INGAP Research Abstracts

Induction of islet neogenesis by cellophane wrapping (CW) reverses streptozotocin-induced (STZ) diabetes. Administration of Ilotropin, a protein extract isolated from CW pancreata, causes recapitulation of normal islet ontogeny and reverses STZ diabetes, reducing mortality by 50%. We investigated the hypothesis that a novel gene encoding a constituent of Ilotropin was expressed in the hamster pancreas undergoing islet neogenesis. Islet neogenesis associated protein (INGAP) is a product of a novel gene expressed in regenerating hamster pancreas. Northern blot analysis showed a strong single transcript of 850bp at 1 and 2 after CW that disappeared by the 6th day and was absent from untreated control pancreata. INGAP gene is expressed in acinar cells, but not in islets. Western blot analysis demonstrated the presence of INGAP in Ilotropin but not in extracts from control pancreata. A synthetic pentadecapeptide, corresponding to a region unique to INGAP, stimulated a 2.4-fold increase in [3H]thymidine incorporation into hamster duct epithelium in primary culture and a rat pancreatic duct cell line but had no effect on a hamster insulinoma tumor cell line. A portion of human INGAP gene was cloned and appears to be highly homologus to the hamster gene. This data suggests that the INGAP gene is a novel pancreatic gene expressed during islet neogenesis whose protein product is a constituent of Ilotropin and is capable of initiating duct cell proliferation, a prerequisite for islet neogenesis.

INGAP is a Human Cytokine Expressed Only in the Presence of Islet Neogenesis.

• Islet neogenesis associated protein (INGAP) expression is increased in hamster pancreata in which islet neogenesis is experimentally induced by partial duct obstruction. The mRNA and protein are initially found in the acinar tissue 2d after stimulation of islet neogenesis and later in the periphery of islets at 10d.

• We hypothesize that INGAP is only expressed in pancreatic islet regeneration in a topographic location compatible with stimulation of differentiation of a proto-differentiated cell.

• To determine whether INGAP is associated with islet neogenesis in humans we obtained pancreatic tissue from patients undergoing total or partial pancreatectomy or PM autopsies. These included: a 65-year old with Type 1 diabetes and futile regeneration, one patient with ALL, and 6 patients with duct obstruction for other various reasons. We localized INGAP using a polycolonal antibody raised against the amino-terminal region of the mature protein that has considerable homology with the human counterpart. This antibody shows no cross-reactivity with pancreatitis-associated protein (PAP) or Reg protein.

• INGAP immunoreactivity was mainly evident in the periphery of islets but was also present in ducts and in ductal-like cells of patients with various conditions in which partial duct obstruction and islet neogenesis were observed. It was also present in Type 1 diabetes and ALL. INGAP immunoreactivity was negative in pancreatic tissue of normal patients and of patients with pancreatic carcinoids or insulinomas.

• These finds indicate that INGAP is a human cytokine, expressed only in pancreata undergoing islet regeneration in a topographic location compatible with stimulation of a protodifferentiated cell.

• The development of the pancreatic anlage into the complex mature pancreas with components of endocrine, ductal and acinar structures is characterized by complex interactions between as yet unknown factors.

• Islet neogenesis associated protein (INGAP) is a putative islet cell growth and differentiation factor whose expression is stimulated by experimentally induced islet neogenesis.

• We hypothesize that INGAP is expressed during normal ontogeny of the pancreas, prior to formation of adult islets.

• To determine whether there was a developmental change in the expression of INGAP we analyzed tissue obtained from baboon fetuses and mouse embryos at known stages of appearance of the pancreatic anlage. We localized INGAP using a polyclonal antibody raised against the amino-terminal region of the mature protein.

• The results show that INGAP is: expressed in both the mouse embryo and baboon fetus; expressed as early as embryonic day 11 in the mouse and day 100 in the baboon; co-localizes with glucagon but not with insulin or somatostatin in some fetal baboon cells on double immunostaining. INGAP immunoreactivity was mainly evident in foci of endocrine cells but was also present in ducts and in ductal-like cells. Its appearance in ductular structures and co-localization with glucagon imply an ontogenic sequence involving ductular and primitive endocrine cells.

• We conclude that the expression of INGAP in the mouse embryo and baboon fetus occurs at a time, which is compatible with a possible role in initiation of growth and differentiation of pancreatic endocrine cells.

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