Anti-Diabetic Effect of Soy-Whey Dual-Protein on Mice with Type 2 Diabetes Mellitus Through INS/IRS1/PI3K Signaling Pathway

Abstract

The effects of soy protein and whey protein supplementation on glycemic control show inconsistency, and the mechanisms underlying the impact of a high-protein diet on blood glucose regulation remain unclear. This study aimed to explore the impact of a dual-protein (DP) blend comprising soy protein isolate (SPI) and whey protein concentrate (WPC), processed through high-pressure homogenization, on mice with Type 2 diabetes mellitus (T2DM) and its potential mechanisms. In the in vitro experiments, an insulin-resistant (IR) HepG2 cell model was treated with DP, resulting in a significant enhancement of glucose uptake and upregulation of IRS1 and GLUT4 expression. For the in vivo experiments, male C57BL/6J mice were randomly assigned into four groups (n = 6) based on body weight: normal control, T2DM model group, Metformin-treated group, and DP-treated group. Following a 5-week feeding period, Metformin and DP significantly reduced levels of blood sugar, AUC, TC, TG, and LDL-C in T2DM mice. Additionally, TP and ALB levels in the DP group were notably higher in the model group. In the liver and pancreas, DP alleviated histopathological changes and promoted liver glycogen synthesis in T2DM mice. Moreover, the levels of IRS1 and PI3K in the livers of mice in the DP group were significantly higher than those in the model group. Compared with the model groups, DP significantly reduced the expression of CD45 and increased the expression of CD206 in the pancreas of mice. Furthermore, 16S rRNA analysis revealed that DP altered the composition of the gut microbiota in diabetic mice, increasing the relative abundance of Lactobacillus, Parvibacter, and Lactobacillaceae. This suggested that DP could alleviate functional metabolic disorders in the gut and potentially reverse the risk of related complications. In conclusion, soy whey dual-protein may have an effective nutritional therapeutic effect on T2DM mice by regulating lipid metabolism, the INS/IRS1/PI3K signaling pathway, and gut microbiota.

Keywords: dual-protein; gut microbiota; insulin resistance; mechanism of action; type 2 diabetes.

Figure 1

Animal experimental design. Con: normal control mice; Mol: diabetic model mice; Met: diabetic mice treated with metformin (200 mg/kg); DP: diabetic mice treated with DP (670 mg/kg). Figure created with Biorender.com.

Figure 2

In vitro DP promotes glucose uptake and activates IRS1/GLUT4 pathway in HepG2 cells. (A) Cell viability of HepG2 treated with DP in 24 h. (B) Glucose intake in different treatment groups in 24 h. (C) Representative CSLM images of IRS1 expression on HepG2 treated with metformin (0.03 mg/mL) and DP (10 mg/mL). The cell nuclei were stained with DAPI (blue) and IRS1 were stained with Alexa Fluor^® 488 (green). (D) Relative IRS1 level in HepG2. (E) Western blot analysis to detect the expression levels of GLUT4 treated by metformin (0.03 mg/mL) and DP (10 mg/mL) for 24 h, and GAPDH was used as an internal reference protein. (F) Quantitative western blot analysis of GLUT4 by Image J software (n = 3, data are presented as mean ± SD). Statistical significances between every two groups were calculated via one-way ANOVA. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 3

Effect of DP on glucose metabolism in T2DM mice. The weekly changes of blood glucose (A) and body weight (B) in T2DM mice during DP intervention. (C) OGTT test performed at the end of the experiment. All mice were fasted overnight, and then orally administered a 2.0 g/kg dose of glucose. The blood glucose levels were measured at 0, 30, 60, 90, and 120 min. (D) The area under the curve (AUC) of each group. (E) Food intake of mice after intervention (n = 6, data are presented as mean ± SD). Statistical significances between every two groups were calculated via one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Effect of DP on lipid metabolism and protein metabolism in mice with T2DM. TC (A), TG (B), HDL-C (C), LDL-C (D), TP (E), and ALB (F) concentration in serum after intervention (n = 3, data are presented as mean ± SD). Statistical significances between every two groups were calculated via one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns—no significant difference.

Figure 5

Effect of DP on liver histopathology and the relative expression of IRS1 and PI3K on liver. (A) HE staining study of liver tissues in each treatment group. (B) PAS staining of liver tissue of mice in each treatment group. Scale bar = 100 μm. (C) The relative expression of IRS1 on liver. Quantitative analysis was conducted using Image J software. (D) The relative expression of PI3K on liver. Scale bar = 20 μm. Quantitative analysis was conducted using Image J software (n = 3, data are presented as mean ± SD). Statistical significances between every two groups were calculated via one-way ANOVA. *** p < 0.001, **** p < 0.0001.

Figure 6

Effect of DP on pancreatic histopathology and in vivo anti-T2DM immune response of DP. (A) Representative images of HE-stained pancreas section. (B) Pancreatic insulin (Green) and glucagon (Red) CLSM. (C) Flow cytometric analysis of CD4+ and CD8+ cells gating on CD3+ cells in the pancreas. (D) Flow cytometric analysis of CD45 cells in the pancreas. (E) Representative CSLM images of CD45 cells (Red) in the pancreas. (F) Representative CSLM images of CD206 cells (Red) in the pancreas. The percentages of populations of cells in each group are presented as histograms. Scale bar = 100 μm (n = 3, data are presented as mean ± SD). Statistical significances between every two groups were calculated via one-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 7

Effects of DP on the diversity of gut microbiota in mice with T2DM. (A) OTUs Venn graph of mice feces in each group. (B) Inter-group difference analysis of β-diversity based on Unweighted Unifrac distance and PCoA analysis diagram. (C) LDA scores of taxa enriched at different taxonomy levels. (D) Taxonomic cladogram generated by LEfSe analysis (n = 6).