Table of Contents

Volume 2, Issue 5

  September/October 2010

  Pages 265 - 338

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REVIEW

Open Access Article

Surgical aspects of human islet isolation

Tatsuya Kin and James Shapiro
Pages 265 - 273
http://dx.doi.org/10.4161/isl.2.5.13019
Abstract | PDF

Islet transplantation is a safe and effective procedure; however, it depends on the critical step of isolating high quality of islets from whole pancreas. Human islet isolation requires considerable experience and expertise, and is frequently seen as ‘an art rather than a science’. However, without scientific knowledge of pancreatic anatomy, real experience can not be gained. In particular, an understanding ductal anatomy is important to perform human islet isolation. This review is based on clinical experience of more than 900 human islet isolations performed over 10 years and aims to highlight pancreatic anatomy and surgical techniques in islet processing.




SHORT REPORT

Open Access Article

Expression of secretory phospholipase A₂ in insulitis of human transplanted pancreas and its insulinotropic effect on isolated rat islets

Miwako Ishida-Oku, Masanori Iwase, Kazuo Sonoki, Nobuhiro Sasaki, Hirofumi Imoto and Yuji Uchizono
Pages 274 - 277
http://dx.doi.org/10.4161/isl.2.5.12728
Abstract | PDF

The expression of secretory phospholipase A₂ (sPLA₂) is induced by inflammatory stimuli in various cells, and sPLA₂ contribute to produce proinflammatory lipid mediators via hydrolyzing plasma membrane phospholipids into free fatty acid and lysophospholipid. We studied the expression of group IIA sPLA₂ in human islets of transplanted pancreas before and after the recurrence of type 1 diabetes mellitus in a case study. In addition, the effects of exogenous sPLA₂ in isolated rat islets were investigated. Expression of group IIA sPLAs was immunohistochemicaly investigated in the pancreas graft biopsy specimens. Insulin secretion was evaluated by static incubation with different concentrations of snake venom sPLA₂. Intracellular free Ca²+ concentration was measured with Fura 2 and lysophosphatidylcholine (LPC) contents in islets were determined by electrospray ionization-liquid chromatography/mass spectrometry. Group IIA sPLA₂ was not expressed in islets without insulitis before the recurrence, whereas it was diffusely expressed in islets after the recurrence with insulitis. There were cells co-expressing group IIA sPLA₂ and insulin. sPLA₂ dose-dependently induced insulin secretion in isolated rat islets, which was completely prevented by a specific sPLA₂ inhibitor indoxam. The application of sPLA₂ did not affect intracellular free Ca²+ concentration in β cells. On the other hand, LPC contents in islets were significantly increased in sPLA₂-treated islets compared with untreated islets. Incubation with indoxam suppressed the sPLA₂-induced increase of LPC. In conclusion, the present study suggests that group IIA sPLA₂ may be expressed in islets during insulitis in humans. Although sPLA₂ induced insulin secretion in vitro probably via the production of lysophospholipid, the significance of this enzyme expression in insulitis remains elusive.




RESEARCH PAPERS

Open Access Article

Experience of islet isolation without neutral protease supplementation

Tatsuya Kin, Doug O’Gorman, Peter Senior and AM James Shapiro
Pages 278 - 282
http://dx.doi.org/10.4161/isl.2.5.12602
Abstract | PDF

Background. We have reported improved islet isolation outcomes using a new digestion protocol where the pancreas is perfused only with collagenase, and neutral protease (NP) is administered during the digestion phase. Since the inception of this protocol, we have had some cases where administration of NP was not required. Methods. Our new protocol was utilized in 94 islet isolations. The timing of adding NP was dependent on the progression of digestion but in 10 cases the progression was rapid and most islets in the assessment samples were free from the exocrine tissue. As a result NP was not added at all for these isolations (no-NP group). In the remaining 84 isolations, NP was added during digestion phase (control group). Results. Pancreata in the each group were digested with a similar collagenase dose. Digestion time was shorter in the no-NP (15.0±1.8 vs 19.5±0.6 min, P=0.004). At post-digestion, the no-NP had fewer trapped islets (10.9±2.8 vs 28.1±2.4%, P=0.009). Post-purification islet yield was similar (355±45 x10³ vs 318±17 x10³ IE, P=0.29). Five preparations in the no-NP were used for clinical transplantation, leading to a 64.3±15.2% reduction in insulin usage. Interestingly, cold ischemia time was longer in the no-NP (10.3±0.9 vs 7.9±0.4 h, P=0.04). One particular collagenase lot having the highest NP activity contamination was used in 7 isolations in the no-NP.

Open Access Article

Mitochondrial proteome analysis reveals altered expression of voltage dependent anion channels in pancreatic β-cells exposed to high glucose

Meftun Ahmed, Sarheed J Muhammed, Benedikt Kessler and Albert Salehi
Pages 283 - 292
http://dx.doi.org/10.4161/isl.2.5.12639
Abstract | PDF

Chronic hyperglycemia leads to deterioration of insulin release from pancreatic β-cells as well as insulin action on peripheral tissues. However, the mechanism underlying β-cell dysfunction resulting from glucose toxicity has not been fully elucidated. The aim of the present study was to define a set of alterations in mitochondrial protein profiles of pancreatic β-cell line in response to glucotoxic condition using 2-DE and tandem mass spectrometry. INS1E cells were incubated in the presence of 5.5 and 20 mM glucose for 72 hrs and mitochondria were isolated. Approximately 75 protein spots displayed significant changes (p < 0.05) in relative abundance in the presence of 20 mM glucose compared to controls. Mitochondrial proteins down regulated under glucotoxic conditions includes ATP synthase α chain and δ chain, malate dehydrogenase, aconitase, trifunctional enzyme β subunit, NADH cytochrome b5 reductase and voltage-dependent anion-selective channel protein (VDAC) 2. VDAC1, 75 kDa glucose-regulated protein, heat shock protein (HSP) 60 and HSP10 were found to be upregulated. The orchestrated changes in expression of VDACs and multiple other proteins involved in nutrient metabolism, ATP synthesis, cellular defense, glycoprotein folding and mitochondrial DNA stability may explain cellular dysfunction in glucotoxicity resulting in altered insulin secretion.

Open Access Article

Expression and function of cannabinoid receptors in mouse islets

Chen Li, James E. Bowe, Peter M. Jones and
Pages 293 - 302
http://dx.doi.org/10.4161/isl.2.5.12729
Abstract | PDF

The endocannabinoid system plays a key role in energy homeostasis, with agonists and antagonists of CB1 receptors acting centrally to stimulate and inhibit food intake, respectively. In addition to their established effects on the central nervous system, cannabinoid receptor agonists also exert peripheral effects by modulating cellular cyclic AMP and calcium levels and there have been reports that they regulate β-cell function. However, the few reports to date on islet expression of cannabinoid receptors and effects of agonists on insulin secretion have failed to reach a consensus. We have therefore investigated cannabinoid receptor expression by mouse islet β-and α-cells and the effects of selective receptor agonists on cyclic AMP and calcium levels, and on dynamic insulin secretory responses. CB1 and CB2 mRNA and protein expression by islets was detected by RT-PCR and western blotting respectively, and cellular location of the receptors was identified by immunohistochemistry with insulin and glucagon antibody co-staining. Cyclic AMP generation was quantified by enzyme immunoassay and changes in calcium levels were measured by microfluorimetry of Fura-2-loaded mouse islet cells. Dynamic insulin secretion was quantified by radioimmunoassay after perifusion of isolated islets. We found that mouse islets expressed both CB1 and CB2 receptors, and they were localised to β-cells. Activation of mouse β-cell CB1 and CB2 receptors resulted in decreased cyclic AMP, increased calcium and potentiation of glucose-stimulated insulin secretion. Thus, activation of islet cannabinoid receptors by locally produced endocannabinoids such as 2-aminoglycerol may be another regulatory pathway by which islets stimulate insulin secretion to maintain glucose homeostasis.

Open Access Article

Electrophysiological effects of osmotic cell shrinkage in rat pancreatic β-Cells

Len Best and Allen P. Yates
Pages 303 - 307
http://dx.doi.org/10.4161/isl.2.5.12748
Abstract | PDF

Electrical and secretory activity in the pancreatic β-cell can be elicited by hypotonic cell swelling, due largely to activation of a volume-regulated anion channel (VRAC) leading to depolarisation and electrical activity. However, β-cell responses to cell shrinkage are less well characterised. The present study has examined the effects of osmotic cell shrinkage on rat pancreatic β-cells. Electrical activity and whole-cell current were studied in isolated β-cells using the perforated patch and conventional whole-cell recording techniques. Insulin release was measured using intact islets by radioimmunoassay. Exposure to a 33% hypertonic bath solution resulted in an initial depolarisation and a period of electrical activity. In several cases, this depolarisation was transient and was followed by a hyperpolarisation. A similar pattern was observed with insulin release. In voltage-clamp experiments, osmotic shrinkage resulted in activation of a non-selective cation channel (NSCC) sensitive to inhibition by flufenamic acid and Gd3+. It is suggested that activation of this NSCC is responsible for the depolarisation evoked by hypertonic media. The secondary hyperpolarisation is likely to be the result of inhibition of VRAC activity. These opposing ionic effects could underlie the biphasic effect on insulin release following exposure to hypertonic media.

Open Access Article

Pim3 negatively regulates glucose-stimulated insulin secretion

Gregory Vlacich, Martijn C. Nawijn, Gene C. Webb and Donald F. Steiner
Pages 308 - 317
http://dx.doi.org/10.4161/isl.2.5.13058
Abstract | PDF

Pancreatic β cell response to glucose stimulation is governed by tightly regulated signaling pathways which have not been fully characterized. A screen for novel signaling intermediates identified Pim3 as a glucose-responsive gene in the β cell, and here, we characterize its role in the regulation of β-cell function. Pim3 expression in the β-cell was first observed through microarray analysis on glucose-stimulated murine insulinoma (MIN6) cells where expression was strongly and transiently induced. In the pancreas, Pim3 expression exhibited similar dynamics and was restricted to the β cell. Perturbation of Pim3 function resulted in enhanced glucose-stimulated insulin secretion, both in MIN6 cells and in isolated islets from Pim3-/- mice, where the augmentation was specifically seen in the second phase of secretion. Consequently, Pim3-/- mice displayed an increased glucose tolerance in vivo. Interestingly, Pim3-/- mice also exhibited increased insulin sensitivity. Glucose stimulation of isolated Pim3-/- islets resulted in increased phosphorylation of ERK1/2, a kinase involved in regulating β-cell response to glucose. Pim3 was also found to physically interact with SOCS6 and SOCS6 levels were strongly reduced in Pim3-/- islets. Overexpression of SOCS6 inhibited glucose-induced ERK1/2 activation, strongly suggesting that Pim3 regulates ERK1/2 activity through SOCS6. These data reveal that Pim3 is a novel glucose-responsive gene in the β cell that negatively regulates insulin secretion by inhibiting the activation of ERK1/2, and through its effect on insulin sensitivity, has potentially a more global function in glucose homeostasis.

Open Access Article

Effect of inborn pancreatic islet deficit in the Munich Wister Frömter rat

Marina Figliuzzi, Barbara Bonandrini, Irene Cattaneo, Giuseppe Remuzzi and Andrea Remuzzi
Pages 318 - 322
http://dx.doi.org/10.4161/isl.2.5.13178
Abstract | PDF

The total mass of pancreatic islet cells is a critical factor in glucose metabolic control. The aim of the present study was to investigate whether in the Munich Wistar Frömter (MWF) rat, beside a reduction in the number of nephrons, there are also alterations in the number of pancreatic islets and of β cell mass. We also examined glucose metabolism, both in normal conditions and following intravenous glucose injection. The number of islets per pancreas, estimated by morphometrical analysis, was significantly lower in MWF rats than in Wistar rats (3,501±1,285 vs. 7,259±2,330 islet/rat, respectively). Also the mean number of islets per gram of body weight was significantly lower in MWF rats than in Wistar rats (18±7 in MWF rats vs. 28±10 islets/g bw in Wistar rats). Morphometric analysis of β cell mass showed an average of 77.1±7% islet cells staining for insulin in MWF rats and 83.9±2.1% in the control Wistar rats. Despite the lower number of islets and β cells, MWF and Wistar rats had comparable fasting blood glucose levels but significant differences in blood glucose following an intraperitoneal glucose tolerance test. In summary, pancreatic islets of MWF and Wistar rats showed a marked difference in morphometrical characteristics. While this difference is not associated with blood glucose levels, glucose metabolism after IPGTT between MWF and Wistar rats is significantly different. These data suggest that an inborn deficit in β cell mass of about 60% is responsible for altered glucose metabolism and could favor the development of diabetes.




COMMENTARIES

Open Access Article

An emerging role for NAADP-mediated Ca²⁺ signaling in the pancreatic β-cell

Abdelilah Arredouani, A. Mark Evans, Jianjie Ma, John Parrington, Michael X. Zhu and Antony Galione
Pages 323 - 330
http://dx.doi.org/10.4161/isl.2.5.12747
Abstract | PDF

Several recent reports, including one in this journal, have reignited the debate about whether the calcium-mobilizing messenger, nicotinic adenine nucleotide diphosphate (NAADP) plays a central role in the regulation of calcium signalling in pancreatic β-cell.^1-5 These studies have highlighted a role for NAADP-induced Ca²⁺ mobilization not only in mediating the effects of the incretin, GLP-1 and the autocrine proliferative effects of insulin, but also possibly a fundamental role in glucose-mediated insulin secretion in the pancreatic β-cell.

Open Access Article

A TRP channel contributes to insulin secretion by pancreatic β-cells

Emily R. Liman
Pages 331 - 333
http://dx.doi.org/10.4161/isl.2.5.12973
Abstract | PDF

The release of insulin by pancreatic beta cells involves a complex interplay of conductances that generate oscillations and drive secretion. A recent report identifies a new player in this process, the ion channel TRPM5. TRPM5 was originally identified in taste cells, where it forms a Ca²⁺-activated cation channel that is required for sensory responses to bitter and sweet tastes. New research now shows that TRPM5 is expressed within the pancreatic islets of Langerhans, where it regulates the frequency of Ca²⁺ oscillations and contributes to insulin release by β-cells.

Open Access Article

Defining the regulation of IL-1β- and CHOP-mediated β-cell apoptosis

Nathan L. Vanderford
Pages 334 - 336
http://dx.doi.org/10.4161/isl.2.5.13095
Abstract | PDF

Diabetes is a multifaceted metabolic disorder that can be caused by pancreatic β-cell destruction (type I diabetes) and/or heightened by β-cell failure (type II diabetes). The gross clinical and physiological characteristics of the disease are well characterized, and viable treatment options can drastically alter the course and effects of the disease. However, the molecular events occurring within the β-cell that cause or contribute to diabetes are not adequately understood, especially in terms of the interplay between the physiological signals that facilitate disease development. A recent report, focused on a mechanism by which IL-1β induces β-cell apoptosis, underscores the complexity of the molecular events that may cause or affect the progression of diabetes. This commentary summarizes aspects of this report, discusses an example of the complexity of β-cell regulation and proposes more frequent use of complex in vitro systems that more closely mimic in vivo conditions so that greater advances can be made toward understanding the molecular mechanisms contributing to diabetes. Understanding the molecular etiology of β-cell dysfunction will likely enhance the possibility of developing novel diabetes therapeutic interventions.




ARTICLE ADDENDUM

Open Access Article

Adding efficiency: The role of the CAN ion channels TRPM4 and TRPM5 in pancreatic islets

Thorsten Enklaar, Lili R. Brixel, Bernhard Zabel and Dirk Prawitt
Pages 337 - 338
http://dx.doi.org/10.4161/isl.2.5.13140
Abstract | PDF

Insulin secretion in β-pancreatic cells after glucose stimulation requires the concerted action of a number of different ion channels. The main players seem to be the ATP sensitive K⁺ (K_ATP-) channels, and voltage gated ion channels that drive Ca²⁺ influx into β-cells. Recently two calcium activated nonselective (CAN) cation channels (TRPM4 and TRPM5) have been shown to influence efficient insulin response upon glucose stimulation. This addendum summarizes the data known for these two TRP channels in β-cells, discusses some of the remaining open questions and addresses a possible scenario that involves and integrates the triggering and amplifying pathway of glucose mediated insulin secretion.