[Live combined Bacillus subtilis and Enterococcus faecium improves glucose and lipid metabolism in type 2 diabetic mice with circadian rhythm disruption via the SCFAs/GPR43/GLP-1 pathway]

Abstract

Objectives: To investigate the effects of live combined Bacillus subtilis and Enterococcus faecium (LCBE) on glucose and lipid metabolism in mice with type 2 diabetes mellitus (T2DM) and circadian rhythm disorder (CRD) and explore the possible mechanisms.

Methods: KM mice were randomized into normal diet (ND) group (n=8), high-fat diet (HFD) group (n=8), and rhythm-intervention with HFD group (n=16). After 8 weeks of feeding, the mice were given an intraperitoneal injection of streptozotocin (100 mg/kg) to induce T2DM. The mice in CRD-T2DM group were further randomized into two equal groups for treatment with LCBE (225 mg/kg) or saline by gavage; the mice in ND and HFD groups also received saline gavage for 8 weeks. Blood glucose level of the mice was measured using a glucometer, and serum levels of Bmal1, PER2, insulin, C-peptide and lipids were determined with ELISA. Colon morphology and hepatic lipid metabolism of the mice were examined using HE staining and Oil Red O staining, respectively, and fecal short-chain fatty acids (SCFAs) was detected using LC-MS; GPR43 and GLP-1 expression levels were analyzed using RT-qPCR and Western blotting.

Results: Compared with those in CRD-T2DM group, the LCBE-treated mice exhibited significant body weight loss, lowered levels of PER2, insulin, C-peptide, total cholesterol (TC) and LDL-C, and increased levels of Bmal1 and HDL-C levels. LCBE treatment significantly increased SCFAs, upregulated GPR43 and GLP-1 expressions at both the mRNA and protein levels, and improved hepatic steatosis and colon histology.

Conclusions: LCBE ameliorates lipid metabolism disorder in CRD-T2DM mice by reducing body weight and improving lipid profiles and circadian regulators possibly via the SCFAs/GPR43/GLP-1 pathway.

Keywords: circadian rhythm; glucolipid metabolism; live combined Bacillus subtilis and Enterococcus faecium; short-chain fatty acids; type 2 diabetes mellitus.

图1

小鼠节律因子水平 Fig. 1 Levels of rhythmic factors in different groups. A: Level of Bmal 1 in the mice. B: Level of Per 2 in the mice. *P<0.05 vs NC.

图2

小鼠体质量变化趋势图 Fig. 2 Trends of body mass changes of the mice. ***P<0.001 vs DM group.

图3

小鼠糖耐量曲线 Fig.3 Glucose tolerance curves in different groups. **P<0.01, ***P<0.001 vs CRD-DM group.

图4

各组小鼠节律因子水平 Fig. 4 Levels of rhythmic factors in the mice in each group. A: Level of Bmal 1 in the mice. B: Level of Per 2 in the mice. **P<0.001 vs NC group; ^## P<0.001.

图5

各组小鼠胰岛素和C肽水平 Fig. 5 Insulin (A) and C-peptide (B) levels of the mice in each group. **P<0.001 vs NC group; ^## P<0.001.

图6

各组小鼠血脂水平比较 Fig.6 Comparison of lipid levels among the groups. A: Total cholesterol levels of the mice in each group. B: Triglyceride levels of the mice in each group. C: Low-density lipoprotein cholesterol levels of the mice in each group. D: High-density lipoprotein cholesterol levels of the mice in each group. **P<0.001 vs NC group; ^## P<0.001.

图7

各组结肠HE染色结果比较 Fig. 7 HE staining of the colon tissue in each group.

图8

各组肝脏油红O染色结果比较 Fig. 8 Oil red O staining of the liver tissue in each group.

图9

各组短链脂肪酸结果比较 Fig. 9 Fecal levels of short-chain fatty acid in each group. A: Acetic acid level. B: Propanoic acid level. C: Butyric acid level. D: Pentanoic acid level. *P<0.05, **P<0.001 vs NC group; ^# P<0.05, ^## P<0.001.

图10

各组结肠GPR43和GLP-1基因表达结果 Fig.10 Colonic GPR43 (A) and GLP-1 (B) mRNA expression in each group. **P<0.001 vs NC group; ^## P<0.001.

图11

各组小鼠结肠GPR43和GLP-1蛋白水平结果 Fig. 11 GPR43 and GLP-1 protein levels in the colon of the mice in each group detected by Western blotting. *P<0.05, **P<0.001 vs NC group; ^## P<0.001.