Restoration of GLP-1 secretion by Berberine is associated with protection of colon enterocytes from mitochondrial overheating in diet-induced obese mice

Abstract

Objective: L-cell dysfunction is reported for GLP-1 reduction in type 2 diabetes. However, the mechanism of dysfunction remains unknown. In this study, we examined mitochondrial function in the mechanistic study in diet-induced obese (DIO) mice.

Subjects: C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks to establish the DIO model for GLP-1 reduction. The mice were then treated with berberine (BBR) (100 mg/kg/day) for 8 weeks to test the impact on GLP-1 expression. Mitochondrial activities of the colon enterocytes were compared among three groups of mice (lean, DIO, and DIO + BBR) at the end of treatment. Gut microbiota and short-chain fatty acids (SCFAs) were examined to understand the mitochondrial responses. A cellular model treated with palmitic acid (PA) was used in the mechanism study.

Results: A reduction in GLP-1 expression was observed in DIO mice with mitochondrial stress responses in the colon enterocytes. The mitochondria exhibited cristae loss, membrane rupture, and mitochondrial swelling, which was observed with an increase in ATP abundance, complex I activity, and deficiency in the activities of complexes II and IV. Those changes were associated with dysbiosis and a reduction in SCFAs in the colon of DIO mice. In the cellular model, an increase in ATP abundance, loss of mitochondrial potential, and elevation of apoptosis were induced by PA. All of the alterations in DIO mice and the cellular model were attenuated by BBR.

Conclusion: The mitochondrial stress responses were observed in the colon enterocytes of DIO mice for GLP-1 reduction. The stress was prevented by BBR in the restoration of GLP-1 expression, in which BBR may act through direct and indirect mechanisms.

Fig. 1

Inhibition of obesity and hyperlipidemia by BBR. a Body weight at 0, 4, and 8 weeks of treatment. b Average daily calorie intake per mouse. c Tissue weight of perirenal fat and epididymal fat pads after 8 weeks of treatment. d Serum lipid levels after 8 weeks of treatment. The BBR treatment was administrated for 8 weeks in DIO mice after 16 weeks on high-fat diet. Data are presented as the mean ± SEM (n = 6). *P < 0.05 HFD versus NCD, ^#P < 0.05 HFD + BBR versus HFD

Fig. 2

Improvement of insulin sensitivity by BBR. a Fasting blood glucose. b Fasting serum insulin. c HOMA-IR. d GTT at 4 weeks of BBR treatment. GTT was performed by intraperitoneal injection of glucose (2 g/kg body weight). e GTT at 8 weeks of BBR treatment. f Area under the curve of GTT assays. g mRNA of G6Pase in liver tissue at 8 weeks of BBR treatment. h mRNA of PEPCK in liver tissue at 8 weeks of BBR treatment. Data are presented as the mean ± SEM (n = 6). *P < 0.05 HFD versus NCD, ^#P < 0.05 HFD + BBR versus HFD

Fig. 3

Upregulation of GLP-1 and GPR43 in colon by BBR. a Plasma GLP-1. b mRNA expression of GCG in colon tissues. c mRNA expression of GPR43 in the mouse colon tissues. Data are presented as the mean ± SEM (n = 6). *P < 0.05 HFD versus NCD, ^#P < 0.05 HFD + BBR versus HFD

Fig. 4

Restoration of mucosal and mitochondrial structure in colon by BBR. a Colon tissue histology. Representative fields of H&E staining and AB-PAS staining were taken from the colon tissue slides (×100, scale bars, 100 μm). In HFD mice, the mucosa was damaged and the mucous content was decreased with epithelial cell detached, and these changes were improved by BBR. b Mitochondrial structure under the transmission electron microscope. The image was taken at 98,000 times of magnitude

Fig. 5

Protection of mitochondrial function in colon by BBR. a Total ATP level in the fresh colon tissue. b Complex activities of mitochondria. The complex activity was determined by the oxygen consumption rate (OCR) in response to the inhibitors in mitochondria isolated from the fresh colon tissue. The order of inhibitor injection was rotenone, succinate, antimycin A, and ascorbate plus N,N,N9,N9-tetramethyl-p-phenylenediamine (TMPD). c Mean value of maximal OCR in the complex assay. d Respiratory control rate (RCR). This was performed with complex II substrate succinate and complex I inhibitor rotenone. Data are presented as the mean ± SEM (n = 6). *P < 0.05 HFD versus NCD, ^#P < 0.05 HFD + BBR versus HFD

Fig. 6

Regulation of mitochondrial stress and apoptosis in palmitate-treated cells by BBR. Palmitate was used to represent the long-chain fatty acid palmitic acid in the induction of mitochondrial stress responses in the intestine epithelial cell line NCI-H716. a Palmitate level in the large intestine. Palmitate was determined in the fecal samples. b ATP elevation in palmitate-treated cells. c Decline of mitochondrial potential in palmitate-treated cells. d Reduction in mitochondrial function in palmitate-treated cells. e Apoptosis in response to palmitate. Data are presented as the mean ± SEM (n = 3). *P < 0.05 PA versus control; ^#P < 0.05 PA + BBR versus PA

Fig. 7

Regulation of SCFAs and gut microflora by BBR. a–f The concentration of butyric acid, acetic acid, propionic acid, isobutyric acid, isovaleric acid, and valeric acid in feces of HFD mice after 8 weeks of BBR treatment. g Bacteria diversity in feces as indicated by ACE richness. h, i The abundance of Firmicutes and Bacteroidetes. j The Firmicutes/Bacteroidetes ratio. k–n The abundances of Clostridiales.g, Oscillospira, Parabacteroides, and Mogibacteriaceae.g. Data are presented as the mean ± SEM (n = 6). *P < 0.05 HFD versus NCD, ^#P < 0.05 HFD + BBR versus HFD