Akari Inada

Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth

The regenerative process in the pancreas is of particular interest because diabetes results from an inadequate number of insulin-producing beta cells and pancreatic cancer may arise from the uncontrolled growth of progenitor/stem cells. Continued and substantial growth of islet tissue occurs after birth in rodents and humans, with additional compensatory growth in response to increased demand. In rodents there is clear evidence of pancreatic regeneration after some types of injury, with proliferation of preexisting differentiated cell types accounting for some replacement. Additionally, neogenesis or the budding of new islet cells from pancreatic ducts has been reported, but the existence and identity of a progenitor cell have been debated. We hypothesized that the progenitor cells are duct epithelial cells that after replication undergo a regression to a less differentiated state and then can form new endocrine and exocrine pancreas. To directly test whether ductal cells serve as pancreatic progenitors after birth and give rise to new islets, we generated transgenic mice expressing human carbonic anhydrase II (CAII) promoter: Cre recombinase (Cre) or inducible CreERTM to cross with ROSA26 loxP-Stop-loxP LacZ reporter mice. We show that CAII-expressing cells within the pancreas act as progenitors that give rise to both new islets and acini normally after birth and after injury (ductal ligation). This identification of a differentiated pancreatic cell type as an in vivo progenitor of all differentiated pancreatic cell types has implications for a potential expandable source for new islets for replenishment therapy for diabetes.

The pancreatic ductal epithelium serves as a potential pool of progenitor cells

Abstract:  With the increasing success of islet transplantation, β‐cell replacement therapy has had renewed interest. To make such a therapy available to more than a few of the thousands of patients with diabetes, new sources of insulin‐producing cells must become readily available. The most promising sources are stem cells, whether embryonic or adult stem cells. Clearly identifiable adult pancreatic stem cells have yet to be characterized. Although considerable evidence suggests their possibility, recent lineage‐tracing experiments challenge their existence. Even in light of these lineage‐tracing experiments, we suggest that evidence for neogenesis or new islet formation after birth remains strong. Our work has suggested that the pancreatic duct epithelium itself serves as a pool for progenitors for both islet and acinar tissues after birth and into adulthood and, thus, that the duct epithelium can be considered ‘facultative stem cells’. We will develop our case for this hypothesis in this perspective.

Transdifferentiation of pancreatic ductal cells to endocrine β-cells

The regenerative process in the pancreas is of particular interest, since diabetes, whether Type 1 or Type 2, results from an inadequate amount of insulin-producing beta-cells. Islet neogenesis, or the formation of new islets, seen as budding of hormone-positive cells from the ductal epithelium, has long been considered to be one of the mechanisms of normal islet growth after birth and in regeneration, and suggested the presence of pancreatic stem cells. Results from the rat regeneration model of partial pancreatectomy led us to hypothesize that differentiated pancreatic ductal cells were the pancreatic progenitors after birth, and that with replication they regressed to a less differentiated phenotype and then could differentiate to form new acini and islets. There are numerous supportive results for this hypothesis of neogenesis, including the ability of purified primary human ducts to form insulin-positive cells budding from ducts. However, to rigorously test this hypothesis, we took a direct approach of genetically marking ductal cells using CAII (carbonic anhydrase II) as a duct-cell-specific promoter to drive Cre recombinase in lineage-tracing experiments using the Cre-Lox system. We show that CAII-expressing pancreatic cells act as progenitors that give rise to both new islets and acini after birth and after injury (ductal ligation). This identification of a differentiated pancreatic cell type as an in vivo progenitor for all differentiated pancreatic cell types has implications for a potential expandable source for new islets for replenishment therapy for diabetes either in vivo or ex vivo.

Overexpression of Inducible Cyclic AMP Early Repressor Inhibits Transactivation of Genes and Cell Proliferation in Pancreatic β Cells

ABSTRACT Transcriptional control mediated by the cyclic AMP-responsive element (CRE) represents an important mechanism of gene regulation. To test our hypothesis that increased inducible cyclic AMP early repressor (ICER) Iγ inhibits function of CRE-binding proteins and thus disrupts CRE-mediated transcription in pancreatic β cells, we generated transgenic mice with β-cell-directed expression of ICER Iγ, a powerful repressor that is greatly increased in diabetes. Three transgenic lines clearly show that increased ICER Iγ expression in β cells results in early severe diabetes. From birth islets were severely disorganized with a significantly increased proportion of α cells throughout the islet. Diabetes results from the combined effects of impaired insulin expression and a decreased number of β cells. The decrease in β cells appears to result from impaired proliferation rather than from increased apoptosis after birth. Cyclin A gene expression is impaired by the strong inhibition of ICER; the suppression of cyclin A results in a substantially decreased proliferation of β cells in the postnatal period. These results suggest that CRE and CRE-binding factors have an important role in pancreatic β-cell physiology not only directly by regulation of gene trans-activation but also indirectly by regulation of β-cell mass.

Establishment of a Diabetic Mouse Model with Progressive Diabetic Nephropathy

Although diabetic animal models exist, no single animal model develops renal changes identical to those seen in humans. Here we show that transgenic mice that overexpress inducible cAMP early repressor (ICER Igamma) in pancreatic beta cells are a good model to study the pathogenesis of diabetic nephropathy. Although ICER Igamma transgenic mice exhibit extremely high blood glucose levels throughout their lives, they survive long enough to develop diabetic nephropathy. Using this model we followed the progress of diabetic renal changes compared to those seen in humans. By 8 weeks of age, the glomerular filtration rate (GFR) was already increased, and glomerular hypertrophy was prominent. At 20 weeks, GFR reached its peak, and urine albumin excretion rate was elevated. Finally, at 40 weeks, diffuse glomerular sclerotic lesions were prominently accompanied by increased expression of collagen type IV and laminin and reduced expression of matrix metalloproteinase-2. Nodular lesions were absent, but glomerular basement membrane thickening was prominent. At this point, GFR declined and urinary albumin excretion rate increased, causing a nephrotic state with lower serum albumin and higher serum total cholesterol. Thus, similar to human diabetic nephropathy, ICER Igamma transgenic mice exhibit a stable and progressive phenotype of diabetic kidney disease due solely to chronic hyperglycemia without other modulating factors.

The cyclic AMP response element modulator family regulates the insulin gene transcription by interacting with transcription factor IID.

We analyzed a mechanism of transcriptional regulation of the human insulin gene by cyclic AMP response element modulator (CREM) through four cyclic AMP response elements (CREs). We isolated two novel CREM isoforms (CREMΔQ1 and CREMΔQ2), which lack one of the glutamine-rich domains, Q1 and Q2 respectively, and six known isoforms (CREMτα, CREMα, inducible cyclic AMP early repressor (ICER) I, ICER Iγ, CREM-17X, and CREM-17) from rat pancreatic islets and the RINm5F pancreatic β-cell line. CREM isoforms functioned as efficient transcriptional activators or repressors to modulate insulin promoter activity by binding to all of the insulin CREs. The binding activity of repressors is higher than that of activators and suppressed not only basal activity but also activator-induced activities. Furthermore, CREM activator interacted directly with the transcription factor IID components hTAFII130 and TATA box-binding protein (TBP). These results suggest that the activation of the insulin gene transcription by CREM activator is mediated by not only direct binding to the CREs but also by recruiting transcription factor IID to the insulin promoter via its interaction with hTAFII130 and TBP. On the other hand, the CREM repressor ICER competitively interrupts the binding of the activators to CREs and does not interact with either TBP or hTAFII130; therefore, it might fail to stabilize the basal transcriptional machinery and repress transactivation.

A model for diabetic nephropathy: advantages of the inducible cAMP early repressor transgenic mouse over the streptozotocin-induced diabetic mouse.

We have previously found progressive diabetic nephropathy in inducible cAMP early repressor (ICER Iγ) transgenic (Tg) mice. The ICER Iγ Tg mouse is an interesting model of sustained hyperglycemia due to its low production of insulin and insulin‐producing β cells. Here in a longitudinal study we further analyzed diabetic nephropathy and structural and functional alterations in other organs, comparing our model with streptozotocin (STZ)‐diabetic model mice. The high‐dose STZ‐diabetic model showed marked variation in blood glucose levels and severe toxicity of STZ in the liver and kidney. The low‐dose STZ‐diabetic model showed less toxicity, but the survival rate was very low. STZ‐diabetic mice had much more variation of glomerular hypertrophy and sclerosis. Furthermore, non‐specific toxicity of STZ or insulin injections to maintain optimal blood glucose levels might have another effect upon the diabetic renal changes. In contrast, ICER Iγ Tg mice exhibited a stable and progressive phenotype of diabetic kidney disease solely due to chronic hyperglycemia without other modulating factors. Thus, ICER Iγ Tg mouse has advantages for examining diabetic renal disease, and offers unique and very different perspectives compared to STZ model. J. Cell. Physiol. 215: 383–391, 2008.

Timing and expression pattern of carbonic anhydrase II in pancreas

In the search for genetic markers for assessing the role of duct cells in pancreas growth, we examined whether carbonic anhydrase II (CAII) has ductal cell specificity. We determined the distribution and timing of CAII expression in mouse pancreas from embryonic stage to adult. The pancreatic ducts only start expressing CAII at embryonic day (E) 18.5, with increases after birth. Around E15.5, glucagon‐positive cells, but not insulin‐positive cells, also express CAII, with further increases by adult. CAII expression was restricted to cells within ductal structures and glucagon‐positive cells with no colocalization with any insulin‐positive cells at any time. In the human pancreas, CAII expression is restricted to the ducts. Furthermore, the activity of a 1.6‐kb fragment of the human promoter with Luciferase assays was moderately strong compared with the cytomegalovirus promoter in human pancreatic duct cell line (PANC‐1). Thus, we believe that the CAII gene could serve as a useful pancreatic duct cell marker. Developmental Dynamics 235:1571–1577, 2006.© 2006 Wiley‐Liss, Inc.

Reduced Tyk2 gene expression in β-cells due to natural mutation determines susceptibility to virus-induced diabetes

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic β-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by β-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic β-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.

PDX1 in Ducts Is Not Required for Postnatal Formation of β-Cells but Is Necessary for Their Subsequent Maturation

Pancreatic duodenal homeobox-1 (Pdx1), a transcription factor required for pancreatic development and maintenance of β-cell function, was assessed for a possible role in postnatal β-cell formation from progenitors in the pancreatic ducts by selectively deleting Pdx1 from the ducts. Carbonic anhydrase II (CAII)Cre;Pdx1Fl mice were euglycemic for the first 2 postnatal weeks but showed moderate hyperglycemia from 3 to 7 weeks of age. By 10 weeks, they had near-normal morning fed glucose levels but showed severely impaired glucose tolerance and insulin secretion. Yet the loss of Pdx1 did not result in decreased islet and β-cell mass at 4 and 10 weeks of age. Within the same pancreas, there was a mixed population of islets, with PDX1 and MAFA protein expression normal in some cells and severely diminished in others. Even at 10 weeks, islets expressed immaturity markers. Thus, we conclude that Pdx1 is not necessary for the postnatal formation of β-cells but is essential for their full maturation to glucose-responsive β-cells.