Type 1 diabetes is a chronic autoimmune disease characterised by the selective destruction of pancreatic beta (β) cells. The understanding of the aetiology of this disease has increased dramatically in recent years by the study of tissue recovered from patients; analysis of the responses of isolated islet and β cells in tissue culture and via the use of animal models. However, knowledge of the immunopathology of type 1 diabetes in humans is still relatively deficient due largely to the difficulty of accessing appropriate samples. This presentation will review the state of current knowledge in relation to the histopathological features of type 1 diabetes in humans. It will focus specifically on recent-onset type 1 diabetes cases since, in such patients, evidence of the ongoing disease process is still present. Our studies have charted the progression of the disease by describing the characteristic features of the pancreas, considering the sequence of immune cell infiltration during islet inflammation and the abnormalities of MHC antigen expression. The possibility that these changes might derive from a persistent enteroviral infection of the islet beta-cells will also be examined and evidence to support this hypothesis presented. We have also studied the possibility that islet endocrine cell proliferation may be initiated as a means to combat the autoimmune attack in human type 1 diabetes, and these data will be discussed.

Biphasic insulin secretion in response to glucose, consisting of a transient first phase followed by a progressive second phase, though a well described feature in mosst species of pancreatic islets, is poorly understood. Using single canine -cells we have compared the time courses of electrical activity and insulin granule exocytosis to biphasic insulin secretion. Short trains of action potentials, similar those found during first phase insulin secretion, trigger phasic exocytosis from a small pool of insulin granules, likely an immediately releasable pool docked near voltage activated Ca2+ channels. In contrast, plateau depolarizations to between -35 and -20 mV resembling those during second phase insulin secretion, trigger tonic exocytosis from a larger pool of insulin granules, likely a highly Ca2+-sensitive pool farther from Ca2+ channels. Both phasic and tonic modes of exocytosis are enhanced by glucose, via its metabolism. However the latter pool, perhaps consisting of “newcomer” granules attracted to transient release sites is particularly enhanced by cAMP, PKC activation and by brief pre-depolarizations that enhance cytosolic Ca2+ without triggering release. Hence, in canine β-cells may present a novel model where two distinct components of exocytosis, tuned to two components of electrical activity, may contribute significantly to biphasic insulin secretion. Aspects of this model may contribute to biphasic secretion in other species such as pigs and humans.

BK and SK4 channel knockout (KO) mice were used to examine the role of KCa channels in glucose homeostasis, beta-cell function and viability. Glucose-tolerance tests of the KO-mouse strains revealed opposed results: SK4-KO mice elicited a lower blood glucose concentration compared to WT littermates. In contrast, BK-KO mice were less glucose tolerant than WT mice. The improved glucose tolerance of SK4-KO mice can be explained by changes in the stimulus-secretion coupling (SSC). Glucose-induced action potential (AP) frequency was increased in SK4-KO beta-cells. This increase in electrical activity was mirrored by enhanced [Ca2+]c. Likewise, the SK4 channel blocker TRAM-34 was able to increase AP frequency and [Ca2+]c. In BK-KO islets glucose-induced insulin secretion was reduced. However, other parameters of SSC were not different between WT and BK-KO beta-cells. Measuring apoptosis revealed that BK-KO mice had a higher rate of apoptotic islet cells vs. WT. BK-KO islet cells were more sensitive to oxidant-induced apoptosis than WT cells. These effects could be mimicked by the BK channel iberiotoxin. It is concluded that KO or inhibition of the SK4 channel increases the sensitivity of beta-cells to glucose. This makes the SK4 channel an interesting new target for antidiabetic drugs. The BK channel seems to be only marginally involved in the SSC of beta-cells, at least in mice, but it may play an important role in beta-cell survival.

Type 1 diabetes (T1D) is a debilitating autoimmune disease with the incidence on the increase in developed countries, suggesting that changes in environment (including changes in microbial environment) may be responsible. Similarly, the incidence of spontaneous T1D in non-obese diabetic (NOD) mice depends on the microbial environment in the animal housing facility. These observations leave no doubt that microbes are capable of controlling the development of T1D. We have initiated and now continue the studies to delineate the role of innate immune receptors in initiation and prevention of T1D. In the course of these studies we have found that: NOD mice lacking MyD88 adaptor protein were resistant to spontaneous T1D when housed in conventional specific-pathogen-free (SPF) conditions; that MyD88 KO NOD mice did not show systemic tolerance to islet antigens, but did show tolerance of T cells to such antigens in the local pancreatic lymph nodes (PLN); and that when these animals were made germ-free (GF), they developed diabetes with the incidence of about 100%. Taken together, the data suggested that commensal (not pathogenic) microbes in the gut influenced the development of T1D. We will discuss further the role of MyD88 in control of the commensal microbes and the mechanisms by which microbes control autoimmunity.

Background and Aims The Wnt signaling pathway has been recently implicated in pancreas development as well as in beta cell biology. Glycogen synthase kinase 3B (GSK3B), a pivotal partner of the Wnt signaling is a multifunctional enzyme that negatively regulates the growth and function of the beta cells. The aim of our study was to assess the impact of GSK3B downregulation on the stimulation of exocrine and endocrine regeneration after subtotal pancreatectomy in rat. Methods: Adult Wistar rats underwent 90% pancreatectomy. In groups of pancreatectomized rats, either antisense oligonucleotides directed against GSK3B (AS-GSK3B) or LiCl were injected directly within the remnant pancreatic tissue immediately after pancreatectomy. Two additional groups were administered with non specific standard oligonucleotides (Std group) or with saline and were used as control for the AS-GSK3B and LiCl treated groups respectively. Beta cell mass was assessed by morphometry 7 days and 4 weeks after pancreatectomy. Beta cell, ductal cells and acinar cell proliferation was measured by BrdU incorporation method 8h and 48h after surgery. Apoptosis was assessed in beta cells, ductal cells and acinar cells by the TUNEL method. Results GSK3B down regulation via administration of LiCl or AS-GSK3B greatly improved the beta cell regeneration 7 days after surgery (p<0.01). Moreover the use of AS-GSK3B had sustained effect on the beta cell mass, since 4 weeks after surgery the beta cell mass in the remnant pancreas was found to be higher (p<0.05) in AS-GSK3B treated group compared to the Std treated group. The effect of GSK3B inactivation on the beta cell mass seemed to be mediated by the stimulation of beta cell proliferation. Regarding the exocrine pancreas, GSK3B knockdown significantly stimulated acinar cell proliferation. Interestingly, GSK3B inactivation reduced the number of apoptotic acinar cells compared to that found in the control Std group. Conclusion Here we show that intra pancreatic in vivo knockdown of GSK3Background and Aims The Wnt signaling pathway has been recently implicated in pancreas development as well as in beta cell biology. Glycogen synthase kinase 3B (GSK3B), a pivotal partner of the Wnt signaling is a multifunctional enzyme that negatively regulates the growth and function of the beta cells. The aim of our study was to assess the impact of GSK3B downregulation on the stimulation of exocrine and endocrine regeneration after subtotal pancreatectomy in rat. Methods: Adult Wistar rats underwent 90% pancreatectomy. In groups of pancreatectomized rats, either antisense oligonucleotides directed against GSK3B (AS-GSK3B) or LiCl were injected directly within the remnant pancreatic tissue immediately after pancreatectomy. Two additional groups were administered with non specific standard oligonucleotides (Std group) or with saline and were used as control for the AS-GSK3B and LiCl treated groups respectively. Beta cell mass was assessed by morphometry 7 days and 4 weeks after pancreatectomy. Beta cell, ductal cells and acinar cell proliferation was measured by BrdU incorporation method 8h and 48h after surgery. Apoptosis was assessed in beta cells, ductal cells and acinar cells by the TUNEL method. Results GSK3B down regulation via administration of LiCl or AS-GSK3B greatly improved the beta cell regeneration 7 days after surgery (p<0.01). Moreover the use of AS-GSK3B had sustained effect on the beta cell mass, since 4 weeks after surgery the beta cell mass in the remnant pancreas was found to be higher (p<0.05) in AS-GSK3B treated group compared to the Std treated group. The effect of GSK3B inactivation on the beta cell mass seemed to be mediated by the stimulation of beta cell proliferation. Regarding the exocrine pancreas, GSK3Bknockdown significantly stimulated acinar cell proliferation. Interestingly, GSK3B inactivation reduced the number of apoptotic acinar cells compared to that found in the control Std group. Conclusion Here we show that intra pancreatic in vivo knockdown of GSK3B promotes both endocrine and exocrine regeneration within the remnant pancreas and could have potential application in the treatment of diabetes and other pancreatic diseases such as pancreatitis. promotes both endocrine and exocrine regeneration within the remnant pancreas and could have potential application in the treatment of diabetes and other pancreatic diseases such as pancreatitis.

Pancreatic beta-cells are extremely sensitive to oxidative stress due to their low expression and activity of antioxidant enzymes. Since excessive production of reactive oxygen species (ROS) critically contributes to beta-cell failure during manifestation of type 2 diabetes up-regulation of antioxidant defense mechanisms is an interesting approach to delay diabetes progression. We investigated whether inhibitors of KATP channels modulate antioxidative capacity of pancreatic beta-cells in addition to their well-known insulinotropic effect. The sulfonylureas gliclazide and tolbutamide increased the activity of catalase (Cat) and superoxide dismutase (SOD) in isolated islets. Both drugs protected islet cells from apoptotic cell death evoked by low concentrations of H2O2. Enzyme up-regulation and prevention of ROS-induced apoptosis was dependent on Ca2+-influx via L-type Ca2+ channels and Ca2+ storage in the endoplasmic reticulum, respectively. The beneficial impact on cell viability obtained with sulfonylureas could be mimicked by genetic ablation of KATP channels in SUR1 knockout (SUR1-KO) mice. Moreover, we could show that stimulus-secretion coupling of SUR1-KO beta-cells was protected against oxidative stress. In agreement with pharmacologic inhibition of KATP channels in wildtype beta-cells, SUR1-KO islets displayed significantly elevated activity of Cat, SOD and glutathione peroxidase. Importantly, SUR1-KO mice were more resistant against streptozotocin-induced diabetes. In summary, these data demonstrate that blocking KATP channels counteracts ROS-mediated impairment of beta-cell function and viability thereby providing a strategy to slow down beta-cell failure during diabetes development.

The pancreatic islet displays diverse patterns of endocrine cell arrangement among species. We studied islet architecture in a wide range of animals including humans, monkeys, pigs, rabbits, birds and mice. Despite differences in overall body and pancreas size as well as total beta-cell mass among these species, the distribution of their islet sizes closely overlaps except in the bird pancreas, which has only a small number of islets. The fraction of alpha-, beta-, and delta-cells within an islet varies between islets in all the species examined. Three-dimensional analysis shows that human islet architecture resembled that of db/db mouse islets. We also examined islets from mice under different physiological and pathophysiological conditions including development, pregnancy, obesity, prediabetes and diabetes. Under conditions of an increased demand for insulin the alpha-cell to beta-cell ratio increases, which is often accompanied with a more central localization of alpha-cells. Furthermore, while alpha-cells in the adult basically only express prohormone convertase 2 (PC2), alpha-cells under these conditions express both PC2 and PC1/3, which results in processing of the proglucagon precursor to GLP-1, thereby leading to local production of this important beta-cell growth factor. Our studies suggest an important role for the endocrine cell network within the islet for beta-cell proliferation.

The endocrine pancreas, consisting of the islets of Langerhans, is the essential organ for glucose homeostasis. The development of this organ is not fully understood with basic questions such as the occurrence of islet neogenesis and fission not definitively addressed. With a novel method to image all the islets in an intact pancreas using transgenic mice expressing a fluorescent protein specifically in beta cells, we obtained islet size distributions at different postnatal days. From the changes of islet size distributions with time, we deduced developmental processes in pancreatic islets with mathematical modeling. We found that (1) new islets were not formed by neogenesis; (2) every beta cell had a potential for proliferation, although beta cells in larger islets had slightly higher proliferation probability; and (3) fission of large islets occurred by postnatal week 4. The combination of precise and complete islet-size measurements and mathematical modeling can be applied to understand changes in the endocrine pancreas under various physiological and pathological conditions such as aging, pregnancy, diabetes, obesity, and cancer. As an example, we examined changes of islet size distributions under insulinoma, and found more accelerated beta-cell proliferation in larger islets.

Adult pancreatic islets contain multiple cell types that produce and secrete well characterized hormones including insulin, glucagon and somatostatin. Although it is becoming increasingly apparent that islets release and respond to more secreted factors than previously thought, systematic analyses are lacking. The aims of the present study were to identify potential autocrine/paracrine islet growth factor loops and to characterize the function of a family of previously unreported islet secreted factors and their receptors. Gene expression databases and microarray analyses of FACS purified human β-cells were used to compile a list of 230 secreted factors and 238 secreted factor receptors (189 factor-receptor pairs) present in islets. This genome-wide analysis led us to characterize context-dependent roles for netrins and their cognate receptors (neogenin and unc5A) in adult pancreatic β-cell apoptosis. These results mirror our previous observations of context-dependent regulation of apoptosis by Notch/Ngn3 signalling in adult islets. We also defined reciprocal roles for activin A and follistatin in the control of β-cell maturity and function. Together, our results highlight the large number of potential islet growth factors and point to a context-dependent pro-survival role for netrins in the adult pancreatic β-cells. Since diabetes results from a deficiency in functional β-cell mass, these studies are an important step towards developing novel therapies to improve β-cell survival.

In many cell types, including pancreatic beta-cells, Bcl-2 and Bcl-xL have been demonstrated to preserve mitochondrial integrity and suppress apoptosis under a variety of cellular stress conditions. Whether these important pro-survival proteins also partake in the regulation of normal beta-cell physiology in vivo or in vitro is not known. In this study we used a combination of genetic and pharmacological loss-of function approaches to test the hypothesis that Bcl-2 and Bcl-xL impact beta-cell energy metabolism, calcium homeostasis and insulin secretion. We generated mice in which Bcl-x was conditionally deleted in adult beta-cells by tamoxifen-injecting of Bcl-xflox/flox;Pdx1-CreER (Bcl-x KO) and littermate Bcl-xflox/flox (Bcl-x WT) control mice. The near complete loss of Bcl-xL expression and protein in islets from these inducible Bcl-x KO mice was confirmed by quantitative real-time PCR and western blot. Loss of islet Bcl-x resulted in a moderate improvement of glucose tolerance in 10-12 week old mice within days of tamoxifen administration. The cellular ATP-to-ADP ratio of Bcl-x KO islet cells was markedly increased in the presence of both basal and stimulatory glucose. Moreover, cytosolic calcium responses were significantly enhanced in glucose-stimulated islets and dispersed beta-cells from Bcl-x KO mice. Similar augmentation of glucose-induced calcium responses was seen in beta-cells from Bcl-2 knockout mice, relative to their heterozygous and wild-type littermates. Small molecule antagonists were used to assess the impact of acute Bcl-2/Bcl-xL inhibition on beta-cell physiology. In accordance with the findings from Bcl-2 and Bcl-x knockout cells, acute treatment of normal mouse and human islet cells with Bcl-2/Bcl-xL antagonists enhanced basal glucose-dependent respiration and induced mitochondrial calcium fluctuations within minutes. This raised ATP/ADP and triggered KATP channel- and voltage-dependent calcium influx and insulin secretion. Our findings demonstrate that anti-apoptotic Bcl proteins exert a tonic suppression of beta-cell metabolic signalling and thus work at the interface of beta-cell survival and physiology. Further study of these survival-regulating proteins and the molecular mechanisms of their metabolic functions may help identify factors to preserve the functional beta-cell mass required to maintain glucose homeostasis.

Endocrine pancreas development, regeneration and function have been very well studied in humans and mouse models. Several molecules and signalling pathways are demonstrated to be involved in these processes. I will discuss our findings related to the role of microRNAs, a class of non-coding RNAs, in regulation of pancreas development and function. We reported earlier that miR-195 family miRNAs targets neurogenin-3, an important pro-endocrine transcription factor, at post-transcriptional level and inhibit its translation into protein during mouse pancreas regeneration. This finding strongly favors the theory of origin of beta cell from existing beta cells. We also determined expression pattern of non-coding RNAs during human pancreas development using the SOLiD platform for next generation sequencing. We observed that few miRs, most notably miR-7 and miR-375 that are important for pancreatic islet function are also important for development of islet beta cells. These 2 microRNAs show exponential increases in their abundance during development, which coincides with that of insulin mRNA expression. I will discuss these and other studies that indicate the importance of non-coding RNAs in endocrine pancreas development and generation of islet progenitor cells. Finally, I will discuss our recent findings related to understanding the role of these ncRNAs in understanding islet beta cell mass and function. These and several other studies carried out until now indicate that non-coding RNAs form an important part of endocrine pancreas development and are also important biomarkers of islet cell death and function.

Current models of stimulus-secretion coupling in pancreatic β-cells emphasize a role for glucose-enhanced plasma membrane ion fluxes as a consequence of the closure of KATP (Kir6.2/SUR1) channels. However, glucose may still evoke Ca2+ signals and insulin exocytosis in β-cells from Kir6.2 or SUR1 knockout mice by KATP-independent pathways. We have investigated the role of the NAADP-targeted acidic Ca2+ stores in glucose-mediated Ca2+ oscillations and insulin secretion in primary mouse β-cells. We have used Ca2+ imaging, electrophysiology and insulin secretion measurements to investigate the role of NAADP in pancreatic β cells in stiumulus-secretion coupling. We found that the release of Ca2+ from acidic Ca2+ stores plays a critical role in triggering and shaping glucose-evoked Ca2+ signals since these signals are abolished by the vacuolar proton pump inhibitor, bafilomycin. Using new membrane-permeant probes, we further show that the intracellular messenger NAADP mobilizes Ca2+ from acidic stores, but not the endoplasmic reticulum, depolarizes the plasma membrane by activating a cation current. Inhibition of NAADP-evoked Ca2+ release, either by Ned- 19, a selective NAADP antagonist, or self-desensitization, abolishes not only glucose-induced depolarization, Ca2+ signals and insulin secretion but also tolbutamide-induced insulin secretion. We have recently discovered that the two-pore channels (TPCs) located on acidic stores are the major target receptors of NAADP. We show that glucose-induced [Ca2+]i signalling is dramatically reduced in TPC1 and TPC2 knockout mice. We propose that NAADP-mediated Ca2+ mobilization from acidic stores via TPCs is a key step in triggering stimulus-secretion coupling in mouse pancreatic β-cells. In turn this activates a calcium-dependent cation current which, when KATP channels close, can depolarize the membrane potential allowing the activation of voltage-gated Ca2+ channels, Ca2+ influx and exocytosis of insulin granules. Supported by: the Wellcome Trust

Diabetes mellitus (DM) is associated with hyperglycemia and impaired β-cell function. Glucagon-like peptide-1 (GLP-1, 50 nmol/Kg body weight) and exenatide (1.0 μg/Kg body weight), a GLP-1 agonist, were given intraperitoneally (i.p.) to normal and diabetic rats daily for 10 weeks. DM was induced by a single (i.p.) injection of streptozotocin (STZ, 60 mg/kg) in Wistar rats. Control animals received a similar quantity of buffer solution. GLP-1 and exenatide-immunoreactive cells were observed in the outer part of pancreatic islets of normal rats. GLP-1 and exenatide-positive cells were located in both the peripheral as well as the central portions of pancreatic islet cells of diabetic rats compared to the peripheral location in control rats. The number of GLP-1 and exenatide-immunopositive cells increased significantly after the onset of STZ-induced diabetes. GLP-1, but not exenatide, significantly (p < 0.05) reduced blood glucose level in diabetic rats compared to normal. Treatment of normal and diabetic rats with either GLP-1 or exenatide induced significant (p < 0.001) increases in the number of insulin, catalase and glutathione-immunopositive cells in the islet of Langerhans. In conclusion, GLP-1 and its agonist, exenatide may improve pancreatic beta cell mass by increasing endogenous antioxidant pool, which may in turn assist in beta cells survival after STZ-induced diabetes. GLP-1 and exenatide may also have a protective effect on pancreatic cell, especially after the onset of diabetes. All of these factors may lead to the hypoglycaemic effect of incretins.

Diabetes is becoming a global epidemic, affecting millions of people worldwide. Cell replacement therapy using cadaveric islets has been shown to be successful for 1-5 years. However, scarcity of available donor pancreas limits this therapy from wide-spread use. Alternate replenishable cell sources such as embryonic stem cells, bone marrow stem cells, umbilical cord blood-derived progenitor cells and tissue-derived stem cells have been thought to be useful for cell replacement therapy in diabetes. We believe that stem / progenitor cells derived from pancreatic islets are “committed” to differentiate into insulin-producing cells. We assessed in vitro differentiation potential of 5 different pancreatic progenitors following a previously established in vitro differentiation protocol and observed that islet-derived progenitor cells show better differentiation to endocrine pancreatic lineage. Analysis of the insulin promoter region confirms a transciptionally active conformation of insulin promoter region in islet-derived progenitor cells as compared to that in other cell types studied. Open chromatin conformation was assessed by using chromatin immunoprecipitation (ChIP) for H3K4me2, H3K4me3, H3Ac, H4Ac. Similarly silencing marks at insulin promoter were assessed by ChIP for H3K9me2 and H3K27me2. These epigenetic marks of active chromatin conformation are believed to be heritable and it is therefore reasonable to think that such islet-derived progenitors are better committed to differentiate into insulin-producing cells. Further studies on clonally expanded pancreatic islet cells will help us in generating endocrine lineage-specific progenitors for cell replacement therapy in diabetes.

Background and aims: Decreased β-cell mass reflects a shift from quiescence/ proliferation into apoptosis, it plays a crucial role in the pathophysiology of type 1 and type 2 diabetes and limits the outcome of islet transplantation. A major apoptotic pathway which triggers glucose- and autoimmunity- mediated β-cell apoptosis is the activation of the Fas/FasL system. Here we show that switching the Fas pathway using Fas apoptotic inhibitory protein (Faim/ TOSO), which regulates apoptosis upstream of caspase 8, is a promising target to block β-cell apoptosis and to increase proliferation. Materials and methods: TOSO expression in pancreatic sections was analyzed by immunofluorescence in 8 patients with T2DM and 8 healthy controls. Human isolated islets were exposed to increasing glucose (5.5-33.3 mM) and IL-1β (0.1-5 ng/ml) with and without TOSO down-regulation by siRNA (siTOSO) or β-cell specific TOSO over-expression by RIP-TOSO plasmid transfection. Apoptosis, proliferation and TOSO expression were analyzed. To answer the question if there is a sub-population of β-cells in human islets in culture, which is especially replication-active, we incorporated the thymidine analogs 5-chloro-2-deoxyuridine (CldU) and 5-iodo-2-deoxyuridine (IdU) into cultured β-cells after TOSO transfection and measured cycles of proliferation. Results: Triple immunostaining for TOSO, insulin and glucagon in pancreatic sections of healthy controls and patients with T2DM revealed that TOSO is clearly expressed in the β-cells and down-regulated in diabetes. We observed a 5.5-fold increase in TOSO by short-term incubation of islets with glucose (12 h), a condition when glucose induced proliferation (1.8-fold increase in Ki67 positive β-cells). In contrast, TOSO was down-regulated to almost undetectable levels after long-term incubation for 72 h, a condition where glucose induced β-cell apoptosis (2.5-fold increased β-cell apoptosis at 33.3 mM compared to 5.5 mM glucose). Previously, we have found that the cytokine IL-1β has a dual role on β-cell turnover; it induces proliferation at low dose and apoptosis at high dose. In line with this finding, low dose IL-1β induced and high dose almost depleted TOSO mRNA expression. These studies lead to the hypothesis that TOSO may regulate a switch between proliferation and apoptosis in the β-cell. To investigate the physiological role of TOSO, we over-expressed and depleted TOSO in the β-cell. Incubation of human islets with siTOSO for 4 days depleted TOSO expression together with 3.5-fold induction of apoptosis. β-cell apoptosis was 4-fold increased by the cytokine mix IL-1β and IFNγ and 3.5-fold by 33.3 mM glucose, which was prevented by RIP-TOSO over-expression. On day 2 and 4 after RIP-TOSO transfection, we incorporated the thymidine analogs 5-chloro-2-deoxyuridine (CldU) and 5-iodo-2-deoxyuridine (IdU), respectively, into cultured islets. RIP-TOSO resulted in a 3.5-fold induction of proliferation. Only a small percentage (~2%) of the proliferating β-cells were co-labeled for CldU and IdU suggesting a very limited capacity of β-cells to undergo multiple rounds of proliferation. Conclusion: Our data suggest that TOSO is an important regulator of β-cell turnover and could be an important tool to switch β-cell apoptosis into proliferation and to maintain and generate a sufficient β-cell mass.

Hörnblad A. 1, Alanentalo T. 1, Eriksson M.1, Mayans S.2, Eriksson A.1, Hill RE.3 Holmberg D.2,4, and Ahlgren U.1 1 Umeå Centre for Molecular Medicine, Umeå University, SE-90187, Umeå, Sweden. 2 Dept. of Medical Biosciences, Umeå University, SE-901 85, Umeå, Sweden. 3 MRC Human Genetics Unit, Western General Hospital, Crewe Rd, Edinburgh, EH4 2XU, UK 4 Dept of Disease Biology, Faculty of Life Science, Copenhagen University, DK-1870, Copenhagen, Denmark Recent advances in the field of biomedical imaging have prompted us to revisit mouse models for development and disease of the pancreas. Optical Projection Tomography (OPT)1, provides unprecedented resolution for whole organ imaging and enables three dimensional (3D) and quantitative assessments of β-cell mass distribution (BCM) throughout the volume of the pancreas, down to the level of the individual islets2. To refine our current information regarding the quantitative and spatial dynamics of type 1 diabetes progression, we assessed the spatial development and progression of insulitis and β-cell destruction in pancreas from diabetes prone NOD mice between 3 and 16 weeks of age. Our data provide evidence for a compensatory growth potential of the larger insulin+ islets during the later stages of the disease. This is in contrast to smaller islets, which appear less resistant to the autoimmune attack. We also provide new information on the spatial dynamics of the insulitis process itself, including its apparently random distribution at onset and the formation of structures resembling tertiary lymphoid organs at later phases of insulitis progression3. In a similar undertaking we recently revisited pancreas development and adult constitution in normal mice. Hereby we describe the organogenesis and developmental prerequisites for formation of the gastric lobe (GL) of the pancreas. Our data demonstrate that the GL forms by perpendicular growth from the stalk of the dorsal pancreatic epithelium into a lateral domain of the dorsal pancreatic mesenchyme. As demonstrated in mice with impaired spleen development, formation of this domain is dependent on spleen organogenesis. In addition, we present data regarding the relative contribution of the gastric, duodenal and splenic lobe to the overall BCM in adult C57BL/6 mice. References: 1 Sharpe et al., Science 2002 2 Alanentalo et al., Nat Methods 2007 3 Alanentalo et al., Diabetes 2010

Microbiological purity of isolated islet cells suspension designed to transplantation is one of the basics conditions which determine transplanted graft function and recipient survival. Before islet cells transplantation it is necessary to confirm lack of microorganisms and determine endotoxin level to avoid exceeding acceptable value. The aim of this study was determining microbiological purity of isolated islet cells suspension acquired from pancreases from dead donors, using and comparing LAL microplate test and PTS (Portable Test System) test. Isolated islet cells was designed to clinical transplantation. The presence of microorganisms and endotoxin level was determined in transport liquid (first product) and in final product preparing to transplantation (final product). Studies showed that using GMP collagenase during isolation and purification islet cell suspension on cell separator give endotoxin value on acceptable level (below 5 EU/kg recipient’s body mass). LAL microplate test and PTS test can be used interchangeably because endotoxin level measurement using this two tests gave similar results. Taking in account time of tests performing and costs associated with transplantation procedure, PTS test is quick and reliable test to determine endotoxin level in isolated islet cells suspension designed to transplantation.

Glucose-stimulated insulin secretion is pulsatile and involves coordinated oscillations of the cytoplasmic Ca2+ and cAMP concentrations beneath the beta-cell plasma membrane. Glucose-induced cAMP oscillations are enhanced by elevations of the cytoplasmic Ca2+ concentration ([Ca2+]i) but conditions raising cytoplasmic ATP trigger cAMP elevations without accompanying [Ca2+]i rise. The cAMP oscillations correlate with pulsatile insulin release from single beta-cells and inhibition of adenylyl cyclases suppresses both cAMP oscillations and pulsatile insulin release. The cAMP-activated guanine nucleotide exchange factor Epac2 is an important mediator of the cAMP action in glucose-stimulated insulin secretion. Accordingly, specific activation of Epac restores pulsatile insulin release in cells treated with adenylyl cyclase inhibitors and siRNA-mediated knock-down of Epac2 reduces the secretory response. Protein kinase A (PKA) inhibitors diminish the secretory response to subsequent glucose stimulation, but do not affect already manifested glucose-induced insulin oscillations. The reduced secretory response is due to a dissociation of the initial [Ca2+]i and [cAMP] elevations such that Ca2+ triggers exocytosis before the amplifying cAMP signal is manifested, and can be restored by activation of Epac. These results demonstrate that temporal coordination of Ca2+ and cAMP signals are important for appropriate induction of glucose-induced pulsatile insulin release. Both PKA and Epac2 partake in initiating insulin secretion, but the cAMP-dependence of established pulsatility is mediated by Epac2.

Acoustically Created “Wall-Less” Test Tubes for single B-Cell Biology in Type 2 Diabetes Related Research

NUCEL (Cell and Molecular Therapy Center www.usp.br/nucel) is a translational research center comprising the Cell and Molecular Biology team, dedicated to the molecular basis of cell proliferation control and neoplasia and the Human Pancreatic Islet Unit, which focuses on innovative approaches to improve the Islet Transplantation success rate. By taking a genomic and proteomic approach, we revealed a number of differentially expressed genes and proteins upon comparing human islets in the absence and in the presence of β-cell mitogens and anti-apoptotic agents, and with human insulinoma tumor samples. Functional genomics was used to probe into the role of some of these gene targets in β-cell proliferation, differentiation and insulin production and secretion. We also designed a new method to scale-up β-cell cultures using bioreactors and a practical and rapid assay to assess the β-cell response to glucose by microcalorimetry. In addition, we isolated and characterized three human β-cell lines and dedicated a large amount of effort to islet encapsulation, using a previously described biomaterial (BiodritinTM). This prompted a number of studies both in vitro and in vivo and the generation of NUCEL´s first spin-off company, CellProtect Biotechnology (www.cellprotect.com.br). Modifications of BiodritinTM led us to a new biomaterial/composite (under patenting), which allowed a significant improvement in pre-clinical tests of Diabetes reversal in immunocompetent diabetic mice, opening new avenues for clinical transplantation of encapsulated islets. However, the scarcity of cadaveric pancreas donors and the cumbersome and costly islet isolation and purification procedure, led us to search for alternative sources of insulin-producing cells. Both murine and human embryonic stem cells and adult progenitor cells from different sources were isolated and fully characterized, aiming at their differentiation into β-cells. The results obtained so far with some of these stem cells are very promising, however, still require optimization to generate large amounts of fully differentiated β-cells. Genetic Engineering was also employed to generate induced pluripotent cells (iPSC) without interfering with the cell genome. This work involves a multidisciplinary group of about 60 people, dedicated to face the great challenges posed by Regenerative Medicine and to continuous training of qualified researchers to impact the Cell and Molecular Therapy field.

Jens Høiriis Nielsen, Louise W. Gaarn, Birgitte Søstrup, Amarnadh Nalla, Department of Biomedical Sciences, University of Copenhagen During pregnancy the increased demand for insulin is compensated by an increase in beta cell mass. In rodents this is accomplished by an increased proliferation of the existing beta cells that can be mimicked in vitro by addition of somatolactogenic hormones (PL, PRL, GH) to islets in culture. This is not the case in human islets but we have found that addition of serum from pregnant women can increase human islet cell replication. In order to identify the active factor(s) we have fractionated pregnancy serum by chromatographic methods, sequencing and mass spectrometry. Using proliferation of INS-1E cells as the first screening we have found both inhibitory and stimulatory fractions. So far we have identified peptides derived from HM kininogen in the stimulatory peptide fractions that are currently being investigated. Gene expression profiling by micro array analysis of three human islet preparations cultured in normal and pregnancy serum with and without addition of GH, PL and GLP-1 revealed a large number of up- and down-regulated genes that have been analyzed by the Ingenuity pathway finder program. Enrichment in factors expressed in pancreatic stellate cells was seen in islets cultured in serum from pregnant women. As the mechanism of expansion of the beta cell mass in human pregnancy is not known we are currently investigating the effect of serum from pregnant women on fetal rat pancreas in culture. Increase in Ngn-3 expression was observed in fibroblast-like cells suggesting that the expansion of the beta cell mass in pregnancy may occur both by proliferation of existing beta cells and by neogenesis from progenitor cells.

The role of the Ca2+-activated non-selective cation channel TRPM5 in the electrical activity of the pancreatic beta cell was investigated using a Trpm5-/- mouse. TRPM5 is expressed in beta cells from the pancreatic islet and Trpm5-/- mice display an impaired glucose tolerance caused by a reduced glucose-induced insulin release from pancreatic islets. We could show that a Ca2+-activated non-selective cation current is significantly reduced in beta cells from Trpm5-/- mice. To gain a mechanistic insight we measured dynamics of the intracellular Ca2+ concentration [Ca2+]IC and membrane potential (Vm) in intact islets. Islets from both phenotypes displayed an oscillatory increase in [Ca2+]IC after stimulation with 10mM glucose, but Trpm5-/- islets clearly showed an overall reduced frequency in [Ca2+]IC oscillations. Through a Fourier analysis, we could distinguish 3 types of oscillating patterns in WT islets: either slow or fast oscillations or a mixed pattern consisting of fast oscillations superimposed on slow ones. Trpm5-/- islets completely lacked the fast oscillations and displayed a significantly and highly reduced frequency of fast oscillations in the mixed pattern. The interburst interval in between fast oscillations is significantly shorter and is characterized by a higher velocity of depolarization. Our data indicate that TRPM5 contributes to the slow depolarization towards the threshold potential in the interburst interval during oscillatory changes of [Ca2+]IC and Vm. Deletion of TRPM5 prolongs this interval, resulting in slower oscillations in Vm and in [Ca2+]IC. This leads to a reduced glucose-induced insulin release and consequently to a less efficient glucose clearance in Trpm5-/- mice. As such, TRPM5 might be a novel target in the treatment of type II diabetes.

Pancreatic stem cells residing within the ductile epithelium have been used to generate islet-like clusters in vitro which has partially reverted diabetes Mellitus (DM) in animal models. Islet cells from pancreatic duct ligation (PDL) induced neogenesis have shown the same efficacy as foetal tissue as a model for transplantation of syngeneic tissues. However, understanding the connection between the morphological changes and the genetic induction in the lineage of endocrine pancreas will be a valuable tool in improving beta cell replacement in patients with diabetes. The aim of this study was: i) to describe the morphological changes of the pancreas during the time periods post-pancreatic duct ligation in rats; ii) to determine the expression pattern and variations of insulin, Pdx1, Ngn3, NeuroD, Pax6 and caspase3 genes across the time period post PDL; iii) to compare the temporal increase in beta cells mass to the highest expression of developmental genes across the time period post PDL. Photomicrographs, morphoimmunofluorescent evaluation of PDL pancreas, and immunohistofluorescent analysis of insulin, Pdx1, Ngn3, NeuroD, Pax6 and caspase3 gene expressions were performed using 78 Sprague-Dawley rats of PDL model. The morphological changes were characterized by the appearance of necrotic tissues at 6 hours post-PDL and the proliferations of endocrine tissue were observed at 84 hours through to 120 hours. The morphoimmunofluorescent evaluation showed the highest reactivity of caspase3 and Pdx1 at 6 hours, Ngn3 at 36 hours, pax6 and insulin at 84 hours while NeuroD expression was at 120 hours. The caspase3 expression was earlier at 6 hours while the insulin expression appeared later at 84 hours. The immunofluorescent analysis demonstrated caspase3 expression early at 6 hours while the insulin expression appeared later at 84 hours. This study confirms the connection between morphological changes and gene expression in PDL pancreas with the same chronobiology rhythm observed during endocrine development in normal rats.

Background and aims: Chronic hyperglycemia leads to deterioration of insulin release as well as insulin action on peripheral tissues. However, the mechanism underlying β cell dysfunction resulting from glucose toxicity has not been fully elucidated. Several studies have examined the toxic effect of high glucose on activities of individual mitochondrial proteins in β cells. Nevertheless there have been no studies of the effects of high glucose on the entire mitochondrial proteome. The aim of the present study was to define a set of alterations in mitochondrial protein profiles of pancreatic β cell line using proteomic approaches. Methods: INS1E cells were incubated in the presence of 5.5 and 20 mM glucose for 72hrs and mitochondria were isolated. Mitochondrial protein profiles were obtained by two-dimensional gel (2-DE) electrophoresis and ESI-LC-MS/MS. Results: Separation of mitochondrial proteins by 2 DE showed higher resolution and a high level of reproducibility was obtained among the gels (correlation coefficient, r = 0.78). More than 400 spots were detected on the colloidal Coomassie stained 2D gels; of these protein spots, 75 displayed two fold or more significant changes (p < 0.05) in relative abundance in the presence of 20 mM glucose compared to the controls. Thirty-three protein spots appear only on the control mitochondrial map. 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. Protein identification revealed contamination of the mitochondrial fraction with proteins from other organelles. These differentially expressed proteins includes proinsulin, calreticulin, Pdia6, PKC substrate 60.1 kDa protein, Orp150, endoplasmin, Hsc70, heterogeneous nuclear ribonucleoproteins D0 and A2/B1, lamin B1, histones H2B, H3.3 and H4 and elongation factor 1 α-1. Conclusions: 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.

Michal Wszola, MD,PhD1, Andrzej Berman, MD1, Michal Fabisiak, PhD2, Rafal Kieszek, MD1, Piotr Domagala, MD,PhD1, Magdalena Zmudzka, PhD2, Marek Sabat, MD,PhD4, Agnieszka Perkowska-Ptasinska, MD,PhD3, Krystian Pawelec, MD,PhD1, Lukasz Kownacki, MD,PhD5, Krzysztof Ostrowski, MD,PhD1, Agnieszka Kownacka, MD,PhD5, Waldemar Klucinski, prof.2, Olgierd Rowinski, prof.5, Artur Kwiatkowski, prof.1 and Andrzej Chmura, prof.1. 1Department of General and Transplantation Surgery, Warsaw Medical University, Warsaw, Poland; 2Department of Veterinary Medicine, University of Life Science, Warsaw, Poland; 3Department of Nephrology and Transplantation Medicine, Warsaw Medical University, Warsaw, Poland; 4Department of Immunology and Internal Diseases, Warsaw Medical University, Warsaw, Poland and 5Department of Radiology, Warsaw Medical University, Warsaw, Poland. Islets transplantation have become standard treatments of patients with diabetic complications but long-term results are still unsatisfactory.Gastric submucosa might be an alternative site for transplantation.The aim of this study was to assess the possibility of endoscopic islets transplantation into the gastric submucosa.Materials and methods:20Landrace pigs weighing 19-24kg were obtained for the study.7 were controls(C-group)and 13 were Transplantation group(TX group).In both groups diabetes was induced by streptozotocine(stz)infusion at 200 mg/kg.At 7 days post stz infusion pigs underwent endoscopy. Immunosuppression consisted of tacrolimus,sirolimus.At 7 days post transplantation, control gastroscopy was performed to assess the gastric mucosa and to take biopsies for histopathology.10 to30 days after eGSM-ITx Magnetic Resonance(MRI)examination was performed.Stomach and pancreas were obtained at autopsy for histopathology.For 10 days after diabetes induction(up to three days after eGSM-ITx)in both groups,insulin was given to reach glycemia between 150-200mg/dl,after that period insulin was given only when glycemia exceeded 600 mg/dl.Results:There were no differences in insulin requirement and glycemia up to the day of eGSM-ITx between the groups.Tx-group received a mean of 6000IEQ/kg. Mean glicemia in the day of Tx was 445 in C-group and 470 in Tx group (p=NS). At 1,3,7,21 and 30 days post-transplantation glicemia in C-group was:452,555, 600,586,573 vs. 215, 278, 220, 213, 123 in Tx-group (p<0.05). Tx-group animals had a significantly lower insulin requirement and significantly lower mean glycemia since the first day post transplantation.This trend was observed till the end of study at 1 month. There were no signs of perforation,ulceration or bleeding after the eGSM-ITx on gastroscopy and histopathological examination.In MRI scans unspecific thickening of gastric wall was observed at sites of islet deposition. Histopathology reviled insulin-producing cells in gastric submucosa in the places of islets injections. Conclusion:Endoscopic islets transplantation into gastric submucosa is feasible and a safe procedure in an experimental setting.Its potential for clinical application needs further studies.

Many herbal medicines have been recommended for the treatment of diabetes. In the present study, an ethanol extract of the olive leaves was investigated for effects on insulin release from the pancreatic beta cells in streptozotocin-induced diabetic rats. Blood samples were drawn from the retro-orbital sinus before and 1.5, 3 and 5 h after administration of the extract. Serum glucose levels were determined by the glucose oxidase method. To determine the insulin releasing activity, after extract treatment the animals were anaesthetized by diethyl ether, the pancreas was excised, fixed in 10% formaldehyde and embedded in paraffin for sectioning. Pancreatic sections of 5 μm were processed for examination of insulin-releasing activity using an immunocytochemistry kit. The results showed that administration of the ethanol extract exhibited a significant reduction in serum glucose. Administration of streptozotocin decreased the number of beta cells with insulin secretory activity in comparison with intact rats, but treatment with the olive leaves increased significantly the activity of the beta cells in comparison with the diabetic control rats. The extract decreased serum glucose in streptozotocin-induced diabetic rats and increased insulin release from the beta cells of the pancreas.

Type 2 diabetes (T2D) is a multifactorial syndrome, with genetic and enviromental factors causing a progressive beta cell dysfunction. The molecular alterations affecting T2D islets are not fully understood. We performed microarray analysis, followed by quantitative PCR of a few selected genes, of isolated islets from 6 T2D (age: 71±9 yrs; gender: 3M/3F; BMI: 26.0±2.2 Kg/m2) and 7 non-diabetic (ND, age: 58±17 yrs; gender: 4M/3F; BMI: 24.8±2.5 Kg/m2) subjects. RNA was hybridized on Affymetrix chips (HG U133A). After quality control by affy and affyPLM, gene expression intensity values were normalized by Robust Multi-array Average (RMA), whereas differential expression was assessed by limma. Functional studies were also performed with isolated islets. When T2D islets were compared to ND, the expression of 1345 probe sets resulted (p<0.01 and fold change of <0.5 and >2.0) different; of these, 59 were up-regulated and 1286 down-regulated. Overall, they identified 1230 genes, related to several beta-cell features. By using Gene Ontology and KEGG databases, we observed that those genes influenced 21 processes and 13 pathways, respectively. All the differences were confirmed by Gene Set Enrichment Analysis, which showed that, of the 1419 gene sets analyzed, 195 were positively and 42 negatively enriched in T2D samples. In all the analyses a reduction of the expression for genes involved in oxidative phosphorylation and citric acid cycle was observed. Accordingly, by qPCR a significant (p<0.02) decrease of succinate dehydrogenase (SDH) subunit B expression was detected. When ND islets were exposed to methyl malonic acid, an inhibitor of SDH activity, a reduction of glucose-stimulated insulin release was observed, that was accompanied by an alteration of the ADP/ATP ratio. In conclusion, type 2 diabetic islets show many alterations of transcriptome, including changes of genes involved in mitochondrial ATP production; it remains to understand which alterations are cause or consequence of the diabetic condition.

Polyamines (PA, in particular putrescine, spermidine and spermine) are ubiquitous chemical entities that play an important role in cell function and turnover, as well as in the synthesis of proteins and nucleic acids. Aim of the study was to assess whether PA may be of importance in affecting beta-cells in non-diabetic subjects and type 2 diabetic individuals. Pancreatic tissue obtained from 17 type 2 diabetic subjects (T2DM; age: 67±9 years; M/F:11/6; BMI: 29±5 kg/m2) and 15 non-diabetic controls (ND; age: 62±15 years; M/F:11/4; BMI: 26±2 kg/m2) was studied. The presence of PA in beta-cells was assessed on electron microscopy by immunogold technique. Gene expression studies were performed with isolated islets or beta-cell enriched samples prepared by laser capture microdissection. Islets insulin secretion was determined in response to acute glucose stimulation after 24 h incubation with or without 2-difluoromethylornithine (DFMO, an inhibitor of ornithine decarboxylase I, 5 mM), putrescine (50 μM) and spermidine (50 μM), alone or in combination. Putrescine and spermine were detected in beta-cells from ND and T2DM by electron microscopy. Localization was mainly in the cytoplasm for putrescine and in both the cytoplasm and the nucleus for spermine. Microarray analyses showed that ornithine decarboxylase I (key enzyme in PA synthesis) was lower in beta-cells (p<0.001) and islets (p=0.03) from T2DM. Data were confirmed by qPCR studies, which detected also a lower expression of arginase II in diabetic samples. DFMO caused a decrease (p<0.05) of glucose-stimulated insulin release (-32 ± 9.5%); the same was observed in presence of spermidine (-48 ± 32%, p=0.05). Putrescine prevented the inhibitory effect of spermidine. This study shows that 1) PA are present in human beta-cells; 2) PA biosynthesis pathway may be different in type 2 diabetic beta-cells; and 3) PA may have a role in insulin secretion.

The aim of this study was to investigate whether functional TRPV1 channels exist in pancreatic -cells. The effect of capsaicin, a known activator of TRPV1, was studied by microfluorometry in single living -cells. Capsaicin increased [Ca2+]i in a concentration-dependent manner. In nominally Ca2+ free buffer there was no capsaicin-induced [Ca2+]i increase, although carbachol gave a [Ca2+]i increase. Capsazepine completely blocked the capsaicin-induced [Ca2+]i increase. Also the effect of temperature was studied. In the range of 43-49 °C [Ca2+]i was increased in 60% of the cells, whereas temperatures < 42 °C did not increase [Ca2+]i . When Ca2+ was omitted from the extracellular environment, the [Ca2+]i increase as response to heat was decreased. This demonstrated that the [Ca2+]i increase partially was due to Ca2+ entry. In the same experiment, carbachol increased the [Ca2+]i and confirmed that intracellular Ca2+ stores were not emptied. No difference in the heat-induced [Ca2+]i increase was seen after and before administration of carbachol. Ruthenium red reduced the heat-induced [Ca2+]i increase in 3/7 cells. We conclude that TRPV1 channels exist in INS-1E cells and are functional.