Swertisin an Anti-Diabetic Compound Facilitate Islet Neogenesis from Pancreatic Stem/Progenitor Cells via p-38 MAP Kinase-SMAD Pathway: An In-Vitro and In-Vivo Study

Abstract

Transplanting islets serves best option for restoring lost beta cell mass and function. Small bio-chemical agents do have the potential to generate new islets mass, however lack of understanding about mechanistic action of these small molecules eventually restricts their use in cell-based therapies for diabetes. We recently reported “Swertisin” as a novel islet differentiation inducer, generating new beta cells mass more effectively. Henceforth, in the present study we attempted to investigate the molecular signals that Swertisin generate for promoting differentiation of pancreatic progenitors into islet cells. To begin with, both human pancreatic progenitors (PANC-1 cells) and primary cultured mouse intra-islet progenitor cells (mIPC) were used and tested for Swertisin induced islet neogenesis mechanism, by monitoring immunoblot profile of key transcription factors in time dependent manner. We observed Swertisin follow Activin-A mediated MEPK-TKK pathway involving role of p38 MAPK via activating Neurogenin-3 (Ngn-3) and Smad Proteins cascade. This MAP Kinase intervention in differentiation of cells was confirmed using strong pharmacological inhibitor of p38 MAPK (SB203580), which effectively abrogated this process. We further confirmed this mechanism in-vivo in partial pancreatectomised (PPx) mice model, where we could show Swertisin exerted potential increase in insulin transcript levels with persistent down-regulation of progenitor markers like Nestin, Ngn-3 and Pancreatic Duodenal Homeobox Gene-1 (PDX-1) expression, within three days post PPx. With detailed molecular investigations here in, we first time report the molecular mode of action of Swertisin for islet neogenesis mediated through MAP Kinase (MEPK-TKK) pathway involving Ngn-3 and Smad transcriptional regulation. These findings held importance for developing Swertisin as potent pharmacological drug candidate for effective and endogenous differentiation of islets in cell based therapy for diabetes.

Pancreatic resident endocrine progenitors demonstrate high islet neogenic fidelity and committed homing towards diabetic mice pancreas

“…Furthermore, this rapid differentiation was observed in both Activin-A and Swertisin that follow the AKT-MEPK-TKK pathway, elucidated in our previous report (Dadheech et al, 2015). Analysing the temporal protein expression from undifferentiated PREPs at day zero to completely mature islet clusters at day four confirmed high islet cluster forming fidelity.…”

“…The islets generated from Activin-A and Swertisin groups were functional that is expressed Insulin transcript and positive for hormones including INSULIN and GLUCAGON, expressed GLUT2 and NKX6.1 (β-cell marker) and demonstrated glucose-stimulated c-peptide secretion. We observed that Swertisin-induced islets were significantly superior across all the functional parameters, thus generating a better quality of islet clusters, which could effectively maintain glucose homeostasis (Dadheech et al, 2013(Dadheech et al, , 2015Lumelsky et al, 2001). We further, wanted to evaluate the efficacy of both PREPs homing toward the damaged pancreas and their islet neogenic potential in the in vivo diabetic mice model.…”

“…2.10 | RNA extraction, first strand cDNA synthesis and real-time quantitative PCR (qRT-PCR) RNA was harvested from the C2C12 cell line, undifferentiated PREPs, and clusters at every time point during islet differentiation across groups followed by cDNA preparation and qRT-PCR, as described in our previous report (Dadheech et al, 2015). The primers used for the RT-PCR are given in Supporting Information Table S7.…”

Direct lineage tracing reveals Activin-a potential for improved pancreatic homing of bone marrow mesenchymal stem cells and efficient ß-cell regeneration in vivo

“…Prior to lineage tracing experiments, we examined the ß-cell differentiation potential of genetically labeled BMSCs using Activin-a growth factor, ex vivo, as previously reported [ 26 , 31 ]. Genome integrated GFP + BMSC showed effective cell clustering at day 2 deciphered into islet-like aggregates by day 4 till day 7, similar to the non-transfected control BMSC (Suppl.…”

“…GFP + BMSCs were assessed for in vitro islet differentiation and formation of ILCC using Activin-a (20 ng/ml) as described previously [ 26 , 27 ].…”