Ronit Rafaeloff

Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters.

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 850 bp at 1 and 2 d 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 homologous 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.

Islet-cell regeneration in the diabetic hamster pancreas with restoration of normoglycaemia can be induced by a local growth factor(s)

SummaryPartial pancreatic duct obstruction in the hamster leads to the induction of endocrine-cell differentiation and new islet formation. We prepared cytosolic extracts from the partially obstructed pancreas and identified one, which when administered i.p., produced significant increases in the incorporation of tritiated thymidine by ductular and islet cells, as well as a corresponding increase in islet mass. In this study, we evaluate the ability of this extract to reverse streptozotocin diabetes mellitus. Hamsters were treated i. p. twice daily for 7 weeks with either 0.9% NaCl (saline) (n=10) or a cytosol extract (n=10) prepared previously from partially obstructed hamster pancreata. All animals in the cytosol group survived vs only 60% of the saline group (p=0.02). Random blood glucose levels were greater than 22.2 mmol/l in 90% of the saline group vs 40% in the cytosol group (p<0.05). Pancreatic tissue from the surviving saline animals and from persistently hyperglycaemic cytosol-treated animals, showed intra-cytoplasmic vacuolation of islet cells, a characteristic lesion of sustained hyperglycaemic states. Vacuolation was not observed in normoglycaemic extract treated animals. Islets in hyperglycaemic animals demonstrated a profound decrease or absence of immunoreactive insulin, compared to an abundance of immunoreactive beta cells in cytosol-treated animals that reverted to normoglycaemia. In this group, single cells or nests of cells stained for insulin or glucagon cells were identified in ductal epithelium in association with cells budding from the duct. Morphometric analysis of pancreata in reverted cytosol-treated animals showed a new population of small islets compared with saline controls and an increased islet mass. In summary, streptozotocin diabetes can be reversed by new islet formation induced by local pancreatic growth factors, the exact nature of which remains to be determined.

Induction of Islet Cell Differentiation and New Islet Formation in the Hamster-Further Support for a Ductular Origin

Partial obstruction of the adult hamster pancreas leads to islet cell differentiation and new islet formation. From morphologic and morphometric observations, we have tentatively identified the source of the new islet tissue to be from cells in the ducts. In this study, in vivo labeling with a single pulse of tritiated thymidine after partial duct obstruction was used to ascertain whether newly formed islet cells were in fact derived from cells in the ductal epithelium. Supportive evidence for this formulation was also sought using immunocyto-chemistry for islet hormones and in situ hybridization for glucagon and insulin mRNA to probe areas of proliferating duct cells. Endocrine cell differentiation was observed as a migration of cells out from small ducts beginning at about 10 days after obstruction. Duct and islet cell labeling indices (LI; %) in control animals remained at a low level (0.35 ± 0.01 and 0.36 ± 0.03, respectively) throughout the experiment. In contrast, at 2 weeks after partial obstruction, the duct and islet cell LI were 4.2 ± 0.7 and 0.80 ± 0.1 (p < 0.05 vs. control). After 2 weeks, there was a rapid and significant 86% decline in the duct cell LI to a low of 0.6 ± 0.2 at 8 weeks, which was accompanied by a comparable, but reciprocal, 113% increase in the islet cell LI to a high of 1.7 ± 0.8 (p < 0.05). In situ hybridization demonstrated glucagon and insulin mRNA-positive cells within intralobular ducts as early as 6 and 8 days, respectively, after obstruction. Glucagon and insulin peptides appeared in these cells at ∼8 and 10 days, respectively, as cells migrated out from the duct wall. This study provides additional evidence that further supports our concept that pancreatic endocrine cell differentiation in this model reiterates the normal ontogeny of β cell differentiation from cells in the ductular epithelium.

Identification of differentially expressed genes induced in pancreatic islet neogenesis

Cellophane wrapping of the hamster pancreas induces islet neogenesis. We have used the mRNA differential display technique to select for genes expressed during islet neogenesis but not in control pancreata. Ten candidate clones have been identified. Upon sequencing, 6 clones showed a high degree of homology to known genes, 1 showed some, and 3 showed no homology to genes of known sequence. Thus, mRNA differential display is a useful technique to identify genes induced during islet neogenesis, and in combination with screening hamster pancreatic cDNA libraries for full length clones, will enhance the likelihood of capturing the participants in this process.

Expression of Reg gene in the Syrian golden hamster pancreatic islet regeneration model

SummaryWe have reported previously that cellophane wrapping of the hamster pancreas is a stimulus that leads to the induction of duct epithelial cell proliferation, followed by endocrine cell differentiation and new islet formation. Reg is a candidate gene that has been reported to be expressed in regenerating pancreatic islets, suggesting a role in islet growth. We examined Reg gene expression in the cellophane-wrap model by isolating total RNA from hamster pancreata at various times after wrapping. Northern blot analysis using a rat cDNA Reg probe showed no expression of Reg in control non-wrapped hamster pancreas, whereas a strong signal was detected in control wrapped rat pancreas. Using reverse transcription of RNA followed by polymerase chain reaction (PCR) we amplified, isolated and sequenced a 194 base pair product which showed homology to rat Reg in both control and wrapped hamster pancreas. When the PCR product was used as a probe for Northern blot analysis, no signal was detected in control non-wrapped pancreata. In contrast, a strong signal was detected 1 and 2 days after wrapping, which then returned to basal between 4 and 6 days after wrapping. A similar temporal pattern was observed using in situ hybridization to localize the Reg gene. One- and 2-day wrapped but not control pancreas expressed Reg in acinar cells, but not in islets. In conclusion, (a) Reg expression is low in the control hamster pancreas; (b) in the cellophane-wrap model of islet neogenesis, increased Reg mRNA is found within acinar tissue; (c) Reg gene may thus be involved as an acinar paracrine effector of duct cell proliferation in the initial step of islet neogenesis, but may also participate in the inflammatory response to traumatic stimuli.