Beta-cell ß1 integrin deficiency affects in utero development of islet growth and vascularization.

The ß1 integrin subunit contributes to pancreatic beta cell growth and function through communication with the extracellular matrix (ECM). The effects of in vitro and in vivo ß1 integrin knockout have been extensively studied in mature islets, yet no study to date has examined how the loss of ß1 integrin during specific stages of pancreatic development impacts beta cell maturation. Beta-cell-specific tamoxifen-inducible Cre recombinase (MIP-CreERT) mice were crossed with mice containing floxed Itgb1 (ß1 integrin) to create an inducible mouse model (MIPß1KO) at the second transition stage (e13.5) of pancreas development. By e19.5-20.5, the expression of beta-cell ß1 integrin in fetal MIPß1KO mice was significantly reduced and these mice displayed decreased beta cell mass, density and proliferation. Morphologically, fetal MIPß1KO pancreata exhibited reduced islet vascularization and nascent endocrine cells in the ductal region. In addition, decreased ERK phosphorylation was observed in fetal MIPß1KO pancreata. The expression of transcription factors needed for beta-cell development was unchanged in fetal MIPß1KO pancreata. The findings from this study demonstrate that ß1 integrin signaling is required during a transition-specific window in the developing beta-cell to maintain islet mass and vascularization.

Long-term c-Kit overexpression in beta cells compromises their function in ageing mice.

AIMS/HYPOTHESIS: c-Kit signalling regulates intracellular pathways that enhance beta cell proliferation, insulin secretion and islet vascularisation in mice up to 28 weeks of age and on short-term high-fat diet. However, long-term c-Kit activation in ageing mouse islets has yet to be examined. This study utilises beta cell-specific c-Kit-overexpressing transgenic (c-KitßTg) ageing mice (~60 weeks) to determine the effect of its activation on beta cell dysfunction and insulin secretion. METHODS: Wild-type and c-KitßTg mice, aged 60 weeks, were examined using metabolic tests to determine glucose tolerance and insulin secretion. Pancreas histology and proteins in isolated islets were examined to determine the expression of beta cell transcription factors, proliferation and intracellular signalling. To determine the role of insulin receptor signalling in ageing c-KitßTg mice, we generated beta cell-specific inducible insulin receptor knockout in ageing c-KitßTg mice (c-KitßTg;ßIRKO mice) and examined the ageing mice for glucose tolerance and islet histology. RESULTS: Ageing c-KitßTg mice progressively developed glucose intolerance, compared with age-matched wild-type littermates, due to impaired insulin secretion. Increased beta cell mass, proliferation and nuclear forkhead box transcription factor O1 (FOXO1) expression and reduced exocytotic protein levels were detected in ageing c-KitßTg mouse islets. Protein analyses of isolated islets showed increased insulin receptor, phosphorylated IRS-1Ser612 and cleaved poly(ADP-ribose) polymerase levels in ageing c-KitßTg mice. Ageing c-KitßTg mouse islets treated ex vivo with insulin demonstrated reduced Akt phosphorylation, indicating that prolonged c-Kit induced beta cell insulin insensitivity. Ageing c-KitßTg;ßIRKO mice displayed improved glucose tolerance and beta cell function compared with ageing c-KitßTg mice. CONCLUSIONS/INTERPRETATION: These findings indicate that long-term c-Kit overexpression in beta cells has a negative impact on insulin exocytosis and that temporally dependent regulation of c-Kit-insulin receptor signalling is important for optimal beta cell function.

Aldehyde dehydrogenase 1 activity in the developing human pancreas modulates retinoic acid signalling in mediating islet differentiation and survival.

AIMS/HYPOTHESIS: Aldehyde dehydrogenase 1 (ALDH1), a human stem-cell marker, is an enzyme responsible for converting retinaldehydes to retinoic acids (RAs) to modulate cell differentiation. However, data on expression levels and functional roles of ALDH1 during human fetal pancreatic development are limited. The focus of this study was to characterise ALDH1 expression patterns and to determine its functional role in islet cell differentiation. METHODS: The presence of ALDH1 in the human fetal pancreas (8-22 weeks) was characterised by microarray, quantitative RT-PCR, western blotting and immunohistological approaches. Isolated human fetal islet-epithelial cell clusters were treated with ALDH1 inhibitors, retinoic acid receptor (RAR) agonists and ALDH1A1 small interfering (si)RNA. RESULTS: In the developing human pancreatic cells, high ALDH1 activity frequently co-localised with key stem-cell markers as well as endocrine transcription factors. A high level of ALDH1 was expressed in newly differentiated insulin(+) cells and this decreased as development progressed. Pharmacological inhibition of ALDH1 activity in human fetal islet-epithelial cell clusters resulted in reduced endocrine cell differentiation and increased cell apoptosis, and was reversed with co-treatment of RAR/RXR agonists. Furthermore, siRNA knockdown of ALDH1A1 significantly decreased RAR expression and induced cell apoptosis via suppression of the phosphoinositide 3-kinase (PI3K) pathway and activation of caspase signals. CONCLUSIONS/INTERPRETATION: Our findings indicate that ALDH1(+) cells represent a pool of endocrine precursors in the developing human pancreas and that ALDH1 activity is required during endocrine cell differentiation. Inhibition of ALDH1-mediated retinoid signalling impairs human fetal islet cell differentiation and survival.

Harnessing Proliferation for the Expansion of Stem Cell-Derived Pancreatic Cells: Advantages and Limitations.

Restoring the number of glucose-responsive ß-cells in patients living with diabetes is critical for achieving normoglycemia since functional ß-cells are lost during the progression of both type 1 and 2 diabetes. Stem cell-derived ß-cell replacement therapies offer an unprecedented opportunity to replace the lost ß-cell mass, yet differentiation efficiencies and the final yield of insulin-expressing ß-like cells are low when using established protocols. Driving cellular proliferation at targeted points during stem cell-derived pancreatic progenitor to ß-like cell differentiation can serve as unique means to expand the final cell therapeutic product needed to restore insulin levels. Numerous studies have examined the effects of ß-cell replication upon functionality, using primary islets in vitro and mouse models in vivo, yet studies that focus on proliferation in stem cell-derived pancreatic models are only just emerging in the field. This mini review will discuss the current literature on cell proliferation in pancreatic cells, with a focus on the proliferative state of stem cell-derived pancreatic progenitors and ß-like cells during their differentiation and maturation. The benefits of inducing proliferation to increase the final number of ß-like cells will be compared against limitations associated with driving replication, such as the blunted capacity of proliferating ß-like cells to maintain optimal ß-cell function. Potential strategies that may bypass the challenges induced by the up-regulation of cell cycle-associated factors during ß-cell differentiation will be proposed.

Postnatal knockout of beta cell insulin receptor impaired insulin secretion in male mice exposed to high-fat diet stress.

The presence of insulin receptor (IR) on insulin-secreting beta cells suggests an autocrine regulatory role for insulin in its own signalling. Congenital beta cell-specific IR knockout (ßIRKO) mouse studies have demonstrated the development of age-dependent glucose intolerance. We investigated the role of beta cell IR signalling specifically during postnatal life following undisturbed prenatal pancreatic development and maturation. We utilized a tamoxifen-inducible mouse insulin 1 promoter (MIP) driven Cre recombinase IR knockout mouse model (MIP-ßIRKO) to achieve partial knockout of IR in islets and determine the functional role of beta cell IR in adult mice fed a control normal diet (ND) or 60% high-fat diet (HFD). At 24 weeks of age, MIP-ßIRKO ND mice maintained glucose tolerance, insulin release, and unchanged beta cell mass when compared to control ND mice. In contrast, 24-week-old MIP-ßIRKO mice demonstrated significant glucose intolerance and lower insulin release after 18 weeks of HFD feeding. A reduction in beta cell soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein expression, phosphorylated AktS473 and P70S6K1T389, and glucose transporter 2 (GLUT2) expression were also identified in MIP-ßIRKO HFD islets. Overall, the postnatal knockout of beta cell IR in HFD-fed mice resulted in decreased expression of beta cell glucose-sensing and exocytotic proteins and a reduction in intracellular signalling. These findings highlight that IR expression in the adult islet is required to maintain beta cell function under hyperglycemic stress.

ß-Cell Receptor Tyrosine Kinases in Controlling Insulin Secretion and Exocytotic Machinery: c-Kit and Insulin Receptor.

Insulin secretion from pancreatic ß-cells is initiated through channel-mediated depolarization, cytoskeletal remodeling, and vesicle tethering at the cell membrane, all of which can be regulated through cell surface receptors. Receptor tyrosine kinases (RTKs) promote ß-cell development and postnatal signaling to improve ß-cell mass and function, yet their activation has also been shown to initiate exocytotic events in ß-cells. This review examines the role of RTK signaling in insulin secretion, with a focus on RTKs c-Kit and insulin receptor (IR). Pathways that control insulin release and the potential interplay between c-Kit and IR signaling are discussed, along with clinical implications of RTK therapy on insulin secretion.

Characterization and Differentiation of Sorted Human Fetal Pancreatic ALDHhi and ALDHhi/CD133+ Cells Toward Insulin-Expressing Cells.

The enzyme aldehyde dehydrogenase (ALDH) is found in developing and multipotent cell populations, and is important for the production and regulation of retinoic acid, which controls ß-cell differentiation in the pancreas. The role of ALDH-expressing cells in the formation of endocrine-like cells and co-localization with the putative stem cell marker CD133 has not been examined during human pancreatic development. This study focuses on the co-expression of CD133 on ALDH+ cells from the human fetal pancreas (18-22 weeks of fetal age) with transcription factors (TFs) central to endocrine cell development. Fluorescence-activated cell sorting demonstrated that cells with high ALDH activity (ALDHhi) had increased co-expression of CD133 and endocrine-lineage TFs when compared with cells with low ALDH (ALDHlo) expression. Hormone-expressing (insulin, somatostatin) and ductal cells (CK19) were noted in the ALDHhi population, while mesenchymal (vimentin) and endothelial (CD31) markers were predominantly found in ALDHlo cells. Culture of sorted ALDHhi or ALDHhi/CD133+ cells resulted in loss of endocrine TF, insulin, and CK19 expression. The formation of cell clusters from cultured ALDHhi or ALDHhi/CD133+ cells led to restored CK19 expression and showed endocrine TFs and insulin expression. In summary, pancreatic ALDHhi cells contain a heterogeneous CD133-enriched population with a subset of ß-cell associated markers in the developing human pancreas.

ß-cell insulin receptor deficiency during in utero development induces an islet compensatory overgrowth response.

The presence of insulin receptor (IR) on ß-cells suggests that insulin has an autocrine/paracrine role in the regulation of ß-cell function. It has previously been reported that the ß-cell specific loss of IR (ßIRKO) leads to the development of impaired glycemic regulation and ß-cell death in mice. However, temporally controlled ßIRKO induced during the distinct transitions of fetal pancreas development has yet to be investigated. We hypothesized that the presence of IR on ß-cells during the 2nd transition phase of the fetal murine pancreas is required for maintaining normal islet development.We utilized a mouse insulin 1 promoter driven tamoxifen-inducible Cre-recombinase IR knockout (MIP-ßIRKO) mouse model to investigate the loss of ß-cell IR during pancreatic development at embryonic day (e) 13, a phase of endocrine proliferation and ß-cell fate determination. Fetal pancreata examined at e19-20 showed significantly reduced IR levels in the ß-cells of MIP-ßIRKO mice. Morphologically, MIP-ßIRKO pancreata exhibited significantly enlarged islet size with increased ß-cell area and proliferation. MIP-ßIRKO pancreata also displayed significantly increased Igf-2 protein level and Akt activity with a reduction in phospho-p53 when compared to control littermates. Islet vascular formation and Vegf-a protein level was significantly increased in MIP-ßIRKO pancreata.Our results demonstrate a developmental role for the ß-cell IR, whereby its loss leads to an islet compensatory overgrowth, and contributes further information towards elucidating the temporally sensitive signaling during ß-cell commitment.

c-Kit Receptor Signaling Regulates Islet Vasculature, ß-Cell Survival, and Function In Vivo.

The receptor tyrosine kinase c-Kit plays an integral role in maintaining ß-cell mass and function. Although c-Kit receptor signaling promotes angiogenesis in multiple cell types, its role in islet vasculature is unknown. This study examines the effects of c-Kit-mediated vascular endothelial growth factor isoform A (VEGF-A) and islet vascularization on ß-cell function and survival using in vitro cell culture and in vivo mouse models. In cultured INS-1 cells and primary islets, c-Kit regulates VEGF-A expression via the Akt/mammalian target of rapamycin (mTOR) signaling pathway. Juvenile mice with mutated c-Kit (c-Kit(Wv/+)) showed impaired islet vasculature and ß-cell dysfunction, while restoring c-Kit expression in ß-cells of c-Kit(Wv/+) mice rescued islet vascular defects through modulation of the Akt/mTOR/VEGF-A pathway, indicating that c-Kit signaling in ß-cells is a required regulator for maintaining normal islet vasculature. Furthermore, ß-cell-specific c-Kit overexpression (c-KitßTg) in aged mice showed significantly increased islet vasculature and ß-cell function, but, when exposed to a long-term high-fat diet, c-Kit signaling in c-KitßTg mice induced substantial vascular remodeling, which resulted in increased islet inflammatory responses and ß-cell apoptosis. These results suggest that c-Kit-mediated VEGF-A action in ß-cells plays a pivotal role in maintaining islet vascularization and function.

The role of SOX9 transcription factor in pancreatic and duodenal development.

Progenitor expansion during development is a highly regulated process dictating the final organ size, while expansion of specific progenitor populators can adjust the final cellular composition of the organ. Understanding factors involved in these pathways is required to develop cell-based therapies such as ß-cell transplantation for conditions such as diabetes mellitus. One versatile factor controlling both processes as well as a network of other proteins involved in pancreatic and duodenal development is the transcription factor SOX9. This review will focus on a comparison of SOX9 function during progenitor expansion and differentiation in the developing pancreas and duodenum with specific focus on endocrine development. During human pancreatic development, SOX9 functions in a dose-dependent manner to regulate epithelial progenitor expansion and endocrine differentiation. SOX9 expression is eventually limited to a subset of ductal and centroacinar cells, hypothesized to be the pancreatic stem cell compartment. Similarly, during duodenal development, SOX9 is expressed in most early epithelial progenitors and becomes gradually restricted to proliferative progenitors in the lower crypts, as well as mature Paneth and enteroendocrine cells indicating some differences in functional roles. However, in both developmental contexts, SOX9 is involved in pathways responsible for cellular proliferation and differentiation, such as Notch and Wnt. With its adaptable and central function in progenitor control, SOX9 represents an attractive target for manipulation for in vitro progenitor expansion and differentiation meriting further investigation.