Intact Pancreas Research Articles

Transcriptional Heterogeneity of Beta Cells in the Intact Pancreas

Pancreatic beta cells have been shown to be heterogeneous at multiple levels. However, spatially interrogating transcriptional heterogeneity in the intact tissue has been challenging. Here, we developed an optimized protocol for single-molecule transcript imaging in the intact pancreas and used it to identify a sub-population of "extreme" beta cells with elevated mRNA levels of insulin and other secretory genes. Extreme beta cells contain higher ribosomal and proinsulin content but lower levels of insulin protein in fasted states, suggesting they may be tuned for basal insulin secretion. They exhibit a distinctive intra-cellular polarization pattern, with elevated mRNA concentrations in an apical ER-enriched compartment, distinct from the localization of nascent and mature proteins. The proportion of extreme cells increases in db/db diabetic mice, potentially facilitating the required increase in basal insulin. Our results thus highlight a sub-population of beta cells that may carry distinct functional roles along physiological and pathological timescales.

Three-Dimensional Analysis of the Human Pancreas.

Pancreatic islets are endocrine micro-organs scattered throughout the exocrine pancreas. Islets are surrounded by a network of vasculature, ducts, neurons, and extracellular matrix. Three-dimensional imaging is critical for such structural analyses. We have adapted transparent tissue tomography to develop a method to image thick pancreatic tissue slices (1 mm) with multifluorescent channels. This method takes only 2 to 3 days from specimen preparation and immunohistochemical staining to clearing tissues and imaging. Reconstruction of the intact pancreas visualizes islets with β, α, and δ cells together with their surrounding networks. Capturing several hundred islets at once ensures sufficient power for statistical analyses. Further surface rendering provides clear views of the anatomical relationship between islets and their microenvironment as well as the basis for volumetric quantification. As a proof-of-principle demonstration, we show an islet size-dependent increase of intraislet capillary density and an inverse decrease in sphericity.

CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep

One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1−/− fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.

Simple and Rapid Tissue Clearing Method for Three-Dimensional Histology of the Pancreas.

Previously, high-resolution three-dimensional imaging of a whole and intact pancreas was not possible, since light is scattered when it passes through cell compartments with different refractive indices. CLARITY is one of the tissue clearing techniques that has yielded success with the central nervous system. To preserve tissue integrity after delipidation, conventional protocols embed tissue in an acrylamide-based hydrogel, which involves the use of specialized equipment. Recently, we determined that the hydrogel-embedding step could be simplified and replaced by passive tissue fixation in 4% paraformaldehyde (PFA). The whole procedure is less time-consuming and less error-prone, and can be completed within a week, compared to conventional CLARITY protocols that may take weeks to complete. Here, the detailed stepwise procedures involved in the simplified CLARITY workflow are applied to the pancreas of wild-type and gene-knockout 6-week old mice expressing green fluorescent protein (GFP) under the mouse insulin 1 promoter (MIP-GFP). This technique could facilitate high-resolution, three-dimensional imaging of pancreatic islets and comparison between different mouse genotypes under different disease and treatment conditions. © 2017 by John Wiley & Sons, Inc.

Bicarbonate-rich fluid secretion predicted by a computational model of guinea-pig pancreatic duct epithelium.

The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO3- concentrations as high as 140mm during hormonal stimulation. A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea-pig pancreatic ducts. The model was readily able to secrete 140mm HCO3- . Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers. We conclude that the main requirement for secreting high HCO3- concentrations is to minimize the secretion of Cl- ions. These findings help to clarify the mechanism responsible for pancreatic HCO3- secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease. A computational model of guinea-pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO3- ions are secreted at concentrations in excess of 140mm. Parameters defining the contributions of the individual ion channels and transporters were estimated by least-squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP-stimulated secretion were well replicated by increasing the activities of the basolateral Na+ -HCO3- cotransporter (NBC1) and apical Cl- /HCO3- exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K+ permeability and apical Cl- and HCO3- permeabilities (CFTR), and reducing the activity of the basolateral Cl- /HCO3- exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted ∼140mm HCO3- at a rate of ∼3nlmin-1 mm-2 , which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl- /HCO3- exchange via SLC26A6 at the apical membrane were able to support a HCO3- -rich secretion. Raising the HCO3- /Cl- permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO3- concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by ∼80% was essential in minimizing the intracellular Cl- concentration following cAMP stimulation and thereby maximizing the secreted HCO3- concentration. The addition of a basolateral Na+ -K+ -2Cl- cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl- and resulted in a lower secreted HCO3- concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl- secretion is the main requirement for secreting 140mm HCO3- .

Intra-islet lesions and lobular variations in β-cell mass expansion in ob/ob mice revealed by 3D imaging of intact pancreas.

The leptin deficient ob/ob mouse is a widely used model for studies on initial aspects of metabolic disturbances leading to type 2 diabetes, including insulin resistance and obesity. Although it is generally accepted that ob/ob mice display a dramatic increase in β-cell mass to compensate for increased insulin demand, the spatial and quantitative dynamics of β-cell mass distribution in this model has not been assessed by modern optical 3D imaging techniques. We applied optical projection tomography and ultramicroscopy imaging to extract information about individual islet β-cell volumes throughout the volume of ob/ob pancreas between 4 and 52 weeks of age. Our data show that cystic lesions constitute a significant volume of the hyperplastic ob/ob islets. We propose that these lesions are formed by a mechanism involving extravasation of red blood cells/plasma due to increased islet vessel blood flow and vessel instability. Further, our data indicate that the primary lobular compartments of the ob/ob pancreas have different potentials for expanding their β-cell population. Unawareness of the characteristics of β-cell expansion in ob/ob mice presented in this report may significantly influence ex vivo and in vivo assessments of this model in studies of β-cell adaptation and function.

The dosimetric impact of image guided radiation therapy by intratumoral fiducial markers

PurposePancreatic fiducials have proven superior over other isocenter localization surrogates, including anatomical landmarks and intratumoral or adjacent stents. The more clinically relevant dosimetric impact of image guided radiation therapy (IGRT) using intratumoral fiducial markers versus bony anatomy has not yet been described and is therefore the focus of the current study. Methods and materialsUsing daily orthogonal kV or cone beam computed tomography (CBCT) images and positional and dosimetric data were analyzed for 12 consecutive patients treated with fiducial based IGRT and volumetric modulated arc therapy to the intact pancreas. The shifts from fiducial to bone (ΔFid-Bone) required to realign the daily fiducial-matched pretreatment images (kV, CBCTs) to the planning computed tomography (CT) using bony anatomic landmarks were recorded. The isocenter was then shifted by (ΔFid-Bone) for 5 evenly spaced treatments, and the dosimetric impact of ΔFid-Bone was calculated for planning target volume coverage (PTV50.4 and PTV47.9) and organs at risk (liver, kidney, and stomach/duodenum). ResultsThe ΔFid-Bone were greatest in the superoinferior direction (ΔFid-Bone anteroposterior, 2.7 ± 3.0; left-right, 2.8 ± 2.8; superoinferior, 6.3 ± 7.9 mm; mean ± standard deviation; P = .03). PTV50.4 coverage was reduced by 13% (fiducial plan 95 ± 2.0 vs bone plan 82 ± 12%; P = .005; range, 5%-52%; >5% loss in all; and >10% loss in 42% of patients), and to a lesser degree for PTV47.9 (difference, -8%; range, 1%-30%; fiducial plan 100 ± 0.3% vs bone plan 92 ± 7.6%; P = .003; with reductions of >5% in 66% and >10% in 33% of patients). The dosimetric impact of ΔFid-Bone on the organs at risk was not significant. Positional shifts for kV- and CBCT-based realignments were nearly identical. ConclusionCompared with matching by fiducial markers, IGRT matched by bony anatomy substantially reduces the PTV50.4 and PTV47.9 coverage, supporting the use of intratumoral pancreatic markers for improved targeting in IGRT for pancreatic cancer.

SU-C-210-06: Quantitative Evaluation of Dosimetric Effects Resulting From Positional Variations of Pancreatic Tumor Volumes

Purpose: The aim of this study is to quantify dosimetric effects resulting from variation in pancreatic tumor position assessed by bony anatomy and implanted fiducial markers Methods: Twelve pancreatic cancer patients were retrospectively analyzed for this study. All patients received modulated arc therapy (VMAT) treatment using fiducial-based Image Guided Radiation Therapy (IGRT) to the intact pancreas. Using daily orthogonal kV and/or Cone beam CT images, the shift needed to co-register the daily pre-treatment images to reference CT from fiducial to bone (Fid-Bone) were recorded as Left-Right (LR), Anterior-Posterior (AP) and Superior-Inferior (SI). The original VMAT plan iso-center was shifted based on KV bone matching positions at 5 evenly spaced fractions. Dose coverage of the planning target volumes (PTVs) (V100%), mean dose to liver, kidney and stomach/duodenum were assessed in the modified plans. Results: A total of 306 fractions were analyzed. The absolute fiducial-bone positional shifts were greatest in the SI direction, (AP = 2.7 ± 3.0, LR = 2.8 ± 2.8, and SI 6.3 ± 7.9 mm, mean ± SD). The V100% was significantly reduced by 13.5%, (Fid-Bone = 95.3 ± 2.0 vs. 82.3 ± 11.8%, p=0.02). This varied widely among patients (Fid-Bone V100% Range = 2–60%), where 33% ofmore » patients had a reduction in V100% of more than 10%. The impact on OARs was greatest to the liver (Fid-Bone= 14.6 vs. 16.1 Gy, 10%), and stomach, (Fid-Bone = 23.9 vx. 25.5 Gy, 7%), however was not statistically significant (p=0.10 both). Conclusion: Compared to matching by fiducial markers, matching by bony anatomy would have substantially reduced the PTV coverage by 13.5%. This reinforces the importance of online position verification based on fiducial markers. Hence, implantation of fiducial markers is strongly recommended for pancreatic cancer patients undergoing intensity modulated radiation therapy treatments.« less

Toward beta cell replacement for diabetes.

The discovery of insulin more than 90 years ago introduced a life-saving treatment for patients with type 1 diabetes, and since then, significant progress has been made in clinical care for all forms of diabetes. However, no method of insulin delivery matches the ability of the human pancreas to reliably and automatically maintain glucose levels within a tight range. Transplantation of human islets or of an intact pancreas can in principle cure diabetes, but this approach is generally reserved for cases with simultaneous transplantation of a kidney, where immunosuppression is already a requirement. Recent advances in cell reprogramming and beta cell differentiation now allow the generation of personalized stem cells, providing an unlimited source of beta cells for research and for developing autologous cell therapies. In this review, we will discuss the utility of stem cell-derived beta cells to investigate the mechanisms of beta cell failure in diabetes, and the challenges to develop beta cell replacement therapies. These challenges include appropriate quality controls of the cells being used, the ability to generate beta cell grafts of stable cellular composition, and in the case of type 1 diabetes, protecting implanted cells from autoimmune destruction without compromising other aspects of the immune system or the functionality of the graft. Such novel treatments will need to match or exceed the relative safety and efficacy of available care for diabetes.

Death and regeneration of the pancreatic islets in relation to the development of fibrosis in patients with chronic pancreatitis

The present study was conducted on biopsy material obtained from 65 patients with complicated chronic pancreatitis and 6 samples of tissue from intact pancreas. Сellular composition, islets’ death and regeneration were analyzed using histological and immunohistochemical methods depending on the fibrous transformation in the development of chronic pancreatitis. Given study provides a detailed description of the histological changes that occur in the pancreas during the development of fibrosis, along with expression patterns of pro-apoptotic nucleases Endonuclease G and DNase, and the proliferation markers PCNA and Neurogenin-3 at different stages of chronic pancreatitis.

Ventromedial hypothalamic lesions change the expression of cell proliferation-related genes and morphology-related genes in rat pancreatic islets

Studies in normal rats and ob/ob mice indicated that islet neogenesis does not occur in the intact rodent pancreas. We previously reported that ventromedial hypothalamic (VMH) lesions stimulated cell proliferation of rat pancreatic islet B and acinar cells primarily through a cholinergic receptor mechanism and examined how gene families involved in cell proliferation in total pancreatic tissue are regulated after VMH lesions formation. This study examined how gene families involved in cell proliferation in pancreatic islets alone are regulated after VMH lesions formation. Pancreatic islet RNA was extracted, and differences in gene expression profiles between rats at day 3 after VMH lesioning and sham-VMH-lesioned rats were investigated using DNA microarray and real-time polymerase chain reaction. VMH lesions regulated genes that were involved in functions related to cell cycle and differentiation, growth, binding, apoptosis and morphology in pancreas islets. Real-time polymerase chain reaction also confirmed that gene expression of polo-like kinase 1 (Plk1) and topoisomerase (DNA) II α 170 kDa (Top2a), and stanniocalcin 1 (Stc1) were upregulated at day 3 after the VMH lesions. Ventromedial hypothalamic lesions may change the expression of cell proliferation-related genes and morphology-related genes in rat pancreatic islets.

Assessing the secretory capacity of pancreatic acinar cells.

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex vivo culture will represent the in vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.

Diabetes Associated Metabolomic Perturbations in NOD Mice.

Non-obese diabetic (NOD) mice are a widely-used model oftype1 diabetes (T1D). However, not all animals develop overt diabetes. This study examined the circulating metabolomic profiles of NOD mice progressing or not progressing to T1D. Total beta-cell mass was quantified in the intact pancreas using transgenic NOD mice expressinggreen fluorescent protein under the control of mouse insulin I promoter.While both progressor and non-progressor animals displayed lymphocyte infiltration and endoplasmic reticulum stress in the pancreas tissue;overt T1D did not develop until animals lost ~70% of the total beta-cell mass.Gas chromatography time of flight mass spectrometry (GC-TOF) was used to measure >470 circulating metabolites in male and female progressor and non-progressor animals (n=76) across a wide range of ages (neonates to >40-wk).Statistical and multivariate analyses were used to identify age and sex independent metabolic markers which best differentiated progressor and non-progressor animals' metabolic profiles. Key T1D-associated perturbations were related with: (1) increased plasma glucose and reduced 1,5-anhydroglucitol markers of glycemic control; (2) increased allantoin, gluconic acid and nitric oxide-derived saccharic acid markers of oxidative stress; (3) reduced lysine, an insulin secretagogue; (4) increased branched-chain amino acids, isoleucine and valine; (5) reduced unsaturated fatty acids including arachidonic acid; and (6)perturbations in urea cycle intermediates suggesting increased arginine-dependent NO synthesis. Together these findings highlight the strength of the unique approach of comparing progressor and non-progressor NOD mice to identify metabolic perturbations involved in T1D progression.

English

The mechanism of anti-diabetic activity of Gongronema latifolium was evaluated. The ethanol extract of the leaves of G. latifolium were fractionated using solvents of increasing polarity, namely n-hexane, chloroform, ethylacetate and ethanol. Phytochemical screening of the dried fractions were carried and then acute toxicity tests on mice carried out. The induction of diabetes mellitus was achieved with 150 mg/kg b.w for mild diabetes and 300 mg/kg b.w for the severe condition. The effects of the crude ethanol extract (CEE) and its fractions on alloxan-induced hyperglycaemia were monitored. The result obtained reveals that crude ethanol extract significantly and dose-dependently reduced hyperglycaemia. The fractions of the ethanol extract equally reduced hyperglycaemia but the level of reduction was affected by the phytochemical content. This suggests that an intact pancreas is required for the hypoglycaemic action which is the mechanism of action of the sulphonylureas.   Key words: Anti-diabetic, Gongronema latifolium, hyperglycaemia, hypoglycaemia, phytochemical screening.

FRII-08 DR. ALEXANDER BRUNSCHWIG (1901-1969) - A FOUNDER OF THE TOTAL PELVIC EXENTERATION AND OTHER RADICAL SURGICAL ADVANCES OF THE 20TH CENTURY.

You have accessJournal of UrologyHistory of Urology II (Moderated Poster)1 Apr 2014FRII-08 DR. ALEXANDER BRUNSCHWIG (1901-1969) - A FOUNDER OF THE TOTAL PELVIC EXENTERATION AND OTHER RADICAL SURGICAL ADVANCES OF THE 20TH CENTURY. Ariel Schulman, James Kashanian, Ervin Teper, and Ciril Godec Ariel SchulmanAriel Schulman More articles by this author , James KashanianJames Kashanian More articles by this author , Ervin TeperErvin Teper More articles by this author , and Ciril GodecCiril Godec More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1746AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives The total pelvic exenteration is the most invasive procedure in the repository of the pelvic surgeon. Though morbid, it provides functional improvement, palliation and in some cases enduring cure in the setting of invasive disease. The basic principles remain from the procedures developed in the 1940’s. We describe the contributions of Dr. Alexander Brunschwig who was among the first to perform this “brutal and cruel,” yet effective procedure. Methods Medical journals and news publications were reviewed regarding the work of Dr. Brunschwig. Results Alexander Brunschwig was born in El Paso, Texas on Sept. 11th, 1901. He attended Rush Medical School followed by training at Boston City Hospital, the University of Chicago and the University of Strasbourg, France. He was on staff at the University of Chicago from 1931 to 1947 when he performed the first one-stage radical pancreatoduodenectomy in 1937 which disproved the prevailing thought that an intact pancreas was required. He subsequently published “The Surgery of Pancreatic Tumors” in 1942, a milestone work in the developing field of hepatobiliary surgery. In 1947, Brunschwig became Chief of Gynecology at Memorial Hospital for Cancer in New York where he remained for the duration of his career. While Bricker performed the first pelvic exenteration in 1940 for rectal cancer, Brunschwig was operating largely on advanced gynecologic cancers and published the first case series of 22 patients in 1948 in the first volume of the journal ‘Cancer.’ Brunschwig’s initial cases had ‘wet colostomies’ with implantation of the ureters into segments of colon. His work was the subject of criticism from some contemporaries who considered him a “butcher,” but largely favorable recognition culminating with the publication of ‘L’Exenteration pelvienne’ in 1964 that reported 5 year survival in 116 of 562 patients. Dr. Brunschwig’s commitment to surgical therapy is exemplified in his aggressive approach, while his feelings about alternatives are reflected by his belief that “more people make a living giving chemotherapy than are cured by it.” Conclusions On August 7th, 1969, Dr. Brunschwig collapsed in the operating room during his final surgical procedure. He died the same night at the age of 67 due to a cardiac event. His early contributions to the surgical management of advanced pelvic malignancy, and multitude of other fields, deserve a place in the History of Urology. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e629-e630 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Ariel Schulman More articles by this author James Kashanian More articles by this author Ervin Teper More articles by this author Ciril Godec More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Islet Transplantation in a Subarachnoid Cavity Surrounding Olfactory Bulb of Diabetic Rats

Islet Transplantation in a Subarachnoid Cavity Surrounding Olfactory Bulb of Diabetic Rats

Multimodal imaging of pancreatic beta cells in vivo by targeting transmembrane protein 27 (TMEM27)

Aims/hypothesisNon-invasive diagnostic tools specific for pancreatic beta cells will have a profound impact on our understanding of the pathophysiology of metabolic diseases such as diabetes. The objective of this study was to use molecular imaging probes specifically targeting beta cells on human samples and animal models using state-of-the-art imaging modalities (fluorescence and PET) with preclinical and clinical perspective.MethodsWe generated a monoclonal antibody, 8/9-mAb, targeting transmembrane protein 27 (TMEM27; a surface N-glycoprotein that is highly expressed on beta cells), compared its expression in human and mouse pancreas, and demonstrated beta cell-specific binding in both. In vivo imaging was performed in mice with subcutaneous insulinomas overexpressing the human TMEM27 gene, or transgenic mice with beta cell-specific hTMEM27 expression under the control of rat insulin promoter (RIP-hTMEM27-tg), using fluorescence and radioactively labelled antibody, followed by tissue ex vivo analysis and fluorescence microscopy.ResultsFluorescently labelled 8/9-mAb showed beta cell-specific staining on human and mouse pancreatic sections. Real-time PCR on islet cDNA indicated about tenfold higher expression of hTMEM27 in RIP-hTMEM27-tg mice than in humans. In vivo fluorescence and PET imaging in nude mice with insulinoma xenografts expressing hTMEM27 showed high 8/9-mAb uptake in tumours after 72 h. Antibody homing was also observed in beta cells of RIP-hTMEM27-tg mice by in vivo fluorescence imaging. Ex vivo analysis of intact pancreas and fluorescence microscopy in beta cells confirmed these findings.Conclusions/interpretationhTMEM27 constitutes an attractive target for in vivo visualisation of pancreatic beta cells. Studies in mouse insulinoma models and mice expressing hTMEM27 demonstrate the feasibility of beta cell-targeted in vivo imaging, which is attractive for preclinical investigations and holds potential in clinical diagnostics.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2605-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Thiopental-induced insulin secretion via activation of IP3-sensitive calcium stores in rat pancreatic β-cells

While glucose-stimulated insulin secretion depends on Ca(2+) influx through voltage-gated Ca(2+) channels in the cell membrane of the pancreatic β-cell, there is also ample evidence for an important role of intracellular Ca(2+) stores in insulin secretion, particularly in relation to drug stimuli. We report here that thiopental, a common anesthetic agent, triggers insulin secretion from the intact pancreas and primary cultured rat pancreatic β-cells. We investigated the underlying mechanisms by measurements of whole cell K(+) and Ca(2+) currents, membrane potential, cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), and membrane capacitance. Thiopental-induced insulin secretion was first detected by enzyme-linked immunoassay, then further assessed by membrane capacitance measurement, which revealed kinetics distinct from glucose-induced insulin secretion. The thiopental-induced secretion was independent of cell membrane depolarization and closure of ATP-sensitive potassium (K(ATP)) channels. However, accompanied by the insulin secretion stimulated by thiopental, we recorded a significant intracellular [Ca(2+)] increase that was not from Ca(2+) influx across the cell membrane, but from intracellular Ca(2+) stores. The thiopental-induced [Ca(2+)](i) rise in β-cells was sensitive to thapsigargin, a blocker of the endoplasmic reticulum Ca(2+) pump, as well as to heparin (0.1 mg/ml) and 2-aminoethoxydiphenyl borate (2-APB; 100 μM), drugs that inhibit inositol 1,4,5-trisphosphate (IP(3)) binding to the IP(3) receptor, and to U-73122, a phospholipase C inhibitor, but insensitive to ryanodine. Thapsigargin also diminished thiopental-induced insulin secretion. Thus, we conclude that thiopental-induced insulin secretion is mediated by activation of the intracellular IP(3)-sensitive Ca(2+) store.

High-Resolution Magnetic Resonance Imaging Quantitatively Detects Individual Pancreatic Islets

OBJECTIVEWe studied whether manganese-enhanced high-field magnetic resonance (MR) imaging (MEHFMRI) could quantitatively detect individual islets in situ and in vivo and evaluate changes in a model of experimental diabetes.RESEARCH DESIGN AND METHODSWhole pancreata from untreated (n = 3), MnCl2 and glucose-injected mice (n = 6), and mice injected with either streptozotocin (STZ; n = 4) or citrate buffer (n = 4) were imaged ex vivo for unambiguous evaluation of islets. Exteriorized pancreata of MnCl2 and glucose-injected mice (n = 6) were imaged in vivo to directly visualize the gland and minimize movements. In all cases, MR images were acquired in a 14.1 Tesla scanner and correlated with the corresponding (immuno)histological sections.RESULTSIn ex vivo experiments, MEHFMRI distinguished different pancreatic tissues and evaluated the relative abundance of islets in the pancreata of normoglycemic mice. MEHFMRI also detected a significant decrease in the numerical and volume density of islets in STZ-injected mice. However, in the latter measurements the loss of β-cells was undervalued under the conditions tested. The experiments on the externalized pancreata confirmed that MEHFMRI could visualize native individual islets in living, anesthetized mice.CONCLUSIONSData show that MEHFMRI quantitatively visualizes individual islets in the intact mouse pancreas, both ex vivo and in vivo.

Elevated Amylase and Lipase Mimicking Pancreatitis: Diagnostic Pitfall

Purpose: Introduction: Serum amylase and lipase levels are standard tests to diagnose acute pancreatitis. However, they can be elevated in various clinical settings, including acute gastroenteritis or chronic alcoholism. Recognition is particularly important because using an elevated amylase or lipase level to diagnose acute pancreatitis may not be appropriate. We herein present a case of elevated amylase and lipase levels in the absence of acute pancreatitis. Case Presentation: A 56-year-old male with chronic alcoholism presented with nausea and vomiting for 5 days. He denied abdominal pain, fever or diarrhea. His past medical history was insignificant. He admitted alcohol abuse, notably hard liquors, which constituted his major dietary intake. His vital signs were T 96.9F, HR 90 /min, BP 142/91 mmHg, SpO2 97% on room air. He appeared disheveled. His heart sounds were regular and lungs were clear to auscultation. His abdomen was soft, bowel sounds were diminished but no tenderness was present. His complete blood count and metabolic panel were normal. His liver panel showed a slightly elevated ALT level of 90 U/L with normal bilirubin, ALP and ALT level. Serum amylase and lipase level was found to be 216 mg/dL and 702 mg/dL respectively. His alcohol level was 404 mg/dL. Ultrasonography showed fatty infiltration in the liver with intact pancreas, spleen and gallbladder. Subsequent esophagogastroduodenoscopy (EGD) revealed nonerosive gastritis. He was treated with intravenous fluids and esomeprazole. The following day, amylase and lipase level went down to 123 mg/dL and 437 mg/dL respectively. Discussion: Serum amylase or lipase levels are recognized as a useful marker for pancreatic inflammation. However, there are several other conditions that can cause hyperamylasemia or hyperlipasemia, such as gut perforation and renal failure. In this case, elevated amylase and lipase was probably due to acute gastritis and chronic alcoholism, considering the totally benign abdominal examination as well as the EGD findings. Although gastroenteritis in not widely considered as a cause of hyperamylasemia or hyperlipasemia, numerous cases have reported with possible specific pathogenesis. It may result from pancreatic involvement in the inflammatory process or from increased absorption of pancreatic enzymes from the intestinal lumen. Also, according to the literature, elevated serum lipase levels have been found in 11 to 33% of asymptomatic alcoholics. Therefore, acute gastroenteritis and chronic alcoholism should be included in the differential diagnosis of hyperamylasemia or hyperlipasemia. Extra caution should be exercised when interpreting elevated amylase and lipase mimicking pancreatitis.