PROBIOTIC STIMULATION OF INCRETIN HORMONE SECRETION AS A POTENTIAL THERAPEUTIC STRATEGY FOR TYPE 2 DIABETES MELLITUS: EXAMINING THE MECHANISM OF ACTION
Panwar H.,Calderwood D., Gillespie A., Graham S., Wylie A., Grant I., Grover S., Green B.
Institute for Global Food Security, School of Biological Sciences, Queen’s University Belfast, Northern Ireland, UK
Incretin hormones are gastrointestinal insulin-releasing peptides involved in the regulation of postprandial nutrient homeostasis. The incretin hormones have been the basis for a number of clinically approved pharmaceutical compounds with good efficacy for the treatment of human type 2 diabetes and its complications. The two established incretin hormones are glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) and they are produced by enteroendocrine cells lining the intestine. Exploiting the effects of human commensal microbiota on intestinal cells could provide novel, natural, safe and cost effective strategies for diabetes therapy. The present study examined the ability of human gut Lactobacillus isolates along with reference probiotic strains to modulate the expression and secretion of incretin hormones besides examining the potential mechanisms involved.
Live bacteria (approximately 1x109) were co-cultured with STC-1pGIPneo murine intestinal cells (2x106) for 3h and incretin hormone secretion was assessed. GLP-1 and GIP concentrations were assessed in cell supernatant using specific and selective immunoassays. Data were analysed by One-Way ANOVA with the Tukey post hoc test. Co-incubation was followed by RNA isolation, cDNA synthesis and RT-qPCR SYBR green based expression study for GLP-1, GIP and free fatty acid receptors. Studies were also conducted with L. rhamnosus alone or in combination with either a Myd88 blocking peptide or an anti-CD14 antibody. Amino acid profiling of cell supernatant was done by GC-MS. Cell cytotoxicity was determined by LDH based cyto-toxicity detection kit (Roche). RT2profiler PCR arrays were used to detect the expression of 84 genes implicated in regulating TLR pathways, in STC-1pGIPneo cells induced with L. rhamnosus.
Acute co-culture of LAB with enteroendocrine cells showed that certain LAB strains elicit GLP-1 and GIP secretion (13-194fold) and upregulate their gene expression. Maximum secretion of GLP-1 and GIP was recorded with L. plantarum subsp. argentototensis (Lb-3), L. johnsonii and L.rhamnosus as determined by RIA and ELISA respectively. However, a varied response for RT-qPCR based expression of respective genes was recorded at transcriptional level with Lb4, Lb6, L. casei, L. plantarum and L. rhamnosus significantly upregulating expression of both GLP-1 and GIP. However, Lb8, Lb9, L. acidophilus and B. bifidum could activate only GIP. LAB induced incretin hormone secretion did not appear to involve nutrient mechanism nor was there any evidence of cytolysis. Instead PCR array studies implicated signalling agents of the toll-like receptor system e.g. L. rhamnosus downregulated MyD88 (23fold) along with over-expression of cell surface antigen, CD14 (17fold). Mechanistic studies found that blockade of MyD88 triggered significant GLP-1 secretion. Furthermore, blocking of CD14 completely attenuated LAB-induced secretion. Besides this, upregulated expression of free fatty acid receptors in presence of selected isolates i.e. GPR-41 and 120 by Lb-6, GPR-40 and 41 by L. casei and only GPR-40 by L.rhamnosus was observed. GC-MS based profiling revealed enhanced level of alanine, proline and histidine.
Shortlisted strains could be explored as prospective candidates for management of diabetes through incretin hormone stimulation, after ensuring their safety and efficacy in animal models and human clinical trials.
Keywords: GIP, Probiotics, Incretin hormones, Type 2 Diabetes, GLP-1
Panwar H., et al. (2016). Probiotic stimulation of incretin hormone secretion as a potential therapeutic strategy for type 2 diabetes mellitus: Examining the mechanism of action. Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 55.). IPC2016