Oral Delivery of Pentameric Glucagon-Like Peptide-1 by Recombinant Lactobacillus in Diabetic Rats

Abstract

Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by intestinal cells and stimulates insulin secretion from the pancreas in a glucose-dependent manner. Exogenously supplied GLP-1 analogues are used in the treatment of type 2 diabetes. An anti-diabetic effect of Lactobacillus in lowering plasma glucose levels and its use as a vehicle for delivery of protein and antibody fragments has been shown previously. The aim of this study was to employ lactobacilli as a vehicle for in situ production and delivery of GLP-1 analogue to normalize blood glucose level in diabetic GK (Goto-Kakizaki) rats. In this study, we designed pentameric GLP-1 (5×GLP-1) analogues which were both expressed in a secreted form and anchored to the surface of lactobacilli. Intestinal trypsin sites were introduced within 5×GLP-1, leading to digestion of the pentamer into an active monomeric form. The E. coli-produced 5×GLP-1 peptides delivered by intestinal intubation to GK rats resulted in a significant improvement of glycemic control demonstrated by an intraperitoneal glucose tolerance test. Meanwhile, the purified 5×GLP-1 (trypsin-digested) from the Lactobacillus cultures stimulated insulin secretion from HIT-T15 cells, similar to the E. coli-produced 5×GLP-1 peptides. When delivered by gavage to GK rats, non-expressor L. paracasei significantly lowered the blood glucose level but 5×GLP-1 expression did not provide an additional anti-diabetic effect, possibly due to the low levels produced. Our results indicate that lactobacilli themselves might be used as an alternative treatment method for type 2 diabetes, but further work is needed to increase the expression level of GLP-1 by lactobacilli in order to obtain a significant insulinotropic effect in vivo.

Lactobacillus delivery of bioactive interleukin-22

“…Selected probiotic Lactobacillus strains can colonize the gastrointestinal tract for week [ 22 ] where they may constitutively produce the desired peptide, reducing their exposure to gastric acid, bile and digestive enzymes. Antibody fragments and incretin hormone have previously been delivered by lactobacilli to treat viral infections or diabetes [ 23 , 24 ]. A major shift in the composition of the intestinal microbiota as well as increased permeability of the intestinal mucosa and bacterial translocation is associated with aggravation of GVHD symptoms [ 25 – 27 ].…”

Advances and potential of gene therapy for type 2 diabetes mellitus

“…A similar study, using Lactobacillus paracasei as host, orally delivered pentameric GLP-1 into diabetic Goto-Kakizaki (GK) rats. However, delivery of 5 Â GLP-1 for 16 days had no added anti-diabetic effect, though L. paracasei itself significantly lowered the blood glucose level [61]. This result might be because of either the low level expression or incorrect enzymatic degradation of 5 Â GLP-1 protein in vivo.…”

“…Crucially, parallel comparisons cannot be made due to the different designs of these reported gene therapies. Although GLP-1 is the most widely studied target for gene therapy for T2DM, contradictory results still exist [61,62]. Hence, we believe parallel studies should be carried out by more groups/centers as reproducibility is crucial for a therapy, for example, using the same gene target, the same delivery method, and the same animal models.…”

“…Probiotics have many health-promoting properties including anti-diabetic properties, and there are different intervention approaches for T2DM using probiotics [74]. As noted, probiotic L. paracasei significantly lowers the blood glucose level [61]. Lactobacillus reuteri SD5865 significantly improves the secretion of GLPs, insulin and C-peptide [75].…”

Engineering bacteria for diagnostic and therapeutic applications

“…Several examples of these mutations and losses have been documented in preclinical studies following delivery of engineered therapeutic bacteria to a host, or even during in vitro growth. 40% -65% of L. paracasei BL23 cells lost engineered plasmids during transit of the rat gut 50 . Similar loss of luminescence gene-carrying plasmids occurred in S. Typhimurium during 60 hours of growth in a mouse tumor model 103 .…”