitus (T2DM); but its mechanism remains incompletely understood.

Objectives: This study aimed to investigate whether RYGB improves glucose metabolism by upregulating hepatic trefoil factor family 3 (TFF3) and thereby activating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway.

Setting: Affiliated Hospital of North Sichuan Medical college in Nanchong city, China.

Methods: Zucker diabetic fatty (ZDF) rats underwent RYGB or sham surgery (SHAM), and Zucker lean (ZL) rats served as controls (CON). TFF3 expression and PI3K/Akt pathway activity were compared between groups using western blot, immunofluorescence, and RT-qPCR. Adeno-associated virus (AAV) was used to specifically overexpress and interfere with hepatic TFF3. Liver fibrosis and steatosis were assessed using Masson trichrome and Oil Red O staining. HepG2 cells overexpressing or knocking out TFF3 were constructed using lentiviral transfection and CRISPR/Cas9 technology. After verifying the activity of the PI3K/Akt pathway by western blot, rescue experiments were performed on HepG2 cell overexpressing and knocking out TFF3 using LY294002 and 740Y-P, respectively. The activities of gluconeogenic enzymes and glucose uptake capacity in different HepG2 cells were evaluated using qPCR and flow cytometry.

Results: Compared with the SHAM group, the blood glucose, body weight, insulin resistance, and lipid metabolism of ZDF rats in the RYGB group were significantly improved. The expression of TFF3 and PI3K/Akt phosphorylation in the liver of the RYGB group were higher than those of the rats that had undergone SHAM. In addition, compared with the SHAM group, the liver fibrosis and fatty degeneration of RYGB rats were milder, and the activity of gluconeogenic enzymes was lower. After tail vein injection of AAV that specifically overexpresses liver TTF3 in rats in the SHAM group, rats' insulin resistance, glucose tolerance, gluconeogenic enzymes, and other glucose metabolism indicators improved. After tail vein injection of AAV that interferes with liver TFF3 in rats in the RYGB group, rats' glucose metabolism indicators deteriorated. In in vitro experiments, the PI3K/Akt activity of TFF3-knocked-out HepG2 cells was lower than that of other groups. Lower glucose concentration were observed in TFF3-overexpressing cell lines. After rescue experiments, differences were found. The glucose metabolism level of the TFF3-expressing HepG2 cell line was positively correlated with the activity of the PI3K/Akt pathway.

Conclusions: RYGB regulates the expression of TFF3 in the liver of ZDF rats, thereby activating the PI3K/Akt pathway and improving T2DM.

Keywords: Akt; PI3K; RYGB; T2DM; TFF3.