Organogenesis of Kidney and Endocrine Pancreas

Growing new organs in situ by implanting developing animal organ primordia (organogenesis) represents a novel solution to the problem of limited supply for human donor organs that offers advantages relative to transplanting embryonic stem (ES) cells or xenotransplantation of developed organs. Successful transplantation of organ primordia depends on obtaining them at defined windows during embryonic development within which the risk of teratogenicity is eliminated, growth potential is maximized, and immunogenicity is reduced. We and others have shown that renal primordia transplanted into the mesentery undergo differentiation and growth, become vascularized by blood vessels of host origin, exhibit excretory function and support life in otherwise anephric hosts. Renal primordia can be transplanted across isogeneic, allogeneic or xenogeneic barriers. Pancreatic primordia can be transplanted across the same barriers undergo growth, and differentiation of endocrine components only and secrete insulin in a physiological manner following mesenteric placement. Insulin-secreting cells originating from embryonic day (E) 28 (E28) pig pancreatic primordia transplanted into the mesentery of streptozotocin-diabetic (type 1) Lewis rats or ZDF diabetic (type 2) rats or STZ-diabetic rhesus macaques engraft without the need for host immune-suppression. Our findings in diabetic macaques represent the first steps in the opening of a window for a novel treatment of diabetes in humans.     

Endocrine Cell Clustering During Human Pancreas Development

The development of efficient, reproducible protocols for directed in vitro differentiation of human embryonic stem (hES) cells into insulin-producing β cells will benefit greatly from increased knowledge regarding the spatiotemporal expression profile of key instructive factors involved in human endocrine cell generation. Human fetal pancreases 7 to 21 weeks of gestational age, were collected following consent immediately after pregnancy termination and processed for immunostaining, in situ hybridization, and real-time RT-PCR expression analyses. Islet-like structures appear from approximately week 12 and, unlike the mixed architecture observed in adult islets, fetal islets are initially formed predominantly by aggregated insulin- or glucagon-expressing cells. The period studied (7–22 weeks) coincides with a decrease in the proliferation and an increase in the differentiation of the progenitor cells, the initiation of NGN3 expression, and the appearance of differentiated endocrine cells. The present study provides a detailed characterization of islet formation and expression profiles of key intrinsic and extrinsic factors during human pancreas development. This information is beneficial for the development of efficient protocols that will allow guided in vitro differentiation of hES cells into insulin-producing cells. (J Histochem Cytochem 57:811–824, 2009)     

Follicles in the Endocrine Pancreas of Some Myxinoid Species

A light microscopic study of the endocrine pancreas in some myxinoids has revealed a striking difference in the occurrence of follicles. Islet tissue follicles are regularly occurring and also abundant in Myxine glutinosa, but are sparse or absent in Eptatretus burgeri and Eptatretus stouti. Whether this is caused by a decreased need for insulin in Myxine glutinosa is discussed but remains an open question.     

Studies on the Endocrine Pancreas of Amphibians and Reptiles

At this time, it can be said that the available data obtainedfrom amphibians and reptiles as regards the endocrine pancreasis consistent with the much more extensive data known for themetabolic effects of insuilin and glucagon in mammals. Thereremain, however, many unexplored facets of the specific responsesof the pancreas and the target tissues in lower animals.     

Elimination of Von Hippel-Lindau Function Perturbs Pancreas Endocrine Homeostasis in Mice

Mutations in the human homolog of the Vhlh gene [encoding the von-Hippel Lindau (VHL) protein] lead to tumor development. In mice, depletion of Vhlh in pancreatic ß-cells causes perturbed glucose homeostasis, but the role of this gene in other pancreatic cells is poorly understood. To investigate the function of VHL/HIF pathway in pancreatic cells, we inactivated Vhlh in the pancreatic epithelium as well as in the endocrine and exocrine lineages. Our results show that embryonic depletion of Vhlh within the pancreatic epithelium causes postnatal lethality due to severe hypoglycemia. The hypoglycemia is recapitulated in mice with endocrine-specific removal of Vhlh, while animals with loss of Vhlh predominantly in the exocrine compartment survive to adulthood with no overt defects in glucose metabolism. Mice with hypoglycemia display diminished insulin release in response to elevated glucose. Significantly, the glucagon response is impaired both in vivo (circulating glucagon levels) as well as in an in vitro secretion assay in isolated islets. Hypoxia also impairs glucagon secretion in a glucagon-expressing cell line in culture. Our results reveal a novel role for the hypoxia/HIF pathway in islet hormone secretion and maintenance of the fine balance that allows for the establishment of normoglycemia.     

Histochimie du Pancreas du Chat Avec Discussion du Tissu Langerhansien Endocrine

Résumé Des recherches histologiques et histochimiques sont faites sur le pancréas du chat avec discussion surtout au sujet du tissu endocrine. Quelques particularités sont signalées: la richesse en îlots ganglionnaires, en corpuscules de Pacini et en complexes neuro-insulaires, aussi bien que l'existence dans la partie apicale de l'épithélium des canaux excréteurs de granulations paraldéhyde positives. En principe l'histoenzymologie du pancréas du chat est semblable à celle des autres mammifères mais ne lui est pas toujours identique. Certaines considérations sont faites sur le rôle probable des enzymes étudiées et du Zn dans la cytophysiologie insulaire.

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Summary Morphological and histochemical investigations on the pancreas of cat have been carried out and discussion on the question of the endocrine tissue. Some particularities are marked down: abundance of ganglion islets, of corpuscules of Pacini and neuro-insular complexes, as well as existence of positive paraldehyde granulations in the top portion of the epithelium of the excretory ducts. In general the histoenzymology of the pancreas of the cat is similar to other mammals, but is not always identical. Some considerations on the probable role of the enzymes investigated and on the zinc in the insular cytophysiology are made.

     

Follicles in the Endocrine Pancreas of Myxine glutinosa Studied by Scanning Electron Microscopy

Abstract Scanning electron microscopic studies of the inner surfaces of the follicular cavities in the endocrine pancreas of Myxine glutinosa have revealed two types of follicles. The predominant type of follicles is lined by cells bearing microvilli, while the rare second type has smooth surfaced cavities. It is postulated, that the microvillous cells absorb material stored in these follicles, while the smooth surfaced follicles are disintegrating.     

Control of Gastric Acid Secretion and the Endocrine Pancreas by Gastrointestinal Regulatory Peptides

Gastrointestinal peptides that perform the dual functions ofregulating gastric acid secretion and secretion by the endocrinepancreas are reviewed. The regulation of release ofgastrin andgastrin releasing peptide and their roles in stimulating acidsecretion are first considered. Inhibitory regulation of acidsecretion is then outlined, with particular emphasis on candidateenterogastrones, such as gastric inhibitory polypeptide (GIP),and the enterogastric reflex. Finally, the contribution of GIP,and other incretins, and the neural component of the enteroinsularaxis to the regulation of insulin secretion by the endocrinepancreas is discussed. Although the major emphasis is on studiesperformed in mammals, the more limited information on thesepathways in non-mammalian vertebrates is also considered.     

Functional and Morphological Changes in Endocrine Pancreas following Cola Drink Consumption in Rats

Aim

We report the effects of long-term cola beverage drinking on glucose homeostasis, endocrine pancreas function and morphology in rats.

Methods

Wistar rats drank: water (group W), regular cola beverage (group C, sucrose sweetened) or “light” cola beverage (group L, artificially sweetened). After 6 months, 50% of the animals in each group were euthanized and the remaining animals consumed water for the next 6 months when euthanasia was performed. Biochemical assays, insulinemia determination, estimation of insulin resistance (HOMA-IR), morphometry and immunohistochemistry evaluations were performed in pancreas.

Results

Hyperglycemia (16%, p<0.05), CoQ₁₀ (coenzyme-Q₁₀) decrease (−52%,p<0.01), strong hypertriglyceridemia (2.8-fold, p<0.01), hyperinsulinemia (2.4 fold, p<0.005) and HOMA-IR increase (2.7 fold, p<0.01) were observed in C. Group C showed a decrease in number of α cells (−42%, p<0.01) and β cells (−58%, p<0.001) and a moderate increase in α cells’ size after wash-out (+14%, p<0.001). Group L showed reduction in β cells’ size (−9%, p<0.001) and only after wash-out (L₁₂) a 19% increase in size (p<0.0001) with 35% decrease in number of α cells (p<0.01). Groups C and L showed increase in α/β-cell ratio which was irreversible only in C (α/β = +38% in C₆,+30% in C₁₂, p<0.001vs.W₆). Regular cola induced a striking increase in the cytoplasmic expression of Trx1 (Thioredoxin-1) (2.25-fold in C₆ vs. W₆; 2.7-fold in C₁₂ vs. W_12, p<0.0001) and Prx2 (Peroxiredoxin-2) (3-fold in C₆ vs. W₆; 2-fold in C₁₂ vs. W_12, p<0.0001). Light cola induced increase in Trx1 (3-fold) and Prx2 (2-fold) after wash-out (p<0.0001, L₁₂ vs. W₁₂).

Conclusion

Glucotoxicity may contribute to the loss of β cell function with depletion of insulin content. Oxidative stress, suggested by increased expression of thioredoxins and low circulating levels of CoQ₁₀, may follow sustained hyperglycemia. A likely similar panorama may result from the effects of artificially sweetened cola though via other downstream routes.     

Cell Degeneration and Cavity Formation in the Endocrine Pancreas of the Hagfish Myxine glutinosa

Abstract Cell degeneration within the islet lobules of the pancreas of Myxine glutinosa appears to be a natural feature of the gland in this species. Degenerating cells bordering a lobule cavity discharge debris directly into the cavity, while cells undergoing degeneration at sites distant from such cavities discharge debris into the surrounding intercellular space. The latter process may well act as a locus for local degeneration within the lobule and the formation of a lobule cavity.     

PERK Regulates the Proliferation and Development of Insulin-Secreting Beta-Cell Tumors in the Endocrine Pancreas of Mice

Background

PERK eIF2α kinase is required for the proliferation of the insulin-secreting beta- cells as well as insulin synthesis and secretion. In addition, PERK signaling has been found to be an important factor in determining growth and angiogenesis of specific types of tumors, and was attributed to PERK-dependent regulation of the hypoxic stress response. In this report we examine the role of PERK in regulating proliferation and angiogenesis of transformed beta-cells in the development of insulinomas.

Methodology

The SV40 Large T-antigen (Tag) was genetically introduced into the insulin secreting beta-cells of Perk KO mice under the control of an inducible promoter. Tumor growth and the related parameters of cell proliferation were measured. In late stage insulinomas the degree of vascularity was determined.

Principal Findings

The formation and growth of insulinomas in Perk-deficient mice was dramatically ablated with much fewer tumors, which averaged 38-fold smaller than seen in wild-type control mice. Beta-cell proliferation was ablated in Perk-deficient mice associated with reduced tumor growth. In the small number of large encapsulated insulinomas that developed in Perk-deficient mice, we found a dramatic reduction in tumor vascularity compared to similar sized insulinomas in wild-type mice. Although insulinoma growth in Perk-deficient mice was largely impaired, beta-cell mass was increased sufficiently by T-antigen induction to rescue the hypoinsulinemia and diabetes in these mice.

Conclusions

We conclude that PERK has two roles in the development of beta-cell insulinomas, first to support rapid cell proliferation during the initial transition to islet hyperplasia and later to promote angiogenesis during the progression to late-stage encapsulated tumors.     

Fetal Rat Pancreas in Organ Culture: Effects of Serum on the Development of the Endocrine Cells

Eighteen-day-old rat fetal pancreas was grown in organ culture for four days on medium consisting of tissue culture Medium 199 and varying concentrations of chicken serum. The glucagon and somatostatin concentration of the explants was decreased when the serum concentration of the medium was reduced from 50 to 10%. There was a further reduction in these hormones when the explants were cultured on Medium 199 alone. Explant insulin content was reduced only when serum was omitted from the medium. A serum factor tripeptide was not able to substitute for this serum requirement. Heat-inactivation of the serum resulted in a significant increase in medium insulin content and an increase in both the insulin and glucagon contents of the explants. This increase in hormone content was directly correlated with increases in the beta and alpha cell volumes of the explants. There was no change in the somatostatin content or delta cell volume of the explants grown on heat-inactivated medium. It is suggested that the serum is an important component of the culture media and is apparently required in high concentration for the development of the islet cells in vitro. The islet cell types differ in their requirement for serum. The alpha and delta require a higher concentration than do the beta cells.     

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.