Berberine Slows the Progression of Prediabetes to Diabetes in Zucker Diabetic Fatty Rats by Enhancing Intestinal Secretion of Glucagon-Like Peptide-2 and Improving the Gut Microbiota

Abstract

Background: Berberine is a plant alkaloid that has multiple beneficial effects against intestine inflammation. In our previous study, we have found that berberine also possesses an antidiabetic effect. However, whether berberine is useful in the prevention of type 2 diabetes mellitus (T2DM) through its effect on intestine endocrine function and gut microbiota is unclear.

Aim: To investigate the effects of berberine in the prevention of T2DM, as well as its effects on intestine GLP-2 secretion and gut microbiota in ZDF rats.

Methods: Twenty Zucker Diabetic Fatty (ZDF) rats were fed a high-energy diet until they exhibited impaired glucose tolerance (IGT). The rats were then divided into two groups to receive berberine (100 mg/kg/d; berberine group) or vehicle (IGT group) by gavage for 3 weeks. Five Zucker Lean (ZL) rats were used as controls. Fasting blood glucose (FBG) was measured, an oral glucose tolerance test was performed, and the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) was calculated. Intestinal expression of TLR-4, NF-κB, TNF-α, mucin, zona occludens-1 (ZO-1) and occludin were assessed (immunohistochemistry). Plasma levels and glutamine-induced intestinal secretion of glucagon-like peptide-1 (GLP-1) and GLP-2 were measured (enzyme-linked immunosorbent assay). The plasma lipopolysaccharide (LPS) level was measured. Fecal DNA extraction, pyrosequencing, and bioinformatics analysis were performed.

Results: After 3 weeks of intervention, diabetes developed in all rats in the IGT group, but only 30% of rats in the berberine group. Treatment with berberine was associated with reductions in food intake, FBG level, insulin resistance, and plasma LPS level, as well as increases in fasting plasma GLP-2 level and glutamine-induced intestinal GLP-2 secretion. Berberine could increase the goblet cell number and villi length, and also reverse the suppressed expressions of mucin, occludin, ZO-1 and the upregulated expressions of TLR-4, NF-κB and TNF-α induced in IGT rats (P<0.05). Berberine also improved the structure of the gut microbiota and restored species diversity.

Conclusion: Berberine may slow the progression of prediabetes to T2DM in ZDF rats by improving GLP-2 secretion, intestinal permeability, and the structure of the gut microbiota.

Keywords: berberine; glucagon-like peptide-2; intestinal microbiota; intestinal permeability; type 2 diabetes mellitus.

Figure 1

Effects of berberine on biochemistry parameters. (A) Oral glucose tolerance test after 3 weeks of intervention. *P < 0.05 vs. control group, ^#P < 0.05 vs. IGT group. (B) Fasting plasma insulin level. (C) Fasting plasma glucagon level. *P < 0.05 vs. control group, ^#P < 0.05 vs. IGT group. (D) Homeostatic Model Assessment of Insulin Resistance (HOMA-IR). *P < 0.05 vs. control group, ^#P < 0.05 vs. IGT group. (E) Homeostatic model assessment of insulin secretion (HOMA-β). *P < 0.05. (F) Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). *P < 0.05. (G) Blood urea nitrogen (BUN), plasma creatinine (Cr) and plasma uric acid (UA). *P < 0.05. (H) Plasma levels of cholesterol (CHO), triglycerides (TG) and high-density lipoprotein (HDL). *P < 0.05. Control group (n=5), IGT group (n=10), and berberine group (n=10).

Figure 2

Effects of berberine on the intestinal mucosal barrier. (A) Representative hematoxylin-eosin staining. Inflammatory cell infiltration, broadening and fusion of villi were indicated using arrows. Magnification 10 ×, 40 ×, and 100 × respectively of each row from top to bottom in (A). (B) Analysis of the villi length and the numbers of goblet cells. *P < 0.05. (C) Immunohistochemistry images of the intestinal epithelium and glandular cells showing the distribution of tight-junction proteins and mucin 3 weeks after the intervention. Magnification 400 × in (C). (D) Statistical analysis of scored immunohistochemistry positive staining for occludin and ZO-1. *P < 0.05. (E) Effects of berberine on mucin by western blotting. (F) Statistical analysis of mucin expression by western blotting. *P < 0.05. Control group (n=5), IGT group (n=10), and berberine group (n=10).

Figure 3

Representative immunohistochemistry images of (A) TNF-α, (B) NF-κB, and (C) TLR-4. (D) Statistical analysis of inflammatory factors expression by immunohistochemistry. *P < 0.05. Control group (n=5), IGT group (n=10), and berberine group (n=10).

Figure 4

Effects of berberine on the levels of glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and lipopolysaccharide (LPS). (A) Fasting plasma GLP-2 levels. (B) Glutamine-induced GLP-2 secretion from ileal tissue. (C) Fasting plasma GLP-1 levels. (D) Glutamine-induced GLP-1 secretion from ileal tissue. (E) Fasting plasma LPS levels. *P < 0.05. Control group (n=5), IGT group (n=10), and berberine group (n=10).

Figure 5

Effects of berberine on the structure of the fecal microbiota. (A) Observed species index. (B) Shannon diversity index. (C) Principal component analysis 3 weeks after the intervention. (D) Weighted UniFrac significance test 3 weeks after the intervention. (E) Unweighted UniFrac significance test 3 weeks after the intervention. Control group (n=3), IGT group (n=4), and berberine group (n=3).

Figure 6

Gut microbial compositions in the different groups. (A) Phylum level. (B) Genus level. (C) Species level. Control group (n=3), IGT group (n=4), and berberine group (n=3).