Oral delivery of GLP-1 peptide using recombinant Lactobacillus gasseri for the treatment of type 2 diabetes mellitus

Abstract

Glucagon-like peptide-1 (GLP-1) is an effective therapeutic peptide for the treatment of type 2 diabetes mellitus (T2DM). Here, we constructed an effective probiotic-based oral GLP-1 delivery system by engineering the probiotic strain of Lactobacillus gasseri (Lgs^GPA) to secrete GLP-1 fusion peptide, which combines GLP-1 with protein transduction domain (PTD) and a serum albumin binding peptide (ABP), GLP-1-PTD-ABP (GPA). The supernatants of Lgs^GPA cultures significantly upregulated the expression of PDX-1 and stimulated insulin release in Min6 cells. Daily oral administration of Lgs^GPA in db/db mice significantly alleviated insulin resistance, hyperglycemia, and dyslipidemia. Simultaneously, the abundance of unclassified_f_Erysipelotrichaceae and Intestinimonas was significantly reduced in db/db mice, while the average abundance of Akkermansia increased in the SD rats. These findings demonstrate that the probiotic-based delivery system represents a versatile and effective strategy for the oral administration of therapeutic peptides. Collectively, our results highlight the potential of this probiotic-based approach as a promising therapeutic and preventive intervention for T2DM.IMPORTANCEIt is important to develop the oral delivery strategy for therapeutic peptides. Due to issues with patient adherence and the low oral bioavailability of current administration methods, researchers have been exploring oral delivery strategies for GLP-1 analogs for many years, including the use of advanced microbiome therapeutics (AMTs). AMTs offer the potential to use engineered microbes for innovative therapeutic applications, such as the oral delivery of GLP-1 analogs. Our approaches offer a general oral delivery strategy for therapeutic peptides. The probiotic-based approach represents a promising method for treating and preventing T2DM.

Keywords: GLP-1 analog; Lactobacillus gasseri; advanced microbiome delivery; glucagon-like peptide-1; gut microbiota; oral delivery; protein transduction domain; serum albumin binding peptide; type 2 diabetes mellitus.

Fig 1

Preparation of GPA peptide and detection of its bioactivity in vivo and in vitro. (a) The schematic of the GPA gene cassette subcloned into the pMFH vector. The amino acid sequences of GLP-1, PTD, and ABP and two linkers, L1 and L2. (b) HPLC profile of purified GPA peptide peak with a retention time between 16.5 and 17.0 min was collected and confirmed by Western blot analysis. (c) The detection of GPA peptide in the blood by HPLC and Western blot analysis, after the rats were injected with 0.02 mg/kg of body weight GPA for 24 h. (d and e) Blood glucose levels (d) and the area under the curve (AUC) (e) in the glucose tolerance test (GTT) of C57BL/6J mice after intraperitoneal injection of 0.2 mg/kg of body weight GPA peptide for 24 h. (f) Random blood glucose of db/db mice (n = 6) after intraperitoneal injection of 0.2 mg/kg of body weight GPA peptide. (g) Fasting blood glucose of db/db mice (n = 6) after intraperitoneal injection of 0.2 mg/kg of body weight GPA peptide for 24 h. (h) The schematic of the gene cassette subcloned into the pMG36e vector. (i and j) The GPA peptide released to the supernatant was identified by Western blot analysis (i) and enzyme-linked immunosorbent assay (ELISA) (j), respectively. (k and l) The β-cell markers PDX-1 (k) and insulin release (l) by Min6 cells, respectively, in the presence of medium of recombinant L. gs. Data were presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, determined by unpaired two-tailed Student’s t test for two-group comparisons, one-way ANOVA followed by Tukey’s multiple comparison test for multiple group comparisons.

Fig 2

Improved the effects of Lgs^GPA on diabetic symptoms in db/db mice. (a) The schematic of the animal experiment. (b) Fasting blood glucose levels. (c) Blood glucose levels and the area under the curve (AUC) in the glucose tolerance test (GTT) of the db/db mice. (d) Blood glucose levels and the AUC in the insulin tolerance test (ITT) of the db/db mice. (e) Serum levels of glycosylated hemoglobin A1c (GHbA1c). (f) Serum levels of aspartate aminotransferase (AST). (g) Serum levels of alanine aminotransferase (ALT). (h) The ratio of AST/ALT. (i) Serum levels of creatinine (CRE). (j) Serum levels of urea (BUN). (k) Serum levels of triglyceride (TG). (l) Plasma levels of total cholesterol (TC). (m) Serum levels of high-density lipoprotein cholesterol (HDL-C). (n) Serum levels of low-density lipoprotein cholesterol (LDL-C). Lgs^36e group, the db/db mice were administered Lgs^36e (n = 6); Lgs^GPA group, the db/db mice were administered Lgs^GPA (n = 5). Data were presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs Lgs^36e group, determined by unpaired two-tailed Student’s t test for two-group comparisons.

Fig 3

Lgs^GPA remodels the gut microbiome in db/db mice. (a) The schematic of the study stool sample collection of each group at the indicated time points. (b) The total number of core operational taxonomic units (OTUs) shared and that are unique in the Venn diagram. (c) β diversity analysis of PCoA analysis on the OTU level. (d) Linear discriminant analysis (LDA) effect size (LEfSe) analysis to identify bacterial genera whose abundance differed significantly between the two groups of mice. Only taxa with LDA scores of more than 2 were presented (n = 3). P value was determined by the Mann-Whitney U test. (e) Student’s t-test bar plot on genus level.

Fig 4

The analysis at the genus level on the stool microbiome of db/db mice. (a) The community heatmap analysis of the top 50 taxonomic composition analysis and the abundance at the genus level. (b) The correlation heatmap analysis of gut microbiota with clinical factors.

Fig 5

The effects of Lgs^GPA on the gut microbiome of SD rats. (a) The schematic of the study stool and blood sample collection of each group at the indicated time points. (b) The content of GPA in the blood of rats was detected by HPLC and Western blot analysis. (c) Community bar plot of the most relevant taxa responsible for the difference at the family level. (d) Community bar plot of the most relevant taxa responsible for the difference at the genus level.