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.     

Human adipose tissue endothelial cells promote preadipocyte proliferation

Adipogenesis is preceded by development of a microvascular network, and optimal functioning of adipose tissue as an energy store and endocrine organ is dependent on extensive vascularization. We have examined the role of endothelial cell-derived factors that influence the proliferation of human preadipocytes. Microvascular endothelial cells and preadipocytes were isolated from human omental and subcutaneous adipose tissue biopsies by use of a developed procedure of collagenase digest, immunoselection, and differential trypsinization. Conditioned medium from microvascular endothelial cell cultures promoted the proliferation of preadipocytes (P = <0.001) and (to a lesser extent) other cell types. No depot-specific differences in mitogenic capacity of microvascular endothelial cell medium or of preadipocyte response were observed. These results indicate that adipose tissue endothelial cells secrete soluble adipogenic factor(s).     

Endocrine cells in gastric carcinoma and adjacent mucosa. An immunohistochemical and ultrastructural study

Endocrine cells are often found in human gastric carcinoma and may be recognized by the immunoreactivity of their chromogranin A, peptides and biogenic amines content. Anti-chromogranin A was used to investigate the morphology of endocrine cells using light and electron microscope immunohistochemical techniques. The hormone content of endocrine cells was examined in both tumour tissue and tumour-adjacent mucosa. It was found that the endocrine cells in tumour tissue were malignant, often had amphocrine differentiation and did not resemble a normal cell type. The hormone content of endocrine cells in tumour tissue seldom corresponded to the hormonal content of endocrine cells in tumour-adjacent mucosa. In intestinal-type carcinoma and in some parts of diffuse-type gastric carcinomas, endocrine cell hyperplasia and an alteration of the differentiation in the tumour-adjacent mucosa were discovered. The distribution of endocrine cells in the tumour tissue was different in both types of gastric carcinoma. The results reported here suggest that endocrine cell differentiation of malignant endocrine cells in human gastric carcinoma develops in a different way from that of endocrine cells in tumour-adjacent mucosa, and as a result, diverse hormonal products may appear in tumour tissue.     

Update on adipose tissue blood flow regulation

According to Fick's principle, any metabolic or hormonal exchange through a given tissue depends on the product of the blood flow to that tissue and the arteriovenous difference. The proper function of adipose tissue relies on adequate adipose tissue blood flow (ATBF), which determines the influx and efflux of metabolites as well as regulatory endocrine signals. Adequate functioning of adipose tissue in intermediary metabolism requires finely tuned perfusion. Because metabolic and vascular processes are so tightly interconnected, any disruption in one will necessarily impact the other. Although altered ATBF is one consequence of expanding fat tissue, it may also aggravate the negative impacts of obesity on the body's metabolic milieu. This review attempts to summarize the current state of knowledge on adipose tissue vascular bed behavior under physiological conditions and the various factors that contribute to its regulation as well as the possible participation of altered ATBF in the pathophysiology of metabolic syndrome.     

Production of islet-like structures from neonatal porcine pancreatic tissue in suspension bioreactors

The aim of this study was to develop a scaleable process to expand pancreatic endocrine tissue (i.e., aggregates or islet-like structures) in suspension bioreactors. Key issues addressed included (i) serum-free media, (ii) cell inoculation density, (iii) medium pH, and (iv) aggregate dissociation. Suspension bioreactors were inoculated with pancreatic neonatal tissue and operated under controlled conditions for a 9-day period. Medium studies showed that a new serum-free medium developed in our laboratory was capable of supporting endocrine cell expansion. An inoculation density of 127,000 cells/mL resulted in more than a 7.5-fold increase in the number of insulin-positive cells after 9 days. The resulting population consisted of single cells and many islet-like aggregates that contained all of the endocrine cell types (including insulin-positive, glucagon-positive, somatostatin-positive, and pancreatic polypeptide-positive cells). Furthermore, the cell aggregates exhibited a glucose-responsive behavior. This study represents a significant milestone on the path to the effective expansion of human islet-like tissue in bioreactors that may be used for cell therapy to treat Type 1 diabetes.     

Synaptophysin immunoreactivity in the mammalian endocrine pancreas

Summary Synaptophysin, a major membrane glycoprotein of small presynaptic vesicles in neurons, has also been found in microvesicles of endocrine cells, e.g., of the endocrine pancreas. In the present study, the endocrine pancreas in 9 mammalian species (man, dog, mink, bovine, rabbit, guinea pig, rat, mouse, gerbil) has been investigated immunohistochemically for synaptophysin immunoreactivity. Synaptophysin-positive cells have been identified and localized on semithin plastic sections. Our study demonstrates that, in all species examined, all pancreatic endocrine cell types are consistently synaptophysin-positive independent of their location within the tissue, or the conditions of tissue processing. In addition, a few cells that cannot be hormonally identified show synaptophysin immunoreactivity. Hence, synaptophysin appears to be a regular constituent of all pancreatic endocrine cells in mammals. In several species, a subpopulation of endocrine cells, consisting of glucagon-containing and/or pancreatic-polypeptide-containing cells, exhibits a significantly higher degree of synaptophysin immunoreactivity. In the gerbil, this heterogeneity can readily be detected from the day of birth onwards. Our findings indicate that closely related endocrine cell types may differ with respect to the content of synaptophysin.     

Development of the endocrine pancreas during larval phases of Rana temporaria

Summary The pancreatic endocrine component was studied at different stages of development in the tadpoles of Rana temporaria. The material was embedded in Epon, and serial semithin and thin sections were made in order to correlate ultrastructural features and tinctorial traits of the endocrine cells. Serial semithin sections were also stained with the peroxidase-antiperoxidase immunocytochemical method and with silver impregnations for argyrophilia and argentaffinity. In early larvae (legless tadpoles), A and B cells are present. Both can be found within ducts and exocrine tissue or, more frequently, in cellular clusters among the ducts and acini. These primitive islets are solid structures, surrounded but not penetrated by capillaries. Mitoses were observed in A and B cells. In the following phase (tadpoles with hindlegs), D and pancreatic polypeptide-immunoreactive cells are also present, as well as numerous endocrine cells scattered among exocrine tissue. There is also a change in the vascular-insular pattern: capillaries not only surround but also penetrate the endocrine group. The structure of the endocrine pancreas in older tadpoles is similar. Tinctorial traits and ultrastructural features of endocrine cells are described, and the origin of primitive islets is discussed.     

Ultrastructure of endocrine cells in the gastric fundal glands at different seasons of the year]

Endocrine cells of gastric glands of Citellus erythrogenus Brandt were studied during various seasons of the year by electron microscopy. ECL, EC, D and D1 cells having different structure of granules were detected. The slightly changed, weakly functioning cells as well as the cells of different alteration degree (up to destructing) were found in hibernating rodents. The presence of actively functioning EC-cells over this period confirms an opinion concerning the influence of peripheral serotonin on the hibernation mechanism. On the 3d--4th day of awakening pronounced activation of endocrine cells, their rejuvenation and normalization of cell ultrastructure, completed two weeks after awakening, were noted.     

Ultrastructure of cells from the liver-endocrine pancreas system in hibernating animals

In hibernated ground squirrel (Citellus erythrogenys) during various periods of hibernation morphological changes in organs participating in insurance of energoplastic homeostasis (liver, endocrine part of the pancreas) have been followed. At the beginning of hibernation certain signs of functional strain in the liver are observed-shortening and swelling of the endoplasmic reticulum (ER) canaliculi, decreases of glycogen resources and activation of the lysosomal apparatus, as well as certain features demonstrating its decreased activity--shortening cisterns in the Golgi complex, poorly manifested granular ER, tightening of mitochondrial matrix. At a deep hibernation, the signs of strain disappear, ultrastructure of hepatocytes corresponds to a new lower level of functioning in comparison to that in the active state and at the beginning of hibernation. During these periods the structure of endocrine cells in the pancreas demonstrates certain changes in the character of functioning of the cells. Ultrastructural transformations in the liver and endocrine part of the pancreas in the ground squirrel are mutually correlated.     

PD-L1 Expression in Normal Endocrine Tissues Is Not Increased Despite High Incidence of PD-1 Inhibitor-Associated Endocrinopathies

ObjectiveTreatment with immune-checkpoint inhibitors often results in endocrine immune-related adverse events (irAEs), affecting the pituitary, thyroid, adrenal, and parathyroid glands and pancreas. The mechanism underlying the endocrine irAEs has not been fully elucidated, and it remains unclear why endocrine organs are so commonly affected. In the present study, we evaluated immunostaining patterns of programmed death-ligand 1 (PD-L1) in normal endocrine tissues to determine whether increased expression may explain the predilection of endocrinopathies in patients treated with programmed cell death-1 inhibitors.MethodsNormal formalin-fixed paraffin-embedded endocrine tissues (pituitary, thyroid, adrenal, pancreas, and parathyroid) were collected from our hospital’s pathology tissue archive. The tissues were assessed for membranous and cytoplasmic PD-L1 immunostaining using the Dako 22C3 pharmDx assay on an automated staining platform.ResultsWe examined 49 endocrine tissues, including 12 thyroid, 5 pancreatic, 17 adrenal, 5 parathyroid, and 10 pituitary samples. Samples with less than 1% membranous PD-L1–positive cells were considered negative, while those with more than 1% of PD-L1 membranous staining were considered positive. Immunostaining result of immune-related cells was also evaluated, considering the cytoplasmic PD-L1–positive cells with the same cutoff of 1%. None of the endocrine tissues demonstrated PD-L1 positivity higher than 1% in the relevant cells.ConclusionWhile our results do not suggest a role of PD-L1 expression in the pathogenesis of endocrine irAEs, they may serve as a basis for future studies further investigating the mechanisms of autoimmune, inflammatory, or malignant endocrine conditions.     

Is gestational diabetes an acquired condition?

Intrvenous injection of 30 mg of streptozotocin per kg body weight induces a mild diabetes in pregnant rats (first generation); the non-fasting blood glucose is increased and the percentage of endocrine tissue and also the percentage of granulated beta cells do not increase. The fetuses of these mildly diabetic pregnant rats have an increased percentage of pancreatic endocrine tissue and there is beta-cell degranulation. The modifications in the endocrine pancreas during intrauterine life causes persistent changes in later adult life (second generation), which are not perceptible in basal conditions, but become apparent in situations stressing the beta-cell activity, such as an intravenous glucose load or pregnancy. During pregnancy in the second generation rats an increased non-fasting blood glucose and no adaptation of the beta cells is seen. This inadequate adaptation to pregnancy causes changes in the fetal endocrine pancreas of the fetuses of the third generation. From these experiments it may be concluded that gestational diabetes is an acquired condition.     

White and brown adipose stem cells: From signaling to clinical implications

Epidemiological studies estimate that by the year 2030, 2.16 billion people worldwide will be overweight and 1.12 billion will be obese [1]. Besides its now established function as an endocrine organ, adipose tissue plays a fundamental role as an energy storage compartment. As such, adipose tissue is capable of extensive expansion or retraction depending on the energy balance or disease state of the host, a plasticity that is unparalleled in other organs and – under conditions of excessive energy intake – significantly contributes to the afore mentioned obesity pandemic. Expansion of adipose tissue is driven by both hypertrophy and hyperplasia of adipocytes, which can renew frequently to compensate for cell death. This underlines the importance of adipocyte progenitor cells within the distinct adipose tissue depots to control both energy storage and endocrine functions of adipose tissue. Here we summarize recent findings on the identity and plasticity of adipose stem cells, the involved signaling cascades, and potential clinical implications of these cells for the treatment of metabolic dysfunction in obesity. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

南方鲇头肾的组织学和超微结构

采用解剖学、组织学、组织化学方法,通过电镜观察,研究了南方鲇(Silurus meridionulis Chen)头肾的形态和超微结构。南方鲇左右头肾不相连,与肾明显分离。头肾组织包括血管系统、淋巴细胞聚集区、粒细胞聚集区、内分泌组织区。血管系统由头肾动脉、头肾静脉、后主静脉及其分枝所组成。淋巴区细胞染色深,紧密聚集成网状结构,主要包括大、小淋巴细胞等细胞类型。粒细胞区域狭窄,染色浅,主要为粒细胞聚集。内分泌组织区域细胞大型,常规染色极浅,包括肾间组织细胞、肾上组织细胞。肾间组织细胞有丰富的线粒体、内质网及分泌颗粒。肾上组织细胞主要分布于后主静脉及其分枝的周围、静脉壁中,细胞中有密集的分泌颗粒。肾上组织细胞和肾间组织细胞集中于头肾,内分泌功能增强,是与其运动能力强、快速捕食相适应的特征。     

Electrical activity in endocrine pituitary cells in situ: a support for a multiple-function coding

The anterior pituitary is an endocrine gland that controls basic body functions. Pituitary cell functioning depends on membrane excitability, which induces cytosolic calcium rises. Here, we reported the first identification of small-amplitude voltage fluctuations that controlled spike firing in endocrine cells recorded in situ. Three patterns of voltage fluctuations were distinguishable by their durations (1-100 s). These patterns could be ordered on top of each other, namely in response to secretagogues. Thus, pituitary endocrine cells express in situ a cell code in which small-amplitude voltage fluctuations lead to a multimodal arrangement of spike firing, which may finely tune calcium-dependent functions.     

Immunocytochemical studies of subtypes of pulmonary endocrine cells in diethylnitrosamine-treated rabbits

New Zealand White rabbits were injected subcutaneously with 20 mg/kg body weight of diethylnitrosamine (DEN), twice per week, starting when they were 1 week old. The animals were sacrificed 6 to 12 months after the first injection and lung tissues were processed for light microscopy. Using serotonin (5HT) and neuron specific enolase (NSE) as markers for the endocrine cells, tissue sections were stained immunocytochemically by the avidin-biotin complex method. Numerous neuroepithelial bodies (NEBs) positive for 5HT, but negative for NSE, were seen in the alveolar duct regions of DEN-treated rabbits. On the other hand, an increased number of solitary endocrine cells immunoreactive for NSE was found in bronchial or bronchiolar epithelia. The results indicate that DEN induced increases in two distinct types of endocrine cells: the component cells of NEBs are positive for 5HT and solitary cells are positive for NSE.     

Towards the development of artificial endocrine tissues: (31)P NMR spectroscopic studies of immunoisolated, insulin-secreting AtT-20 cells

Transformed, insulin-secreting endocrine cells have been proposed as an alternative to islets for the development of a bioartificiat pancreas. With appropriate immunoprotection, such cells may be implanted without the need for patient immunosuppression. Use of continuous cell lines alleviates the cell availability limitation, but poses questions regarding thestability and biochemical and secretory function of the preparation, especially in the longterm. We have developed a bioreactor/perfusion system, compatible with a horizontal-bore NMR instrument, that can maintain immunoprotected endocrine cells for prolonged periods of time. (31)P NMR spectroscopy was used to study the bioenergetics of recombinant, insulin-secreting mouse pituitary AtT-20 cells entrapped as spheroids in calcium alginate/poly-L-lysine/alginate beads. NMR provided data verifying the macroscopic homogeneity within the bioreactor and allowing the evaluation of changes in cellular bioenergetics for a period of 70 days under different culture conditions. Levels of high-energy phosphates changed slightly during the first 40 days of the experiment, then decreased considerably as cell death occurred. Rates of glucose consumption and insulin-related peptide secretion also remained constant for 40 days and decreased rapidly thereafter. This study constitutes the beginning of an extensive quantitative analysis of the biochemistry of transformed endocrine cell lines in a sequestered, artificial tissue environment. (c) 1995 John Wiley & Sons Inc.     

The endocrine pancreas of a squamate reptile, the desert lizard (Chalcides ocellatus). A histological and immunocytochemical investigation

The endocrine pancreas of the desert lizard (Chalcides ocellatus) was investigated histologically and immunocytochemically. The endocrine tissue was concentrated in the dorsal lobe, where it constituted about 7% of the total volume. In the ventral lobe the endocrine tissue formed approximately 1% of the total volume. Four endocrine cell types were observed in the pancreas of this species, namely insulin-, glucagon-, somatostatin- and pancreatic polypeptide (PP)-immunoreactive cells. The volume occupied by these cells was 1, 1, 0.6 and 0.3% of the total volume of the pancreas, respectively. Insulin-immunoreactive cells were located in the islet centre and comprised 3% of dorsal and 0.2% of the ventral lobe volume. Glucagon cells occurred at the islet periphery and amounted to 3 and 0.2% of the volume of the dorsal and ventral lobes, respectively. Somatostatin-immunoreactive cells were located at the islet periphery as well as in between the exocrine parenchyma. They constituted 1 and 0.2% of the volume of the dorsal and ventral lobes, respectively. PP-immunoreactive cells occurred mainly among the exocrine parenchyma as solitary cells. They formed only 0.03% of the volume of the dorsal lobe. The corresponding figure in the ventral lobe was 0.6%.