Increased alpha and beta cell mass during mouse pregnancy is not dependent on transdifferentiation

Maternal pancreatic beta-cell mass (BCM) increases during pregnancy to compensate for relative insulin resistance. If BCM expansion is suboptimal, gestational diabetes mellitus can develop. Alpha-cell mass (ACM) also changes during pregnancy, but there is a lack of information about α-cell plasticity in pregnancy and whether α- to β-cell transdifferentiation can occur. To investigate this, we used a mouse model of gestational glucose intolerance induced by feeding low-protein (LP) diet from conception until weaning and compared pregnant female offspring to control diet-fed animals. Control and LP pancreata were collected for immunohistochemical analysis and serum glucagon levels were measured. In order to lineage trace α- to β-cell conversion, we utilized transgenic mice expressing yellow fluorescent protein behind the proglucagon gene promoter (Gcg-Cre/YFP) and collected pancreata for histology at various gestational timepoints. Alpha-cell proliferation increased significantly at gestational day (GD) 9.5 in control pregnancies resulting in an increased ACM at GD18.5, and this was significantly reduced in LP animals. Despite these changes, serum glucagon was higher in LP mice at GD18.5. Pregnant Gcg-Cre/YFP mice showed no increase in the abundance of insulin⁺YFP⁺glucagon^– cells (phenotypic β-cells). A second population of insulin⁺YFP⁺glucagon⁺ cells was identified which also did not alter during pregnancy. However, there was an altered anatomical distribution within islets with fewer insulin⁺YFP⁺glucagon^– cells but more insulin⁺YFP⁺glucagon⁺ cells being present in the islet mantle at GD18.5. These findings demonstrate that dynamic changes in ACM occur during normal pregnancy and were altered in glucose-intolerant pregnancies.     

Enhanced islet expansion by β‐cell proliferation in young diabetes‐prone rats fed a protective diet

Type 1 diabetes is inhibited in diabetes‐prone BioBreeding (BBdp) rats fed a low‐antigen hydrolyzed casein (HC) diet. In cereal‐fed BBdp rats, islet expansion is defective accompanied by a futile upregulation of islet neogenesis without increased islet mass, due to a subtle blockage in islet cell cycle. We hypothesized that islet growth is enhanced before insulitis in HC‐fed young BBdp rats and that islet neogenesis could be stimulated by a trophic factor, islet neogenesis‐associated protein (INGAP). β‐Cell homeostasis was analyzed using immunohistochemistry, morphometry, laser capture microdissection and RT‐PCR in BBdp rats fed HC or cereal diets. β‐cell proliferation in small and medium islets, and the number and area fraction of medium and large islets were increased in HC‐fed animals. In situ islet cell cycle analysis revealed an increased proportion of proliferating S + G2 cells in medium and large islets of 25–45 day HC‐fed rats. Expression of the cell cycle inhibitor, p16^INK4a correlated with islet size and the percentage of p16^INK4a+ β‐cells increased in HC‐fed BBdp rats, likely reflecting an increase in large islet area fraction. In HC‐fed rats, extra‐islet insulin⁺ clusters (EIC), insulin⁺ duct cells, large islet area fraction, and β‐cell mass were increased. Neurogenin‐3 and Pdx‐1, markers of β‐cell progenitors, were increased in EIC of weanling HC‐fed rats. Daily injection of INGAP (30–45 days) increased the number of small islets, total islets, and insulin⁺ cells in small ducts. Thus, in BBdp rats fed a protective HC diet, β‐cell expansion is enhanced through increased β‐cell proliferation and stimulation of islet neogenesis. J. Cell. Physiol. 224: 501–508, 2010. © 2010 Wiley‐Liss, Inc.     

Determination of Optimal Sample Size for Quantification of β-Cell Area,Amyloid Area and β-Cell Apoptosis in Isolated Islets

Culture of isolated rodent islets is widely used in diabetes research to assess different endpoints, including outcomes requiring histochemical staining. As islet yields during isolation are limited, we determined the number of islets required to obtain reliable data by histology. We found that mean values for insulin-positive β-cell area/islet area, thioflavin S-positive amyloid area/islet area and β-cell apoptosis do not vary markedly when more than 30 islets are examined. Measurement variability declines as more islets are quantified, so that the variability of the coefficient of variation (CV) in human islet amyloid polypeptide (hIAPP) transgenic islets for β-cell area/islet area, amyloid area/islet area and β-cell apoptosis are 13.20% ± 1.52%, 10.03% ± 1.76% and 6.78% ± 1.53%, respectively (non-transgenic: 7.65% ± 1.17% β-cell area/islet area and 8.93% ± 1.56% β-cell apoptosis). Increasing the number of islets beyond 30 had marginal effects on the CV. Using 30 islets, 6 hIAPP-transgenic preparations are required to detect treatment effects of 14% for β-cell area/islet area, 30% for amyloid area/islet area and 23% for β-cell apoptosis (non-transgenic: 9% for β-cell area/islet area and 45% for β-cell apoptosis). This information will be of value in the design of studies using isolated islets to examine β cells and islet amyloid.     

Human Islets Have Fewer Blood Vessels than Mouse Islets and the Density of Islet Vascular Structures Is Increased in Type 2 Diabetes

Human and rodent islets differ substantially in several features, including architecture, cell composition, gene expression and some aspects of insulin secretion. Mouse pancreatic islets are highly vascularized with interactions between islet endothelial and endocrine cells being important for islet cell differentiation and function. To determine whether human islets have a similar high degree of vascularization and whether this is altered with diabetes, we examined the vascularization of islets from normal human subjects, subjects with type 2 diabetes (T2D), and normal mice. Using an integrated morphometry approach to quantify intra-islet capillary density in human and mouse pancreatic sections, we found that human islets have five-fold fewer vessels per islet area than mouse islets. Islets in pancreatic sections from T2D subjects showed capillary thickening, some capillary fragmentation and had increased vessel density as compared with non-diabetic controls. These changes in islet vasculature in T2D islets appeared to be associated with amyloid deposition, which was noted in islets from 8/9 T2D subjects (and occupied 14% ± 4% of islet area), especially around the intra-islet capillaries. The physiological implications of the differences in the angioarchitecture of mouse and human islets are not known. Islet vascular changes in T2D may exacerbate β cell/islet dysfunction and β cell loss.     

Isolation,Characterization and Potential Role in Beta Cell-Endothelium Cross-Talk of Extracellular Vesicles Released from Human Pancreatic Islets

The cross-talk between beta cells and endothelium plays a key role in islet physiopathology and in the revascularization process after islet transplantation. However, the molecular mechanisms involved in this cross-talk are not fully elucidated. Extracellular vesicles (EVs) are secreted membrane nanoparticles involved in inter-cellular communication through the transfer of proteins and nucleic acids. The aims of this study were: 1) isolation and characterization of EVs from human islets; 2) evaluation of the pro-angiogenic effect of islet-derived EVs on human islet endothelial cells (IECs). EVs were isolated by ultracentrifugation from conditioned medium of human islets and characterized by nanotrack analysis (Nanosight), FACS, western blot, bioanalyzer, mRNA/microRNA RT-PCR array. On IECs, we evaluated EV-induced insulin mRNA transfer, proliferation, resistance to apoptosis, in vitro angiogenesis, migration, gene and protein profiling. EVs sized 236±54 nm, expressed different surface molecules and islet-specific proteins (insulin, C-peptide, GLP1R) and carried several mRNAs (VEGFa, eNOS) and microRNAs (miR-27b, miR-126, miR-130 and miR-296) involved in beta cell function, insulin secretion and angiogenesis. Purified EVs were internalized into IECs inducing insulin mRNA expression, protection from apoptosis and enhancement of angiogenesis. Human islets release biologically active EVs able to shuttle specific mRNAs and microRNAs (miRNAs) into target endothelial cells. These results suggest a putative role for islet-derived EVs in beta cell-endothelium cross-talk and in the neoangiogenesis process which is critical for engraftment of transplanted islets.     

T Lymphocyte Density and Distribution in Human Colorectal Mucosa,and Inefficiency of Current Cell Isolation Protocols

Mucosal tissues are critical immune effector sites containing complex populations of leukocytes in a tissue microenvironment that remains incompletely understood. We identify and quantify in human distal colorectal tissue absolute mucosal CD3⁺ lymphocytes, including CD4⁺ and CD8⁺ subsets, by direct visualization using immunohistochemistry (IHC), immunofluorescence (IF), and an automated counting protocol (r²=0.90). Sigmoid and rectal mucosal tissues are both densely packed with T lymphocytes in the mucosal compartment. Both compartments had similar densities of CD3⁺ T lymphocytes with 37,400 ± 2,801 cells/mm³ and 33,700 ± 4,324 cell/mm³, respectively. Sigmoid mucosa contained 57% CD3⁺CD4⁺ and 40% CD3⁺CD8⁺ T lymphocytes which calculates to 21,300 ± 1,476/mm³ and 15,000 ± 275/mm³ T lymphocytes, respectively. Rectal mucosa had 57% CD3⁺CD4⁺ and 42% CD3⁺CD8⁺ or 21,577 ± 332, and 17,090 ± 1,206 cells/mm³, respectively. By comparison, sigmoid mucosal biopsies subjected to conventional collagenase digestion, mononuclear cell (MMC) isolation and staining for flow cytometry yielded 4,549 ± 381/mm³ and 2,708 ± 245/mm³ CD4+ and CD8+ T lymphocytes. These data suggest only ~20.7% recovery compared to IHC results for these markers. Further studies will determine if this reflects a selective bias in only CD3⁺, CD4⁺ and CD8⁺ T cells or can be generalized to all flow-analyzed cells from mucosal tissues for phenotyping and functional testing.     

Reg3α Overexpression Protects Pancreatic β Cells from Cytokine-Induced Damage and Improves Islet Transplant Outcome

The process of islet transplantation for treating type 1 diabetes has been limited by the high level of graft failure. This may be overcome by locally delivering trophic factors to enhance engraftment. Regenerating islet-derived protein 3α (Reg3α) is a pancreatic secretory protein which functions as an antimicrobial peptide in control of inflammation and cell proliferation. In this study, to investigate whether Reg3α could improve islet engraftment, a marginal mass of syngeneic islets pretransduced with adenoviruses expressing Reg3α or control EGFP were transplanted under the renal capsule of streptozotocin-induced diabetic mice. Mice receiving islets with elevated Reg3α production exhibited significantly lower blood glucose levels (9.057 ± 0.59 mmol/L versus 13.48 ± 0.35 mmol/L, P < 0.05) and improved glucose-stimulated insulin secretion (1.80 ± 0.17 ng/mL versus 1.16 ± 0.16 ng/mL, P < 0.05) compared with the control group. The decline of apoptotic events (0.57% ± 0.15% versus 1.06% ± 0.07%, P < 0.05) and increased β-cell proliferation (0.70% ± 0.10% versus 0.36% ± 0.14%, P < 0.05) were confirmed in islet grafts overexpressing Reg3α by morphometric analysis. Further experiments showed that Reg3α production dramatically protected cultured islets and pancreatic β cells from cytokine-induced apoptosis and the impairment of glucose-stimulated insulin secretion. Moreover, exposure to cytokines led to the activation of MAPKs in pancreatic β cells, which was reversed by Reg3α overexpression in contrast to control group. These results strongly suggest that Reg3α could enhance islet engraftments through its cytoprotective effect and advance the therapeutic efficacy of islet transplantation.     

The Effect of Nrf2 Pathway Activation on Human Pancreatic Islet Cells

Background

Pancreatic islets are known to contain low level of antioxidants that renders them vulnerable to oxidative stress. Nrf2 is the master regulator of numerous genes, encoding antioxidant, detoxifying, and cytoprotective molecules. Activation of Nrf2 pathway induces up-regulation of numerous genes encoding antioxidant and phase II detoxifying enzymes and related proteins. However, little is known regarding the role of this pathway in human islet cells. The aim was to investigate the effect of Nrf2 activator (dh404, CDDO-9,11-dihydro-trifluoroethyl amide) on human islet cells.

Methods

Human islets were obtained from cadaveric donors. After dh404 treatment, Nrf2 translocation, mRNA expression, and protein abundance of its key target gene products were examined. The proportion of dh404-treated or non-treated viable islet beta cells was analyzed using flowcytemetry. The cytoprotective effects against oxidative stress and production of inflammatory mediators, and in vivo islet function after transplantation were determined.

Results

Nrf2 nuclear translocation was confirmed by con-focal microscope within 2 hours after treatment, which was associated with a dose-dependent increase in mRNA expression of anti-oxidants, including NQO1, HO-1, and GCLC. Enhanced HO-1 expression in dh404 treated islets was confirmed by Western Blot assay. Islet function after transplantation (2000 IEQ/mouse) to diabetic nude mice was not affected with or without dh404 treatment. After induction of oxidative stress with hydrogen peroxide (200 μM) the proportion of dh404-treated viable islet cells was significantly higher in the dh404-treated than untreated islets (74% vs.57%; P<0.05). Dh404 significantly decreased production of cytokines/chemokines including IL-1β, IL-6, IFN-γ and MCP-1.

Conclusion

Treatment of human pancreatic islets with the potent synthetic Nrf2 activator, dh404, significantly increased expression of the key anti-oxidants enzymes, decreased inflammatory mediators in islets and conferred protection against oxidative stress in beta cells.     

Incorporation of Bone Marrow Cells in Pancreatic Pseudoislets Improves Posttransplant Vascularization and Endocrine Function

Failure of revascularization is known to be the major reason for the poor outcome of pancreatic islet transplantation. In this study, we analyzed whether pseudoislets composed of islet cells and bone marrow cells can improve vascularization and function of islet transplants. Pancreatic islets isolated from Syrian golden hamsters were dispersed into single cells for the generation of pseudoislets containing 4×10³ cells. To create bone marrow cell-enriched pseudoislets 2×10³ islet cells were co-cultured with 2×10³ bone marrow cells. Pseudoislets and bone marrow cell-enriched pseudoislets were transplanted syngeneically into skinfold chambers to study graft vascularization by intravital fluorescence microscopy. Native islet transplants served as controls. Bone marrow cell-enriched pseudoislets showed a significantly improved vascularization compared to native islets and pseudoislets. Moreover, bone marrow cell-enriched pseudoislets but not pseudoislets normalized blood glucose levels after transplantation of 1000 islet equivalents under the kidney capsule of streptozotocin-induced diabetic animals, although the bone marrow cell-enriched pseudoislets contained only 50% of islet cells compared to pseudoislets and native islets. Fluorescence microscopy of bone marrow cell-enriched pseudoislets composed of bone marrow cells from GFP-expressing mice showed a distinct fraction of cells expressing both GFP and insulin, indicating a differentiation of bone marrow-derived cells to an insulin-producing cell-type. Thus, enrichment of pseudoislets by bone marrow cells enhances vascularization after transplantation and increases the amount of insulin-producing tissue. Accordingly, bone marrow cell-enriched pseudoislets may represent a novel approach to increase the success rate of islet transplantation.     

The Co-Transplantation of Bone Marrow Derived Mesenchymal Stem Cells Reduced Inflammation in Intramuscular Islet Transplantation

Aims/HypothesisAlthough the muscle is one of the preferable transplant sites in islet transplantation, its transplant efficacy is poor. Here we attempted to determine whether an intramuscular co-transplantation of mesenchymal stem cells (MSCs) could improve the outcome.MethodsWe co-cultured murine islets with MSCs and then analyzed the morphological changes, viability, insulin-releasing function (represented by the stimulation index), and gene expression of the islets. We also transplanted 500 islets intramuscularly with or without 5 × 10⁵ MSCs to diabetic mice and measured their blood glucose level, the glucose changes in an intraperitoneal glucose tolerance test, and the plasma IL-6 level. Inflammation, apoptosis, and neovascularization in the transplantation site were evaluated histologically.ResultsThe destruction of islets tended to be prevented by co-culture with MSCs. The stimulation index was significantly higher in islets co-cultured with MSCs (1.78 ± 0.59 vs. 7.08 ± 2.53; p = 0.0025). In terms of gene expression, Sult1c2, Gstm1, and Rab37 were significantly upregulated in islets co-cultured with MSCs. Although MSCs were effective in the in vitro assays, they were only partially effective in facilitating intramuscular islet transplantation. Co-transplanted MSCs prevented an early inflammatory reaction from the islets (plasma IL-6; p = 0.0002, neutrophil infiltration; p = 0.016 inflammatory area; p = 0.021), but could not promote neovascularization in the muscle, resulting in the failure of many intramuscular transplanted islets to engraft.ConclusionsIn conclusion, co-culturing and co-transplanting MSCs is potentially useful in islet transplantation, especially in terms of anti-inflammation, but further augmentation for an anti-apoptosis effect and neovascularization is necessary.     

Cytokines Inducing Bone Marrow SCA+ Cells Migration into Pancreatic Islet and Conversion into Insulin-Positive Cells In Vivo

We hypothesize that specific bone marrow lineages and cytokine treatment may facilitate bone marrow migration into islets, leading to a conversion into insulin producing cells in vivo. In this study we focused on identifying which bone marrow subpopulations and cytokine treatments play a role in bone marrow supporting islet function in vivo by evaluating whether bone marrow is capable of migrating into islets as well as converting into insulin positive cells. We approached this aim by utilizing several bone marrow lineages and cytokine-treated bone marrow from green fluorescent protein (GFP) positive bone marrow donors. Sorted lineages of Mac-1⁺, Mac-1⁻, Sca⁺, Sca⁻, Sca⁻/Mac-1⁺ and Sca⁺/Mac-1⁻ from GFP positive mice were transplanted to irradiated C57BL6 GFP negative mice. Bone marrow from transgenic human ubiquitin C promoter GFP (uGFP, with strong signal) C57BL6 mice was transplanted into GFP negative C57BL6 recipients. After eight weeks, migration of GFP positive donor'' bone marrow to the recipient''s pancreatic islets was evaluated as the percentage of positive GFP islets/total islets. The results show that the most effective migration comes from the Sca⁺/Mac⁻ lineage and these cells, treated with cytokines for 48 hours, were found to have converted into insulin positive cells in pancreatic islets in vivo. This study suggests that bone marrow lineage positive cells and cytokine treatments are critical factors in determining whether bone marrow is able to migrate and form insulin producing cells in vivo. The mechanisms causing this facilitation as well as bone marrow converting to pancreatic beta cells still need to be investigated.     

Glucose Metabolism,Islet Architecture,and Genetic Homogeneity in Imprinting of [Ca2+]i and Insulin Rhythms in Mouse Islets

We reported previously that islets isolated from individual, outbred Swiss-Webster mice displayed oscillations in intracellular calcium ([Ca²⁺]_i) that varied little between islets of a single mouse but considerably between mice, a phenomenon we termed “islet imprinting.” We have now confirmed and extended these findings in several respects. First, imprinting occurs in both inbred (C57BL/6J) as well as outbred mouse strains (Swiss-Webster; CD1). Second, imprinting was observed in NAD(P)H oscillations, indicating a metabolic component. Further, short-term exposure to a glucose-free solution, which transiently silenced [Ca²⁺]_i oscillations, reset the oscillatory patterns to a higher frequency. This suggests a key role for glucose metabolism in maintaining imprinting, as transiently suppressing the oscillations with diazoxide, a K_ATP-channel opener that blocks [Ca²⁺]_i influx downstream of glucose metabolism, did not change the imprinted patterns. Third, imprinting was not as readily observed at the level of single beta cells, as the [Ca²⁺]_i oscillations of single cells isolated from imprinted islets exhibited highly variable, and typically slower [Ca²⁺]_i oscillations. Lastly, to test whether the imprinted [Ca²⁺]_i patterns were of functional significance, a novel microchip platform was used to monitor insulin release from multiple islets in real time. Insulin release patterns correlated closely with [Ca²⁺]_i oscillations and showed significant mouse-to-mouse differences, indicating imprinting. These results indicate that islet imprinting is a general feature of islets and is likely to be of physiological significance. While islet imprinting did not depend on the genetic background of the mice, glucose metabolism and intact islet architecture may be important for the imprinting phenomenon.     

Pancreatic Islets Exhibit Dysregulated Adaptation of Insulin Secretion after Chronic Epinephrine Exposure

Chronic adrenergic stimulation is the dominant factor in impairment of the β-cell function. Sustained adrenergic exposure generates dysregulated insulin secretion in fetal sheep. Similar results have been shown in Min6 under the elevated epinephrine condition, but impairments after adrenergic removal are still unknown and a high rate of proliferation in Min6 has been ignored. Therefore, we incubated primary rats’ islets with half maximal inhibitory concentrations of epinephrine for three days, then determined their insulin secretion responsiveness and related signals two days after removal of adrenaline via radioimmunoassay and qPCR. Insulin secretion was not different between the exposure group (1.07 ± 0.04 ng/islet/h) and control (1.23 ± 0.17 ng/islet/h), but total islet insulin content after treatment (5.46 ± 0.87 ng/islet/h) was higher than control (3.17 ± 0.22 ng/islet/h, p < 0.05), and the fractional insulin release was 36% (p < 0.05) lower after the treatment. Meanwhile, the mRNA expression of Gαs, Gαz and Gβ1-2 decreased by 42.8% 19.4% and 24.8%, respectively (p < 0.05). Uncoupling protein 2 (Ucp2), sulphonylurea receptor 1 (Sur1) and superoxide dismutase 2 (Sod2) were significantly reduced (38.5%, 23.8% and 53.8%, p < 0.05). Chronic adrenergic exposure could impair insulin responsiveness in primary pancreatic islets. Decreased G proteins and Sur1 expression affect the regulation of insulin secretion. In conclusion, the sustained under-expression of Ucp2 and Sod2 may further change the function of β-cell, which helps to understand the long-term adrenergic adaptation of pancreatic β-cell.