Thioredoxin Interacting Protein Expressed in Osteoblasts Mediates the Anti-Proliferative Effects of High Glucose and Modulates the Expression of Osteocalcin.

BACKGROUND: Hyperglycemia is associated with impaired bone health in patients with diabetes mellitus. Although a direct detrimental effect of hyperglycemia on the bone has been previously reported, the specific molecular mediator(s) responsible for the inhibitory effect of high glucose levels on the bone remains unclear. We hypothesized that thioredoxin-interacting protein (Txnip), an essential mediator of oxidative stress, is such a mediator. METHODS: We cultured MG-63 cells (immortalized human osteoblasts) with normal or high glucose concentrations and transfected them with scrambled or Txnip-specific small interfering RNA (siRNA). RESULTS: High glucose levels increased Txnip expression and reduced MG-63 cell proliferation. The high-glucose level mediated reduction in cell proliferation was prevented in Txnip siRNA-transfected cells. In addition, we demonstrated that silencing Txnip mRNA expression in osteoblasts reduced the expression of the osteocalcin gene. Our results suggest that high glucose levels or silencing of Txnip mRNA expression may induce apoptosis in osteoblasts. CONCLUSIONS: Our findings indicate that Txnip is an intracellular mediator of the anti-proliferative effects of extracellular high glucose levels on osteoblasts.

Intermittent mechanical loading on mouse tibia accelerates longitudinal bone growth by inducing PTHrP expression in the female tibial growth plate.

It is not clear as to whether weight bearing and ambulation may affect bone growth. Our goal was to study the role of mechanical loading (one of the components of ambulation) on endochondral ossification and longitudinal bone growth. Thus, we applied cyclical, biologically relevant strains for a prolonged time period (4 weeks) to one tibia of juvenile mice, while using the contralateral one as an internal control. By the end of the 4-week loading period, the mean tibial growth of the loaded tibiae was significantly greater than that of the unloaded tibiae. The mean height and the mean area of the loaded tibial growth plates were greater than those of the unloaded tibiae. In addition, in female mice we found a greater expression of PTHrP in the loaded tibial growth plates than in the unloaded ones. Lastly, microCT analysis revealed no difference between loaded and unloaded tibiae with respect to the fraction of bone volume relative to the total volume of the region of interest or the tibial trabecular bone volume. Thus, our findings suggest that intermittent compressive forces applied on tibiae at mild-moderate strain magnitude induce a significant and persistent longitudinal bone growth. PTHrP expressed in the growth plate appears to be one growth factor responsible for stimulating endochondral ossification and bone growth in female mice.

A Green Light-Activated Insulin Depot with Ultrafast In Vivo Efficiency in the Subcutaneous Space.

In this work, for the first time, we demonstrate light control of a therapeutic protein's release from a depot in the subcutaneous layer of the skin. The subcutaneous layer is a standard location for therapeutic protein depots due to its large size and ease of access, but prior attempts to utilize this space failed because insufficient light can reach this deeper layer. An analysis of existing biophysical literature suggested that an increase of photoactivation wavelength from 365 to 500 nm could allow an increase of depot irradiation in the subcutaneous by >100-fold. We therefore used a green light-activated thio-coumarin-based material and demonstrated robust release of a therapeutic, insulin, in response to skin illumination with an LED light source. We further demonstrated that this release is ultrafast, as fast or faster than any commercially used insulin, while maintaining the native insulin sequence. This release of insulin was then accompanied by a robust reduction in blood glucose, demonstrating the retention of bioactivity despite the synthetic processing required to generate the material. In addition, we observed that the material exhibits slow basal release of insulin, even in the absence of light, potentially through biochemical or photochemical unmasking of insulin. Thus, these materials can act much like the healthy pancreas does: releasing insulin at a slow basal rate and then, upon skin irradiation, releasing an ultrafast bolus of native insulin to reduce postprandial blood glucose excursions.

In vivo variable and multi-day response from an insulin-releasing photoactivated depot.

Previously we have demonstrated that light can be used to control the release of insulin in diabetic animals, followed by a reduction in blood glucose. This is accomplished using a photoactivated depot (PAD) of insulin injected into the skin, and irradiated by a small external LED light source. In this work for the first time we demonstrate dose-response, showing that we can vary insulin release and commensurate blood glucose reduction by varying the amount of light administered. In addition to demonstrating dose-response, we have shown multi-day depot response, with insulin being released on two different days from the same depot. The material used in these studies was CD-insulin, a form of insulin that has a highly non-polar cyclododecyl group attached, markedly reducing the solubility of the modified material, and allowing it to form a depot upon injection. Upon photolysis, the cyclododecyl group is removed, releasing fully native, soluble insulin. Variable response and multi-day response as demonstrated strongly support the potential utility of the PAD approach for the variable and extended release of therapeutic peptides.

Elevated glucose acts directly on osteocytes to increase sclerostin expression in diabetes.

Bone quality in diabetic patients is compromised, leading to weaker bones and increased fracture risk. However, the mechanism by which this occurs in diabetic bone remains to be fully elucidated. We hypothesized that elevated glucose and glucose variation would affect the function of osteocytes, essential regulators of bone homeostasis and quality. To first test this hypothesis, we used the IDG-SW3 osteocyte-like cell line to examine the effects of glucose levels on osteocyte function and viability in vitro. We confirmed our in vitro findings using the in vivo streptozotocin-induced (STZ) diabetic rat model and ex-vivo cultured osteocytes from these rats. IDG-SW3 cells cultured under high glucose conditions displayed significantly increased Sost mRNA(100-fold) and sclerostin protein, a negative regulator of bone formation(5000-fold), compared to cells in control media. mRNA expression of osteoblast markers such as Osx, Ocn and Col1a1 was unaffected by glucose. Factors associated with osteoclast activation were affected by glucose, with Rankl being upregulated by low glucose. Opg was also transiently upregulated by high glucose in mature IDG-SW3 cells. Induction of diabetes in Sprague-Dawley rats via a single dose of STZ (70 mg/kg) resulted in elevated maximum glucose and increased variability compared to control animals (670/796 vs. 102/142 mg/dL). This was accompanied by increased Sost/sclerostin expression in the osteocytes of these animals. These results show that glucose levels directly regulate osteocyte function through sclerostin expression and suggest a potential mechanism for the negative impact of diabetes on bone quality.

Visible-Light-Activated High-Density Materials for Controlled in Vivo Insulin Release.

In this work, we describe the synthesis, characterization, and ultimate in vivo assessment of second-generation insulin photoactivated depot (PAD) materials. These are the first to use visible light to stimulate insulin release and have an in vivo performance that is 28-fold improved relative to first-generation materials. This improvement is due to two major factors linked to the utilized chemistry: (1) we have incorporated the coumarin photocleavable group, which increases the photorelease wavelength into the visible range, enhancing tissue penetration of the light; (2) phototoggling of insulin solubility is produced by linking three insulin molecules to a central bridge via light cleaved groups, and not by bonding to a large polymer. The resulting trimer is, therefore, highly dense (87% insulin dry w/w) but retains the insolubility required of the approach. Only after irradiation with visible light is native, soluble insulin is released from the dermal depot. This high density increases the amount and ease of insulin release, as the density of photolytic groups is 10-20-fold higher than in polymer-based first-generation materials. We have synthesized new azide-terminated coumarin linkers that we react with the amine groups of insulin. Using mass spectrometry methods, we identify the sites of reaction and purify individual isomers, which we demonstrate have in vitro photolysis rates that are within a factor of 2 of each other. We then reacted these terminal azide groups with a tridentate strained alkyne linker. We show that the resulting insulin trimer is highly insoluble, but can be milled into injectable particles that release insulin only in response to light from a 406 nm light source. Finally, we demonstrate that these materials have a significantly improved in vivo performance, releasing 28-fold more insulin on a per energy basis than first-generation materials.

Thioredoxin-interacting protein promotes high-glucose-induced macrovascular endothelial dysfunction.

Thioredoxin-interacting protein (TXNIP) emerges as a central regulator for glucose homeostasis, which goes awry in diabetic subjects. Endothelial dysfunction is considered the earliest detectable stage of cardiovascular disease (CVD), a major complication of diabetes. Here, we hypothesize that TXNIP may promote endothelial dysfunction seen in Type 1 diabetes mellitus (T1D). Using a T1D-like rat model, we found that diabetic rats showed significantly higher TXNIP mRNA and protein levels in peripheral blood, compared to their non-diabetic counterparts. Those changes were accompanied by decreased production of nitric oxide (NO) and vascular endothelial growth factor (VEGF), concurrent with increased expression of reactive oxygen species (ROS) and vascular cell adhesion molecule 1 (VCAM-1) in the aortic endothelium. In addition, TXNIP overexpression in primary human aortic endothelial cells (HAECs) induced by either high glucose or overexpression of carbohydrate response element binding protein (ChREBP), a major transcriptional activator of TXNIP, promoted early apoptosis and impaired NO bioactivity. The correlation between TXNIP expression levels and endothelial dysfunction suggests that TXNIP may be a potential biomarker for vascular complications in T1D patients.

Light Control of Insulin Release and Blood Glucose Using an Injectable Photoactivated Depot.

In this work we demonstrate that blood glucose can be controlled remotely through light stimulated release of insulin from an injected cutaneous depot. Human insulin was tethered to an insoluble but injectable polymer via a linker, which was based on the light cleavable di-methoxy nitrophenyl ethyl (DMNPE) group. This material was injected into the skin of streptozotocin-treated diabetic rats. We observed insulin being released into the bloodstream after a 2 min trans-cutaneous irradiation of this site by a compact LED light source. Control animals treated with the same material, but in which light was blocked from the site, showed no release of insulin into the bloodstream. We also demonstrate that additional pulses of light from the light source result in additional pulses of insulin being absorbed into circulation. A significant reduction in blood glucose was then observed. Together, these results demonstrate the feasibility of using light to allow for the continuously variable control of insulin release. This in turn has the potential to allow for the tight control of blood glucose without the invasiveness of insulin pumps and cannulas.

New Insight Into Metformin Action: Regulation of ChREBP and FOXO1 Activities in Endothelial Cells.

Metformin has been considered a potential adjunctive therapy in treating poorly controlled type 1 diabetes with obesity and insulin resistance, owing to its potent effects on improving insulin sensitivity. However, the underlying mechanism of metformin's vascular protective effects remains obscure. Thioredoxin-interacting protein (TXNIP), a key regulator of cellular redox state induced by high-glucose concentration, decreases thioredoxin reductase activity and mediates apoptosis induced by oxidative stress. Here we report that high glucose-induced endothelial dysfunction is associated with induction of TXNIP expression in primary human aortic endothelial cells exposed to high-glucose conditions, whereas the metformin treatment suppresses high-glucose-induced TXNIP expression at mRNA and protein levels. We further show that metformin decreases the high-glucose-stimulated nuclear entry rate of two transcription factors, carbohydrate response element-binding protein (ChREBP) and forkhead box O1 (FOXO1), as well as their recruitment on the TXNIP promoter. An AMP-activated protein kinase inhibitor partially compromised these metformin effects. Our data suggest that endothelial dysfunction resulting from high-glucose concentrations is associated with TXNIP expression. Metformin down-regulates high-glucose-induced TXNIP transcription by inactivating ChREBP and FOXO1 in endothelial cells, partially through AMP-activated protein kinase activation.

Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions.

Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H2O2 assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H2O2 levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival.

Expression and regulation of nampt in human islets.

Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the mammalian NAD+ biosynthesis of a salvage pathway and exists in 2 known forms, intracellular Nampt (iNampt) and a secreted form, extracellular Nampt (eNampt). eNampt can generate an intermediate product, nicotinamide mononucleotide (NMN), which has been reported to support insulin secretion in pancreatic islets. Nampt has been reported to be expressed in the pancreas but islet specific expression has not been adequately defined. The aim of this study was to characterize Nampt expression, secretion and regulation by glucose in human islets. Gene and protein expression of Nampt was assessed in human pancreatic tissue and isolated islets by qRT-PCR and immunofluorescence/confocal imaging respectively. Variable amounts of Nampt mRNA were detected in pancreatic tissue and isolated islets. Immunofluorescence staining for Nampt was found in the exocrine and endocrine tissue of fetal pancreas. However, in adulthood, Nampt expression was localized predominantly in beta cells. Isolated human islets secreted increasing amounts of eNampt in response to high glucose (20 mM) in a static glucose-stimulated insulin secretion assay (GSIS). In addition to an increase in eNampt secretion, exposure to 20 mM glucose also increased Nampt mRNA levels but not protein content. The secretion of eNampt was attenuated by the addition of membrane depolarization inhibitors, diazoxide and nifedipine. Islet-secreted eNampt showed enzymatic activity in a reaction with increasing production of NAD+/NADH over time. In summary, we show that Nampt is expressed in both exocrine and endocrine tissue early in life but in adulthood expression is localized to endocrine tissue. Enzymatically active eNampt is secreted by human islets, is regulated by glucose and requires membrane depolarization.

CREB participates in the IGF-I-stimulation cyclin D1 transcription.

IGF-I stimulates proliferation and cell cycle progression in progenitor cells of a number of neural cell types, including neuronal and glial progenitors. The precise mechanisms of this regulation, however, have not been fully defined. To elucidate the mechanism of IGF-I actions on neural cell proliferation, we utilized a rat oligodendroglial cell line (OL-1) and primary oligodendrocyte precursors (OPC) and studied IGF-I regulation of cyclin D1 expression and its promoter activity, because cyclin D1 is critical to the promotion of cell proliferation and cell cycle progression. Transient transfection of a reporter driven by the rat cyclin D1 promoter showed that IGF-I stimulates cyclin D1 promoter activity. Furthermore, 5'-end deletions and mutation analysis of this promoter revealed that a cAMP response element (CRE) within -174 base pair (bp) upstream of the transcription start site is crucial to the IGF-induced increase in cyclin D1 transcription. Consistently, Western immunoblot analysis demonstrated that IGF-I induced CREB (CRE binding protein) phosphorylation, while ablation of CREB expression with small interfering RNAs (siRNA) blocked IGF-I actions on cyclin D1 mRNA expression and cell proliferation. Additionally, IGF-I-stimulated CREB phosphorylation was blunted by the MAP kinase inhibitor, PD98059, but not by the PI3 kinase inhibitor, wortmannin. ChIP assays revealed that IGF-1 increased the association of CREB with the cyclin D1 promoter. Taken together, our data indicate that IGF-I upregulates cyclin D1 transcription partially by inducing CREB phosphorylation through the ERK-MAP kinase pathway, and thus increasing its recruitment to the cyclin D1 promoter. These results provide an important mechanism of IGF-I-induced glial cell growth and proliferation. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 559-570, 2013.

Beta-Cell Injury in Ncb5or-null Mice is Exacerbated by Consumption of a High-Fat Diet.

NADH-cytochrome b5 oxidoreductase (Ncb5or) in endoplasmic reticulum (ER) is involved in fatty acid metabolism, and Ncb5or(-/-) mice fed standard chow (SC) are insulin-sensitive but weigh less than wild type (WT) littermates. Ncb5or(-/-) mice develop hyperglycemia at about age 7 weeks due to ß-cell dysfunction and loss associated with saturated fatty acid accumulation and manifestations of ER and oxidative stress. Here we report that when Ncb5or(-/-) mice born to heterozygous mothers fed a high fat (HF) diet continue to ingest HF, they weigh as much as SC-fed WT at age 5 weeks. By age 7 weeks, diabetes mellitus develops in all HF-fed vs. 68% of SC-fed Ncb5or(-/-) mice. Islet ß-cell content in age 5-week Ncb5or(-/-) mice fed HF for 7 days is lower (53%) than for those fed SC (63%), and both are lower than for WT (75%, SC, vs. 69%, HF). Islet transcript levels for markers of mitochondrial biogenesis (PGC-1α) and ER stress (ATF6α) are higher in Ncb5or(-/-) than WT mice but not significantly affected by diet. Consuming a HF diet exacerbates Ncb5or(-/-) ß-cell accumulation of intracellular saturated fatty acids and increases the frequency of ER distention from 11% (SC) to 47% (HF), thus accelerates ß-cell injury in Ncb5or(-/-) mice.

Bone marrow cavity: a supportive environment for islet engraftment.

An important goal in advancing islet transplantation for the treatment for type 1 diabetes, is to discover transplantation sites that promote long-term islet engraftment. Here, we investigate the bone marrow cavity in rats as a potential site for islet transplantation. Dark agouti streptozotocin diabetic recipients received DA islets to one of three sites: to the renal subcapsular, intrahepatic or bone marrow cavity site. Assessment of graft function was made by measuring blood glucose concentrations using a wireless continuous glucose monitoring system (CGM), performing a glucose tolerance test (GTT), and histological analysis. To determine if bone tissue secretes factors supportive to islet function and survival, human islets were cultured in the presence of osteoblast conditioned medium. Gene expression, insulin secretion and content were assessed in islets after culture. All transplant recipients with islets transplanted to the bone marrow cavity site had reversal of hyperglycemia and remained diabetes free until the end of the experiment at four months. Mean blood glucose concentrations, glucose variability and GTT, using CGM in recipients, yielded similar results between all transplantation sites. Histological assessments at four months after transplantation showed viable islets within the bone marrow space. Incubation of human islets in the presence of osteoblast conditioned medium resulted in positive changes in gene expression, insulin secretion and content. These positive changes were mediated by osteocalcin which was present in the conditioned medium. In summary, islets transplanted to the bone marrow cavity in diabetic rats showed good engraftment. In addition, the bone marrow cavity may provide an environment that is protective against post-transplant cellular stress thus increasing the chances of long-term islet function and survival.

Reduction of diffusion barriers in isolated rat islets improves survival, but not insulin secretion or transplantation outcome.

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter >150 µm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter <100 µm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 µm/min in small islets and 2.8 µm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150 µm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.

Prevention of diabetes: effect of mycophenolate mofetil and anti-CD25 on onset of diabetes in the DRBB rat.

BACKGROUND: Anti-CD25 and mycophenolate mofetil (MMF) treatment of patients with new-onset diabetes is currently being tested as one of the trials in TrialNet. We tested the effectiveness of MMF and anti-CD25 in preventing autoimmune diabetes in the diabetes-resistant biobreeding (DRBB) rat. METHODS: Autoimmune diabetes in the DRBB rat was induced with a Treg cell depletion regimen starting at 24-26 d of age. Treatment was started on the first day of the depletion regimen in the following groups: (i) control (vehicle); (ii) MMF 25 mg/kg/d intramuscularly daily for 8 wk; (iii) anti-CD25 0.8 mg/kg/d intraperitoneally 5 d/wk for 3 wk; and (iv) combination of MMF and anti-CD25. In a second set of experiments, treatments were started on day 5 of the depletion regimen (delayed treatment) with groups 1, 3, and 4. Rats that had diabetes-free survival for at least 30 d after the treatment was stopped underwent a second Treg depletion (redepletion). RESULTS: In each of the three treatment groups (n = 10/group), onset of diabetes was delayed or prevented in 20, 40 and 80% in groups 2, 3, and 4, respectively. After redepletion, diabetes-free survival was unchanged in group 2 and decreased to 10 and 30% in groups 3 and 4, respectively. With delayed treatment, groups 3 and 4 had 33 and 50% diabetes-free survival that decreased to 0 and 33% after redepletion. SUMMARY: MMF and anti-CD25 alone or in combination are effective in delaying and preventing diabetes in the DRBB rat especially if treatment is started before stimulation and expansion of the autoreactive T cells.

The effect of immunomodulators on prevention of autoimmune diabetes is stage dependent: FTY720 prevents diabetes at three different stages in the diabetes-resistant biobreeding rat.

BACKGROUND: Autoimmune diabetes of the diabetes-resistant biobreeding (DRBB) rat shares similarities with diabetes in humans and has stages of diabetes that can be controlled and compared. FTY720 is an immunomodulator that has been efficacious in transplant and autoimmune models without inducing an immunosuppressed state. We determined the stages of diabetes that are affected by FTY720 in the DRBB rat. METHODS: Autoimmune diabetes was induced with RT6.1 T-cell-depleting antibody and polyIC starting at 4 weeks of age. FTY720 (1 mg/kg/d) was started at day 0, 5, 7, and 14 following the start of depletion. The rats that did not develop diabetes were maintained for 60 d following the last dose of FTY720 before undergoing a second course of depletion. RESULTS: FTY720 starting at day 0, 5, 7, and 14 of depletion prevented diabetes in 100, 100, 50, and 20% of the DRBB rats compared to 0% of the control rats. The surviving rats in the 5-, 7-, and 14-d groups developed diabetes after FTY720 treatment was stopped. Histological examination indicated insulitis in the control rats between day 7 and 11 of depletion and end-stage insulitis by day 18 of depletion compared to negligible insulitis in rats without diabetes. Redepletion in the surviving day 0 rats resulted in development of diabetes in 25% of these rats compared to none of the age-matched controls. SUMMARY: FTY720 can prevent autoimmune diabetes, if administered before and/or during stimulation and expansion of the autoreactive T cells or in the early stages of insulitis. The effectiveness diminishes with each successive stage of diabetes.

Prevention of autoimmune diabetes in the DRBB rat by CD40/154 blockade.

The autoimmune diabetes of the DRBB rat shares important similarities with autoimmune diabetes in humans. We have tested the ability of CD40/154 blockade using an anti-CD154 antibody (AH.F5) to prevent autoimmune diabetes in DRBB rats. The rats were treated with two intravenous doses/wk of AH.F5 (15mg/kg/dose) starting at 2-6wks of age. RT6.1 T-cell depletion and poly I/C was started at 4wks of age. Control rats developed diabetes within 25 days after start of depletion therapy. Six of 7, 11 of 13, 7 of 12, and 4 of 11 rats treated with AH.F5 did not develop diabetes when treatment was started at 2-3, 4, 5, and 6wks of age, respectively. The rats that did not develop diabetes were maintained for a minimum of 72 days to >150 days following the last dose of AH.F5. Eleven rats maintained for >150 days underwent an additional depletion and 5/11 developed diabetes within 8-19 days following start of depletion.Histological examination indicated that AH.F5 prevented and possibly reversed insulitis. Islets in about 50% of the treated rats remained free of inflammation following a second course of RT 6.1 T-cell depletion after the serum concentration of AH.F5 was negligible. In summary, CD40/154 blockade with AH.F5 prevents development of autoimmune diabetes if treatment is started prior to overt signs of beta cell destruction. The results indicate that the CD40/154 blockade can prevent diabetes by modifying the expansion or effector phase of the autoimmune diabetes.

Decreased survival of islet allografts in rats with advanced chronic complications of diabetes.

Successful islet transplantation has been possible in experimental animals in contrast to humans. One difference between animal models of diabetes and human islet transplantation is the presence of advanced chronic complications in humans. Even longer-term follow-up of islet transplantation in humans according to the Edmonton protocol suggests that advanced chronic complications may adversely affect allograft survival with the glucocorticoid-free immunosuppressive regimen as well. We developed a rat model of chronic complications of diabetes and compared islet allograft survival in rats with advanced chronic complications to age-matched control rats with acute onset diabetes. Islets were transplanted at either the renal supcapsular, intrahepatic, or intramuscular location. The survival of islet allografts in rats with chronic complications was decreased at all sites compared with the age-matched controls. The best survival in the rats with advanced chronic complications occurred at the renal subcapsular site. Blood sugar measurements indicated impaired glucose tolerance in most of the rats with chronic complications and surviving renal subcapsular islet allograft. Histological and gross examination of the surviving renal subcapsular islet allografts indicated disordered angiogenesis in the rats with chronic complications. Rats with successful intrahepatic islet allografts and the respective age-matched controls had comparable blood sugars. Survival of islet allografts at the intramuscular site was poor in rats with chronic complications or acute onset diabetes. We conclude that the structural or metabolic abnormalities associated with chronic poor control of diabetes impair islet allograft survival and function. This should be considered as a possible explanation for failure of islet allograft survival in human islet transplantation.