Pig bile powder maintains blood glucose homeostasis by promoting glucagon-like peptide-1 secretion via inhibiting farnesoid X receptor

Figure 1 Bile powder improved blood glucose in a diabetic mouse model. A: Schematic diagram. Mice were fed a high-fat diet (HFD) for 6 weeks and were injected with streptozotocin (STZ) (50 mg/kg, intravenous) to induce blood glucose disorder. Mice continued a HFD for an additional 6 weeks. The mice were randomly divided into four groups (n = 5 per group) and treated with different doses of bile powder (BP): The HFD + STZ group (control group); The low-dose BP group (25 mg/kg/day, intragastric gavage); The medium-dose BP group (50 mg/kg/day, intragastric gavage); The high-dose BP group (75 mg/kg/day, intragastric gavage). The oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed after 30 days and 45 days of BP administration; B: OGTT after 30 days of BP treatment; C: Area under the curve (AUC) of the OGTT after 30 days of BP treatment; D: OGTT after 45 days of BP treatment; E: AUC of the OGTT after 45 days of BP treatment; F: ITT after 45 days of BP treatment; G: AUC of the ITT after 45 days of BP treatment; H: Serum glucagon-like peptide-1 levels in the HFD + STZ group and the high-dose BP group after 45 days of treatment. Data are shown as mean ± SEM. NS: Not significant (P > 0.05) compared between groups. Statistical analysis was performed using the Student’s t-test. ^aP < 0.05. ¹High-fat diet + streptozotocin vs low-dose bile powder. ²High-fat diet + streptozotocin vs medium-dose bile powder. ³High-fat diet + streptozotocin vs high-dose bile powder. HFD: High-fat diet; STZ: Streptozotocin; BP: Bile powder; OGTT: Oral glucose tolerance test; ITT: Insulin tolerance test; AUC: Area under the curve; GLP-1: Glucagon-like peptide-1.

Figure 2 Bile powder alleviated hyperglycemia through glucagon-like peptide-1 signaling. A: Schematic diagram. Mice were fed a high-fat diet (HFD) for 6 weeks and were injected with streptozotocin (STZ) (50 mg/kg, intravenous) to induce blood glucose disorder. The HFD was continued for an additional 6 weeks. The mice were randomly divided into three groups (n = 5 per group): The HFD + STZ group; the bile powder (BP) group (75 mg/kg/day BP, intragastric gavage); and the BP + glucagon-like peptide-1 receptor agonist group [75 mg/kg/day BP, intragastric gavage + 25 nmol/kg/day glucagon-like peptide-1 receptor antagonist, exendin-3 (3-39) amide, intraperitoneal]. The oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed after the 30-day treatment; B: OGTT; C: Area under the curve (AUC) of the OGTT after the 30-day treatment; D: ITT; E: AUC of the ITT after the 30-day treatment. Data are shown as mean ± SEM. P < 0.05 compared between groups. Statistical analysis was performed using the Student’s t-test. ^aP < 0.05. ¹High-fat diet-streptozotocin vs bile powder. ²Bile powder vs bile powder + glucagon-like peptide-1 receptor antagonist. HFD: High-fat diet; STZ: Streptozotocin; BP: Bile powder; OGTT: Oral glucose tolerance test; ITT: Insulin tolerance test; AUC: Area under the curve; GLP-1: Glucagon-like peptide-1; GLP1-RA: Glucagon-like peptide-1 receptor antagonist.

Figure 3 Bile acid mixture stimulated glucagon-like peptide-1 secretion in STC-1 cells. A: Schematic diagram. STC-1 cells were treated with 0, 10, and 20 μg/mL of mixed bile acids for 1 hour or 24 hours to determine glucagon-like peptide-1 secretion and the proglucagon (Gcg) expression level; B: Glucagon-like peptide-1 levels in the medium after 1 hour of treatment; C: Glucagon-like peptide-1 levels in the medium after 24 hours of treatment; D: Relative expression levels of Gcg in STC-1 cells after 24 hours of treatment. Data are shown as mean ± SEM. P < 0.05 compared between groups. Statistical analysis was performed using the Student’s t-test. ^aP < 0.05. BA: Bile acid; ELASA: Enzyme-linked immunosorbent assay; RT-PCR: Real-time polymerase chain reaction; GLP-1: Glucagon-like peptide-1.

Figure 4 Bile powder regulated blood glucose homeostasis via inhibiting intestinal farnesoid X receptor signaling pathway. A: Schematic diagram. Mice were fed a high-fat diet (HFD) for 6 weeks and were injected with streptozotocin (STZ) (50 mg/kg, intravenous) to induce blood glucose disorder. Mice were fed a HFD for an additional 6 weeks. The mice were randomly divided into three groups (n = 5 per group): The HFD + STZ group; The bile powder (BP) group (75 mg/kg/day BP, intragastric gavage); The BP + fexaramine group (75 mg/kg/day BP, intragastric gavage + 100 mg/kg/day fexaramine, intragastric gavage); B: Intestinal immunohistochemistry analysis of the farnesoid X receptor (scale bars = 100 μm); C: Relative expression of fibroblast growth factor 15 transcription in the ileum; D: Serum fibroblast growth factor 15 levels; E: Oral glucose tolerance test after the 30-day treatment; F: The area under the curve of the oral glucose tolerance test after the 30-day treatment. Data are shown as mean ± SEM. P < 0.05 compared between groups. Statistical analysis was performed using Student’s t-test. ^aP < 0.05. ¹High-fat diet + streptozotocin vs bile powder. ²Bile powder vs bile powder + fexaramine. FXR: Farnesoid X receptor; FEX: Fexaramine; HFD: High-fat diet; STZ: Streptozotocin; BP: Bile powder; OGTT: Oral glucose tolerance test; RT-PCR: Real-time polymerase chain reaction; IHC: Immunohistochemistry; AUC: Area under the curve.

Figure 5 Long-term feeding of bile powder prevented blood glucose disorders. A: Schematic diagram. Mice were randomly divided into two groups (n = 5 per group). All of the mice were fed a high-fat diet (HFD) for 6 weeks and were injected with streptozotocin (STZ) (50 mg/kg, intravenous) to induce blood glucose disorder. An HFD was continued for an additional 6 weeks. Mice in the bile powder group were treated with bile powder (75 mg/kg/day, intragastric gavage) for 12 weeks, and those in the HFD + STZ group were treated with the vehicle; B: Oral glucose tolerance test after 12 weeks of treatment; C: The area under the curve of the oral glucose tolerance test after 12 weeks of treatment; D: Insulin tolerance test after 12 weeks; E: The area under the curve of the insulin tolerance test after 12 weeks of treatment; F: Serum glucagon-like peptide-1 levels; G: Relative expression of farnesoid X receptor and fibroblast growth factor 15 gene expression in the ileum. Data are shown as mean ± SEM. P < 0.05. ns: Not significant (P > 0.05) compared between groups. Statistical analysis was performed using the Student’s t-test. HFD: High-fat diet; STZ: Streptozotocin; BP: Bile powder; OGTT: Oral glucose tolerance test; ITT: Insulin tolerance test; AUC: Area under the curve; GLP-1: Glucagon-like peptide-1; RT-PCR: Real-time polymerase chain reaction.

Figure 6 Mechanism of glucose disorder amelioration by bile powder-mediated intestinal glucagon-like peptide-1 secretion. Our study revealed that bile powder interacts with the intestinal enteroendocrine L cells to inhibit farnesoid X receptor signaling and to promote glucagon-like peptide-1 production. Bile powder is a glucagon-like peptide-1 stimulator and attenuates blood glucose impairment. GLP-1: Glucagon-like peptide-1; FXR: Farnesoid X receptor.