Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.     

Distinct distribution of laminin and its integrin receptors in the pancreas.

Tissue function is regulated by the extracellular microenvironment including cell basement membranes, in which laminins are a major component. Previously, we found that laminin-1 promotes differentiation and survival of pancreatic islet cells. Here we characterize the expression pattern of laminins and their integrin receptors in adult pancreas. Although they are expressed in the basement membrane of acinar cells and duct epithelium, no laminin chains examined were detected extracellularly in the pancreatic islets. In contrast to laminin beta(1)- and gamma(1)-chains, the alpha(1)-chain, unique to laminin-1, was not detected. Laminin-10 (alpha(5)beta(1)gamma(1)) was expressed in acinar tissue, whereas laminins-2 (alpha(2)beta(1)gamma(1)) and -10 were expressed in the blood vessels. The laminin connector molecule, nidogen-1, had a distribution similar to that of laminin beta(1) and gamma(1), whereas fibulin-1 and -2, which compete with nidogen-1, were mostly confined to blood vessels. Integrin subunits alpha(6) and alpha(3) were detected in acinar cells and duct epithelial cells, but alpha(6) was absent in islet cells. Integrin alpha(6)beta(4) was detected only in duct cells, alpha(6)beta(1) in both acinar and ductal cells, and alpha(3)beta(1) in acinar, duct, and islet cells. These findings are a basis for further investigation of the role of extracellular matrix molecules and their receptors in pancreas function.     

Cytochemical localization of NADPH-diaphorase in the four types of pancreatic islet cell

NADPH-diaphorase activity, which has been previously reported to be associated with the enzyme nitric oxide synthase (NOS), was localized cytochemically in the pancreatic islets of normal rats. All islet cells types, i.e. insulin-, glucagon-, somatostatin- and pancreatic polypeptide-immunoreactive cells, expressed NADPH-diaphorase histochemical activity, whereas the exocrine tissue was almost negative. In streptozotocin-treated rats, only the surviving non-beta cells in the islet periphery were stained. Isolated beta and non-beta cells also expressed intense NADPH-diaphorase activity. By electron microscopy, the enzyme was localized primarily on membranes of the endoplasmic reticulum and nuclear envelope, as previously reported for neurons. In addition the enzyme activity was found in the cis-region of the Golgi complex. These results suggest that the four types of endocrine cells of the islets of Langerhans may contain the NOS-enzyme and thus constitutively produce nitric oxide.     

胰腺发育相关maf基因在胰腺导管和胰岛的表达

为探讨胰岛功能和发育相关maf基因在胰腺导管上皮中的表达情况,对新鲜小鼠胰腺组织切片进行显微切割,分离纯化胰腺组织中的导管和胰岛,以及外分泌腺组织细胞作为对照,利用荧光实时定量PCR的方法完成对目的基因的相对定量.结果显示,mafa mRNA,mafb mRNA水平在胰岛及导管中非常接近,无统计学差异.而c-maf在导管的表达高于胰岛(P<0.05),外分泌腺则无上述基因的表达.胰腺导管中mafa,mafb,cmaf均有表达,肯定了导管上皮细胞向内分泌细胞分化的潜能,而c-maf在导管中的表达高于胰岛,提示导管上皮c-maf的下调可能有助于导管上皮细胞向内分泌细胞的分化成熟.     

Differential localization of "brain-specific" S-100 and its subunits in rat salivary glands

In the rat, the S-100 antigens in the submandibular gland were found to be immunochemically identical with those in the brain (glial cells) when compared using crossed immunoelectrophoresis. Specific antibodies against the S-100a non-beta and against the S-100 beta subunit were prepared from antibodies against crude S-100 protein and from S-100 components (S-100a and b) by affinity chromatography. In the rat salivary glands a differential distribution of subunit immunoreactivity was clearly evidenced using indirect immunofluorescence. Certain intercalated duct cells of the submandibular gland as well as Schwann cells contained the S-100 beta subunit immunoreactivity exclusively, while other duct cells in parotid, submandibular, and sublingual glands contained S-100a non-beta subunit immunoreactivity. Both subunits were present in astrocytes and ependymal cells. The immunocytochemical localization of alpha and beta subunits is a promising technique for the classification of various types of S-100-containing cells.     

Can pancreatic duct-derived progenitors be a source of islet regeneration?

The regenerative process of the pancreas is of interest because the main pathogenesis of diabetes mellitus is an inadequate number of insulin-producing β-cells. The functional mass of β-cells is decreased in type 1 diabetes, so replacing missing β-cells or triggering their regeneration may allow for improved type 1 diabetes treatment. Therefore, expansion of the β-cell mass from endogenous sources, either in vivo or in vitro, represents an area of increasing interest. The mechanism of islet regeneration remains poorly understood, but the identification of islet progenitor sources is critical for understanding β-cell regeneration. One potential source is the islet proper, via the dedifferentiation, proliferation, and redifferentiation of facultative progenitors residing within the islet. Neogenesis, or that the new pancreatic islets can derive from progenitor cells present within the ducts has been reported, but the existence and identity of the progenitor cells have been debated.In this review, we focus on pancreatic ductal cells, which are islet progenitors capable of differentiating into islet β-cells. Islet neogenesis, seen as budding of hormone-positive cells from the ductal epithelium, is considered to be one mechanism for normal islet growth after birth and in regeneration, and has suggested the presence of pancreatic stem cells. Numerous results support the neogenesis hypothesis, the evidence for the hypothesis in the adult comes primarily from morphological studies that have in common the production of damage to all or part of the pancreas, with consequent inflammation and repair. Although numerous studies support a ductal origin for new islets after birth, lineage-tracing experiments are considered the “gold standard” of proof. Lineage-tracing experiments show that pancreatic duct cells act as progenitors, giving rise to new islets after birth and after injury. The identification of differentiated pancreatic ductal cells as an in vivo progenitor for pancreatic β-cells has implications for a potentially important, expandable source of new islets for diabetic replenishment therapy.     

Ngn3(+) endocrine progenitor cells control the fate and morphogenesis of pancreatic ductal epithelium

During pancreas development, endocrine and exocrine cells arise from a common multipotent progenitor pool. How these cell fate decisions are coordinated with tissue morphogenesis is poorly understood. Here we have examined ductal morphology, endocrine progenitor cell fate and Notch signaling in Ngn3^−/− mice, which do not produce islet cells. Ngn3 deficiency results in reduced branching and enlarged pancreatic duct-like structures, concomitant with Ngn3 promoter activation throughout the ductal epithelium and reduced Notch signaling. Conversely, forced generation of surplus endocrine progenitor cells causes reduced duct caliber and an excessive number of tip cells. Thus, endocrine progenitor cells normally provide a feedback signal to adjacent multipotent ductal progenitor cells that activates Notch signaling, inhibits further endocrine differentiation and promotes proper morphogenesis. These results uncover a novel layer of regulation coordinating pancreas morphogenesis and endocrine/exocrine differentiation, and suggest ways to enhance the yield of beta cells from stem cells.     

Persistent expression of HNF6 in islet endocrine cells causes disrupted islet architecture and loss of beta cell function

We used transgenesis to explore the requirement for downregulation of hepatocyte nuclear factor 6 (HNF6) expression in the assembly, differentiation, and function of pancreatic islets. In vivo, HNF6 expression becomes downregulated in pancreatic endocrine cells at 18. 5 days post coitum (d.p.c.), when definitive islets first begin to organize. We used an islet-specific regulatory element (pdx1(PB)) from pancreatic/duodenal homeobox (pdx1) gene to maintain HNF6 expression in endocrine cells beyond 18.5 d.p.c. Transgenic animals were diabetic. HNF6-overexpressing islets were hyperplastic and remained very close to the pancreatic ducts. Strikingly, alpha, delta, and PP cells were increased in number and abnormally intermingled with islet beta cells. Although several mature beta cell markers were expressed in beta cells of transgenic islets, the glucose transporter GLUT2 was absent or severely reduced. As glucose uptake/metabolism is essential for insulin secretion, decreased GLUT2 may contribute to the etiology of diabetes in pdx1(PB)-HNF6 transgenics. Concordantly, blood insulin was not raised by glucose challenge, suggesting profound beta cell dysfunction. Thus, we have shown that HNF6 downregulation during islet ontogeny is critical to normal pancreas formation and function: continued expression impairs the clustering of endocrine cells and their separation from the ductal epithelium, disrupts the spatial organization of endocrine cell types within the islet, and severely compromises beta cell physiology, leading to overt diabetes.     

The ductal origin of structural and functional heterogeneity between pancreatic islets

Islets form in the pancreas after the first endocrine cells have arisen as either single cells or small cell clusters in the epithelial cords. These cords constitute the developing pancreas in one of its earliest recognizable stages. Islet formation begins at the time the cords transform into a branching ductal system, continues while the ductal system expands, and finally stops before the exocrine tissue of ducts and acini reaches its final expansion. Thus, islets continuously arise from founder cells located in the branching and ramifying ducts. Islets arising from proximal duct cells locate between the exocrine lobules, develop strong autonomic and sensory innervations, and pass their blood to efferent veins (insulo-venous efferent system). Islets arising from cells of more distal ducts locate within the exocrine lobules, respond to nerve impulses ending at neighbouring blood vessels, and pass their blood to the surrounding acini (insulo-acinar portal system). Consequently, the section of the ductal system from which an islet arises determines to a large extent its future neighbouring tissue, architecture, properties, and functions. We note that islets interlobular in position are frequently found in rodents (rats and mice), whereas intralobularly-located, peripheral duct islets prevail in humans and cattle. Also, we expound on bovine foetal Laguesse islets as a prominent foetal type of type 1 interlobular neuro-insular complexes, similar to neuro-insular associations frequently found in rodents. Finally, we consider the probable physiological and pathophysiological implications of the different islet positions within and between species.     

Pancreatic cell proliferation in normal rats studied by in vivo autoradiography with 3H-thymidine

In vivo 3H-Thymidine autoradiographic investigations of DNA synthesis in acinar, islet and duct cells in the pancreas of normal rats showed that activity was dependent on age. The proliferation of acinar and islet cells, which was high in young animals, decreased exponentially with age; proliferation of the ductal cells on the other hand, increased until the animals became mature. These findings suggest that the physiological regeneration of acinar and islet cells, as well as their replacement after injury in adult animals commences from pancreatic ducts.     

Pancreatic beta cell-specific transcription of the pdx-1 gene. The role of conserved upstream control regions and their hepatic nuclear factor 3beta sites

To identify potential transactivators of pdx-1, we sequenced approximately 4.5 kilobases of the 5' promoter region of the human and chicken homologs, assuming that sequences conserved with the mouse gene would contain critical cis-regulatory elements. The sequences associated with hypersensitive site 1 (HSS1) represented the principal area of homology within which three conserved subdomains were apparent: area I (-2694 to -2561 base pairs (bp)), area II (-2139 to -1958 bp), and area III (-1879 to -1799 bp). The identities between the mouse and chicken/human genes are very high, ranging from 78 to 89%, although only areas I and III are present within this region in chicken. Pancreatic beta cell-selective expression was shown to be controlled by mouse and human area I or area II, but not area III, from an analysis of pdx-1-driven reporter activity in transfected beta- and non-beta cells. Mutational and functional analyses of conserved hepatic nuclear factor 3 (HNF3)-like sites located within area I and area II demonstrated that activation by these regions was mediated by HNF3beta. To determine if a similar regulatory relationship might exist within the context of the endogenous gene, pdx-1 expression was measured in embryonic stem cells in which one or both alleles of HNF3beta were inactivated. pdx-1 mRNA levels induced upon differentiation to embryoid bodies were down-regulated in homozygous null HNF3beta cells. Together, these results suggest that the conserved sequences represented by areas I and II define the binding sites for factors such as HNF3beta, which control islet beta cell-selective expression of the pdx-1 gene.     

Cellular and subcellular localization of an Mr 64,000 protein autoantigen in insulin-dependent diabetes

Antibodies to an Mr 64,000 protein from human or rat islets have been detected at high frequency in newly diagnosed insulin-dependent diabetic patients. In this study, we show that the antigenic and amphiphilic properties of the rat islet Mr 64,000 protein resemble that of the human protein. We have analyzed the expression of the Mr 64,000 protein in populations of pancreatic beta and non-beta cells and in selected rat tissues by immunoprecipitation of [35S]methionine-radiolabeled proteins with sera from diabetic patients or from healthy control individuals. When islet cell populations enriched in beta or non-beta cells were tested for the expression of the Mr 64,000 antigen, the protein was primarily observed in the beta cells. On analyzing preparations of islets, liver, kidney, thyroid, adrenal, pituitary, spleen, and thymus, the protein could only be detected in islets. The protein was also characterized in terms of its subcellular localization by Percoll density gradient centrifugation and was recovered in a fraction enriched in the plasma membrane marker, 5'-nucleotidase. These results are consistent with a beta cell-restricted plasma membrane expression of the protein and support the hypothesis that this protein is a target antigen of beta cell-specific autoimmunity in insulin-dependent diabetes.     

In vitro transdifferentiation of adult pancreatic acinar cells into insulin-expressing cells

Despite a recent breakthrough in human islet transplantation for treating diabetes mellitus, the limited availability of insulin-producing tissue is still a major obstacle. Here, we studied whether adult pancreatic acinar cells have the potential to transdifferentiate into islet or beta cells. Pancreatic acini were isolated from 7- to 8-weeks-old male Sprague-Dawley rats and cultured in suspension. Within 1 week, most of the acinar cells lost amylase expression and converted to cells with a duct cell phenotype. Insulin-positive cells were also observed, mainly at the periphery of the acini-derived spheroids. Insulin gene and protein expression was increased. Presence of a few insulin-positive cells coexpressing cytokeratins suggests that a spontaneous acinar to ductal cell transdifferentiation process was further going on towards beta cells. This study provides the first evidence that adult pancreatic acinar cells could be differentiated into insulin-expressing cells in vitro.     

Gene therapy for diabetes mellitus

There are diverse strategies for gene therapy of diabetes mellitus. Prevention of beta-cell autoimmunity is a specific gene therapy for prevention of type 1 (insulin-dependent) diabetes in a preclinical stage, whereas improvement in insulin sensitivity of peripheral tissues is a specific gene therapy for type 2 (non-insulin-dependent) diabetes. Suppression of beta-cell apoptosis, recovery from insulin deficiency, and relief of diabetic complications are common therapeutic approaches to both types of diabetes. Several approaches to insulin replacement by gene therapy are currently employed: 1) stimulation of beta-cell growth, 2) induction of beta-cell differentiation and regeneration, 3) genetic engineering of non-beta cells to produce insulin, and 4) transplantation of engineered islets or beta cells. In type 1 diabetes, the therapeutic effect of beta-cell proliferation and regeneration is limited as long as the autoimmune destruction of beta cells continues. Therefore, the utilization of engineered non-beta cells free from autoimmunity and islet transplantation with immunological barriers are considered potential therapies for type 1 diabetes. Proliferation of the patients' own beta cells and differentiation of the patients' own non-beta cells to beta cells are desirable strategies for gene therapy of type 2 diabetes because immunological problems can be circumvented. At present, however, these strategies are technically difficult, and transplantation of engineered beta cells or islets with immunological barriers is also a potential gene therapy for type 2 diabetes.     

Rac1 regulates pancreatic islet morphogenesis

Background  

Pancreatic islets of Langerhans originate from endocrine progenitors within the pancreatic ductal epithelium. Concomitant with differentiation of these progenitors into hormone-producing cells such cells delaminate, aggregate and migrate away from the ductal epithelium. The cellular and molecular mechanisms regulating islet cell delamination and cell migration are poorly understood. Extensive biochemical and cell biological studies using cultured cells demonstrated that Rac1, a member of the Rho family of small GTPases, acts as a key regulator of cell migration.     

Malignancy-associated changes in lactiferous duct epithelium

OBJECTIVE: To determine whether cells from histologically normal appearing epithelium of the lactiferous duct from women with a remote ductal lesion in the breast provide any clues indicating the existence of such a lesion. STUDY DESIGN: Tissue sections cut to 4 microns and stained with hematoxylin and eosin were prepared from duct tissue of 20 women with breast lesions and of 20 women free of any such lesion who had undergone mammoplastic procedures or resection for benign reasons. One hundred nuclei were measured from each case. Measures of nuclear deviation from normal were computed, discriminant functions were derived, and multivariate significance tests were conducted. RESULTS: Nuclei from histologically normal appearing regions of lactiferous duct epithelium from women harboring distant lesions exhibited changes in the distribution pattern of their nuclear chromatin, indicating the presence of these lesions. The statistical significance of these changes was documented. The changes were clearly evident in all 20 subjects with lesions and were not observed in 19 of the 20 subjects without lesions. CONCLUSION: The results suggest that studies aimed at detecting malignancy-associated changes in cells collected by ductal lavage might lead to a minimally invasive screening procedure for breast lesions.     

Recent Research on the Development of Microbial Strains for Amino-Acid Production

Abstract

Since the advent of islet transplantation, there has been a significant emphasis on the importance of islet purity despite an inevitable associated loss of islet mass during the purification process. One of the key elements of the 'Edmonton Protocol' for islet transplantation published in 2000 was an emphasis on the need for sequential transplants of highly purified islets (averaging 24% beta cell purity) and the close correlation between the numbers of islets transplanted and the success of the procedure. However, the emphasis on islet purity may warrant further consideration as auto transplantation of non-purified islets currently provides the most successful insulin independence rates within the field of islet transplantation. While the role of auto and allo immunity could contribute to the differences in the success rates it is clear that within the clinical setting, significant acinar and ductal contamination is well tolerated. However, one could go further and hypothesize that extra-insular tissue including acinar tissue, ductal tissue, peri-pancreatic lymph nodes and vascular tissue actually confer an advantage to islet survival/function and may even contribute to the insulin secreting capacity of the graft post transplant. As such this review will assess the influence of extra-insular pancreatic tissue on the results of islet transplantation based on published evidence and will also explore the possibility that non-islet pancreatic cells are capable of differentiating into a beta cell phenotype in vivo contributing to an ongoing regeneration of endocrine mass during the period following transplantation.     

Production of Transgenic Tilapia with Brockmann Bodies Secreting [desThrB30] Human Insulin

BACKGROUND: Tilapia are commercially important tropical fish which, like many teleosts, have anatomically discrete islet organs called Brockmann bodies. When transplanted into diabetic nude mice, tilapia islets provide long-term normoglycemia and mammalian-like glucose tolerance profiles. METHODS: Using site-directed mutagenesis and linker ligation we have "humanized" the tilapia insulin gene so that it codes for [desThrB30] human insulin while maintaining the tilapia regulatory sequences. Following microinjection into fertilized eggs, we screened DNA isolated from whole fry shortly after hatching by PCR. Positive fish were grown to sexual maturity and mated to wild-types and positive Fl's were further characterized. RESULTS: Human insulin was detected in both serum and in the clusters of beta cells scattered throughout the Brockmann bodies. Surrounding non-beta cells as well as other tissues were negative indicating beta cell specific expression. Purification and sequencing of both A-and B-chains verified that the insulin was properly processed and humanized. CONCLUSIONS: After extensive characterization, transgenic tilapia could become a suitable, inexpensive source of islet tissue that can be easily mass-produced for clinical islet xenotransplantation. Because tilapia islets are exceedingly resistant to hypoxia by mammalian standards, transgenic tilapia islets should be ideal for xenotransplantation using immunoisolation techniques.