Beta-cell Volume Research Articles

Pathological evaluation of the pathogenesis of diabetes mellitus and diabetic peripheral neuropathy.

Currently, there are more than 10 million patients with diabetes mellitus in Japan. Therefore, the need to explore the pathogenesis of diabetes and the complications leading to its cure is becoming increasingly urgent. Pathological examination of pancreatic tissues from patients with type 2 diabetes reveals a decrease in the volume of beta cells because of a combination of various stresses. In human type 2 diabetes, islet amyloid deposition is a unique pathological change characterized by proinflammatory macrophage (M1) infiltration into the islets. The pathological changes in the pancreas with islet amyloid were different according to clinical factors, which suggests that type 2 diabetes can be further subclassified based on islet pathology. On the other hand, diabetic peripheral neuropathy is the most frequent diabetic complication. In early diabetic peripheral neuropathy, M1 infiltration in the sciatic nerve evokes oxidative stress or attenuates retrograde axonal transport, as clearly demonstrated by in vitro live imaging. Furthermore, islet parasympathetic nerve density and beta cell volume were inversely correlated in type 2 diabetic Goto-Kakizaki rats, suggesting that diabetic peripheral neuropathy itself may contribute to the decrease in beta cell volume. These findings suggest that the pathogenesis of diabetes mellitus and diabetic peripheral neuropathy may be interrelated.

Stevia aquatic extract protects the pancreas from streptozocin (STZ) induced damage: A stereological study

In recent years, among antidiabetic medicinal herbs, Stevia received a lot of attention due to its diverse therapeutic applications. Despite extensive reports on the effects of Stevia on the pancreas, its molecular mechanism is not clear yet. In this study, we investigated the protective and preventive effects of oral extracts of Stevia on the pancreas through the stereological methods in streptozocin (STZ) induced diabetic rats. Thus, 66 adult male rats were assigned to six groups (n = 11) viz., healthy control, healthy Stevia (400 mg /kg), diabetic-control, diabetic-metformin (500 mg/kg), diabetic-Stevia and pre-Stevia-diabetic group. Treatment with Stevia significantly reduced fasting blood sugar (FBS) and MDA compared to the diabetic control group (P <0.05). The results indicated that the weight and the volume of the pancreas increased significantly in all our treated groups compared to the diabetic one (P <0.05). The volume density of the pancreatic islands and the number of beta cells increased in healthy and diabetic groups treated with Stevia (P <0.05). However, the pre-treated diabetic rats with Stevia did not show significant preventive effects on the volume and number of beta cells as well as the volume of islets against destructive effects of STZ. More specifically, our results confirmed the protective effects of Stevia through restoring pancreatic cells and repairing the stereological damage induced by STZ.

Increased NKX6.1 expression and decreased ARX expression in alpha cells accompany reduced beta-cell volume in human subjects

Pancreatic islet cells have plasticity, such as the abilities to dedifferentiate and transdifferentiate. Islet cell conversion to other characteristic cell is largely determined by transcription factors, but significance of expression patterns of these transcription factors in human islet cells remained unclear. Here, we present the NKX6.1-positive ratio of glucagon-positive cells (NKX6.1+/GCG+ ratio) and the ARX-negative ratio of glucagon-positive cells (ARX−/GCG+ ratio) in 34 patients who were not administered antidiabetic agents. Both of NKX6.1+/GCG+ ratio and ARX−/GCG+ ratio negatively associated with relative beta cell area. And these ratios did not have significant correlation with other parameters including age, body mass index, hemoglobin A1c, fasting plasma glucose level or relative alpha-cell area. Our data demonstrate that these expression ratios of transcription factors in glucagon-positive cells closely correlate with the reduction of beta-cell volume in human pancreas.

3D quantification of changes in pancreatic islets in mouse models of diabetes type I and II.

ABSTRACTDiabetes is characterized by rising levels of blood glucose and is often associated with a progressive loss of insulin-producing beta cells. Recent studies have demonstrated that it is possible to regenerate new beta cells through proliferation of existing beta cells or trans-differentiation of other cell types into beta cells, raising hope that diabetes can be cured through restoration of functional beta cell mass. Efficient quantification of beta cell mass and islet characteristics is needed to enhance drug discovery for diabetes. Here, we report a 3D quantitative imaging platform for unbiased evaluation of changes in islets in mouse models of type I and II diabetes. To determine whether the method can detect pharmacologically induced changes in beta cell volume, mice were treated for 14 days with either vehicle or the insulin receptor antagonist S961 (2.4 nmol/day) using osmotic minipumps. Mice treated with S961 displayed increased blood glucose and insulin levels. Light-sheet imaging of insulin and Ki67 (also known as Mki67)-immunostained pancreata revealed a 43% increase in beta cell volume and 21% increase in islet number. S961 treatment resulted in an increase in islets positive for the cell proliferation marker Ki67, suggesting that proliferation of existing beta cells underlies the expansion of total beta cell volume. Using light-sheet imaging of a non-obese diabetic mouse model of type I diabetes, we also characterized the infiltration of CD45 (also known as PTPRC)-labeled leukocytes in islets. At 14 weeks, 40% of the small islets, but more than 80% of large islets, showed leukocyte infiltration. These results demonstrate how quantitative light-sheet imaging can capture changes in individual islets to help pharmacological research in diabetes.

SUN-654 Dynamic and Regional Variation of Pancreatic Innervation in Diabetes

Background: The pancreas is a highly heterogeneous organ, with regional anatomical, developmental and functional differences. The endocrine pancreas is densely innervated, and neural signals play a significant role in glucose regulation by modulating pancreatic hormone release. However, relatively little is known about the anatomical relationships between islets and nerves across the whole pancreas. Since thin filamentous structures, such as nerves, are difficult to quantify and trace over large volumes using thin section histology, there is a need for high resolution imaging and rendering of intact pancreatic tissue in 3D. Aim: To use optical clearing, whole organ imaging, and 3D rendering to quantify islets and innervation across the whole pancreas in healthy mice, in two mouse models of diabetes, and in pancreatic samples from nondiabetic and diabetic human donors. Methods: Whole-mount staining and clearing was performed using iDISCO+ to quantify innervation, defined by the neuronal marker NF200, and beta cells in pancreata from C57Bl/6 mice, non-obese diabetic (NOD) mice, streptozotocin (STZ)-treated mice, and in pancreatic samples from nondiabetic and diabetic human donors. Z-stacked optical sections were acquired with an Ultramicroscope II at 4x or 12x magnification. Imaris was used to create digital surfaces covering the NF200+ innervation and islets to automatically determine innervation density and islet/nerve interactions. Results: Beta cell volumes were 1-4% in the human pancreas, and 1-2% in the healthy mouse pancreas, with regional variations in islet volume and insulin intensity. There were also significant differences in islet biology between the diabetes models. Innervation of the endocrine pancreas was significantly enriched compared to the surrounding exocrine pancreas, with regional variation. Islets were closely associated with pancreatic innervation and decreased in size with increasing distance from nerves in both mouse and human pancreatic tissue. Innervated islets were relatively preserved in models of diabetes. Finally, islet innervation and expression of neural markers were higher in human samples from diabetic patients and in mouse models of diabetes, with temporal and regional differences. Conclusions: 3D imaging and unbiased analysis across the whole pancreas provides comprehensive measurement of pancreatic nerve volumes and distribution. It allows detailed analysis of the anatomical relationship between nerves and islets, and reveals a close association that is maintained across species. The relative enrichment of innervated islets in diabetes and dynamic changes in islet innervation during the development of diabetes suggest further work is needed to examine the role of pancreatic nerves in preserving and protecting beta cells.

2127-P: Reduced Islet Parasympathetic Nerve Density Correlates with Decrease in Beta-Cell Volume of Islet in Lean Type 2 Diabetic Goto-Kakizaki Rat

Pancreatic branch of vagus nerve (pvn) signaling plays a pivotal role in the maintenance of islet β cell volume density (Vβ). In type 2 diabetes (T2D), small fibers in the epidermis is decreased by diabetic insult. It is not still uncovered yet, however, that the reduction of terminal pvn fibers is associated with Vβ in T2DM. Herein, we explored the association between islet pvn fiber density and Vβ. We recruited non-obese spontaneous T2D Goto-Kakizaki (GK) rats and control Wistar rats (W) at 5 weeks of age. GK was divided into DPP-4 inhibitor (DPi)-treated group (GTe) (10mg/kg teneligliptine), SGLT2 inhibitor (SGi)-treated group (GCa) (10mg/kg canagliflozin) and both treated group (GTeCa). During experimental period, 2g/kg oral glucose tolerance test (OGTT) and glucose-stimulated insulin secretion test (GSIS) was performed. At end, pancreata were underwent for the pathological evaluation. OGTT in GK was attenuated (p&amp;lt;0.01 vs. W). Although DPi and SGi in each significantly improved OGTT compared to GK, the effect was the most robust in GTeCa. Low GSIS in GK (p&amp;lt;0.01 vs. W) was significantly improved in GTe and GCaTe (p&amp;lt;0.05 and p&amp;lt;0.01 vs. GK, respectively), although it was comparable between GK and GCa. Pathological evaluation disclosed a significant decrease in Vβ in GK (p&amp;lt;0.01 vs. W). Vβ was the most well-preserved in GCaTe (p&amp;lt;0.05 vs. GTe), followed by GTe (p&amp;lt;0.05 vs. GK), while it was comparable between GK and GCa. The density of pvn fibers labeled by the antibody for vesicular acetylcholine transporter (VachT) were significantly decreased in the islet of GK (p&amp;lt;0.01 vs. W). It was significantly improved in GCa, GTe and GCaTe compared to GK (p&amp;lt;0.05 vs. GCa and GTe; p&amp;lt;0.01 GCaTe, respectively). pvn fiber density in the islets significantly correlated with Vβ (r=0.53, p&amp;lt;0.01). Our results shed light on the association between terminal fiber density of pvn in the islet and Vβ, and the maintenance of the fibers may be beneficial for the prevention from Vβ loss in T2D. Disclosure H. Mizukami: None. D. Guo: None. K. Takahashi: None. S. Osonoi: None. K. Kudo: None. S. Yagihashi: None.

Possible Prevention of Diabetes with a Gluten-Free Diet.

Gluten seems a potentially important determinant in type 1 diabetes (T1D) and type 2 diabetes (T2D). Intake of gluten, a major component of wheat, rye, and barley, affects the microbiota and increases the intestinal permeability. Moreover, studies have demonstrated that gluten peptides, after crossing the intestinal barrier, lead to a more inflammatory milieu. Gluten peptides enter the pancreas where they affect the morphology and might induce beta-cell stress by enhancing glucose- and palmitate-stimulated insulin secretion. Interestingly, animal studies and a human study have demonstrated that a gluten-free (GF) diet during pregnancy reduces the risk of T1D. Evidence regarding the role of a GF diet in T2D is less clear. Some studies have linked intake of a GF diet to reduced obesity and T2D and suggested a role in reducing leptin- and insulin-resistance and increasing beta-cell volume. The current knowledge indicates that gluten, among many environmental factors, may be an aetiopathogenic factors for development of T1D and T2D. However, human intervention trials are needed to confirm this and the proposed mechanisms.

Crucial role of Reg I from acinar-like cell cluster touching with islets (ATLANTIS) on mitogenesis of beta cells in EMC virus-induced diabetic mice

Recently, we reported the presence of distinct cell clusters named acinar-like cell clusters touching Langerhans islets with thin interstitial surrounding (ATLANTIS) in human pancreas. A morphological study in humans demonstrated that ATLANTIS and islet cell clusters are found together in the microenvironment enclosed by a common basement membrane, and ATLANTIS releases vesicles containing Regenerating gene protein (REG Iα) to islet cell clusters.We examined 1) the presence or absence of ATLANTIS in homozygous Reg I (mouse homologue of human REG Iα) deficient (Reg I−/-) and wild-type mice, and 2) the possible role of ATLANTIS in the regeneration of beta cell clusters after encephalomyocarditis (EMC) virus (D-variant) infection in Reg I−/- and wild-type mice.ATLANTIS was found in both wild-type and Reg I−/- mice. In both groups, mean blood glucose increased transiently to greater than 14.0 mmol/L at 5 days after EMC virus infection and recovered to baseline at 12 days. At 12 days after EMC virus infection, lower BrdU labeling indices were observed in islet beta cells of Reg I−/- mice compared to wild-type mice. Beta cell volume 12 days after EMC virus infection in Reg I−/- mice did not differ from that of wild-type mice.These results suggest that Reg I, which is released from ATLANTIS to islet beta cell clusters, has a crucial role in beta cell regeneration in EMC virus-induced diabetes. The presence of mechanism(s) other than that mediated by Reg I in beta cell restoration after destruction by EMC virus was also suggested.

Synergistic Effects of Long-Term Combination Therapy of DPP-4 Inhibitor and SGLT2 Inhibitor on the Preservation of Beta-Cell Volume in Rats with Type 2 Diabetes

One of the main pathogenetic features of type 2 diabetes (T2D) is reduction of β cell volume (Vβ). Several antidiabetic oral agents were shown to improve Vβ in experimental studies. While DPP-4 inhibitor (DPi) ameliorated Vβ possibly via the action of GLP-1, recent studies reported that SGLT2 inhibitor (SGi) may facilitate GLP-1 secretion from L cells. These findings raised a hypothesis that the combination therapy (CT) of DPi and SGi may potentiate the effects of DPi on Vβ. To explore this possibility, we recruited non-obese spontaneous T2D Goto-Kakizaki (GK) rats and control Wistar rats (W) at 5 weeks of age. GK was divided into DPi treated group (GTe) (10mg/kg teneligliptine), SGi-treated group (GCa) (10mg/kg canagliflozin) and CT group (GTeCa). During experimental period, diabetic state was evaluated by glycated Hb (gHb), 2g/kg oral glucose tolerance test (OGTT), glucose-stimulated insulin secretion test (GSIS) and serum active GLP-1. At end, pancreata were underwent for the pathological evaluation of each endocrine cell volume by tetra-immnohistochemistry. Over 24 weeks of treatment, GK showed higher gHb and deteriorated OGTT (p&amp;lt;0.01 vs. W). Although DPi and SGi in each significantly improved gHb and OGTT compared to GK, the effect was the most marked in GTeCa. Of note, active GLP-1 was the highest in GCaTe among all groups (p&amp;lt;0.vs. GTe), while the level in GTe was higher than in W, GK, and GCa (p&amp;lt;0.01). GK showed attenuated GSIS (p&amp;lt;0.01 vs. W). It was significantly improved in GTe and GCaTe compared to GK (p&amp;lt;0.vs. GTe, p&amp;lt;0.01 vs. GCaTe), although it was comparable between GK and GCa. Pathological evaluation disclosed a significant decrease in Vβ in GK (p&amp;lt;0.01 vs. W). Vβ was the most well-preserved in GCaTe (p&amp;lt;0.vs. GTe), followed by GTe (p&amp;lt;0.vs. GK), while it was comparable between GK and GCa. Our findings suggest that CT of DPi and SGi is more effective for β cell function and pathology in T2D than the monotherapy of DPi or SGi. Disclosure H. Mizukami: Research Support; Self; Sanwa Kagaku Kenkyusho Co., Ltd., Mitsubishi Tanabe Pharma Corporation. D. Guo: None. K. Takahashi: None. S. Osonoi: None. S. Ogasawara: None. W. Inaba: None. S. Yagihashi: None.

Vegf-A mRNA transfection as a novel approach to improve mouse and human islet graft revascularisation.

The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment. Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice. At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean ± SEM: 0.118 ± 0.01 [n = 3] in Vegf-A mRNA transfected group (VEGF) vs 0.056 ± 0.01 [n = 3] in no RNA [p < 0.05] vs 0.063 ± 0.02 [n = 4] in Gfp mRNA transfected group (GFP) [p < 0.05]); EndoC-bH3 grafts (2.85- and 2.48-fold. respectively) (0.085 ± 0.02 [n = 4] in VEGF vs 0.030 ± 0.004 [n = 4] in no RNA [p < 0.05] vs 0.034 ± 0.01 [n = 5] in GFP [p < 0.05]); and human islet grafts (3.17- and 3.80-fold, respectively) (0.048 ± 0.013 [n = 3] in VEGF vs 0.015 ± 0.0051 [n = 4] in no RNA [p < 0.01] vs 0.013 ± 0.0046 [n = 4] in GFP [p < 0.01]). At day 30 post transplantation, human islet grafts maintained a vascularisation benefit (1.70- and 1.82-fold, respectively) (0.049 ± 0.0042 [n = 8] in VEGF vs 0.029 ± 0.0052 [n = 5] in no RNA [p < 0.05] vs 0.027 ± 0.0056 [n = 4] in GFP [p < 0.05]) and a higher beta cell volume (1.64- and 2.26-fold, respectively) (0.0292 ± 0.0032 μl [n = 7] in VEGF vs 0.0178 ± 0.0021 μl [n = 5] in no RNA [p < 0.01] vs 0.0129 ± 0.0012 μl [n = 4] in GFP [p < 0.001]). Vegf-A mRNA transfection before transplantation provides a promising and safe strategy to improve engraftment of islets and other cell-based implants.

Abstract 441: Zebrafish Larvae as a Model System for High-throughput, Image-based Genetic Screens in Dyslipidemia and Atherosclerosis

Background: Genome-wide association (GWAS) have identified hundreds of loci that are robustly associated with the risk of cardiometabolic risk factors and diseases. With few exceptions, the causal genes through which these loci influence disease risk remain uncharacterized. Since murine model systems are not suitable for systematic characterization of candidate genes in hundreds of loci, novel in vivo model systems are desirable. Methods: My group has developed and validated zebrafish model systems that make optimal use of: 1) the zebrafish’ well-annotated genome, with orthologues of at least 71.4% of human genes; 2) recent developments in multiplex CRISPR-Cas9-based mutagenesis; 3) advances in automated positioning of non-embedded zebrafish larvae; 4) fluorescent transgenes and dyes; and 5) custom-written image-quantification pipelines. Results: Five days of overfeeding, dietary cholesterol supplementation and/or exposure to glucose induce atherogenic and insulin resistant phenotypes (higher/more whole-body LDL cholesterol (LDLc) and triglyceride levels; vascular foam cell formation and inflammation; beta-cell number and volume; subcutaneous and hepatic accumulation of fat) that can largely be prevented by concomitant treatment with lipid-lowering or diabetes medication (N&gt;4000). In line with recent results in humans, treatment with atorvastatin and ezetimibe results in higher whole-body glucose levels (N~835). Each additional mutated allele in zebrafish orthologues of APOE results in higher LDLc levels, while mutations in LDLR affect vascular infiltration by lipids and macrophages (N~330). Ongoing characterization of 37 prioritized genes in GWAS-identified loci for triglyceride levels identified genes that influence triglycerides, LDLc, and/or vascular inflammatory and atherogenic traits, while characterizing prioritized genes in loci associated with heart rate variability yielded genes that influence cardiac rate and rhythm. Conclusions: Systematic characterization of candidate genes for cardiometabolic traits in zebrafish model systems will help increase our understanding of human disease and identify novel targets that can be translated into efficient therapeutics.

Sequential Coating of Insulin Secreting Beta Cells within Multilayers of Polysaccharide Nanogels

Pancreatic islet transplantation has emerged as a promising treatment for type-1 diabetes (T1D); however, its clinical application is still limited by the life-long use of immunosuppressive drugs, insufficient number of islets to achieve normoglycemia, and large transplantation volume. This paper reports a unique approach for nanothin coating of insulin secreting beta cell aggregates. The coating is based on hydrophobic and covalent interactions between natural acrylate modified cholesterol bearing pullulan (CHPOA) nanogels and MIN6 beta cell aggregates. Beta cell aggregates are prepared as spheroids through hanging drop method, which is optimized with respect to hanging drop volume and initial number of beta cells. These aggregates, defined as pseudoislets, are coated with sequential layers of nanogels and are evaluated as viable and functional for insulin secretion. Coating experiments are carried out using physiologically compatible medium, where pseudoislets are not brought in contact with toxic prepolymer solutions used in existing approaches. This study offers new opportunities through coating of islets with advanced functional materials under completely physiological conditions for clinical translation of cell transplantation technology. The technique developed here will establish a new paradigm for creating tolerable grafts for other chronic diseases such as anemia, cancer, central nervous system (CNS) diseases.

Gluten-free diet during pregnancy alleviates signs of diabetes and celiac disease in NOD mouse offspring.

Gluten-free (GF) diet during pregnancy ameliorates autoimmune diabetes in nonobese diabetic (NOD) mouse offspring. Due to comorbidity of celiac disease in type 1 diabetes, we hypothesized that GF diet in utero alleviates the humoral and histopathological signs of celiac disease in NOD mice. We aimed to establish the mechanisms behind the diabetes-protective effect of GF diet in utero. Breeding pairs of NOD mice were fed a GF or gluten-containing standard (STD) diet until parturition. The offspring were nursed by mothers on STD diet and continued on this diet until ages 4 and 13weeks. Analyses of serum antitissue transglutaminase (anti-tTG) intestine and islet histology, islet transglutaminase (TG) activity, and cytokine expression in T cells from lymphoid organs were performed. GF versus STD diet in utero led to reduced serum anti-tTG titre and increased villus-to-crypt ratio at both ages. Insulitis along with systemic and local inflammation were decreased, but islet TG activity was unchanged in 13-week-old GF mice. These mice had unchanged beta-cell volumes, but increased islet numbers throughout the prediabetic period. Collectively, GF diet administered during pregnancy improves signs of celiac disease and autoimmune diabetes in the offspring. The diabetes-ameliorative effect of GF diet in utero is followed by dampening of inflammation, unchanged beta-cell volume, but increased islet numbers.

Subchronic exposure to acrylamide leads to pancreatic islet remodeling determined by alpha cell expansion and beta cell mass reduction in adult rats

Acrylamide (AA) is a toxic substance, used to synthesize polymers for industrial and laboratory processes. Also, AA is a food contaminant formed during the high temperature preparation of carbohydrate-rich food. The main subject of this study was to examine effects of subchronic AA treatment on the islets of Langerhans of adult rats. Adult male Wistar rats were orally treated with 25 or 50 mg/kg bw of AA for 3 weeks. Qualitative and quantitative immunohistochemical evaluation of glucagon and insulin expression and stereological analyses of pancreatic alpha and beta cells were performed. Serum insulin and glucose levels were measured. Analysis of glucagon-immunostained sections revealed a dose-dependent increase of intensity of glucagon immunopositive signal, alpha cell surface and numerical densities, volume density of alpha cell nuclei and nucleocytoplasmic ratio in AA-treated groups compared to the control. In insulin-immunolabeled pancreatic sections in AA-treated animals was observed decrease of intensity of insulin immunopositive signal, beta cell surface, numerical and volume densities and volume density of beta cell cytoplasm. Serum insulin and glucose concentrations remained unchanged after both AA treatments. The number of islets of Langerhans was not affected by AA treatment. Our results suggest that AA subchronic treatment of adult rats leads to remodeling of islet of Langerhans characterized by alpha cell expansion and beta cell mass reduction.

Semi-automated digital measurement as the method of choice for beta cell mass analysis.

Pancreas injury by partial duct ligation (PDL) activates beta cell differentiation and proliferation in adult mouse pancreas but remains controversial regarding the anticipated increase in beta cell volume. Several reports unable to show beta cell volume augmentation in PDL pancreas used automated digital image analysis software. We hypothesized that fully automatic beta cell morphometry without manual micrograph artifact remediation introduces bias and therefore might be responsible for reported discrepancies and controversy. However, our present results prove that standard digital image processing with automatic thresholding is sufficiently robust albeit less sensitive and less adequate to demonstrate a significant increase in beta cell volume in PDL versus Sham-operated pancreas. We therefore conclude that other confounding factors such as quality of surgery, selection of samples based on relative abundance of the transcription factor Neurogenin 3 (Ngn3) and tissue processing give rise to inter-laboratory inconsistencies in beta cell volume quantification in PDL pancreas.

The Munich MIDY Pig Biobank – A unique resource for studying organ crosstalk in diabetes

ObjectiveThe prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates. MethodsFemale MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics. ResultsMIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples. ConclusionsThe broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension.

Conditional islet hypovascularisation does not preclude beta cell expansion during pregnancy in mice.

Endothelial-endocrine cell interactions and vascular endothelial growth factor (VEGF)-A signalling are deemed essential for maternal islet vascularisation, glucose control and beta cell expansion during mouse pregnancy. The aim of this study was to assess whether pregnancy-associated beta cell expansion was affected under conditions of islet hypovascularisation. Soluble fms-like tyrosine kinase 1 (sFLT1), a VEGF-A decoy receptor, was conditionally overexpressed in maternal mouse beta cells from 1.5 to 14.5days post coitum. Islet vascularisation, glycaemic control, beta cell proliferation, individual beta cell size and total beta cell volume were assessed in both pregnant mice and non-pregnant littermates. Conditional overexpression of sFLT1 in beta cells resulted in islet hypovascularisation and glucose intolerance in both pregnant and non-pregnant mice. In contrast to non-pregnant littermates, glucose intolerance in pregnant mice was transient. sFLT1 overexpression did not affect pregnancy-associated changes in beta cell proliferation, individual beta cell size or total beta cell volume. Reduced intra-islet VEGF-A signalling results in maternal islet hypovascularisation and impaired glycaemic control but does not preclude beta cell expansion during mouse pregnancy.

Sequential intravital imaging reveals in vivo dynamics of pancreatic tissue transplanted under the kidney capsule in mice.

Aims/hypothesisDynamic processes in pancreatic tissue are difficult to study. We aimed to develop an intravital imaging method to longitudinally examine engraftment, vascularisation, expansion and differentiation in mature islets or embryonic pancreases transplanted under the kidney capsule.MethodsIsolated pancreatic islets from adult mice and murine embryonic day (E)12.5 pancreases containing fluorescent biomarkers were transplanted under the kidney capsule of immunodeficient recipient mice. Human islet cells were dispersed, transduced with a lentivirus expressing a fluorescent label and reaggregated before transplantation. Graft-containing kidneys were positioned subcutaneously and an imaging window was fitted into the skin on top of the kidney. Intravital imaging using multiphoton microscopy was performed for up to 2 weeks. Volumes of fluorescently labelled cells were determined as a measure of development and survival.ResultsTransplanted islets and embryonic pancreases showed good engraftment and remained viable. Engraftment and vascularisation could be longitudinally examined in murine and human islet cells. Murine islet beta cell volume was unchanged over time. Transplanted embryonic pancreases increased to up to 6.1 times of their original volume and beta cell volume increased 90 times during 2 weeks.Conclusions/interpretationThis method allows for repeated intravital imaging of grafts containing various sources of pancreatic tissue transplanted under the kidney capsule. Using fluorescent markers, dynamic information concerning engraftment or differentiation can be visualised and measured.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-4049-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Longitudinal three-dimensional visualisation of autoimmune diabetes by functional optical coherence imaging.

It is generally accepted that structural and functional quantitative imaging of individual islets would be beneficial to elucidate the pathogenesis of type 1 diabetes. We here introduce functional optical coherence imaging (FOCI) for fast, label-free monitoring of beta cell destruction and associated alterations of islet vascularisation. NOD mouse and human islets transplanted into the anterior chamber of the eye (ACE) were imaged with FOCI, in which the optical contrast of FOCI is based on intrinsic variations of the index of refraction resulting in a faster tomographic acquisition. In addition, the phase sensitivity allows simultaneous label-free acquisition of vascularisation. We demonstrate that FOCI allows longitudinal quantification of progressive autoimmune insulitis, including the three-dimensional quantification of beta cell volume, inflammation and vascularisation. The substantially increased backscattering of islets is dominated by the insulin-zinc nanocrystals in the beta cell granules. This translates into a high specificity for the functional beta cell volume of islets. Applying FOCI to a spontaneous mouse model of type 1 diabetes, we quantify the modifications of the pancreatic microvasculature accompanying the progression of diabetes and reveal a strong correlation between increasing insulitis and density of the vascular network of the islet. FOCI provides a novel imaging technique for investigating functional and structural diabetes-induced alterations of the islets. The label-free detection of beta cell volume and infiltration together with vascularisation offers a unique extension to study ACE-transplanted human islets. These results are contributing to a deeper understanding of human islet transplant rejection and label-free in vivo monitoring of drug efficacy.

Delta cell death in the islet of Langerhans and the progression from normal glucose tolerance to type 2 diabetes in non-human primates (baboon, Papio hamadryas).

The cellular composition of the islet of Langerhans is essential to ensure its physiological function. Morphophysiological islet abnormalities are present in type 2 diabetes but the relationship between fasting plasma glucose (FPG) and islet cell composition, particularly the role of delta cells, is unknown. We explored these questions in pancreases from baboons (Papio hamadryas) with FPG ranging from normal to type 2 diabetic values. We measured the volumes of alpha, beta and delta cells and amyloid in pancreatic islets of 40 baboons (Group 1 [G1]: FPG < 4.44 mmol/l [n = 10]; G2: FPG = 4.44-5.26 mmol/l [n = 9]; G3: FPG = 5.27-6.94 mmol/l [n = 9]; G4: FPG > 6.94 mmol/l [n = 12]) and correlated islet composition with metabolic and hormonal variables. We also performed confocal microscopy including TUNEL, caspase-3, and anti-caspase cleavage product of cytokeratin 18 (M30) immunostaining, electron microscopy, and immuno-electron microscopy with anti-somatostatin antibodies in baboon pancreases. Amyloidosis preceded the decrease in beta cell volume. Alpha cell volume increased ∼ 50% in G3 and G4 (p < 0.05), while delta cell volume decreased in these groups by 31% and 39%, respectively (p < 0.05). In G4, glucagon levels were higher, while insulin and HOMA index of beta cell function were lower than in the other groups. Immunostaining of G4 pancreatic sections with TUNEL, caspase-3 and M30 showed apoptosis of beta and delta cells, which was also confirmed by immuno-electron microscopy with anti-somatostatin antibodies. In diabetic baboons, changes in islet composition correlate with amyloid deposition, with increased alpha cell and decreased beta and delta cell volume and number due to apoptosis. These data argue for an important role of delta cells in type 2 diabetes.