Stachyose Improves the Effects of Berberine on Glucose Metabolism by Regulating Intestinal Microbiota and Short-Chain Fatty Acids in Spontaneous Type 2 Diabetic KKAy Mice

Abstract

Berberine (BBR) has the beneficial effects of anti-inflammation, anti-bacteria, and anti-diabetes. The clinical application of BBR has been hindered by its poor gastrointestinal absorption. Stachyose (Sta), a prebiotic agent, improves the composition of gut microbiota and benefits for diabetes. We therefore investigated whether Sta improves the anti-diabetic actions of BBR using KKAy mice. Here, we find that the combination of BBR and Sta is more effective than BBR alone in blood glucose control, improvement of insulin resistance and islet functions, inflammatory mediators decrease, and maintenance of intestinal barrier integrity. Gut microbiota analysis demonstrates that both BBR and combined administration enhance the abundance of Bacteroidaceae and Akkermansiaceae and decrease Lachnospiraceae levels, whereas Akkermansiaceae elevation due to the administration of BBR with Sta is more significant than BBR alone. Interestingly, the proportion of Lactobacillaceae increases with combination treatment, but is diminished by BBR. Additionally, BBR with Sta significantly reduces the concentrations of fecal short-chain fatty acids compared to BBR. Collectively, these results indicate that the combination of BBR and Sta imparts better effects on the maintenance of glycemia and intestinal homeostasis than BBR alone by modulating gut microbiota and short-chain fatty acids, thereby providing a novel approach for the treatment of type 2 diabetes mellitus.

Keywords: berberine; glucose metabolism; gut microbiota; short-chain fatty acids; stachyose; type 2 diabetes.

FIGURE 1

BBR combined with Sta improves glucose metabolism in KKAy mice. (A) Fasting blood glucose. (B) HbA1c levels. (C) Oral glucose tolerance test (OGTT). (D) Insulin tolerance test (ITT). Area under the curve (AUC) of OGTT or ITT. (E) Homeostasis model assessment of insulin resistance (HOMA-IR) index. The index of the validated HOMA-IR was calculated as follows: HOMA-IR = fasting blood glucose (mmol/L) × fasting plasma insulin (μU/mL)/22.5. (F) Body weight of mice. Data are expressed as mean ± SEM, n = 10–12. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con; ^# p < 0.05 vs. BBR. BBR, berberine; Con, control; Sta, stachyose.

FIGURE 2

BBR combined with Sta modifies islet functions in KKAy mice. (A) Fasting insulin levels in plasma. (B) Plasma insulin levels after glucose administration for 15 min (C) The percentage of insulin elevation after glucose administration for 15 min (D) Plasma glucagon concentrations. (E) Representative images of insulin and glucagon immunofluorescence staining in pancreatic islets. Insulin is shown in red, and glucagon in green. Magnification of all images is ×400. (F) Statistical analysis of the mean fluorescence intensities of insulin and glucagon in islets, and the ratio of insulin to glucagon. Data are presented as mean ± SEM, n = 10–12 for (A–D); n = four to six per group for (E, F). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con; ^# p < 0.05, ^## p < 0.01 vs. BBR. BBR, berberine; Con, control; Sta, stachyose.

FIGURE 3

BBR combined with Sta improves inflammatory status in KKAy mice. (A) Cytokine levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, IL-6, C-reactive protein (CRP), and IL-10 in plasma were detected by ELISA. (B) Gene expression levels of IL-1β, TNF-α, MCP-1, IL-6, IL-10, and TLR4 in intestine were analyzed by qPCR. (C, D) Protein levels of TLR4, p38MAPK, p-p38MAPK, ERK1/2, p-ERK1/2, JNK, and p-JNK were determined by Western blot analysis. β-Actin was detected as the internal reference. Data are expressed as mean ± SEM, n = 10–12 for (A); n = four to five for (B–D). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con. BBR, berberine; Con, control; Sta, stachyose.

FIGURE 4

Effects of BBR with Sta on the proportion and function of macrophage. (A) Representative FACS staining images for CD11b, CD11c, F4/80, and MHC-II on gated CD45.2⁺ cells in mesentery. (B) Statistical analysis of the percentage of macrophage (CD11b⁺F4/80⁺), M1 macrophage (F4/80⁺CD11c⁺), and MHC-II expressed on macrophage surface (F4/80⁺MHC-II⁺). (C) Gene expression levels of F4/80 and CD11c in intestine tissues were measured by quantitative real-time PCR. (D) Protein levels of CD11c and F4/80 in intestine were detected by Western blot. β-Actin as the internal reference. Data are presented as mean ± SEM, n = 3–5. *p < 0.05, **p < 0.01 vs. Con. BBR, berberine; Con, control; Sta, stachyose.

FIGURE 5

BBR with Sta maintains intestinal barrier integrity of KKAy mice. (A) Protein levels of ZO-1 and occludin in intestine tissues were analyzed by Western blot. (B) Quantization of ZO-1 and occludin proteins in intestine. (C, D) Gene expression levels of occludin, ZO-1, and Reg3g in intestine were evaluated by quantitative real-time PCR. β-Actin was detected as the internal reference. Data are represented as mean ± SEM, n = 4–5. *p < 0.05, **p < 0.01 vs. Con; ^# p < 0.05 vs. BBR. BBR, berberine; Con, control; Sta, stachyose.

FIGURE 6

BBR combined with Sta alters the composition of gut microbiota of KKAy mice. (A) Total OTU numbers. (B) Shannon index. (C) Chao index. (D) Principal coordinate analysis (PCoA). (E) Relative abundance of microbiota at the phylum level. (F) Relative abundance of microbiota at the family level. (G) Heatmap analysis of relative abundance of microbiota at the genus level. The heatmap shows the top 50 genera ranked on the basis of abundance. Each column in the heatmap represents one sample, and each row represents one genus. The color bar showing blue (low) to red (high) indicates the relative abundance of each genus. Data are shown as mean ± SEM, n = 9–10. ***p < 0.001 vs. Con; ^## p < 0.01 vs. BBR. BBR, berberine; Con, control; Sta, stachyose.

FIGURE 7

Effects of BBR with Sta on fecal short-chain fatty acids (SCFAs) of KKAy mice. (A) Fecal SCFAs, including acetic acid, propionic acid, butyric acid, isobutyric acid, pentanoic acid, isopentanoic acid, hexanoic acid, isohexanoic acid, were analyzed by GC-MS method. (B) Correlation analysis of SCFAs and specific microbiota at the family level. The R values were shown in different colors in the diagram. The blue represents negative correlation, and red represents positive correlation. n = 8–10 per group. For (A), *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. BBR. For (B), *p < 0.05, **p < 0.01, ***p < 0.001. BBR, berberine; Con, control; Sta, stachyose.