Combination therapy with glucagon-like peptide-1 and gastrin restores normoglycemia in diabetic NOD mice

OBJECTIVE—Glucagon-like peptide-1 (GLP-1) and gastrin promote pancreatic β-cell function, survival, and growth. Here, we investigated whether GLP-1 and gastrin can restore the β-cell mass and reverse hyperglycemia in NOD mice with autoimmune diabetes.RESEARCH DESIGN AND METHODS—Acutely diabetic NOD mice were treated with GLP-1 and gastrin, separately or together, twice daily for 3 weeks. Blood glucose was measured weekly and for a further 5 weeks after treatments, after which pancreatic insulin content and β-cell mass, proliferation, neogenesis, and apoptosis were measured. Insulin autoantibodies were measured, and adoptive transfer of diabetes and syngeneic islet transplant studies were done to evaluate the effects of GLP-1 and gastrin treatment on autoimmunity.RESULTS—Combination therapy with GLP-1 and gastrin, but not with GLP-1 or gastrin alone, restored normoglycemia in diabetic NOD mice. The GLP-1 and gastrin combination increased pancreatic insulin content, β-cell mass, and insulin-positive cells in pancreatic ducts, and β-cell apoptosis was decreased. Insulin autoantibodies were reduced in GLP-1–and gastrin-treated NOD mice, and splenocytes from these mice delayed adoptive transfer of diabetes in NOD-scid mice. Syngeneic islet grafts in GLP-1–and gastrin-treated NOD mice were infiltrated by leukocytes with a shift in cytokine expression from interferon-γ to transforming growth factor-β1, and β-cells were protected from apoptosis.CONCLUSIONS—Combination therapy with GLP-1 and gastrin restores normoglycemia in diabetic NOD mice by increasing the pancreatic β-cell mass and downregulating the autoimmune response.Pancreatic β-cells can regenerate in response to experimental injury in adult animals (1–3) and can increase in humans in response to conditions such as pregnancy (4) and obesity (5). In addition, there is histological evidence of attempts at β-cell regeneration in humans with type 1 diabetes (6,7). Similarly, β-cell proliferation is increased before diabetes onset in NOD mice, an animal model for human type 1 diabetes, but not sufficiently to keep up with the ongoing autoimmune response that decreases the β-cell mass (8). Therefore, therapies directed at stimulating β-cell regeneration in addition to arresting autoimmunity may restore the β-cell mass and reverse type 1 diabetes.Many putative β-cell growth factors have been identified, one of the most promising being glucagon-like peptide-1 (GLP-1), a peptide secreted from intestinal L-cells in response to nutrient ingestion (9). The actions of GLP-1 to stimulate glucose-dependent insulin secretion and inhibit glucagon release, gastric emptying, and food intake (10) have led to its application as a therapy for type 2 diabetes (11). GLP-1 has additional actions that suggest a therapeutic role in conditions with a deficit in β-cell mass. GLP-1 and long-acting GLP-1 receptor agonists, such as exendin-4, increase the β-cell mass in rodents with surgically or chemically induced diabetes through stimulation of β-cell proliferation and islet neogenesis and inhibition of β-cell apoptosis (12–15). Also, GLP-1 (16) and exendin-4 (17) reduce insulitis and protect β-cells in NOD mice when given before diabetes onset. Exendin-4 has also been reported to reverse diabetes in NOD mice; however, this required combination of exendin-4 with immunosuppressive therapy using antilymphocyte serum (18).Gastrin is a gastrointestinal peptide reported to induce β-cell neogenesis from pancreatic exocrine duct cells in rodents (19,20). Combined gastrin and epidermal growth factor (EGF) treatment induces islet regeneration and restores normoglycemia in alloxan-treated mice (21) and ameliorates hyperglycemia after diabetes onset in NOD mice (22). Here, we report that addition of gastrin to GLP-1 treatment restored normoglycemia in acutely diabetic NOD mice by increasing the pancreatic β-cell mass and downregulating the autoimmune response.     

Recognition of Human Proinsulin Leader Sequence by Class I�CRestricted T-Cells in HLA-A*0201 Transgenic Mice and in Human Type 1 Diabetes

OBJECTIVE— A restricted region of proinsulin located in the B chain and adjacent region of C-peptide has been shown to contain numerous candidate epitopes recognized by CD8⁺ T-cells. Our objective is to characterize HLA class I–restricted epitopes located within the preproinsulin leader sequence.RESEARCH DESIGN AND METHODS— Seven 8- to 11-mer preproinsulin peptides carrying anchoring residues for HLA-A1, -A2, -A24, and -B8 were selected from databases. HLA-A2–restricted peptides were tested for immunogenicity in transgenic mice expressing a chimeric HLA-A*0201/β2-microglobulin molecule. The peptides were studied for binding to purified HLA class I molecules, selected for carrying COOH-terminal residues generated by proteasome digestion in vitro and tested for recognition by human lymphocytes using an ex vivo interferon-γ (IFN-γ) ELISpot assay.RESULTS— Five HLA-A2–restricted peptides were immunogenic in transgenic mice. Murine T-cell clones specific for these peptides were cytotoxic against cells transfected with the preproinsulin gene. They were recognized by peripheral blood mononuclear cells (PBMCs) from 17 of 21 HLA-A2 type 1 diabetic patients. PBMCs from 25 of 38 HLA-A1, -A2, -A24, or -B8 patients produced IFN-γ in response to six preproinsulin peptides covering residues 2–25 within the preproinsulin region. In most patients, the response was against several class I–restricted peptides. T-cells recognizing preproinsulin peptide were characterized as CD8⁺ T-cells by staining with peptide/HLA-A2 tetramers.CONCLUSIONS— We defined class I–restricted epitopes located within the leader sequence of human preproinsulin through in vivo (transgenic mice) and ex vivo (diabetic patients) assays, illustrating the possible role of preproinsulin-specific CD8⁺ T-cells in human type 1 diabetes.Type 1 diabetes involves the activation of lymphocytes against β-cell autoantigens. In animals, the predominant role of T-cells is supported by experiments in which diabetes is transferred by diabetogenic T-cells, is prevented by antibodies that interfere with T-cell activation, or fails to develop in diabetes-prone mice in which key genes in T-cell differentiation or activation are deficient. In humans, T-cells are predominant within insulitis at early stages of diabetes. Moreover, type 1 diabetes has been reported in an immunodeficient patient deprived of B-cells (1).Major histocompatibility complex (MHC) class II–restricted CD4⁺ T-cells are central in the diabetes process, but CD8⁺ T-cells play a pivotal role in its initiation in NOD mice (2). In human, CD8⁺ T-cells are predominant, and a high percentage of interferon-γ (IFN-γ)-positive cells is detected within insulitis in recent-onset diabetes in most observations (3,4,5,6). Recurrent diabetes in recipients of isografts from a discordant twin is accompanied by predominant CD8⁺ T-cell infiltration (7).Among β-cell autoantigens, proinsulin has been ascribed a key role in diabetes. In humans, insulin and proinsulin are common targets of autoantibodies (8,9) and T-cells (10,11,12,13,14,15,16,17) in diabetic and pre-diabetic individuals. Anti-insulin antibodies are the first to be detected in children at risk for diabetes and carry a high positive predictive value for diabetes (9). In NOD mice, injection of insulin-specific T-cell clones accelerate diabetes (18). Protection from diabetes is obtained by injecting insulin in pre-diabetic mice (19). In addition, proinsulin 1^−/− or 2^−/− NOD mice show delayed or accelerated diabetes, respectively (20,21).Several new β-cell HLA class I–restricted epitopes have been reported recently (22,23,24,25,26,27). We and others have shown that a restricted region of human proinsulin located in the B chain and adjacent C-peptide clusters proteasome cleavage sites generating correct COOH-termini of putative MHC class I peptides and many epitopes that are recognized by diabetic CD8⁺ T-cells (22,23). Recognition of epitopes that are located within the C-peptide and C-peptide–B chain junction, including residues that are excised during the secretion process, makes a strong case for proinsulin as an autoantigen in diabetes. Despite strong evidence that leader sequence peptides are presented by class I HLA molecules, especially HLA-A2.1 (28), only two HLA A2.1 preproinsulin leader sequence peptides have been identified (26,27).To characterize class I–restricted epitopes within the preproinsulin leader sequence, we selected 8- to 11-mer peptides carrying anchoring residues for class I molecules. These peptides were studied for immunogenicity in HLA-A*0201 transgenic mice (29). Mouse CD8⁺ T-cell clones specific to HLA-A*0201–restricted peptides were tested for cytotoxicity against HLA-A2 target cells transfected with the preproinsulin gene. In human, peptides were studied for binding to common class I molecules, for carrying COOH-terminal residues generated by proteasome digestion, and for recognition by peripheral blood mononuclear cells (PBMCs) from diabetic patients.     

Thymic stromal lymphopoietin and thymic stromal lymphopoietin-conditioned dendritic cells induce regulatory T-cell differentiation and protection of NOD mice against diabetes

OBJECTIVE—Autoimmune diabetes in the nonobese diabetic (NOD) mouse model results from a breakdown of T-cell tolerance caused by impaired tolerogenic dendritic cell development and regulatory T-cell (Treg) differentiation. Re-establishment of the Treg pool has been shown to confer T-cell tolerance and protection against diabetes. Here, we have investigated whether murine thymic stromal lymphopoietin (TSLP) re-established tolerogenic function of dendritic cells and induced differentiation and/or expansion of Tregs in NOD mice and protection against diabetes.RESEARCH DESIGN AND METHODS—We examined the phenotype of TSLP-conditioned bone marrow dendritic cells (TSLP-DCs) of NOD mice and their functions to induce noninflammatory Th2 response and differentiation of Tregs. The functional relevance of TSLP and TSLP-DCs to development of diabetes was also tested.RESULTS—Our results showed that bone marrow dendritic cells of NOD mice cultured in the presence of TSLP acquired signatures of tolerogenic dendritic cells, such as an absence of production of pro-inflammatory cytokines and a decreased expression of dendritic cell costimulatory molecules (CD80, CD86, and major histocompatibility complex class II) compared with LPS-treated dendritic cells. Furthermore, TSLP-DCs promoted noninflammatory Th2 response and induced the conversion of naïve T-cells into functional CD4⁺CD25⁺Foxp3⁺ Tregs. We further showed that subcutaneous injections of TSLP for 6 days or a single intravenous injection of TSLP-DCs protected NOD mice against diabetes.CONCLUSIONS—Our study demonstrates that TSLP re-established a tolerogenic immune response in NOD mice and protects from diabetes, suggesting that TSLP may have a therapeutic potential for the treatment of type 1 diabetes.Dendritic cells are professional antigen-presenting cells (APCs) that have the potential to induce immune response and T-cell tolerance (1). Immature or semimature tolerogenic dendritic cells have been shown to induce and maintain peripheral T-cell tolerance, whereas terminally differentiated mature dendritic cells induce the development of effector T-cells (1). Tolerogenic dendritic cells (tDCs) produce interleukin (IL)-10 and have impaired abilities to synthesize IL-12p70 and indolamine 2,3-dioxygenase and to activate T-cells in vitro (2). Conditioning dendritic cells with granulocyte macrophage–colony-stimulating factor (GM-CSF) (3), IL-10, and/or transforming growth factor-β (TGF-β) (4,5) as well as 1,25-dihydroxyvitamin D3 (6) has been shown to promote tDCs that induce Th2 response and/or differentiation of CD4⁺CD25⁺Foxp3⁺ regulatory T-cells (Tregs). When injected in mice, tDCs were able to suppress acute graft-versus-host disease (7) and autoimmunity (8). Recently, we and others have shown that injections of GM-CSF prevented the development of autoimmune diseases by increasing the number of semimature tDCs and by inducing Treg differentiation (9–11).Tregs arise during the normal process of T-cell maturation in the thymus, and their differentiation can be induced in the periphery by conversion of CD4⁺CD25⁻Foxp3⁻ into CD4⁺CD25⁺Foxp3⁺ Tregs (12–14). Tregs are crucial for suppressing autoimmune responses and maintaining peripheral immunological tolerance (15). The influence of Tregs in maintaining T-cell tolerance is strongly supported by the observations of the development of autoimmune syndromes in mice lacking Tregs and by the findings that defects in Foxp3 gene expression in humans and mice lead to autoimmune syndromes in early life (16,17). In agreement with these observations, prevention of rheumatoid arthritis, inflammatory bowel disease, and type 1 diabetes has been achieved by reconstitution of autoimmune-prone mice with Tregs (18).Autoimmune diabetes in the nonobese diabetic (NOD) mouse model results from a breakdown of T-cell tolerance due to impaired development of tDCs and Treg differentiation (19,20). In addition, bone marrow–derived dendritic cells (BM-DCs) of NOD mice were shown to express abnormal levels of costimulatory molecules under pro-inflammatory conditions and increased capacity to secrete IL-12p70 and to stimulate CD4⁺ and CD8⁺ T-cells (21–23). Consequently, the capacity of dendritic cells in NOD mice to sustain the pool and suppressive function of Tregs is altered, which leads to progression of type 1 diabetes (24,25).Thymic stromal lymphopoietin (TSLP) was first identified in conditioned medium supernatants of the mouse thymic stromal cell line Z210R.1 (26). TSLP, a member of the IL-7 cytokine family, is preferentially expressed by epithelial cells mainly in the lung, skin, and gut (27,28). Recently, clues for a function of TSLP in humans came from two observations. TSLP was found to be selectively expressed by thymic epithelial cells of Hassall''s corpuscles, and TSLP-activated dendritic cells (TSLP-DCs) induced differentiation of CD4⁺Foxp3⁻ thymocytes into CD4⁺Foxp3⁺ Tregs (29). Recently, Jiang et al. (30) have reported that TSLP produced by mouse medullary thymic epithelial cells contribute to Foxp3⁺ expression and Treg maturation. In addition, Lee et al. (31) have shown that TSLP triggered the conversion of thymic Foxp3⁻CD4⁺ T-cells into Foxp3⁺ T-cells in a dendritic cell–independent manner.Here, we have investigated whether murine TSLP-DCs and TSLP induce differentiation and/or expansion of Tregs in the NOD mouse model and protection against diabetes. We found that TSLP-DCs acquire signatures of tDCs and induce the conversion of naïve T-cells into functional Tregs. We have further shown that subcutaneous injections of TSLP or a single intravenous injection of TSLP-DCs protects NOD mice against diabetes. Our data are the first to report that TSLP induces a tolerogenic immune response and protects against diabetes in NOD mice.     

Improved vascular engraftment and graft function after inhibition of the angiostatic factor thrombospondin-1 in mouse pancreatic islets

OBJECTIVE—Insufficient development of a new intra-islet capillary network after transplantation may be one contributing factor to the failure of islet grafts in clinical transplantation. The present study tested the hypothesis that the angiostatic factor thrombospondin-1 (TSP-1), which is normally present in islets, restricts intra-islet vascular expansion posttransplantation.RESEARCH DESIGN AND METHODS—Pancreatic islets of TSP-1–deficient (TSP-1^−/−) mice or wild-type islets transfected with siRNA for TSP-1 were transplanted beneath the renal capsule of syngeneic or immunocompromised recipient mice.RESULTS—Both genetically TSP-1^−/− islets and TSP-1 siRNA-transfected islet cells demonstrated an increased vascular density when compared with control islets 1 month after transplantation. This was also reflected in a markedly increased blood perfusion and oxygenation of the grafts. The functional importance of the improved vascular engraftment was analyzed by comparing glucose-stimulated insulin release from islet cells transfected with either TSP-1 siRNA or scramble siRNA before implantation. These experiments showed that the increased revascularization of grafts composed of TSP-1 siRNA-transfected islet cells correlated to increments in both their first and second phase of glucose-stimulated insulin secretion.CONCLUSIONS—Our findings demonstrate that inhibition of TSP-1 in islets intended for transplantation may be a feasible strategy to improve islet graft revascularization and function.Despite improvements in immunosuppression protocols over the last years, pancreatic islets from at least two donor pancreata are still needed to reverse type 1 diabetes in clinical islet transplantation (1,2). This is far more than the alleged 10–20% of the total islet volume suggested to be enough to maintain normoglycemia in humans. Moreover, in contrast to the results for whole-organ transplantation, there seems to be a continuous decline in islet graft function, and very few patients remain insulin-independent at 5 years posttransplantation (2,3). Because the histocompatibility barrier, the underlying autoimmune disease, and the immunosuppressive agents used are the same for both transplantation procedures, it is likely that issues related to the adaptation of the implanted islets to their new microenvironment play a role for the differences in results.Pancreatic islets become disconnected from their vascular supply during collagenase digestion before transplantation. Revascularization of transplanted islets has been shown to be concluded within 7–14 days (4). However, the resulting vascular density remains lower than in endogenous islets (5–7) and is associated with an impaired oxygenation (6,8) and endocrine function (7,9,10).We have recently observed that freshly isolated rodent islets become better revascularized and function better than islets cultured for several days before transplantation (11), although the islet vascular system, also when using freshly isolated islets for transplantation, is far from fully restored. One possible explanation for the improved vascular engraftment in such islets is that not only host blood vessels but also remnant donor islet endothelial cells may participate in the formation of a new islet vascular network (12–14). However, despite the presence of several mitogens for endothelial cells within the islets, such as vascular endothelial growth factor (VEGF), fibroblast growth factor, and matrix metalloproteinases (15–17), intra-islet endothelial cells normally have a very low proliferation rate (18,19). This endothelial quiescence is presumably due to the fact that pro-angiogenic factors normally are counteracted by anti-angiogenic factors present in the islets (20), including the islet endothelial cells themselves (21,22). A possible key factor in this context is thrombospondin-1 (TSP-1), because it is not downregulated by hypoxia (20), which occurs posttransplantation. Moreover, animals deficient of this glycoprotein are characterized by hypervascular islets (23). The present study tested the hypothesis that use of genetically TSP-1^−/− islets or transfection of islets in vitro with siRNA for TSP-1 would create a microenvironment permissive for blood vessel growth within islets and improve vascular engraftment and function after transplantation.     

Cannabinoid type 1 receptor blockade promotes mitochondrial biogenesis through endothelial nitric oxide synthase expression in white adipocytes

OBJECTIVE—Cannabinoid type 1 (CB1) receptor blockade decreases body weight and adiposity in obese subjects; however, the underlying mechanism is not yet fully understood. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) induces mitochondrial biogenesis and function in adipocytes. This study was undertaken to test whether CB1 receptor blockade increases the espression of eNOS and mitochondrial biogenesis in white adipocytes.RESEARCH DESIGN AND METHODS—We examined the effects on eNOS and mitochondrial biogenesis of selective pharmacological blockade of CB1 receptors by SR141716 (rimonabant) in mouse primary white adipocytes. We also examined eNOS expression and mitochondrial biogenesis in white adipose tissue (WAT) and isolated mature white adipocytes of CB1 receptor–deficient (CB1^−/−) and chronically SR141716-treated mice on either a standard or high-fat diet.RESULTS—SR141716 treatment increased eNOS expression in cultured white adipocytes. Moreover, SR141716 increased mitochondrial DNA amount, mRNA levels of genes involved in mitochondrial biogenesis, and mitochondrial mass and function through eNOS induction, as demonstrated by reversal of SR141716 effects by small interfering RNA–mediated decrease in eNOS. While high-fat diet–fed wild-type mice showed reduced eNOS expression and mitochondrial biogenesis in WAT and isolated mature white adipocytes, genetic CB1 receptor deletion or chronic treatment with SR141716 restored these parameters to the levels observed in wild-type mice on the standard diet, an effect linked to the prevention of adiposity and body weight increase.CONCLUSIONS—CB1 receptor blockade increases mitochondrial biogenesis in white adipocytes by inducing the expression of eNOS. This is linked to the prevention of high-fat diet–induced fat accumulation, without concomitant changes in food intake.Adipose tissue is not merely an energy store but rather an endocrine organ playing an important role in fuel metabolism (1,2). Recent studies have demonstrated that mitochondrial biogenesis increases during adipocyte differentiation (3), and this is presumably due at least in part to the presence of the endothelial nitric oxide (NO) synthase (eNOS). In fact, eNOS protein is not expressed in either undifferentiated 3T3-L1 cells or preadipocytes, whereas its expression markedly increases with adipocyte differentiation (4). It is noteworthy that NO increases mitochondrial biogenesis in white adipocytes via cyclic guanosine monophosphate (cGMP)-dependent pathways, including peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) gene expression (5,6). Consistently, mitochondrial biogenesis is reduced in fat of eNOS-null mutant mice (eNOS^−/−), with decreased energy expenditure and increased body weight (5,6). This suggests that the eNOS-dependent mitochondrial biogenesis is relevant to adipocyte maturation and fuel metabolism.Cannabinoid type 1 (CB1) receptors participate in the physiological modulation of many central and peripheral functions related to the control of energy metabolism (7). They are the most abundant G protein–coupled receptors expressed in the brain, where CB1 receptor activation promotes feeding and modulates the rewarding properties of food (7–9). CB1 receptors also control metabolic functions by acting on peripheral organs, including white adipose tissue (WAT) (7–9). Notably, in adipocytes, CB1 receptor expression increases with cell differentiation (10,11). Moreover, CB1 receptor–deficient mice (CB1^−/−) are lean (12) and resistant to a high-fat diet (13). Similarly, the selective CB1 receptor antagonist SR141716 (rimonabant) persistently reduces body weight during the treatment of obese animals, although food intake renormalizes after an initial 1- to 2-week weight reduction (14,15), suggesting that CB1 receptor blockade stimulates fat metabolism, leading to a decreased fat content.We now demonstrate a prominent role for the CB1 receptors to modulate eNOS gene expression and mitochondrial biogenesis in white adipocytes. Collectively, these data suggest that targeting the endocannabinoid system improves mitochondrial function, implying a novel, unrecognized mechanism of action for the antiobesity drug rimonabant.     

Deficiency in B7-H1 (PD-L1)/PD-1 Coinhibition Triggers Pancreatic ��-Cell Destruction by Insulin-Specific, Murine CD8 T-Cells

OBJECTIVE

RIP-B7.1 mice expressing the costimulator molecule B7.1 (CD80) on pancreatic β-cells are a well established model to characterize preproinsulin-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD). Different immunization strategies could prime preproinsulin-specific CD8 T-cells in wild-type C57BL/6 (B6) mice, but did not induce diabetes. We tested whether altering the B7-H1 (PD-L1) coinhibition on pancreatic β-cells can reveal a diabetogenic potential of preproinsulin-specific CD8 T-cells.

RESEARCH DESIGN AND METHODS

DNA-based immunization and adoptive T-cell transfers were used to characterize the induction of preproinsulin-specific CD8 T-cell responses and EAD in RIP-B7.1, B6, B7-H1^−/−, PD-1^−/− or bone marrow chimeric mice.

RESULTS

Preproinsulin-specific CD8 T-cells primed in B6 mice revealed their diabetogenic potential after adoptive transfer into congenic RIP-B7.1 hosts. Furthermore, preproinsulin-specific CD8 T-cells primed in anti-B7-H1 antibody-treated B6 mice, or primed in B7-H1^−/− or PD-1^−/− mice induced EAD. Immunization of bone marrow chimeric mice showed that deficiency of either B7-H.1 in pancreatic β-cells or of PD-1 in autoreactive CD8 T-cells induced EAD.

CONCLUSIONS

An imbalance between costimulator (B7.1) and coinhibitor (B7-H1) signals on pancreatic β-cells can trigger pancreatic β-cell-destruction by preproinsulin-specific CD8 T-cells. Hence, regulation of the susceptibility of the β-cells for a preproinsulin-specific CD8 T-cell attack can allow or suppress EAD.Insulin-producing β-cells in the pancreatic islets are destroyed by an immune attack in autoimmune type 1 diabetes. Type 1 diabetes is triggered by a poorly defined breakdown in central or peripheral tolerance that allows activation of diabetogenic T-cells (1,2). Preclinical animal models have elucidated some aspects of the priming and effector phase of a diabetogenic immune response (3,4). Mice develop diabetes either spontaneously in the NOD model (5), or in response to transgene-encoded “neo-self” antigens selectively expressed in pancreatic β-cells (6–8). These studies indicated that priming of self-reactive T-cells and β-cell susceptibility to an autoaggressive T-cell attack are distinct steps in the pathogenesis of the disease.Costimulating B7/CD28 family molecules provide critical signals for T-cell activation (9,10). RIP-B7.1 mice express the B7.1 costimulator in pancreatic β-cells under rat insulin promoter (RIP) control (11). We have shown that RIP-B7.1 mice develop CD8 T-cell–dependent experimental autoimmune diabetes (EAD) after immunization with preproinsulin-encoding vectors (12–14). Transgene-driven B7.1 expression in pancreatic β-cells thus makes them susceptible to T-cell–mediated immune attack.Coinhibitory signals generated by “programmed death-1” (PD-1)/“programmed death-ligand-1” (B7-H1 or PD-L1) interaction downmodulated T-cell responses and maintain self-tolerance in autoimmune diabetes (15,16). Inducible or constitutive expression of B7-H1 is found in many peripheral tissues, including the β-cells of the pancreatic islets (17,18). Ligation of PD-1 (expressed by activated T-cells) to B7-H1 (expressed by epitope-presenting cells) downmodulates T-cell proliferation and IFNγ production (19). Furthermore, B7-H1 interacts specifically with the costimulatory B7.1 (CD80) molecule upregulated by activated T-cells and inhibits their responses (20). PD-1/B7-H1 interaction facilitates establishment of self-tolerance, thereby partially controlling diabetes development in NOD mice (15,21–23). Selective, transgene-driven overexpression of B7-H1 by pancreatic β-cells can, however, result in EAD, suggesting operation of a costimulatory B7-H1 pathway (24).We investigated the impact of inhibitory (B7-H1, PD-1) molecules on the pathogenicity of preproinsulin-specific CD8 T-cells. We used RIP-B7.1 mice to characterize the specificity and diabetogenic potential of preproinsulin-specific CD8 T-cell responses. RIP-B7.1 mice were immunized with preproinsulin-encoding vectors, or used as hosts for adoptive T-cell transfers. We further analyzed preproinsulin-specific CD8 T-cell responses and EAD development in C57BL/6 (B6), B7-H1^−/− (25), or PD-1^−/− knockout mice (26), as well as bone marrow chimeras (using different donor T-cell and host β-cell phenotypes of B7-H1/PD-1).     

T-cell promiscuity in autoimmune diabetes

OBJECTIVE—It is well established that the primary mediators of β-cell destruction in type 1 diabetes are T-cells. Nevertheless, the molecular basis for recognition of β-cell–specific epitopes by pathogenic T-cells remains ill defined; we seek to further explore this issue.RESEARCH DESIGN AND METHODS—To determine the properties of β-cell–specific T-cell receptors (TCRs), we characterized the fine specificity, functional and relative binding avidity/affinity, and diabetogenicity of a panel of GAD65-specific CD4⁺ T-cell clones established from unimmunized 4- and 14-week-old NOD female mice.RESULTS—The majority of GAD65-specific CD4⁺ T-cells isolated from 4- and 14-week-old NOD female mice were specific for peptides spanning amino acids 217–236 (p217) and 290–309 (p290). Surprisingly, 31% of the T-cell clones prepared from 14-week-old but not younger NOD mice were stimulated with both p217 and p290. These promiscuous T-cell clones recognized the two epitopes when naturally processed and presented, and this dual specificity was mediated by a single TCR. Furthermore, promiscuous T-cell clones demonstrated increased functional avidity and relative TCR binding affinity, which correlated with enhanced islet infiltration on adoptive transfer compared with that of monospecific T-cell clones.CONCLUSIONS—These results indicate that promiscuous recognition contributes to the development of GAD65-specific CD4⁺ T-cell clones in NOD mice. Furthermore, these findings suggest that T-cell promiscuity reflects a novel form of T-cell avidity maturation.Type 1 diabetes is characterized by the autoimmune-mediated destruction of the insulin-producing β-cells of the islets of Langerhans (1–3). Based on studies in the nonobese diabetic (NOD) mouse, a spontaneous model of type 1 diabetes, the primary effectors of β-cell destruction are CD4⁺ and CD8⁺ T-cells (1,4–6). Early during β-cell autoimmunity, a select panel of autoantigens, including proinsulin, insulin, GAD65, islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP), and dystrophia myotonica kinase, are targeted by CD4⁺ and CD8⁺ T-cells in NOD mice (7–12). As the diabetogenic response proceeds, β-cell–specific T-cell reactivity “spreads” in a relatively defined pattern (13,14). Additional autoantigenic determinants are sequentially recognized within a single protein (intramolecularly) and among different antigens (intermolecularly) to effectively amplify the diabetogenic response.The key events involved in the breakdown of self-tolerance within the T-cell compartment and which shape the T-cell receptor (TCR) repertoire of β-cell–specific T-cells remain ill defined. Studies have suggested that defective thymic negative selection contributes to increased production of β-cell–specific T precursors (15–17). Furthermore, the peptide-binding properties of major histocompatibility complex (MHC) class II and class I molecules that are associated with type 1 diabetes susceptibility are thought to shape the TCR repertoire of diabetogenic T-cell effectors (18–22). Properties intrinsic to β-cell–specific TCRs may also contribute to the pathogenicity of T-cell effectors. For instance, avidity maturation promotes the expansion of IGRP-specific CD8⁺ T-cells that in turn display increased TCR avidity/affinity and enhanced pathogenicity (23). One intriguing possibility is that the pathogenicity of an autoreactive T-cell is influenced by the degree of TCR cross-reactivity. In this model, a T-cell expressing a TCR that cross-reacts with multiple β-cell–derived epitopes would be selectively expanded and exhibit increased pathogenicity.Antigen recognition by TCRs is inherently degenerate (24–26). Furthermore, a number of studies have reported cross-reactive T-cell responses between synthetic peptides that exhibit little if any sequence homology with the natural ligand (27–30). Allogeneic recognition by T-cells provides a biologically relevant example of the flexibility associated with TCR recognition (31,32). Goverman and colleagues (33) demonstrated the presence of CD4⁺ T-cells recognizing two nonoverlapping epitopes of myelin basic protein (MBP) in a murine model of experimental autoimmune encephalomyelitis (EAE). This finding suggests that cross-reactive or “promiscuous” T-cell clonotypes may promote tissue-specific autoimmunity. Other studies have reported promiscuous recognition of nonoverlapping epitopes within the same viral or foreign antigen by CD4⁺ and CD8⁺ T-cells (34–36). The molecular basis and functional impact of T-cell promiscuity remains largely undefined.In an effort to determine the properties of β-cell–specific TCRs, we prepared a large panel of GAD65-specific CD4⁺ T-cell clones isolated from unimmunized 4- and 14-week-old NOD female mice. We demonstrate that a significant frequency of these T-cell clones recognize nonoverlapping peptides derived from GAD65, and that this promiscuity correlates with increased pathogenicity.     

High glucose induces toll-like receptor expression in human monocytes: mechanism of activation

OBJECTIVE—Hyperglycemia-induced inflammation is central in diabetes complications, and monocytes are important in orchestrating these effects. Toll-like receptors (TLRs) play a key role in innate immune responses and inflammation. However, there is a paucity of data examining the expression and activity of TLRs in hyperglycemic conditions. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression and mechanism of their induction in monocytic cells under high-glucose conditions.RESEARCH DESIGN AND METHODS—High glucose (15 mmol/l) significantly induced TLR2 and TLR4 expression in THP-1 cells in a time- and dose-dependent manner (P < 0.05). High glucose increased TLR expression, myeloid differentiation factor 88, interleukin-1 receptor–associated kinase-1, and nuclear factor-κB (NF-κB) p65-dependent activation in THP-1 cells. THP-1 cell data were further confirmed using freshly isolated monocytes from healthy human volunteers (n = 10).RESULTS—Pharmacological inhibition of protein kinase C (PKC) activity and NADPH oxidase significantly decreased TLR2 and TLR4 mRNA and protein (P < 0.05). Knocking down both TLR2 and TLR4 in the cells resulted in a 76% (P < 0.05) decrease in high-glucose–induced NF-κB activity, suggesting an additive effect. Furthermore, PKC-α knockdown decreased TLR2 by 61% (P < 0.05), whereas inhibition of PKC-δ decreased TLR4 under high glucose by 63% (P < 0.05). Small inhibitory RNA to p47Phox in THP-1 cells abrogated high-glucose–induced TLR2 and TLR4 expression. Additional studies revealed that PKC-α, PKC-δ, and p47Phox knockdown significantly abrogated high-glucose–induced NF-κB activation and inflammatory cytokine secretion.CONCLUSIONS—Collectively, these data suggest that high glucose induces TLR2 and -4 expression via PKC-α and PKC-δ, respectively, by stimulating NADPH oxidase in human monocytes.The major cause of death in type 1 and type 2 diabetic patients is atherosclerosis (1–3). The pathogenesis of the accelerated atherosclerosis is multifactorial. Inflammation is pivotal in the development of atherosclerosis. Recent studies have shown that diabetes is a proinflammatory state (4–6). We and others have shown that the proinflammatory phenotype in diabetes is characterized by elevated plasma C-reactive protein (CRP), cytokines, chemokines, adhesion molecules, monocytic activity, etc. (4–6). Hyperglycemia contributes to vascular complications of diabetes. High glucose has been shown to induce inflammatory cytokines, chemokines, p38 mitogen-activated protein kinase, reactive oxygen species (ROS), protein kinase C (PKC), and nuclear factor-κB (NF-κB) activity in both clinical and experimental systems (7–12). Several lines of evidence support a role for oxidative stress in the development of diabetes complications (13,14). Diabetic patients have increased O₂⁻ production in monocytes and neutrophils (8,13,15); however, the mechanism of the interactions among these mediators remain unclear.Toll-like receptors (TLRs) recognize conserved pathogen-associated molecular patterns and induce innate immune responses that are essential for host defenses (16). TLRs are activated by both endogenous and exogenous agonists of microbial and nonmicrobial origin. TLR activation by their agonists triggers a signaling cascade, leading to cytokine production and initiation of an adaptive immune response (17). TLR expression is increased in a plethora of inflammatory disorders, including atherosclerosis and diabetes (18–20). Some of the endogenous ligands for TLR2 and TLR4 include high-mobility group B1, biglycan, hyaluronic acid fragments, necrotic cells, serum amyloid A, advanced glycation end products, and extracellular matrix components (18). Among the TLRs, TLR2 and TLR4 play an important role in atherosclerosis. TLR2 and TLR4 bind to components of the Gram-positive and -negative bacteria, respectively (17). They are expressed in multiple cells and tissues, primarily in monocytes. TLR2 and TLR4 expression is increased in atherosclerotic plaque macrophages and in animal models of atherosclerosis (21–25). Plaques of TLR4 knockout mice on a high-fat diet show reduced lesion size, lipid content, and macrophage infiltration (22). TLR2/LDLR^−/− and TLR2/ApoE^−/− double knockout mice are protected from the development of atherosclerosis (24). In addition, total loss of myeloid differentiation factor 88 (MyD88), a common adapter molecule of TLR2 and TLR4 in the cell, results in reduced plaque size, lipid content, inflammation, and plasma interleukin (IL)-1 and tumor necrosis factor-α (TNF-α) (25).The interactions among inflammation, hyperglycemia, and diabetes have clear implications for the immune system. Mohammad et al. (26) reported increased TLR2 and TLR4 expression in type 1 diabetic NOD mice, correlating with increased NF-κB activation in response to endotoxin, and increased proinflammatory cytokines. Kim et al. (27) using TLR2^−/−, TLR4^−/− knockouts, and NOD mice have demonstrated that TLR2 senses β-cell death and contributes to the instigation of autoimmune diabetes. Devaraj et al. (20) showed increased TLR2 and TLR4 expression, intracellular signaling, and TLR-mediated inflammation in monocytes with significant correlation to A1C levels in type 1 diabetic patients. Also, Song et al. (28) reported increased TLR4 mRNA expression in differentiating adipose tissue of db/db mice. Creely et al. (29) showed increased TLR2 expression in the adipose tissue of type 2 diabetic patients with strong correlates to endotoxin levels. These observations taken together suggest a potential role for TLR2 and TLR4 in the pathology of diabetes with limited mechanistic details.However, data examining the mechanism of increased TLR2 and TLR4 expression in diabetes are unknown. Therefore, this study aimed to test the ability of high glucose, one of the key abnormalities of the diabetic condition, to induce TLR expression in human monocytes.     

Spontaneous diabetes in hemizygous human amylin transgenic mice that developed neither islet amyloid nor peripheral insulin resistance

OBJECTIVES—We sought to 1) Determine whether soluble-misfolded amylin or insoluble-fibrillar amylin may cause or result from diabetes in human amylin transgenic mice and 2) determine the role, if any, that insulin resistance might play in these processes.RESEARCH DESIGN AND METHODS—We characterized the phenotypes of independent transgenic mouse lines that display pancreas-specific expression of human amylin or a nonaggregating homolog, [^25,28,29Pro]human amylin, in an FVB/n background.RESULTS—Diabetes occurred in hemizygous human amylin transgenic mice from 6 weeks after birth. Glucose tolerance was impaired during the mid- and end-diabetic phases, in which progressive β-cell loss paralleled decreasing pancreatic and plasma insulin and amylin. Peripheral insulin resistance was absent because glucose uptake rates were equivalent in isolated soleus muscles from transgenic and control animals. Even in advanced diabetes, islets lacked amyloid deposits. In islets from nontransgenic mice, glucagon and somatostatin cells were present mainly at the periphery and insulin cells were mainly in the core; in contrast, all three cell types were distributed throughout the islet in transgenic animals. [^25,28,29Pro]human amylin transgenic mice developed neither β-cell degeneration nor glucose intolerance.CONCLUSIONS—Overexpression of fibrillogenic human amylin in these human amylin transgenic mice caused β-cell degeneration and diabetes through mechanisms independent from both peripheral insulin resistance and islet amyloid. These findings are consistent with β-cell death evoked by misfolded but soluble cytotoxic species, such as those formed by human amylin in vitro.Increasing evidence indicates that decreased β-cell mass contributes to the impaired insulin secretion characteristic of type 2 diabetes (1–3). Amylin, also referred to as islet amyloid polypeptide, is a 37-amino acid polypeptide (4,5) secreted from pancreatic islet β-cells whose aggregation results in islet amyloid formation in type 2 diabetes (6). Islet amyloid has been reported in 40–90% of pancreases from type 2 diabetic subjects studied post mortem (7–11) and has been linked to both decreased β-cell mass and β-cell dysfunction (12,13). In vitro, human amylin causes apoptosis of islet β-cells, and there is growing evidence that this pathogenic process may contribute to the β-cell deficit in type 2 diabetes (1,2,14,15). However, it remains unresolved whether islet amyloid contributes to the etiopathogenesis of type 2 diabetes or, by contrast, occurs only as a consequence of the disease.Several independent lines of human amylin transgenic mice have been developed to investigate the role of amylin and islet amyloid in the pathogenesis of type 2 diabetes (16–19). The findings and conclusions from phenotypic characterization studies are wide ranging and sometimes at variance. Transgenic animals developed by several research groups did not develop spontaneous diabetes or insulin resistance or exhibit evidence of islet amyloid formation, suggesting that overexpression of human amylin alone was not sufficient to contribute to diabetes development and islet amyloid formation in those models (16–18). In contrast, Janson et al. (19) showed development of spontaneous diabetes in the absence of islet amyloid in homozygous individuals from a further transgenic mouse model, consistent with the view that overexpression of human amylin is sufficient for diabetes development but not islet amyloid formation in that model. It was previously thought that overexpression of human amylin might be sufficient for islet amyloid formation, but some studies have suggested that insulin resistance might also be necessary (20–22).Evidence concerning the role of human amylin in the processes that lead to or cause diabetes remains conflicting, and a clear role for human amylin–mediated β-cell death has not been established at this time, at least in part due to conflicting evidence from the different lines of human amylin transgenic mice. Previous reports have described the noticeable lack of correlation between amyloid deposition and hyperglycemia in other transgenic models of amylin-induced diabetes (21,23). Islets from homozygous individuals from the FVB/n-based line reported by Janson et al. (19) demonstrated a pattern of β-cell loss that closely reflects that in islets from human type 2 diabetic patients (1,3,9), but hemizygous animals from that line reportedly do not develop diabetes.Here, we report a transgenic human amylin mouse model (L13) in which hemizygous individuals developed early-onset diabetes without peripheral insulin resistance and islet amyloid formation. We demonstrate that the disappearance of functional β-cells during the progression of diabetes in this model contributes to the pathogenesis of diabetes. The absence of islet amyloid in the pancreas of transgenic mice before diabetes onset and during its progression, despite the high secretion rates of human amylin, shows that islet amyloid is not required for islet β-cell degeneration and loss of physiological insulin secretion. These findings are consistent with the reports of Janson et al. (19) and provide strong support for continuing exploration of the mechanism by which human amylin evokes β-cell death and contributes to the failure of insulin secretion in type 2 diabetes.     

Selective small-molecule agonists of G protein-coupled receptor 40 promote glucose-dependent insulin secretion and reduce blood glucose in mice

OBJECTIVE— Acute activation of G protein–coupled receptor 40 (GPR40) by free fatty acids (FFAs) or synthetic GPR40 agonists enhances insulin secretion. However, it is still a matter of debate whether activation of GPR40 would be beneficial for the treatment of type 2 diabetes, since chronic exposure to FFAs impairs islet function. We sought to evaluate the specific role of GPR40 in islets and its potential as a therapeutic target using compounds that specifically activate GPR40.RESEARCH DESIGN AND METHODS— We developed a series of GPR40-selective small-molecule agonists and studied their acute and chronic effects on glucose-dependent insulin secretion (GDIS) in isolated islets, as well as effects on blood glucose levels during intraperitoneal glucose tolerance tests in wild-type and GPR40 knockout mice (GPR40^−/−).RESULTS— Small-molecule GPR40 agonists significantly enhanced GDIS in isolated islets and improved glucose tolerance in wild-type mice but not in GPR40^−/− mice. While a 72-h exposure to FFAs in tissue culture significantly impaired GDIS in islets from both wild-type and GPR40^−/− mice, similar exposure to the GPR40 agonist did not impair GDIS in islets from wild-type mice. Furthermore, the GPR40 agonist enhanced insulin secretion in perfused pancreata from neonatal streptozotocin-induced diabetic rats and improved glucose levels in mice with high-fat diet–induced obesity acutely and chronically.CONCLUSIONS— GPR40 does not mediate the chronic toxic effects of FFAs on islet function. Pharmacological activation of GPR40 may potentiate GDIS in humans and be beneficial for overall glucose control in patients with type 2 diabetes.Loss of glucose-dependent insulin secretion (GDIS) from the pancreatic β-cell is responsible for the onset and progression of type 2 diabetes (1,2). Oral agents that stimulate insulin secretion, such as sulfonylureas and related ATP-sensitive K⁺ channel blockers, reduce blood glucose and have been used as a first-line type 2 diabetes therapy for nearly 30 years (3,4). However, these agents act to force the β-cell to secrete insulin continuously regardless of prevailing glucose levels, thereby promoting hypoglycemia and accelerating the loss of islet function and, eventually, diminished efficacy (5,6). Despite the availability of a range of agents for type 2 diabetes, many diabetic patients fail to achieve or to maintain glycemic targets (7–9). In addition, stricter glycemic guidelines have been proposed to help define a path toward diabetes prevention through identifying and treating the pre-diabetes state (10). Agents that induce GDIS have great potential to replace sulfonylureas as a first-line therapy for the treatment of type 2 diabetes. In particular, agents that have positive effects on arresting or even reversing β-cell demise would represent a major therapeutic advance toward addressing the lack of durability seen with current therapies and perhaps obviate the need for eventual insulin intervention (11–13). The recent emergence of glucagon-like peptide 1–based GDIS agents (14–16), including inhibitors of dipeptidyl peptidase-4 (17) and peptidase-stable analogs such as exendin-4 (18), is undoubtedly a major advance in such a direction. Nevertheless, it remains to be observed whether glucagon-like peptide 1–related agents truly exert durable beneficial effects on β-cell mass and function.The molecular pharmacology of lipid and lipid-like mediators that signal through G protein–coupled receptors (GPCRs) has expanded significantly over the past few years. To date, several orphan GPCRs have been paired with lysophospholipids, bile acids, arachidonic acid metabolites, dioleoyl phosphatidic acid, and short-, medium-, and long-chain free fatty acids (FFAs) (19–21). From these discoveries, GPCR 40 (GPR40), GPR119, and GPR120 have been reported to play a role in regulating GDIS and therefore have potential as novel targets for the treatment of type 2 diabetes (22–26). GPR40 is a G_q-coupled, family A GPCR that is highly expressed in β-cells of human and rodent islets. Several naturally occurring medium- to long-chain FFAs and some thiazolidinedione peroxisome proliferator–activated receptor-γ agonists specifically activate GPR40 (27,28). Activation of GPR40 by FFAs (29–32) or synthetic compounds (23,33) enhances insulin secretion through the amplification of intracellular calcium signaling.The pleiotropic effects of FFAs on the pancreatic β-cell are well known. The fact that FFAs are in vitro ligands for GPR40 is suggestive of the link to the wealth of existing literature data on the acute, stimulatory effects of FFAs on insulin release (34,35). However, FFAs also exert suppressive or detrimental effects on β-cells. Lipotoxicity of β-cells, a condition observed with chronic exposure to high FFA levels, results in impairment in their function and a resulting diminution in their insulin secretory capacity (36,37). Currently, there is an ongoing debate on whether GPR40 mediates the deleterious effects of FFAs on islet function (lipotoxicity) and whether an antagonist of GPR40 is preferable to an agonist for the treatment of type 2 diabetes (38,39). Since FFAs can both be metabolized within cells to act as intracellular signaling molecules (35) and activate more than one receptor (20), they cannot be used as specific and selective tools to unravel the role that GPR40 plays in the β-cell. It is therefore necessary to identify small molecules that specifically activate GPR40.In the following discussion, we will detail the identification and in vitro pharmacology of a novel series of synthetic GPR40 agonists. Using isolated islets from wild-type and homozygous GPR40 knockout (GPR40^−/−) mice (to confirm the on-target activity of small-molecule activators), we not only extended previous findings that acute activation of GPR40 enhances GDIS in pancreatic β-cells but also showed that long-term exposure to the GPR40 agonist, in contrast to FFAs, did not impair β-cell function, thus dissociating the activation of GPR40 from β-cell lipotoxicity. Finally, acute and subchronic dosing of the GPR40 agonist robustly reduced the blood glucose excursion during an intraperitoneal glucose tolerance test (IPGTT) in wild-type, but not GPR40^−/−, mice.     

Unexpected Acceleration of Type 1 Diabetes by Transgenic Expression of B7-H1 in NOD Mouse Peri-Islet Glia

OBJECTIVE

Autoimmune target tissues in type 1 diabetes include pancreatic β-cells and peri-islet Schwann cells (pSC)—the latter active participants or passive bystanders in pre-diabetic autoimmune progression. To distinguish between these alternatives, we sought to suppress pSC autoimmunity by transgenic expression of the negative costimulatory molecule B7-H1 in NOD pSC.

RESEARCH DESIGN AND METHODS

A B7-H1 transgene was placed under control of the glial fibrillary acidic protein (GFAP) promoter. Transgenic and wild-type NOD mice were compared for transgene PD-1 affinities, diabetes development, insulitis, and pSC survival. Mechanistic studies included adoptive type 1 diabetes transfer, B7-H1 blockade, and T-cell autoreactivity and sublineage distribution.

RESULTS

Transgenic and endogenous B7-H1 bound PD-1 with equal affinities. Unexpectedly, the transgene generated islet-selective CD8⁺ bias with accelerated rather than suppressed diabetes progression. T-cells of diabetic transgenics transferred type 1 diabetes faster. There were no earlier pSC losses due to conceivable transgene toxicity, but transgenic pSC loss was enhanced by 8 weeks, preceded by elevated GFAP autoreactivity, with high-affinity T-cells targeting the major NOD K^d-GFAP epitope, p253–261. FoxP3⁺ regulatory T- and CD11c⁺ dendritic cell pools were unaffected.

CONCLUSIONS

In contrast with transgenic B7-H1 in NOD mouse β-cells, transgenic B7-H1 in pSC promotes rather than protects from type 1 diabetes. Here, ectopic B7-H1 enhanced the pathogenicity of effector T-cells, demonstrating that pSC can actively impact diabetes progression—likely through modification of intraislet T-cell selection. Although pSC cells emerge as a new candidate for therapeutic targets, caution is warranted with regard to the B7-H1–PD1 axis, where B7-H1 overexpression can lead to accelerated autoimmune disease.The NOD mouse is a spontaneous model of type 1 diabetes, with genetic and pathophysiological roots comparable with the human disease (1). Pancreatic islets of Langerhans are tightly enveloped by peri-islet Schwann cells (pSC) that express glial fibrillary acidic protein (GFAP), a marker of Schwann cells and astrocytes (2). During pre-diabetes progression, T-cell infiltrates accumulate at the endocrine/exocrine border, constituted by the pSC mantle, where lengthy “peri”-insulitis lasts for weeks to months in NOD mice and likely for years in humans with islet autoimmunity. Eventual breakdown of the pSC mantle initiates pathogenic islet invasion, progressive β-cell loss, insulin deficiency, and overt diabetes development. In NOD mice, CD8⁺ T-cells predominate islet attack until late in this process (3).Islet T-cell infiltrations are heterogeneous in their target autoantigen specificities for not only β-cell–selective autoantigens (e.g., insulin) but also autoantigens shared by β-cells and nervous system tissue, islet-associated autoantigens shared by pSC and β-cells (e.g., S100β) or those that are pSC specific (e.g., GFAP) (4). pSC functions and their importance in type 1 diabetes development have yet to be fully characterized. In NOD mice, pSC-specific T-cell autoreactivities are present by 5 weeks of age. GFAP target epitopes were recently mapped to residues 79–87 and 253–261 for K^d and 96–110, 116–130, and 216–230 for NOD-IA^g7, and fresh ex vivo CD8⁺ cells mediate direct lysis of primary pSC cultures from diabetic NOD mice (5).pSC cells likely have physiological functions similar to conventional Schwann cells of the peripheral nervous system, providing neurotrophic support for islet-innervating neurons as well as the neural crest-derived β-cell (2). For example, nerve growth factor, glial cell—derived neurotrophic factor, and insulin-like growth factor-1 promote β-cell survival and probably regeneration (6–8). Loss of these factors with pSC destruction may amplify β-cell stress, enhancing β-cell susceptibility to inflammatory insults (7). Anatomically, pSC provide a physical barrier to infiltrating T-cells, accumulating at the endo-exocrine islet border and impeding direct β- and T-cell contact.B7-H1, a ligand for programmed death (PD)-1, is expressed by CD4⁺ and CD8⁺ T-cells, B-cells, dendritic cells (DCs), macrophages, mast cells, and nonhemopoietic tissues (9). In nonlymphoid tissue, DC-B7-H1 supports peripheral tolerance, limiting randomly arising autoaggressive lymphocytes and their inflammatory tissue damage (10,11). In tumors, expression of B7-H1 contributes to immune evasion, inducing anergy or apoptosis of tumor-specific T-cells (12–14). Consistently with an inhibitory role, treatment of NOD mice with blocking antibodies to either PD-1 or B7-H1 accelerates diabetes (15), with analogous scenarios in autoimmune (16) and other (12,17,18) models. These systemic manipulations of the PD-1/B7-H1 axis generated the consensus view that B7-H1 ligation keeps potentially damaging autoimmune T-cells in check and serves to downregulate lymphoid effector functions (19).However, conflicting data exist. The B7-H1 pathway can promote T-cell activation and autoimmunity in certain experimental settings, including transgenic expression of B7-H1 in β-cells of C57Bl/six mice (20–22). For these exceptions, an alternative receptor for B7-H1 has been proposed but not identified to date (23,24). We nevertheless felt that the weight of evidence, specifically in NOD mice, suggested that B7-H1 might serve as a tool to selectively suppress NOD pSC autoimmunity, allowing us to learn whether and how pSC cells impact on the β-cell autoimmune progression program: transgenic expression of B7-H1 in NOD β-cells protects from type 1 diabetes (19). We here describe the effects of a pSC B7-H1 transgene. Our finding of type 1 diabetes acceleration emphasizes the complexity of this costimulatory pathway, while the selective, intraislet CD8⁺ bias of high-affinity T-cells demonstrates that pSC cells do impact the β-cell destruction program, culminating in type 1 diabetes.     

Hepatic stearoyl-CoA desaturase (SCD)-1 activity and diacylglycerol but not ceramide concentrations are increased in the nonalcoholic human fatty liver

OBJECTIVE—To determine whether 1) hepatic ceramide and diacylglycerol concentrations, 2) SCD1 activity, and 3) hepatic lipogenic index are increased in the human nonalcoholic fatty liver.RESEARCH DESIGN AND METHODS—We studied 16 subjects with (n = 8) and without (n = 8) histologically determined nonalcoholic fatty liver (NAFL⁺ and NAFL⁻) matched for age, sex, and BMI. Hepatic concentrations of lipids and fatty acids were quantitated using ultra-performance liquid chromatography coupled to mass spectrometry and gas chromatography.RESULTS—The absolute (nmol/mg) hepatic concentrations of diacylglycerols but not ceramides were increased in the NAFL⁺ group compared with the NAFL⁻ group. The livers of the NAFL⁺ group contained proportionally less long-chain polyunsaturated fatty acids as compared with the NAFL⁻ group. Liver fat percent was positively related to hepatic stearoyl-CoA desaturase 1 (SCD1) activity index (r = 0.70, P = 0.003) and the hepatic lipogenic index (r = 0.54, P = 0.030). Hepatic SCD1 activity index was positively related to the concentrations of diacylglycerols (r = 0.71, P = 0.002) but not ceramides (r = 0.07, NS).CONCLUSIONS—We conclude that diacylglycerols but not ceramides are increased in NAFL. The human fatty liver is also characterized by depletion of long polyunsaturated fatty acids in the liver and increases in hepatic SCD1 and lipogenic activities.Nonalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver (≥10% of liver weight), which cannot be attributed to alcohol consumption or any other liver disease (1). NAFLD covers a range from simple nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) and fibrosis (1). The fatty liver is resistant to the action of insulin to inhibit hepatic glucose (2,3) and VLDL (4) production, resulting in hyperglycemia and hypertriglyceridemia. The mechanisms underlying insulin resistance in human NAFLD are unclear. While triacylglycerols themselves are inert, lipid intermediates may act as important regulators of both oxidative stress (5) and insulin signaling (6). In vitro studies as well as studies in animals suggest that diacylglycerols, which are immediate precursors of triacylglycerols (7), can induce insulin resistance by activating specific isoforms of protein kinase C (PKC) (8,9). The concentrations of diacylglycerols have recently been shown to be increased in human NAFLD compared with subjects with normal liver histology (10). Ceramides are another class of reactive lipids that mediate saturated fat–induced insulin resistance (6). There are no data comparing ceramide and diacylglycerol concentrations in the human liver or relating them to hepatic fat content.Sources of hepatic lipids include dietary chylomicron remnants, free fatty acids released from either adipose tissue triacylglycerols or chylomicrons hydrolyzed at a rate in excess of what can be taken up by tissues (spillover), and de novo lipogenesis (11). Increased lipolysis is a major contributor to hepatic fat accumulation (12–14). In addition, when estimated using tracer techniques, de novo lipogenesis has been found to be significantly increased in subjects with NAFLD compared with normal subjects (12,15,16). De novo lipogenesis produces saturated fatty acids (17,18). Stearoyl-CoA desaturase 1 (SCD1) converts saturated fatty acids to monounsaturated fatty acids, which are major substrates for synthesis of triacylglycerols and other lipids (19). SCD1 knockout mice are resistant to the development of obesity and hepatic steatosis (20,21), whereas the activity of SCD1 is significantly increased in the fatty livers of ob/ob mice (20,22). These data thus suggest that hepatic SCD1 activity may contribute to lipid accumulation in NAFLD. There are, however, no data on hepatic SCD1 activity in human NAFLD.To address the above questions, we quantified the full range of lipids and fatty acids using ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry (MS) and gas chromatography in the human liver. These analyses were performed in two groups of subjects matched for age, sex, and BMI but with either a normal liver fat content (≤10% macrovesicular steatosis) or a nonalcoholic fatty liver (NAFL) (≥20% macrovesicular steatosis [1]).     

Increased expression of CCL2 in insulin-producing cells of transgenic mice promotes mobilization of myeloid cells from the bone marrow, marked insulitis, and diabetes

OBJECTIVE—To define the mechanisms underlying the accumulation of monocytes/macrophages in the islets of Langerhans.RESEARCH DESIGN AND METHODS—We tested the hypothesis that macrophage accumulation into the islets is caused by overexpression of the chemokine CCL2. To test this hypothesis, we generated transgenic mice and evaluated the cellular composition of the islets by immunohistochemistry and flow cytometry. We determined serum levels of CCL2 by enzyme-linked immunosorbent assay, determined numbers of circulating monocytes, and tested whether CCL2 could mobilize monocytes from the bone marrow directly. We examined development of diabetes over time and tested whether CCL2 effects could be eliminated by deletion of its receptor, CCR2.RESULTS—Expression of CCL2 by β-cells was associated with increased numbers of monocytes in circulation and accumulation of macrophages in the islets of transgenic mice. These changes were promoted by combined actions of CCL2 at the level of the bone marrow and the islets and were not seen in animals in which the CCL2 receptor (CCR2) was inactivated. Mice expressing higher levels of CCL2 in the islets developed diabetes spontaneously. The development of diabetes was correlated with the accumulation of large numbers of monocytes in the islets and did not depend on T- and B-cells. Diabetes could also be induced in normoglycemic mice expressing low levels of CCL2 by increasing the number of circulating myeloid cells.CONCLUSIONS—These results indicate that CCL2 promotes monocyte recruitment by acting both locally and remotely and that expression of CCL2 by insulin-producing cells can lead to insulitis and islet destruction.Type 1 diabetes is a chronic inflammatory disorder characterized by infiltration of the islets of Langerhans by mononuclear cells and autoimmune destruction of insulin-producing β-cells (1,2). Several lines of evidence suggest that macrophages play a role in the development of diabetes. Macrophages are the first cells that appear within the islets of NOD mice (3) and are also implicated in late phases of disease development (4). Administration of clodronate-loaded liposomes, which leads to disappearance of macrophages from the endocrine pancreas and periphery of NOD mice, delays the onset of diabetes (5). Moreover, macrophage depletion inhibits the development of β-cell–cytotoxic T-cells and prevents autoimmune diabetes (6). It has been suggested that the presentation of self-antigens to autoreactive T-cells by dendritic cells and macrophages recruited and activated by transgenic tumor necrosis factor-α (TNF-α) expression accounts for the development of diabetes (7). Macrophages and dendritic cells are found within islets from recent-onset type 1 diabetic patients (8).Tissue macrophages originate from monocytes produced in the bone marrow. Current studies suggest that bone marrow–derived monocytes give rise to two subsets of peripheral blood monocytes (9). One subset (GR-1⁻, CX3CR1^high, CCR2⁻, and CCL62L⁻ monocytes) gives origin to tissue macrophages (splenic macrophages, Kupffer cells, alveolar macrophages, microglia, and osteoclasts). The second subset (GR-1⁺, CX3CR1^low, CCR2⁺, and CD62L⁺ monocytes) is preferentially recruited to inflamed tissues and gives rise to macrophages and dendritic cells. This inflammatory subset also expresses CD115 (granulocyte macrophage colony–stimulating factor receptor) and Ly6C (10,11).Several studies implicate the chemokine CCL2 in monocyte recruitment in vivo (12). CCL2 promotes recruitment of monocytes, macrophages, dendritic cells, and activated T-cells via its receptor, CCR2 (13). Numerous cell types, including fibroblasts, endothelial cells, epithelial cells, leukocytes, and smooth muscle cells express CCL2 in the presence of serum or specific stimuli (14,15). In addition to chemotaxis, CCL2 contributes to activation of monocytes and macrophages because CCL2 induces production of TNF-α and interleukin-1β in murine peritoneal macrophages (16). Macrophage recruitment caused by CCL2 expression has been strongly linked to several inflammatory conditions, such as atherosclerosis (17), development of intimal hyperplasia after arterial injury (18), obesity, and insulin resistance (19).CCL2 expression has also been related to diabetes. Primary cultures of murine and human pancreatic islets express and secrete CCL2 (20). CCL2 expression has been detected in islets of NOD mice during cyclophosphamide treatment (21), and CCL2 expression parallels disease progression in NOD mice (22,23). Low-level secretion of CCL2 by islets before transplantation is associated with a higher rate of insulin independence, suggesting an important role for CCL2 in the clinical outcome of islet transplantation in patients with type 1 diabetes (24).Our laboratory and others have shown that CCL2 expression by insulin-producing cells induces the accumulation of macrophages in the islets of transgenic mice (25,26). We have also found that transgenic expression of CCL2 induces migration of dendritic cells to the islets and that the number of inflammatory cells recruited is dependent on the levels of CCL2 produced by the β-cells (26). The mechanisms controlling accumulation of monocytes in nonlymphoid tissues in response to increased levels of CCL2 have not been examined. It is unclear whether the accumulation of monocytes in inflamed tissues in response to CCL2 reflects increased recruitment of circulating monocytes or monocyte precursors, changes in the number of circulating monocytes, or both. Finally, it is unclear whether changes in local levels of CCL2 may promote development of diabetes.Here, we show that transgenic expression of CCL2 by insulin-producing cells is associated with changes in the number of monocytes in circulation. The monocytes are mobilized from the bone marrow in a CCR2-dependent mechanism. We also found spontaneous development of diabetes in two of the four transgenic lines expressing the highest levels of CCL2 in the pancreas. The development of diabetes required the accumulation of a large number of monocytes in the islets and did not depend on T- or B-cells. Animals expressing low levels of CCL2 in the islets did not spontaneously develop diabetes but became diabetic if the number of circulating monocytes was increased. These results indicate that CCL2 promotes monocyte recruitment by acting both locally and remotely and that dysregulated expression of CCL2 by insulin-producing cells can lead to insulitis and islet destruction.     

Fatty acid synthase inhibitors modulate energy balance via mammalian target of rapamycin complex 1 signaling in the central nervous system

OBJECTIVE—Evidence links the hypothalamic fatty acid synthase (FAS) pathway to the regulation of food intake and body weight. This includes pharmacological inhibitors that potently reduce feeding and body weight. The mammalian target of rapamycin (mTOR) is an intracellular fuel sensor whose activity in the hypothalamus is also linked to the regulation of energy balance. The purpose of these experiments was to determine whether hypothalamic mTOR complex 1 (mTORC1) signaling is involved in mediating the effects of FAS inhibitors.RESEARCH DESIGN AND METHODS—We measured the hypothalamic phosphorylation of two downstream targets of mTORC1, S6 kinase 1 (S6K1) and S6 ribosomal protein (S6), after administration of the FAS inhibitors C75 and cerulenin in rats. We evaluated food intake in response to FAS inhibitors in rats pretreated with the mTOR inhibitor rapamycin and in mice lacking functional S6K1 (S6K1^−/−). Food intake and phosphorylation of S6K1 and S6 were also determined after C75 injection in rats maintained on a ketogenic diet.RESULTS—C75 and cerulenin increased phosphorylation of S6K1 and S6, and their anorexic action was reduced in rapamycin-treated rats and in S6K1^−/− mice. Consistent with our previous findings, C75 was ineffective at reducing caloric intake in ketotic rats. Under ketosis, C75 was also less efficient at stimulating mTORC1 signaling.CONCLUSIONS—These findings collectively indicate an important interaction between the FAS and mTORC1 pathways in the central nervous system for regulating energy balance, possibly via modulation of neuronal glucose utilization.Energy balance is achieved when caloric intake is matched to expenditure. A complex neuroendocrine system underlies this process and regulates energy homeostasis in mammals. In addition to sensing hormonal signals of stored fuels, such as leptin (1), specific populations of neurons in the central nervous system (CNS), and particularly within the hypothalamus, have the ability to sense locally available nutrients, including glucose (2), fatty acids (3), and amino acids (4,5).Recent evidence has highlighted the role of intracellular fuel sensing in the regulation of energy balance (6). In particular, the biochemical pathway underlying fatty acid metabolism has been involved in the regulation of both feeding and glucose homeostasis (6–10). Fatty acid synthase (FAS) catalyzes the condensation of malonyl-CoA and acetyl-CoA to generate long-chain fatty acids (LCFAs). Acetyl-CoA carboxylase (ACC) and FAS are expressed in the hypothalamus (7), where malonyl-CoA (11) and LCFA-CoA levels (8) decrease during fasting and increase after refeeding. Studies using FAS inhibitors and other pharmacological or genetic tools that modify the activity of different enzymes regulating fatty acid metabolism support a role for this pathway in the regulation of feeding (9,12–15).Peripheral administration of the natural FAS inhibitor cerulenin (2,3-epoxy-4-oxo-6-dodecadienoylamide) or the synthetic inhibitor C75 (trans-4-carboxy-5-octyl-3-methylenebutyrolactone) causes profound dose-dependent anorexia and weight loss in several rodent models (12,13,15,16). Reduced food intake is also observed with central administration of much lower doses of C75, suggesting that the brain is the key site of action (12). Increased hypothalamic malonyl-CoA is necessary for the anorexic and weight-reducing effects of FAS inhibitors (9,15,17). Interestingly, the ability of leptin to reduce food intake depends on increased hypothalamic malonyl-CoA (8) and possibly palmitoyl-CoA (18), which are achieved through the concomitant inhibition of AMP-activated protein kinase (AMPK) and activation of ACC (18). Therefore, the hypothalamic fatty acid synthesis pathway appears to process different fuel signals and convert them into efferent outputs that prevent further consumption of nutrients.The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that controls critical aspects of cell growth (19). mTOR is a component of at least two multiprotein complexes: mTOR complex 1 (mTORC1), which includes raptor, and mTOR complex 2 (mTORC2), which includes rictor. Whereas mTORC2 regulates phosphorylation of Akt, mTORC1 modulates the activity of S6 kinase 1 (S6K1) and 4E binding protein 1 (20). Notably, the phosphorylation of S6K1 at Thr 389 is one of the markers commonly used to evaluate mTORC1 activity in vivo (21). Insulin, IGF, amino acids, and glucose all activate intracellular cascades that lead to activation of mTORC1 (22). We reported that the anorexic action of central leptin and leucine are both dependent on the activation of mTORC1 signaling in the hypothalamus (4). Given the ability of mTORC1 to sense and integrate fuel signals, and the role it plays in controlling food intake (4), we hypothesized that mTORC1 signaling is involved in monitoring the biochemical changes induced by the modulation of hypothalamic fatty acid metabolism.     

Inducible overexpression of sFlt-1 in podocytes ameliorates glomerulopathy in diabetic mice

OBJECTIVE—Podocyte-specific, doxycycline (DOX)-inducible overexpression of soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1) in adult mice was used to investigate the role of the VEGF-A/VEGF receptor (VEGFR) system in diabetic glomerulopathy.RESEARCH DESIGN AND METHODS—We studied nondiabetic and diabetic transgenic mice and wild-type controls treated with vehicle (VEH) or DOX for 10 weeks. Glycemia was measured by a glucose-oxidase method and blood pressure by a noninvasive technique. sFlt-1, VEGF-A, VEGFR2, and nephrin protein expression in renal cortex were determined by Western immunoblotting; urine sFlt-1, urine free VEGF-A, and albuminuria by enzyme-linked immunosorbent assay; glomerular ultrastructure by electron microscopy; and VEGFR1 and VEGFR2 cellular localization with Immunogold techniques.RESULTS—Nondiabetic DOX-treated transgenic mice showed a twofold increase in cortex sFlt-1 expression and a fourfold increase in sFlt-1 urine excretion (P < 0.001). Urine free VEGF-A was decreased by 50%, and cortex VEGF-A expression was upregulated by 30% (P < 0.04). VEGFR2 expression was unchanged, whereas its activation was reduced in DOX-treated transgenic mice (P < 0.02). Albuminuria and glomerular morphology were similar among groups. DOX-treated transgenic diabetic mice showed a 60% increase in 24-h urine sFlt-1 excretion and an ∼70% decrease in urine free VEGF-A compared with VEH-treated diabetic mice (P < 0.04) and had lower urine albumin excretion at 10 weeks than VEH-treated diabetic (d) mice: d-VEH vs. d-DOX, geometric mean (95% CI), 117.5 (69–199) vs. 43 (26.8–69) μg/24 h (P = 0.003). Diabetes-induced mesangial expansion, glomerular basement membrane thickening, podocyte foot-process fusion, and transforming growth factor-β1 expression were ameliorated in DOX-treated diabetic animals (P < 0.05). Diabetes-induced VEGF-A and nephrin expression were not affected in DOX-treated mice.CONCLUSIONS—Podocyte-specific sFlt-1 overexpression ameliorates diabetic glomerular injury, implicating VEGF-A in the pathogenesis of this complication.Vascular endothelial growth factor (VEGF)-A is constitutively expressed in glomerular visceral cells (podocytes). Paracrine VEGF-A signaling occurs between podocytes and adjacent endothelial and mesangial cells, which express VEGF receptors (VEGFRs) 1 and 2 (1–3), and both autocrine and paracrine signaling may occur in podocytes themselves (4).VEGF-A has been implicated in the regulation of glomerular barrier properties to protein filtration. In normal animals (5,6) and in cancer patients (7), inhibition of VEGF-A results in proteinuria; while in proteinuric conditions, which are associated with glomerular VEGF-A upregulation such as diabetes, systemic inhibition of VEGF ameliorates albuminuria (8–10). This evidence suggests that a tight regulation of VEGF-A expression level is required to maintain the physiological permselective properties of the glomerular filter.The results of previous studies conducted using either VEGF gene targeting techniques (5) or by administration of inhibitory agents of the VEGF/VEGFR system such as antibodies (6,8,9) or chemicals (10) may have been affected by potential interference with animal organ development and lack of tissue specificity in the mechanisms of action of systemic inhibitors. Soluble VEGF receptor-1 (sFlt-1), a splice variant of the VEGFR1, lacks the transmembrane and complete intracellular tyrosine kinase domain of VEGFR1 but binds to VEGF with the same affinity and specificity as that of the full-length receptor (11,12) and has potent and selective VEGF inhibitory action (11). sFlt-1 acts in two major ways: it can sequester VEGF competing for its binding to the VEGF receptors or can form heterodimers with the extracellular region of the membrane spanning VEGFR1 and VEGFR2, thus inhibiting the activation of downstream signaling pathways (11,12).To target the action of the podocyte-expressed VEGF-A, we developed a transgenic mouse model to overexpress sFlt-1 specifically at the podocyte level with an inducible expression system that is induced only after complete development, in the adult animal, by the administration of doxycycline (Tet-on). The aim of this study was to investigate the role of VEGF-A upregulation in the pathogenesis of diabetic glomerulopathy by locally inhibiting podocyte-expressed VEGF-A activity.     

PDGF-C is a proinflammatory cytokine that mediates renal interstitial fibrosis

PDGF-C is a potent mitogen for fibroblasts in vitro. Transgenic PDGF-C overexpression in the heart or liver induces organ fibrosis, and PDGF-C expression is upregulated at sites of interstitial fibrosis in human and rat kidneys; however, the effect of inhibiting PDGF-C on the development of renal fibrosis in vivo is unknown. Renal fibrosis was induced in C57BL/6 mice by unilateral ureteral obstruction (UUO), and then mice were treated with neutralizing anti–PDGF-C antiserum or nonspecific IgG. An increase in PDGF-C expression was observed in fibrotic areas after UUO, contributed in large part by infiltrating macrophages. Treatment with anti–PDGF-C reduced renal fibrosis by 30% at day 5 and reduced interstitial myofibroblast accumulation by 57%. In vitro, PDGF-C was a potent mitogen for renal fibroblasts and induced chemokine expression. In vivo, anti–PDGF-C treatment produced a decrease in the expression of the renal chemokines CCL2 and CCL5 (85 and 67% reductions, respectively), accompanied by a significant decrease in leukocyte infiltration and CCR2 mRNA expression. Further supporting a role of PDGF-C in renal fibrosis, PDGF-C^−/− mice demonstrated a reduction in fibrosis and leukocyte infiltration in response to UUO compared with wild-type littermates. In conclusion, specific neutralization or lack of PDGF-C reduces the development of renal inflammation and fibrosis in obstructed mouse kidneys. Leukocyte-derived PDGF-C induces chemokine expression, which may lead to the recruitment of additional leukocytes, creating an amplification loop for renal inflammation and fibrosis.PDGF-C is a recently identified cytokine that acts via the PDGF-α receptor and is a potent mitogen for human fibroblasts and vascular smooth muscle cells in vitro.^1,2 Observations in different organs suggest that PDGF-C plays an important role in the regulation of fibrosis. First evidence was derived from a mouse model in which transgenic overexpression of PDGF-C in the heart induced fibroblast proliferation, resulting in cardiac fibrosis, hypertrophy, and ultimately cardiomyopathy.^1,3 Transgenic mice with liver-specific PDGF-C overexpression of the transgene developed liver fibrosis and hepatocellular carcinoma.⁴ Finally, transgenic mice with a lung-specific PDGF-C overexpression developed massive mesenchymal cell hyperplasia and died from respiratory insufficiency immediately after birth.⁵ Increased PDGF-C expression has additionally been observed in different experimental models of organ fibrosis in heart and lung tissues.^6,7Additional functions of PDGF-C include the stimulation of angiogenesis^8,9 and acceleration of wound-healing processes.² A dynamic expression of PDGF-C during organogenesis in mice and humans points to a critical role of this growth factor in organ development,^10,11 which is further substantiated by studies in PDGF-C^−/− mice that die from feeding and respiratory difficulties in the perinatal period when grown on an SV129 background.¹² The phenotype of these PDGF-C^−/− mice is characterized by defective tube formation (facial clefts, spina bifida occulta), subepidermal blisters, and deficiency of connective tissues in the dorsal midline.¹²Little is known about the functional role of PDGF-C within the kidney. We recently identified a constitutive expression of PDGF-C within arterial vascular smooth muscle cells and within collecting duct cells in rat and human kidney.^13,14 We detected a de novo expression and/or overexpression of PDGF-C in fibrotic interstitial areas in different rat models of renal disease as well as in diseased human renal biopsy tissues.^13,14 We therefore hypothesized that PDGF-C plays a functional role in the pathogenesis of tubulointerstitial fibrosis. We subsequently studied the potential profibrotic role of PDGF-C both by using a neutralizing anti–PDGF-C antiserum in murine renal fibrosis induced by unilateral ureteral obstruction (UUO) and by inducing renal fibrosis in PDGF-C^−/− mice.     

Dipeptidyl Peptidase IV Inhibition With MK0431 Improves Islet Graft Survival in Diabetic NOD Mice Partially via T-Cell Modulation

OBJECTIVE—The endopeptidase dipeptidyl peptidase-IV (DPP-IV) has been shown to NH₂-terminally truncate incretin hormones, glucose-dependent insulinotropic polypeptide, and glucagon-like peptide-1, thus ablating their ability to potentiate glucose-stimulated insulin secretion. Increasing the circulating levels of incretins through administration of DPP-IV inhibitors has therefore been introduced as a therapeutic approach for the treatment of type 2 diabetes. DPP-IV inhibitor treatment has also been shown to preserve islet mass in rodent models of type 1 diabetes. The current study was initiated to define the effects of the DPP-IV inhibitor sitagliptin (MK0431) on transplanted islet survival in nonobese diabetic (NOD) mice, an autoimmune type 1 diabetes model.RESEARCH DESIGN AND METHODS—Effects of MK0431 on islet graft survival in diabetic NOD mice were determined with metabolic studies and micropositron emission tomography imaging, and its underlying molecular mechanisms were assessed.RESULTS—Treatment of NOD mice with MK0431 before and after islet transplantation resulted in prolongation of islet graft survival, whereas treatment after transplantation alone resulted in small beneficial effects compared with nontreated controls. Subsequent studies demonstrated that MK0431 pretreatment resulted in decreased insulitis in diabetic NOD mice and reduced in vitro migration of isolated splenic CD4⁺ T-cells. Furthermore, in vitro treatment of splenic CD4⁺ T-cells with DPP-IV resulted in increased migration and activation of protein kinase A (PKA) and Rac1.CONCLUSIONS—Treatment with MK0431 therefore reduced the effect of autoimmunity on graft survival partially by decreasing the homing of CD4⁺ T-cells into pancreatic β-cells through a pathway involving cAMP/PKA/Rac1 activation.The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), exert a number of actions that improve glucose homeostasis, including potentiation of glucose-stimulated insulin secretion (GSIS), promotion of β-cell proliferation and survival, and inhibition of glucagon secretion (1–7). Because GIP and GLP-1 are rapidly degraded by the endopeptidase dipeptidyl peptidase IV (DPP-IV; CD26), it has not been possible to directly take advantage of their beneficial actions for the treatment of type 2 diabetes (8,9). Therefore, a number of strategies have been explored to circumvent this problem, including the development of small molecule (10,11) and DPP-IV–resistant peptide (12,13) incretin receptor agonists, and DPP-IV inhibitors (14,15). Members of two classes of compounds have recently been approved by the Food and Drug Administration as type 2 diabetes therapeutics: the DPP-IV–resistant GLP-1 receptor agonist (incretin mimetic) exenatide (Byetta) and the DPP-IV inhibitor sitagliptin (Januvia).Although DPP-IV inhibitors have been extensively studied in treatment of type 2 diabetes, little is known about their potential for treatment of type 1 diabetes. Infusion of GLP-1 was shown to reduce glycemic excursions in type 1 diabetic patients, and this result was attributed to reduced glucagon levels and delayed gastric emptying (16,17). In preclinical studies, the DPP-IV inhibitor isoleucine thiazolidide was shown to improve glucose tolerance in both streptozotocin (STZ)-induced (18,19) and BioBreeding (BB) (19) diabetic rats, associated with increased β-cell survival and possibly islet neogenesis (18). Additionally, we recently showed that the DPP-IV inhibitor MK0431 prolonged islet graft survival in STZ-induced diabetic mice (20). In the current study, we show that MK0431 pretreatment resulted in the prolongation of islet graft survival in an autoimmune type 1 diabetes model, the nonobese diabetic (NOD) mouse, through a mechanism that includes modulation of CD4⁺ T-cell migration.