Stimulating beta cell replication and improving islet graft function by GPR119 agonists

G protein-coupled receptor 119 (GPR119) is predominantly expressed in β cells and intestinal L cells. In this study, we investigated whether oleoylethanolamide (OEA), a GPR119 endogenous ligand, and PSN632408, a GPR119 synthetic agonist, can stimulate β-cell replication in vitro and in vivo and improve islet graft function in diabetic mice. We found that OEA and PSN632408 significantly increased numbers of insulin(+)/5-bromo-2'-deoxyuridine (BrdU)(+) β cells in cultured mouse islets in a dose-dependent manner. All diabetic recipient mice, given marginal syngeneic islet transplants with OEA or PSN632408 or vehicle, achieved normoglycemia at 4 weeks after transplantation. However, normoglycemia was achieved significantly faster in OEA- or PSN632408-treated diabetic mice than in vehicle-treated diabetic mice (P < 0.05). The percentage of insulin(+)/BrdU(+) β cells in islet grafts in OEA- and PSN632408-treated mice was significantly higher than in vehicle-treated mice (P < 0.01). Our data demonstrated that OEA and PSN632408 can stimulate β-cell replication in vitro and in vivo and improve islet graft function. Targeting GPR119 is a novel therapeutic approach to increase β-cell mass and to improve islet graft function by stimulating β-cell replication.     

Effect of glucagon-like peptide-1 gene expression on graft function in mouse islet transplantation

This study investigated the effect of local glucagon‐like peptide‐1 (GLP‐1) production within mouse islets on cytoprotection in vitro and in vivo by gene transfer of GLP‐1. Transduction of recombinant adenovirus vector expressing GLP‐1 (rAd‐GLP‐1) induced a significant increase in bioactive GLP‐1 in the mouse islet culture, whereas transduction with adenovirus vector expressing β‐galactosidase (rAd‐LacZ), as a control, had no effect on GLP‐1 secretion. Islets transduced with rAd‐GLP‐1 were protected from H₂O₂‐induced cell damage in vitro. In addition, glucose‐stimulated insulin secretion was significantly increased in rAd‐GLP‐1‐transduced islets. When transplanted under the kidney capsule of diabetic syngeneic mice, islet grafts retrieved 4 or 7 days after transplantation revealed that the rAd‐GLP‐1‐transduced group had significantly more Ki67‐positive cells as compared with the rAd‐LacZ‐transduced group. Regarding blood glucose control, diabetic mice transplanted with a marginal mass of rAd‐GLP‐1‐transduced islets became normoglycemic more rapidly and 78% of the recipients were normoglycemic at 35 days post‐transplant, whereas only 48% of the mice transplanted with rAd‐LacZ‐transduced islets were normoglycemic (P < 0.05). In conclusion, delivery of the GLP‐1 gene to islets enhanced islet cell survival during the early post‐transplant period, and preserved islet mass and functions over time in the transplants.     

Assessment of simple indices based on a single fasting blood sample as a tool to estimate beta‐cell function after total pancreatectomy with islet autotransplantation – a prospective study

We investigated six indices based on a single fasting blood sample for evaluation of the beta‐cell function after total pancreatectomy with islet autotransplantation (TP‐IAT). The Secretory Unit of Islet Transplant Objects (SUITO), transplant estimated function (TEF), homeostasis model assessment (HOMA‐2B%), C‐peptide/glucose ratio (CP/G), C‐peptide/glucose creatinine ratio (CP/GCr) and BETA‐2 score were compared against a 90‐min serum glucose level, weighted mean C‐peptide in mixed meal tolerance test (MMTT), beta score and the Igls score adjusted for islet function in the setting of IAT. We analyzed values from 32 MMTTs in 15 patients after TP‐IAT with a follow‐up of up to 3 years. Four (27%) individuals had discontinued insulin completely prior to day 75, while 6 out of 12 patients (50%) did not require insulin support at 1‐year follow‐up with HbA1c 6.0% (5.5–6.8). BETA‐2 was the most consistent among indices strongly correlating with all reference measures of beta‐cell function (r = 0.62–0.68). In addition, it identified insulin independence (cut‐off = 16.2) and optimal/good versus marginal islet function in the Igls score well, with AUROC of 0.85 and 0.96, respectively. Based on a single fasting blood sample, BETA‐2 score has the most reliable discriminant value for the assessment of graft function in patients undergoing TP‐IAT.     

Natural human antibody-mediated destruction of porcine neonatal islet cell grafts

Abstract: Porcine pancreata may be considered a potential source of islets for transplantation into diabetic recipients; however, whether porcine islet grafts will be susceptible to damage by natural antibody-mediated hyperacute rejection remains unknown. In this study, we performed Western blots to determine whether membrane proteins present on porcine neonatal islet cells (NIC) are recognized by xenoreactive antibodies present in human sera. Western blots of freshly isolated porcine NICs with AB sera detected the presence of 14 antigens (MW 24–164 kDa) and 4 antigens (MW 101–150 kDa) to which antiserum against human IgM and IgG bound, respectively. The most prominent antigens with IgM reactivity had MWs of 36, 63, and 120 kDa, whereas for IgG, the most intensely reactive antigen had a MW of 120 kDa. When membrane fractions prepared from purified porcine aortic endothelial cells and LLC-PK1 cells were analyzed, the major antigens had molecular weights comparable to those seen for NICs. After culturing the NICs for 5 days, the number of detected xenoreactive antigens binding IgM or IgG decreased and the antigens present at 36, 63, and 120 kDa with IgM reactivity were shown to have a decreased intensity of binding. Incubation of cultured porcine NICs for 18 hr in the presence of human AB serum containing complement resulted in a 55% loss of cellular insulin content (P < 0.0001), a 45% reduction in recoverable DNA (P < 0.0001), and a marked reduction in insulin secretory response to an in vitro glucose challenge. Recovery and viability of porcine NICs was not affected when incubated with AB serum depleted of anti-Gal antibodies with Synsorb 90. These results demonstrate that natural human antibodies of both IgM and IgG subtypes bind to antigens present on Department of Laboratory Medicine and complement reduces islet cell survival and functional viability. Adsorbing serum with the αGal(1–3)βGal(1–4)βGlc carbohydrate removes natural human antibody-mediated destruction of porcine neonatal islet cell grafts.     

Enhanced effect of human mesenchymal stem cells expressing human TNF‐αR‐Fc and HO‐1 gene on porcine islet xenotransplantation in humanized mice

Background

Porcine islet xenotransplantation is considered an attractive alternative treatment for type 1 diabetes mellitus. However, it is largely limited because of initial rejection due to Instant Blood‐Mediated Inflammatory Reaction (IBMIR), oxidative stress, and inflammatory responses. Recently, soluble tumor necrosis factor‐ɑ receptor type I (sTNF‐αR) and heme oxygenase (HO)‐1 genes (HO‐1/sTNF‐αR) have been shown to improve the viability and functionality of porcine islets after transplantation.

Methods

In this study, genetically modified mesenchymal stem cells (MSCs) expressing the HO‐1/sTNF‐αR genes (HO‐1/sTNF‐αR‐MSC) were developed using an adenoviral system, and porcine islet viability and function were confirmed by in vitro tests such as GSIS, AO/PI, and the ADP/ATP ratio after coculturing with HO‐1/sTNF‐αR‐MSCs. Subsequently, isolated porcine islets were transplanted underneath the kidney capsule of diabetic humanized mice without MSCs, with MSCs or with HO‐1/sTNF‐αR‐MSCs.

Results

According to the results, the HO‐1/sTNF‐αR‐MSC‐treated group exhibited improved survival of porcine islets and could reverse hyperglycemia more than porcine islets not treated with MSCs or islets cotransplanted with MSCs. Moreover, the HO‐1/sTNF‐αR‐MSC group maintained its morphological characteristics and the insulin secretion pattern of transplanted porcine islets similar to endogenous islets in immunocompetent humanized mice.

Conclusions

Our results suggest that HO‐1/sTNF‐αR‐MSCs are efficient tools for porcine islet xenotransplantation, and this study may provide basic information for pre‐clinical animal models and future clinical trials of porcine islet xenotransplantation.     

Improved transplantation outcome through delivery of DNA encoding secretion signal peptide‐linked glucagon‐like peptide‐1 into mouse islets

Glucagon‐like peptide‐1 (GLP‐1) stimulates cell proliferation and has anti‐apoptotic effects on pancreatic islet β cells. In our previous study, the transduction of mouse islets with a recombinant adenovirus containing GLP‐1 cDNA enhanced islet graft survival. In this study, we sought to deliver the GLP‐1 gene using a nonviral vector, which raises fewer safety issues in clinical application. We constructed a plasmid, pβ‐SP‐GLP‐1, in which a secretion signal peptide (SP) was inserted to increase GLP‐1 secretion, and transfected mouse islets using the nonviral carrier Effectene. Transfection of pβ‐SP‐GLP‐1 induced a significant increase in bioactive GLP‐1 levels in islet cultures. Islets transfected with pβ‐SP‐GLP‐1 were protected from H₂O₂‐induced cell damage in vitro. In addition, glucose‐stimulated insulin secretion was significantly increased in pβ‐SP‐GLP‐1‐transfected islets. Diabetic syngeneic mice transplanted under the kidney capsule with a marginal mass of pβ‐SP‐GLP‐1‐transfected islets rapidly became normoglycemic, with 88% of recipients being normoglycemic at 30 days post‐transplantation compared with 52% of mice that received pβ‐transfected islet grafts (P < 0.05). Islet grafts retrieved 7 days after transplantation revealed that the pβ‐SP‐GLP‐1‐transfected group had significantly more Ki67‐positive cells as compared with the pβ‐transfected group. In conclusion, delivery of a plasmid containing a secretion SP and GLP‐1 cDNA using a nonviral carrier leads to efficient secretion of GLP‐1 in mouse islet cells, enhances islet cell survival during the early post‐transplant period, and improves islet transplantation outcome.     

The capsular overgrowth on microencapsulated pancreatic islet grafts in streptozotocin and autoimmune diabetic rats

This study investigates whether capsular overgrowth on alginate-polylysine microencapsulated islets is influenced by (1) the presence of islet tissue, (2) MHC incompatibility between donor and recipient, or (3) the presence of autoimmune diabetes. Encapsulated Albino Oxford (AO, n=6, isografts) and Lewis (n=6, allografts) rat islets, and encapsulated human islets (n=5, xenografts) were implanted intraperitoneally into streptozotocin-diabetic AO rats. Also, encapsulated AO islets were implanted into autoimmune diabetic Bio Breeding/Organon (BB/O) rats (n=5, allografts). Five isografts, five allografts, and three xenografts in AO recipients and five allografts in BB/O recipients resulted in normoglycemia. Two weeks after implantation, islets containing capsules were retrieved by peritoneal lavage, after which all animals that had become normoglycemic after transplantation returned to a state of hyperglycemia. Recovery rates of the capsules of these successful grafts, expressed as percentages of the initially implanted graft volume, varied from 72%±7% to 80%±9%. The associated pericapsular infiltrates (PCI) were similar in all groups and varied from 3.2%±1.4% to 8.3%±2.6%. Similar recovery rates and PCI were also found with empty capsules. However, the recovery rates of recipients with graft failures were lower and showed more PCI. Immunohistological staining of PCI showed no differences in the types of cells in the PCI on capsules with or without islets. We conclude that this early PCI is a capsule-induced foreign body reaction that is not influenced by MHC incompatibility or by the presence of autoimmune diabetes, and it should be avoided by improving the biocompatibility of the capsules.     

Cost and clinical outcome of islet transplantation in Norway 2010‐2015

Islet transplantation is a minimally invasive β‐cell replacement strategy. Islet transplantation is a reimbursed treatment in Norway. Here, we summarize the cost and clinical outcome of 31 islet transplantations performed at Oslo University Hospital (OUS) from January 2010 to June 2015. Patients were retrospectively divided into three groups. Thirteen patients received either one or two islet transplantation alone (ITA), while five patients received islet transplantation after previous solid organ transplantation. For the group receiving 2 ITA, Kaplan‐Meier estimates show an insulin independence of 20% more than 4 years after their last transplantation. An estimated 70% maintain at least partial graft function, defined as fasting C‐peptide >0.1 nmol L^?1, and 47% maintain a HbA1c below 6.5% or 2 percent points lower than before ITA. For all groups combined, we estimate that 44% of the patients have a 50% reduction in insulin requirement 4 years after the initial islet transplantation. The average cost for an islet transplantation procedure was 347 297±60 588 NOK, or 35 424±6182 EUR, of which isolation expenses represent 34%. We hereby add to the common pool of growing experience with islet transplantation and also describe the cost of the treatment at our center.     

Low gravity rotational culture and the integration of immunomodulatory stem cells reduce human islet allo‐reactivity

Modification of human islets prior to transplantation may improve long‐term clinical outcome in terms of diabetes management, by supporting graft function and reducing the potential for allo‐rejection. Intragraft incorporation of stem cells secreting beta (β)‐cell trophic and immunomodulatory factors represents a credible approach, but requires suitable culture methods to facilitate islet alteration without compromising integrity. This study employed a three‐dimensional rotational cell culture system (RCCS) to achieve modification, preserve function, and ultimately influence immune cell responsiveness to human islets. Islets underwent intentional dispersal and rotational culture‐assisted aggregation with amniotic epithelial cells (AEC) exhibiting intrinsic immunomodulatory potential. Reassembled islet constructs were assessed for functional integrity, and their ability to induce an allo‐response in discrete T‐cell subsets determined using mixed islet:lymphocyte reaction assays. RCCS supported the formation of islet:AEC aggregates with improved insulin secretory capacity compared to unmodified islets. Further, the allo‐response of peripheral blood mononuclear cell (PBMC) and purified CD4+ and CD8+ T‐cell subsets to AEC‐bearing grafts was significantly (p < 0.05) attenuated. Rotational culture enables pre‐transplant islet modification involving their integration with immunomodulatory stem cells capable of subduing the allo‐reactivity of T cells relevant to islet rejection. The approach may play a role in achieving acute and long‐term graft survival in islet transplantation.     

Xenoreactive CD4+ memory T cells resist inhibition by anti-CD44 mAb and reject islet grafts via a Th2-dependent pathway

Peng YZ, Chen JB, Shao W, Wang FY, Dai HL, Cheng PP, Xia JJ, Wang F, Huang R, Zhu Q, Qi Z. Xenoreactive CD4⁺ memory T cells resist inhibition by anti‐CD44 mAb and reject islet grafts via a Th2‐dependent pathway. Xenotransplantation 2011; 18: 252–261. © 2011 John Wiley & Sons A/S. Abstract: Background: Memory T cells are a significant barrier to the induction of transplant tolerance. Our previous study demonstrated that multiple applications of anti‐CD44 monoclonal antibody (mAb) could significantly inhibit CD4⁺ memory T cells from mediating rejection of cardiac allografts. Now, we sought to explore the effect and mechanism of anti‐CD44 mAb on the rejection of islet allografts and xenografts mediated by CD4⁺ memory T cells. Methods: In this study, we first engrafted skin grafts of C57BL/6 (B6) mice or Dark Agouti (DA) rats onto BALB/c mice to induce donor‐reactive memory T cells. We adoptively transferred purified CD4⁺ memory T cells to BALB/c origin nude mice and then transplanted islet allografts and xenografts to produce the Allo‐Tx and Xeno‐Tx models, respectively. We subsequently administered multiple anti‐CD44 mAb and observed changes in the survival times of the islet grafts. Results: In the Allo‐Tx model, the mean survival time (MST) of the grafts was 7.7 days in the isotype group, and 20.3 days in the anti‐CD44 group. In the Xeno‐Tx model, the MST of the grafts was 7.2 days in the isotype group and 8.2 days in the anti‐CD44 group. Compared with the isotype group, CD4⁺ T cells on the grafts in the anti‐CD44 group were significantly decreased in both the Allo‐Tx and Xeno‐Tx models, but the proportion of CD4⁺ memory T cells in the spleens and draining lymph nodes of the recipient nude mice in the anti‐CD44 group was significantly decreased in the Allo‐Tx model, while it was increased in the Xeno‐Tx model. The production of donor‐specific IgG antibody in the anti‐CD44 group did not vary in the Allo‐Tx model, while it was markedly elevated in the Xeno‐Tx model. Furthermore, the expression of interferon gamma in the anti‐CD44 group was markedly decreased in both the Allo‐Tx and Xeno‐Tx models, while the expression of IL‐4 in the anti‐CD44 group was significantly increased only in the Xeno‐Tx model. Conclusion: Multiple applications of the anti‐CD44 mAb could significantly inhibit donor‐reactive CD4⁺ memory T cells from rejecting grafts via a Th1‐dependent pathway, but xenoreactive CD4⁺ memory T cells can avoid the effects of anti‐CD44 mAb to reject islet xenografts via a Th2‐dependent pathway.     

Novel cell‐permeable p38‐MAPK inhibitor efficiently prevents porcine islet apoptosis and improves islet graft function

During islet transplantation, mitogen‐activated protein kinase (MAPK) p38 is preferentially activated in response to the isolation of islets and the associated inflammation. Although therapeutic effects of p38 inhibitors are expected, the clinical application of small‐molecule inhibitors of p38 is not recommended because of their serious adverse effects on the liver and central nervous system. Here we designed peptides to inhibit p38, which were derived from the sites on p38 that mediate binding to proteins such as MAPK kinases. Peptide 11R‐p38I₁₁₀ significantly inhibited the activation of p38. To evaluate the effects of 11R‐p38I₁₁₀, porcine islets were incubated with 10 µmol/L 11R‐p38I₁₁₀ or a mutant form designated 11R‐mp38I₁₁₀. After islet transplantation, blood glucose levels reached the normoglycemic range in 58.3% and 0% of diabetic mice treated with 11R‐p38I₁₁₀ or 11R‐mp38I₁₁₀, respectively. These data suggest that 11R‐p38I₁₁₀ inhibited islet apoptosis and improved islet function. Peptide p38I₁₁₀ is a noncompetitive inhibitor of ATP and targets a unique docking site. Therefore, 11R‐p38I₁₁₀ specifically inhibits p38 activation, which may avoid the adverse effects that have discouraged the clinical use of small‐molecule inhibitors of p38. Moreover, our methodology to design “peptide inhibitors” could be used to design other inhibitors derived from the binding sites of proteins.     

Exendin‐4 shows no effects on the prostatic index in high‐fat‐diet‐fed rat with benign prostatic hyperplasia by improving insulin resistance

Benign prostatic hyperplasia (BPH) is a prevalent disease globally, and accumulating evidence has indicated an association between BPH, insulin resistance (IR) and diabetes. Exendin‐4 is widely used in clinics, which could enhance the proliferation of pancreatic β cells. The ability of exendin‐4 to promote tumorigenesis has been of concern, and whether exendin‐4 would enhance the propagation of BPH is not fully understood. We aimed to determine whether glucagon‐like peptide‐1 receptors (GLP‐1Rs) were expressed in rat prostate and to determine the effect of exendin‐4 on prostate of BPH. Male Wistar rats were used and assigned to six groups: normal diet (ND), high‐fat diet (HFD), HFD + exendin‐4, HFD + BPH, HFD + BPH + exendin‐4 and HFD + BPH + rosiglitazone group. After castration, steroids were injected subcutaneously for 4 weeks to induce BPH. Rats were kept on high‐fat diet to induce IR. Treatment groups were treated with exendin‐4 and rosiglitazone. Prostatic index and HOMA‐IR index were used to evaluate the prostatic hyperplasia status and the degree of IR respectively. The expression of GLP‐1R was indicated not only by immunohistochemistry, but also by Western blot analysis. The expression of GLP‐1R was significantly higher, and HOMA‐IR index and body weight significantly decreased after administration of exendin‐4. However, no significant differences in the prostatic index were observed between exendin‐4 treatment groups and non‐exendin‐4 treatment groups. Prostatic index was not influenced by exendin‐4 maybe by improving IR and weight loss.     

Three‐dimensional ex vivo imaging and analysis of intraportal islet transplants

In clinical islet transplantation, because the long‐term insulin‐independence rate is still poor, a method for detailed analysis of the transplanted islets in the liver after transplantation is required. We have established a novel imaging technique suitable for analysis of transplanted islets in liver using an optical projection tomography (OPT) method. A three‐dimensional tomographic image of the transplanted islets in liver was reconstructed. The number of islets transplanted and the number of transplanted islets observed using OPT showed good correlation. The OPT method was used to compare the numbers of transplanted islets in mouse syngeneic and allogeneic transplantation models. Blood glucose concentrations of streptozotocin (STZ)‐induced diabetic mice transplanted with syngeneic islets remained normoglycemic and the number of transplanted islets was largely preserved 11 days after transplantation. In mice transplanted with allogeneic islets, hyperglycemia recurred from 7 days after transplantation and the number and the volume of transplanted islets was significantly reduced 11 days after transplantation. These results indicate that OPT imaging and analysis may be a useful tool to quantitatively and sterically evaluation of transplanted islets in liver at the cellular level.     

Exendin‐4, a Glucagon‐Like Peptide‐1 Receptor Agonist,Prevents Osteopenia by Promoting Bone Formation and Suppressing Bone Resorption in Aged Ovariectomized Rats

Osteoporosis mainly affects postmenopausal women and older men. Gastrointestinal hormones released after meal ingestion, such as glucose‐dependent insulinotropic peptide (GIP) and glucagon‐like peptide (GLP)‐2, have been shown to regulate bone turnover. However, whether GLP‐1, another important gastrointestinal hormone, and its analogues also have antiosteoporotic effects, especially in aged postmenopausal situation, has not been confirmed. In the present study, we evaluated the effects of the GLP‐1 receptor agonist exendin‐4 on ovariectomy (OVX)‐induced osteoporosis in old rats. Twelve‐month‐old female Sprague‐Dawley rats were subjected to OVX, and exendin‐4 was administrated 4 weeks after the surgery and lasted for 16 weeks. Bone characters and related serum and gene biomarkers were analyzed. Sixteen weeks of treatment with exendin‐4 slowed down body weight gain by decreasing fat mass and prevented the loss of bone mass in old OVX rats. Exendin‐4 also enhanced bone strength and prevented the deterioration of trabecular microarchitecture. Moreover, exendin‐4 decreased the urinary deoxypyridinoline (DPD)/creatinine ratio and serum C‐terminal cross‐linked telopeptides of type I collagen (CTX‐I) and increased serum alkaline phosphatase (ALP), osteocalcin (OC), and N‐terminal propeptide of type 1 procollagen (P1NP) levels, key biochemical markers of bone turnover. Interestingly, gene expression results further showed that exendin‐4 not only inhibited bone resorption by increasing the osteoprotegerin (OPG)/receptor activator of NF‐κB ligand (RANKL) ratio, but also promoted bone formation by increasing the expression of OC, Col1, Runx2, and ALP, which exhibited dual regulatory effects on bone turnover as compared with previous antiosteoporotic agents. In conclusion, these findings demonstrated for the first time the antiosteoporotic effects of exendin‐4 in old OVX rats and that it might be a potential candidate for treatment of aged postmenopausal osteoporosis.