Islet neogenesis-associated protein signaling in neonatal pancreatic rat islets: involvement of the cholinergic pathway

Abstract

Islet neogenesis associated protein (INGAP) increases islet mass and insulin secretion in neonatal and adult rat islets. In the present study, we measured the short-and long-term effects of INGAP-PP (a pentadecapeptide having the 104-118 amino acid sequence of INGAP) upon islet protein expression and phosphorylation of components of the PI3K, MAPK and cholinergic pathways, and on insulin secretion. Short-term exposure of neonatal islets to INGAP-PP (90 s, 5, 15, and 30 min) significantly increased Akt1 -Ser473 and MAPK3/1 -Thr202/Tyr204 phosphorylation and INGAP-PP also acutely increased insulin secretion from islets perifused with 2 and 20 mM glucose. Islets cultured for 4 days in the presence of INGAP-PP showed an increased expression of Akt1, Frap1, and Mapk1 mRNAs as well as of the muscarinic M3 receptor subtype, and phospholipase C (PLC)-b2 proteins. These islets also showed increased Akt1 and MAPK3/1 protein phosphorylation. Brief exposure of INGAP-PP-treated islets to carbachol (Cch) significantly increased P70S6K -Thr389 and MAPK3/1 phosphorylation and these islets released more insulin when challenged with Cch that was prevented by the M3 receptor antagonist 4-DAMP, in a concentration-dependent manner. In conclusion, these data indicate that short-and long-term exposure to INGAP-PP significantly affects the expression and the phosphorylation of proteins involved in islet PI3K and MAPK signaling pathways. The observations of INGAPP-PP-stimulated up-regulation of cholinergic M3 receptors and PLC-b2 proteins, enhanced P70S6K and MAPK3/1 phosphorylation and Cch-induced insulin secretion suggest a participation of the cholinergic pathway in INGAP-PP-mediated effects.

Mechanisms of action of islet neogenesis-associated protein: comparison of the full-length recombinant protein and a bioactive peptide

“…A rapid activation of ERK1/2 (MAPK3/1) by INGAP-P has also been shown in neonatal rat islets (5), and involvement of cAMP-PKA in the INGAP-P-induced neurite outgrowth has been reported (51). However, although our data do not indicate the involvement of PI3K/Akt in the proliferative action of INGAP (Fig.…”

“…Since INGAP-P has been shown previously to activate the PI3K/Akt signaling pathway (5,25), and because this pathway can be involved in cell proliferation, we measured phosphoAkt (Ser 473 ) in a time course experiment and observed a weak increase (not statistically significant) by INGAP-P at 30 min but not by rINGAP (Fig. 7D).…”

“…A number of signaling pathways activated by INGAP-P have been reported, with the PI3K/Akt pathway being the most common (5,25). A rapid activation of ERK1/2 (MAPK3/1) by INGAP-P has also been shown in neonatal rat islets (5), and involvement of cAMP-PKA in the INGAP-P-induced neurite outgrowth has been reported (51).…”

“…Biological effects of a synthetic INGAP-P have been studied extensively both in vitro and in vivo in our laboratory and by others. To date, the most compelling data show that INGAP-P 1) induces in vitro regeneration of functional human islets from dedifferentiated, islet-derived, duct-like structures (25), 2) dose-dependently stimulates expansion of ␤-cell mass in rodents, dogs, and cynomolgus monkeys (29,38,43), and 3) increases insulin secretion and ␤-cell size and upregulates the expression of several genes related to ␤-cell function in rat neonatal islets in vitro (4,5,7). These important results were followed by clinical trials to investigate its efficacy and safety in humans, in which INGAP-P was found to have a signal effect, with an improvement of glucose homeostasis confirmed by A1C reduction at 90 days in patients with type 2 diabetes and by a significant increase in C-peptide secretion in patients with type 1 diabetes (18,54).…”

Pancreatic acinar-specific overexpression of Reg2 gene offered no protection against either experimental diabetes or pancreatitis in mice

“…The PI3K/Akt pathway plays a critical role in the proliferation and regeneration of the endocrine and exocrine pancreas (6): an increase in cellular phosphorylated Akt was reported during acinar regeneration in the remnant pancreas following partial pancreatectomy (31), and cyclin D1 was shown to be increased by PI3K/Akt activation in the ␤-cells (7). In Reg1-induced ␤-cell regeneration, PI3K/activating transcription factor (ATF)-2/cyclin D1 signaling was activated, and INGAP peptide also increased Akt phosphorylation (1,25). Class I PI3Ks are heterodimeric enzymes consisting of a 110-kDa catalytic subunit in complex with a regulatory subunit (e.g., p85).…”