Similarities between bacterial GAD and human GAD65: Implications in gut mediated autoimmune type 1 diabetes.

A variety of islet autoantibodies (AAbs) can predict and possibly dictate eventual type 1 diabetes (T1D) diagnosis. Upwards of 75% of those with T1D are positive for AAbs against glutamic acid decarboxylase (GAD65 or GAD), a producer of gamma-aminobutyric acid (GABA) in human pancreatic beta cells. Interestingly, bacterial populations within the human gut also express GAD and produce GABA. Evidence suggests that dysbiosis of the microbiome may correlate with T1D pathogenesis and physiology. Therefore, autoimmune linkages between the gut microbiome and islets susceptible to autoimmune attack need to be further elucidated. Utilizing in silico analyses, we show that 25 GAD sequences from human gut bacterial sources show sequence and motif similarities to human beta cell GAD65. Our motif analyses determined that most gut GAD sequences contain the pyroxical dependent decarboxylase (PDD) domain of human GAD65, which is important for its enzymatic activity. Additionally, we showed overlap with known human GAD65 T cell receptor epitopes, which may implicate the immune destruction of beta cells. Thus, we propose a physiological hypothesis in which changes in the gut microbiome in those with T1D result in a release of bacterial GAD, thus causing miseducation of the host immune system. Due to the notable similarities we found between human and bacterial GAD, these deputized immune cells may then target human beta cells leading to the development of T1D.

Simvastatin mitigates streptozotocin-induced type 1 diabetes in mice through downregulation of ADAM10 and ADAM17.

BACKGROUND: T cell mediates immune response in type 1 diabetes mellitus (T1DM) through its trafficking into pancreatic islets. The role of A Disintigrin And Metalloproteinase 10 (ADAM10) and 17 (ADAM17) in pancreatic T-cells recruitment into the pancreatic islets during T1DM is not known. AIM: Explore the role of ADAM10 and ADAM17 in the processing of CXCL16 in T1DM and possible protective effect of simvastatin (SIM) in streptozotocin (STZ)-induced T1DM. MAIN METHODS: Balb/c mice were classified into 4 groups, 10 each. Control group received buffer while SIM group received 50 mg/kg, i.p daily for 12 days starting from day 4 of the experiment. Diabetic group; received STZ (55 mg/kg, i.p.) for 5 consecutive days starting from day 1 of the experiment. SIM + STZ group; received SIM (50 mg/kg, i.p.) daily for 12 days and STZ (55 mg/kg, i.p.) for 5 consecutive days. Biochemical, inflammatory and apoptotic markers as well as expression of CXCL16, ADAM10, NF-κB and pancreatic T-cells expression were analyzed. KEY FINDINGS: Significant increase in biochemical, inflammatory, apoptotic parameters, expression of ADAM10, ADAM17, CXCL16, NF-κB, and infiltrated T-cells to the pancreatic islets were found in STZ group. SIM treatment in the presence of STZ improved biochemical and inflammatory parameters as well as it reduced the expression of CXCL16, ADAM10, ADAM17, NF-κΒ, T-cells migration and apoptosis in the pancreatic islets. SIGNIFICANCE: SIM mitigated pancreatic ß-cell death induced by STZ through down regulation of ADAM10, ADAM17and CXCL16. Therefore, ADAM10/ADAM17 and CXCL16 may serve as novel therapeutic targets for T1DM.

Role of Innate Immunity and Oxidative Stress in the Development of Type 1 Diabetes Mellitus. Peroxiredoxin 6 as a New Anti-Diabetic Agent.

The review discusses information on the development of type 1 diabetes mellitus (T1D) as a systemic autoimmune and inflammatory disease. Focus of the review is on the role of innate immune system, including activation of some signaling cascades, cytokine response, and activity of the Toll-like receptors in the development of T1D. Dysfunction of innate immunity is the cause of the attack of pancreatic beta cells by the host T-lymphocytes, which leads to the death of pancreatic beta cells that produce insulin. Lack of insulin causes hyperglycemia and the need for lifelong injections of insulin in patients with T1D, which, nevertheless, does not exclude damage to many organs and tissues, given particular vulnerability of the blood vessels under conditions of hyperglycemia. The review discusses the role of oxidative stress as a factor that plays a major role in damage of vascular system and pancreatic tissue during the development of T1D. Considering high sensitivity of pancreatic beta cells to the action of reactive oxygen species (ROS), the possibility of using antioxidants for reducing the level of pathological consequences in the course of T1D development is discussed. New information on anti-diabetic activity of the exogenous antioxidant enzyme peroxiredoxin 6, which is capable of penetrating cells, activating insulin production in beta cells, reducing ROS levels, as well as decreasing activation of some signaling cascades, production of pro-inflammatory cytokines, and expression of Toll-like receptors in beta cells and in immune cells during T1D development is discussed.

ALDH2/SIRT1 Contributes to Type 1 and Type 2 Diabetes-Induced Retinopathy through Depressing Oxidative Stress.

Clinical observations found vision-threatening diabetic retinopathy (DR) occurs in both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) patients, but T1DM may perform more progressive retinal abnormalities at the same diabetic duration with or without clinical retinopathy. In the present study, T1DM and T2DM patients without manifestations of DR were included in our preliminary clinical retrospective observation study to investigate the differentiated retinal function at the preclinical stage. Then, T1DM and T2DM rat models with 12-week diabetic duration were constructed to explore the potential mechanism of the discrepancy in retinal disorders. Our data demonstrated T1DM patients presented a poor retinal function, a higher allele frequency for ALDH2GA/AA, and a depressed aldehyde dehydrogenase 2 (ALDH2) activity and silent information regulator 1 (SIRT1) level, compared to T2DM individuals. In line with this, higher amplitudes of neurovascular function-related waves of electroretinograms were found in T2DM rats. Furthermore, the retinal outer nuclear layers were reduced in T1DM rats. The levels of retinal oxidative stress biomarkers including total reactive oxygen species, NADPH oxidase 4 and mitochondrial DNA damage, and inflammatory indicators covering inducible/endothelial nitric acid synthase ratio, interleukin-1, and interleukin-6 were obviously elevated. Notably, the level of retinal ALDH2 and SIRT1 in T1DM rats was significantly diminished, while the expression of neovascularization factors was dramatically enhanced compared to T2DM. Together, our data indicated that the ALDH2/SIRT1 deficiency resulted in prominent oxidative stress and was in association with DR progression. Moreover, a differentiating ALDH2/SIRT1 expression may be responsible for the dissimilar severity of DR pathological processes in chronic inflammatory-related T1DM and T2DM.

MERTK on mononuclear phagocytes regulates T cell antigen recognition at autoimmune and tumor sites.

Understanding mechanisms of immune regulation is key to developing immunotherapies for autoimmunity and cancer. We examined the role of mononuclear phagocytes during peripheral T cell regulation in type 1 diabetes and melanoma. MERTK expression and activity in mononuclear phagocytes in the pancreatic islets promoted islet T cell regulation, resulting in reduced sensitivity of T cell scanning for cognate antigen in prediabetic islets. MERTK-dependent regulation led to reduced T cell activation and effector function at the disease site in islets and prevented rapid progression of type 1 diabetes. In human islets, MERTK-expressing cells were increased in remaining insulin-containing islets of type 1 diabetic patients, suggesting that MERTK protects islets from autoimmune destruction. MERTK also regulated T cell arrest in melanoma tumors. These data indicate that MERTK signaling in mononuclear phagocytes drives T cell regulation at inflammatory disease sites in peripheral tissues through a mechanism that reduces the sensitivity of scanning for antigen leading to reduced responsiveness to antigen.

Inactivation of cysteine 674 in the sarcoplasmic/endoplasmic reticulum calcium ATPase 2 causes retinopathy in the mouse.

Diabetic retinopathy is a multifactorial microvascular complication, and its pathogenesis hasn't been fully elucidated. The irreversible oxidation of cysteine 674 (C674) in the sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) was increased in the type 1 diabetic retinal vasculature. SERCA2 C674S knock-in (SKI) mouse line that half of C674 was replaced by serine 674 (S674) was used to study the effect of C674 inactivation on retinopathy. Compared with wild type (WT) mice, SKI mice had increased number of acellular capillaries and pericyte loss similar to those in type 1 diabetic WT mice. In the retina of SKI mice, pro-apoptotic proteins and intracellular Ca2+-dependent signaling pathways increased, while anti-apoptotic proteins and vessel density decreased. In endothelial cells, C674 inactivation increased the expression of pro-apoptotic proteins, damaged mitochondria, and induced cell apoptosis. These results suggest that a possible mechanism of retinopathy induced by type 1 diabetes is the interruption of calcium homeostasis in the retina by oxidation of C674. C674 is a key to maintain retinal health. Its inactivation can cause retinopathy similar to type 1 diabetes by promoting apoptosis. SERCA2 might be a potential target for the prevention and treatment of diabetic retinopathy.

CDK11 Promotes Cytokine-Induced Apoptosis in Pancreatic Beta Cells Independently of Glucose Concentration and Is Regulated by Inflammation in the NOD Mouse Model.

Background: Pancreatic islets are exposed to strong pro-apoptotic stimuli: inflammation and hyperglycemia, during the progression of the autoimmune diabetes (T1D). We found that the Cdk11(Cyclin Dependent Kinase 11) is downregulated by inflammation in the T1D prone NOD (non-obese diabetic) mouse model. The aim of this study is to determine the role of CDK11 in the pathogenesis of T1D and to assess the hierarchical relationship between CDK11 and Cyclin D3 in beta cell viability, since Cyclin D3, a natural ligand for CDK11, promotes beta cell viability and fitness in front of glucose. Methods: We studied T1D pathogenesis in NOD mice hemideficient for CDK11 (N-HTZ), and, in N-HTZ deficient for Cyclin D3 (K11HTZ-D3KO), in comparison to their respective controls (N-WT and K11WT-D3KO). Moreover, we exposed pancreatic islets to either pro-inflammatory cytokines in the presence of increasing glucose concentrations, or Thapsigargin, an Endoplasmic Reticulum (ER)-stress inducing agent, and assessed apoptotic events. The expression of key ER-stress markers (Chop, Atf4 and Bip) was also determined. Results: N-HTZ mice were significantly protected against T1D, and NS-HTZ pancreatic islets exhibited an impaired sensitivity to cytokine-induced apoptosis, regardless of glucose concentration. However, thapsigargin-induced apoptosis was not altered. Furthermore, CDK11 hemideficiency did not attenuate the exacerbation of T1D caused by Cyclin D3 deficiency. Conclusions: This study is the first to report that CDK11 is repressed in T1D as a protection mechanism against inflammation-induced apoptosis and suggests that CDK11 lies upstream Cyclin D3 signaling. We unveil the CDK11/Cyclin D3 tandem as a new potential intervention target in T1D.

Macrophage inflammatory state in Type 1 diabetes: triggered by NLRP3/iNOS pathway and attenuated by docosahexaenoic acid.

Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease characterized by insulin-producing pancreatic ß-cell destruction and hyperglycemia. While monocytes and NOD-like receptor family-pyrin domain containing 3 (NLRP3) are associated with T1D onset and development, the specific receptors and factors involved in NLRP3 inflammasome activation remain unknown. Herein, we evaluated the inflammatory state of resident peritoneal macrophages (PMs) from genetically modified non-obese diabetic (NOD), NLRP3-KO, wild-type (WT) mice and in peripheral blood mononuclear cells (PBMCs) from human T1D patients. We also assessed the effect of docosahexaenoic acid (DHA) on the inflammatory status. Macrophages from STZ-induced T1D mice exhibited increased inflammatory cytokine/chemokine levels, nitric oxide (NO) secretion, NLRP3 and iNOS protein levels, and augmented glycolytic activity compared to control animals. In PMs from NOD and STZ-induced T1D mice, DHA reduced NO production and attenuated the inflammatory state. Furthermore, iNOS and IL-1ß protein expression levels and NO production were lower in the PMs from diabetic NLRP3-KO mice than from WT mice. We also observed increased IL-1ß secretion in PBMCs from T1D patients and immortalized murine macrophages treated with advanced glycation end products and palmitic acid. The present study demonstrated that the resident PMs are in a proinflammatory state characterized by increased NLRP3/iNOS pathway-mediated NO production, up-regulated proinflammatory cytokine/chemokine receptor expression and altered glycolytic activity. Notably, ex vivo treatment with DHA reverted the diabetes-induced changes and attenuated the macrophage inflammatory state. It is plausible that DHA supplementation could be employed as adjuvant therapy for treating individuals with T1D.

Endothelium-restricted endothelin-1 overexpression in type 1 diabetes worsens atherosclerosis and immune cell infiltration via NOX1.

AIMS: NADPH oxidase (NOX) 1 but not NOX4-dependent oxidative stress plays a role in diabetic vascular disease, including atherosclerosis. Endothelin (ET)-1 has been implicated in diabetes-induced vascular complications. We showed that crossing mice overexpressing human ET-1 selectively in endothelium (eET-1) with apolipoprotein E knockout (Apoe-/-) mice enhanced high-fat diet-induced atherosclerosis in part by increasing oxidative stress. We tested the hypothesis that ET-1 overexpression in the endothelium would worsen atherosclerosis in type 1 diabetes through a mechanism involving NOX1 but not NOX4. METHODS AND RESULTS: Six-week-old male Apoe-/- and eET-1/Apoe-/- mice with or without Nox1 (Nox1-/y) or Nox4 knockout (Nox4-/-) were injected intraperitoneally with either vehicle or streptozotocin (55 mg/kg/day) for 5 days to induce type 1 diabetes and were studied 14 weeks later. ET-1 overexpression increased 2.5-fold and five-fold the atherosclerotic lesion area in the aortic sinus and arch of diabetic Apoe-/- mice, respectively. Deletion of Nox1 reduced aortic arch plaque size by 60%; in contrast, Nox4 knockout increased lesion size by 1.5-fold. ET-1 overexpression decreased aortic sinus and arch plaque alpha smooth muscle cell content by ∼35% and ∼50%, respectively, which was blunted by Nox1 but not Nox4 knockout. Reactive oxygen species production was increased two-fold in aortic arch perivascular fat of diabetic eET-1/Apoe-/- and eET-1/Apoe-/-/Nox4-/- mice but not eET-1/Apoe-/-/Nox1y/- mice. ET-1 overexpression enhanced monocyte/macrophage and CD3+ T-cell infiltration ∼2.7-fold in the aortic arch perivascular fat of diabetic Apoe-/- mice. Both Nox1 and Nox4 knockout blunted CD3+ T-cell infiltration whereas only Nox1 knockout prevented the monocyte/macrophage infiltration in diabetic eET-1/Apoe-/- mice. CONCLUSION: Endothelium ET-1 overexpression enhances the progression of atherosclerosis in type 1 diabetes, perivascular oxidative stress, and inflammation through NOX1.

Oxidized CaMKII and O-GlcNAcylation cause increased atrial fibrillation in diabetic mice by distinct mechanisms.

Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.

Therapeutic Potential of Tpl2 (Tumor Progression Locus 2) Inhibition on Diabetic Vasculopathy Through the Blockage of the Inflammasome Complex.

OBJECTIVE: Diabetic retinopathy, one of retinal vasculopathy, is characterized by retinal inflammation, vascular leakage, blood-retinal barrier breakdown, and neovascularization. However, the molecular mechanisms that contribute to diabetic retinopathy progression remain unclear. Approach and Results: Tpl2 (tumor progression locus 2) is a protein kinase implicated in inflammation and pathological vascular angiogenesis. Nε-carboxymethyllysine (CML) and inflammatory cytokines levels in human sera and in several diabetic murine models were detected by ELISA, whereas liquid chromatography-tandem mass spectrometry analysis was used for whole eye tissues. The CML and p-Tpl2 expressions on the human retinal pigment epithelium (RPE) cells were determined by immunofluorescence. Intravitreal injection of pharmacological inhibitor or NA (neutralizing antibody) was used in a diabetic rat model. Retinal leukostasis, optical coherence tomography, and H&E staining were used to observe pathological features. Sera of diabetic retinopathy patients had significantly increased CML levels that positively correlated with diabetic retinopathy severity and foveal thickness. CML and p-Tpl2 expressions also significantly increased in the RPE of both T1DM and T2DM diabetes animal models. Mechanistic studies on RPE revealed that CML-induced Tpl2 activation and NADPH oxidase, and inflammasome complex activation were all effectively attenuated by Tpl2 inhibition. Tpl2 inhibition by NA also effectively reduced inflammatory/angiogenic factors, retinal leukostasis in streptozotocin-induced diabetic rats, and RPE secretion of inflammatory cytokines. The attenuated release of angiogenic factors led to inhibited vascular abnormalities in the diabetic animal model. CONCLUSIONS: The inhibition of Tpl2 can block the inflammasome signaling pathway in RPE and has potential clinical and therapeutic implications in diabetes-associated retinal microvascular dysfunction.

Overexpression of D-amino acid oxidase prevents retinal neurovascular pathologies in diabetic rats.

AIMS/HYPOTHESIS: Diabetic retinopathy is characterised by retinal neurodegeneration and retinal vascular abnormalities, affecting one third of diabetic patients with disease duration of more than 10 years. Accumulated evidence suggests that serine racemase (SR) and D-serine are correlated with the pathogenesis of diabetic retinopathy and the deletion of the Srr gene reverses neurovascular pathologies in diabetic mice. Since D-serine content is balanced by SR synthesis and D-amino acid oxidase (DAAO) degradation, we examined the roles of DAAO in diabetic retinopathy and further explored relevant therapy. METHODS: Rats were used as a model of diabetes by i.p. injection of streptozotocin at the age of 2 months and blood glucose was monitored with a glucometer. Quantitative real-time PCR was used to examine Dao mRNA and western blotting to examine targeted proteins in the retinas. Bisulphite sequencing was used to examine the methylation of Dao mRNA promoter in the retinas. Intravitreal injection of DAAO-expressing adenovirus (AAV8-DAAO) was conducted one week before streptozotocin administration. Brain specific homeobox/POU domain protein 3a (Brn3a) immunofluorescence was conducted to indicate retinal ganglion cells at 3 months after virus injection. The permeability of the blood-retinal barrier was examined by Evans blue leakage from retinal capillaries. Periodic acid-Schiff staining and haematoxylin counterstaining were used to indicate retinal vasculature, which was further examined with double immunostaining at 7 months after virus injection. RESULTS: At the age of 12 months, DAAO mRNA and protein levels in retinas from diabetic animals were reduced to 66.2% and 70.4% of those from normal (control) animals, respectively. The Dao proximal promoter contained higher levels of methylation in diabetic than in normal retinas. Consistent with the observation, DNA methyltransferase 1 was increased in diabetic retinas. Injection of DAAO-expressing virus completely prevented the loss of retinal ganglion cells and the disruption of blood-retinal barrier in diabetic rats. Diabetic retinas contained retinal ganglion cells at a density of 54 ± 4/mm2, which was restored to 68 ± 9/mm2 by DAAO overexpression, similar to the levels in normal retinas. The ratio between the number of endothelial cells and pericytes in diabetic retinas was 6.06 ± 1.93/mm2, which was reduced to 3.42 ± 0.55/mm2 by DAAO overexpression; the number of acellular capillaries in diabetic retinas was 10 ± 5/mm2, which was restored to 6 ± 2/mm2 by DAAO overexpression, similar to the levels in normal retinas. Injection of the DAAO-expressing virus increased the expression of occludin and reduced gliosis, which were examined to probe the mechanism by which the disrupted blood-retinal barrier in diabetic rats was rescued and retinal neurodegeneration was prevented. CONCLUSIONS/INTERPRETATION: Altogether, overexpression of DAAO before the onset of diabetes protects against neurovascular abnormalities in retinas from diabetic rats, which suggests a novel strategy for preventing diabetic retinopathy. Graphical abstract.

A cannabinoid type 2 (CB2) receptor agonist augments NOS-dependent responses of cerebral arterioles during type 1 diabetes.

While activation of cannabinoid (CB2) receptors has been shown to be neuroprotective, no studies have examined whether this neuroprotection is directed at cerebral arterioles and no studies have examined whether activation of CB2 receptors can rescue cerebrovascular dysfunction during a chronic disease state such as type 1 diabetes (T1D). Our goal was to test the hypothesis that administration of a CB2 agonist (JWH-133) would improve impaired endothelial (eNOS)- and neuronal (nNOS)-dependent dilation of cerebral arterioles during T1D. In vivo diameter of cerebral arterioles in nondiabetic and T1D rats was measured in response to an eNOS-dependent agonist (adenosine 5'-diphosphate; ADP), an nNOS-dependent agonist (N-methyl-d-aspartate; NMDA), and an NOS-independent agonist (nitroglycerin) before and 1 h following JWH-133 (1 mg/kg IP). Dilation of cerebral arterioles to ADP and NMDA was greater in nondiabetic than in T1D rats. Treatment with JWH-133 increased responses of cerebral arterioles to ADP and NMDA in both nondiabetic and T1D rats. Responses of cerebral arterioles to nitroglycerin were similar between nondiabetic and T1D rats, and JWH-133 did not influence responses to nitroglycerin in either group. The restoration in responses to the agonists by JWH-133 could be inhibited by treatment with a specific inhibitor of CB2 receptors (AM-630; 3 mg/kg IP). Thus, activation of CB2 receptors can potentiate reactivity of cerebral arterioles during physiologic and pathophysiologic states. We speculate that treatment with CB2 receptor agonists may have potential therapeutic benefits for the treatment of cerebral vascular diseases via a mechanism that can increase cerebral blood flow.

Diabetic nephropathy associates with deregulation of enzymes involved in kidney sulphur metabolism.

Nephropathy is a major chronic complication of diabetes. A crucial role in renal pathophysiology is played by hydrogen sulphide (H2 S) that is produced excessively by the kidney; however, the data regarding H2 S bioavailability are inconsistent. We hypothesize that early type 1 diabetes (T1D) increases H2 S production by a mechanism involving hyperglycaemia-induced alterations in sulphur metabolism. Plasma and kidney tissue collected from T1D double transgenic mice were subjected to mass spectrometry-based proteomic analysis, and the results were validated by immunological and gene expression assays.T1D mice exhibited a high concentration of H2 S in the plasma and kidney tissue and histological, showed signs of subtle kidney fibrosis, characteristic for early renal disease. The shotgun proteomic analyses disclosed that the level of enzymes implicated in sulphate activation modulators, H2 S-oxidation and H2 S-production were significantly affected (ie 6 up-regulated and 4 down-regulated). Gene expression results corroborated well with the proteomic data. Dysregulation of H2 S enzymes underly the changes occurring in H2 S production, which in turn could play a key role in the initiation of renal disease. The new findings lead to a novel target in the therapy of diabetic nephropathy. Mass spectrometry data are available via ProteomeXchange with identifier PXD018053.

Neutrophil elastase triggers the development of autoimmune diabetes by exacerbating innate immune responses in pancreatic islets of non-obese diabetic mice.

Type 1 diabetes is an autoimmune disease resulted from self-destruction of insulin-producing pancreatic ß cells. However, the pathological pathways that trigger the autoimmune destruction remain poorly understood. Clinical studies have demonstrated close associations of neutrophils and neutrophil elastase (NE) with ß-cell autoimmunity in patients with Type 1 diabetes. The present study aims to investigate the impact of NE inhibition on development of autoimmune diabetes in NOD mice. NE pharmacological inhibitor (sivelestat) or biological inhibitor (elafin) was supplemented into NOD mice to evaluate their effects on islet inflammation and diabetogenesis. The impact of NE inhibition on innate and adaptive immune cells was measured with flow cytometry and immunohistochemistry. A significant but transient increase in neutrophil infiltration accompanied with elevated NE activity was observed in the neonatal period of NOD mice. Treatment of NOD mice with sivelestat or elafin at the early age led to a marked reduction in spontaneous development of insulitis and autoimmune diabetes. Mechanistically, inhibition of NE significantly attenuated infiltration of macrophages and islet inflammation, thus ameliorating cytotoxic T cell-mediated autoimmune attack of pancreatic ß cells. In vitro studies showed that NE directly induced inflammatory responses in both min6 ß cells and RAW264.7 macrophages, and promoted macrophage migration. These findings support an important role of NE in triggering the onset and progression of ß-cell autoimmunity, and suggest that pharmacological inhibition of NE may represent a promising therapeutic strategy for treatment of autoimmune diabetes.

Role of Arginase in Selective Impairment of Endothelium-Dependent Nitric Oxide Synthase-Mediated Dilation of Retinal Arterioles during Early Diabetes.

Purpose: Retinal vasomotor activity can be regulated by two major endothelial enzymes, nitric oxide synthase (NOS) and cyclooxygenase (COX). The vascular arginase also consumes a NOS substrate and thus impedes NOS-mediated vasodilation. Diabetes mellitus exhibits vascular complications in the retina with elevated oxidative stress and compromised NOS-mediated vasodilation. However, the underlying molecular mechanisms remain unclear, and the effect of diabetes on COX-mediated vasodilation is unknown. Herein, we examined the relative impact of diabetes on retinal arteriolar dilations to COX and NOS activation and the roles of arginase and superoxide in diabetes-induced vasomotor dysfunction. Methods: Retinal arterioles were isolated from streptozocin-induced diabetic pigs (2 weeks of hyperglycemia, 433 ± 27 mg/dL) or age-matched control pigs (97 ± 4 mg/dL). The vasodilations to bradykinin (NOS activator) and histamine (NOS/COX activator) were examined in vitro. Results: Retinal arteriolar dilations to histamine and bradykinin were significantly reduced after 2 weeks of diabetes. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) attenuated the dilations of control vessels, but not diabetic vessels, to histamine. In the presence of L-NAME and COX inhibitor indomethacin, histamine-induced dilations of control and diabetic vessels were reduced similarly. Treatment of diabetic vessels with arginase inhibitor nor-NOHA, but not superoxide dismutase mimetic TEMPOL, preserved both histamine- and bradykinin-induced dilations in an L-NAME-sensitive manner. Conclusions: Arginase, rather than superoxide, impairs endothelium-dependent NOS-mediated dilation of retinal arterioles during diabetes, whereas vasodilation mediated by COX remains intact. Blockade of vascular arginase may improve endothelial function of retinal arterioles during early onset of diabetes.

Heparanase and Type 1 Diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing beta cells in pancreatic islets. The degradation of the glycosaminoglycan heparan sulfate (HS) by the endo-ß-D-glycosidase heparanase plays a critical role in multiple stages of the disease process. Heparanase aids (i) migration of inflammatory leukocytes from the vasculature to the islets, (ii) intra-islet invasion by insulitis leukocytes, and (iii) selective destruction of beta cells. These disease stages are marked by the solubilization of HS in the subendothelial basement membrane (BM), HS breakdown in the peri-islet BM, and the degradation of HS inside beta cells, respectively. Significantly, healthy islet beta cells are enriched in highly sulfated HS which is essential for their viability, protection from damage by reactive oxygen species (ROS), beta cell function and differentiation. Consequently, mouse and human beta cells but not glucagon-producing alpha cells (which contain less-sulfated HS) are exquisitely vulnerable to heparanase-mediated damage. In vitro, the death of HS-depleted mouse and human beta cells can be prevented by HS replacement using highly sulfated HS mimetics or analogues. T1D progression in NOD mice and recent-onset T1D in humans correlate with increased expression of heparanase by circulating leukocytes of myeloid origin and heparanase-expressing insulitis leukocytes. Treatment of NOD mice with the heparanase inhibitor and HS replacer, PI-88, significantly reduced T1D incidence by 50%, impaired the development of insulitis and preserved beta cell HS. These outcomes identified heparanase as a novel destructive tool in T1D, distinct from the conventional cytotoxic and apoptosis-inducing mechanisms of autoreactive T cells. In contrast to exogenous catalytically active heparanase, endogenous heparanase may function in HS homeostasis, gene expression and insulin secretion in normal beta cells and immune gene expression in leukocytes. In established diabetes, the interplay between hyperglycemia, local inflammatory cells (e.g. macrophages) and heparanase contributes to secondary micro- and macro-vascular disease. We have identified dual activity heparanase inhibitors/HS replacers as a novel class of therapeutic for preventing T1D progression and potentially for mitigating secondary vascular disease that develops with long-term T1D.

Xanthine oxidoreductase activity correlates with vascular endothelial dysfunction in patients with type 1 diabetes.

AIMS: Xanthine oxidoreductase (XOR) is an enzyme regulating uric acid synthesis and generation of reactive oxygen species. Several studies suggested relationship between XOR and atherosclerotic diseases; however, few previous studies have directly examined the relationship between XOR and vascular endothelial dysfunction in patients with type 1 diabetes mellitus (T1DM). The aim of this study was to evaluate the relationship between XOR activity and vascular endothelial function in patients with T1DM. METHODS: Seventy-one patients with T1DM participated in the study and underwent assessments, including plasma XOR activity and flow-mediated dilation (FMD), to measure vascular endothelial function. RESULTS: The natural logarithm value of XOR activity (ln-XOR) was 3.03 ± 0.99 pmol/h/mL, and FMD was 5.5% ± 2.4%. FMD was inversely and significantly correlated with ln-XOR (correlation coefficient: r = - 0.396, P < 0.001), UA (r = - 0.252, P = 0.034), and asymmetric dimethylarginine (ADMA) (r = - 0.414, P < 0.001). ln-XOR showed positive correlation with HbA1c (r = 0.292, P = 0.013), ALT (r = 0.658, P < 0.001), and ADMA (r = 0.363, P = 0.002). Stepwise multiple regression analysis showed that ln-XOR (standard partial regression coefficient: ß = - 0.254, P = 0.018) was an independent explanatory variable of FMD. CONCLUSIONS: The results of this study showed for the first time that XOR activity is associated with glycemic control in patients with T1DM and that XOR activity is associated with vascular endothelial dysfunction.

Clinical and genetic characteristics of people with type 1 diabetes who have discrepancies in titers of anti-glutamic acid decarboxylase antibody measured by radioimmunoassay and enzyme-linked immunosorbent assay.

AIMS/INTRODUCTION: The aim of the present study was to compare the clinical and genetic characteristics between people with type 1 diabetes who were positive and negative for autoantibodies against glutamic acid decarboxylase (GADA) measured by enzyme-linked immunosorbent assay (ELISA) with low-titer GADA measured by radioimmunoassay. MATERIALS AND METHODS: Among Japanese people with type 1 diabetes in whom GADA were measured by both ELISA and radioimmunoassay, those who had low titers of GADA measured by radioimmunoassay (1.5-10 U/mL), regardless of positivity for GADA measured by ELISA, were studied. There were 65 participants with acute-onset type 1 diabetes and 30 participants with slowly progressive insulin-dependent diabetes mellitus. Clinical characteristics and human leukocyte antigen types were compared in ELISA-positive (≥5 U/mL) and ELISA-negative participants. Endogenous insulin secretion was evaluated by C-peptide index. RESULTS: Among participants with slowly progressive insulin-dependent diabetes mellitus, postprandial C-peptide index was significantly higher in ELISA-negative participants than in ELISA-positive participants (r = 0.619, P = 0.002). Among 52 participants whose human leukocyte antigen typing was carried out, all of the participants with slowly progressive insulin-dependent diabetes mellitus who had DRB1*09:01 were positive by GADA-ELISA (P = 0.021). In acute-onset type 1 diabetes participants, there were no significant differences for the C-peptide index and human leukocyte antigen genotypes. CONCLUSIONS: The difference in the positivity for GADA-ELISA might reflect cytotoxicity toward pancreatic ß-cells and preservation of endogenous insulin secretion in people with slowly progressive insulin-dependent diabetes mellitus. We also suggest that the difference in the GADA-ELISA-specific epitope depends on the human leukocyte antigen genotype.

Gastrin secretion in normal subjects and diabetes patients is inhibited by glucagon-like peptide 1: a role in the gastric side effects of GLP-1-derived drugs?

Background: Randomized and controlled trials of glucagon-like peptide-1 (GLP-1) derived drugs have shown that the most frequent adverse symptoms are gastrointestinal. Some of the side effects such as dyspepsia, nausea and upper abdominal pain may well be of gastric origin. Since the antral hormone gastrin regulates gastric secretion of acid and enzymes and contributes to the regulation of gastric motility, we examined the effect of GLP-1 on the secretion of gastrin in normal subjects and diabetes patients.Method: Plasma was sampled from ten healthy subjects and ten patients with diabetes mellitus type 1 with glucose clamped between 6 and 9 mM. GLP-1 or saline were infused for 4 h during and after a meal. Plasma concentrations of gastrin and GLP-1 were measured using specific radioimmunoassays.Results: Basal plasma concentrations of gastrin were similar in controls and patients. After the meal, the gastrin concentrations rose significantly during saline infusion, whereas the GLP-1 infusion suppressed the secretion of gastrin significantly, most pronounced in the diabetes patients.Conclusions: The results show that GLP-1 infusion suppresses the postprandial secretion of gastrin in normal subjects and even more so in the diabetes patients. The results may therefore shed further light on the upper gastrointestinal side effects of GLP-1-derived drugs in diabetic patients.