Influence of sphingolipid enzymes on blood glucose levels, development of diabetes, and involvement of pericytes.

AIMS: Sulfatide is a chaperone for insulin manufacturing in beta cells. Here we explore whether the blood glucose values normally could be associated with this sphingolipid and especially two of its building enzymes CERS2 and CERS6. Both T1D and T2D have low blood sulfatide levels, and insulin resistance on beta cells at clinical diagnosis. Furthermore, we examined islet pericytes for sulfatide, and beta-cell receptors for GLP-1, both of which are related to the insulin production. MATERIALS AND METHODS: We examined mRNA levels in islets from the DiViD and nPOD studies, performed genetic association analyses, and histologically investigated pericytes in the islets for sulfatide. RESULTS: Polymorphisms of the gene encoding the CERS6 enzyme responsible for synthesising dihydroceramide, a precursor to sulfatide, are associated with random blood glucose values in non-diabetic persons. This fits well with our finding of sulfatide in pericytes in the islets, which regulates the capillary blood flow in the islets of Langerhans, which is important for oxygen supply to insulin production. In the islets of newly diagnosed T1D patients, we observed low levels of GLP-1 receptors; this may explain the insulin resistance in their beta cells and their low insulin production. In T2D patients, we identified associated polymorphisms in both CERS2 and CERS6. CONCLUSIONS: Here, we describe several polymorphisms in sulfatide enzymes related to blood glucose levels and HbA1c in non-diabetic individuals. Islet pericytes from such persons contain sulfatide. Furthermore, low insulin secretion in newly diagnosed T1D may be explained by beta-cell insulin resistance due to low levels of GLP-1 receptors.

A pilot study of a virtually delivered dissonance-based eating disorder prevention program for young women with type 1 diabetes: within-subject changes over 6-month follow-up.

INTRODUCTION: In an uncontrolled study, we previously demonstrated the feasibility and preliminary efficacy of our virtual diabetes-specific version (Diabetes Body Project) of the eating disorder (ED) prevention program the Body Project. The aim of the current study was to evaluate further this program for women with type 1 diabetes (T1D) by assessing within-subject changes in outcomes from pretest over 6-month follow-up. METHODS: Young women with T1D aged 16-35 years were invited to participate in Diabetes Body Project groups. A total of 35 participants were allocated to five Diabetes Body Project groups (six meetings over 6 weeks). Primary outcome measures included ED risk factors and symptoms, and secondary outcomes included three T1D-specific constructs previously found to be associated with ED pathology: glycemic control as measured by HbA1c level, diabetes distress, and illness perceptions. RESULTS: Within-subject reductions, with medium-to-large effect sizes, were observed for the primary (ED pathology, body dissatisfaction, thin-ideal internalization, and appearance ideals and pressures) and secondary outcomes (within-condition Cohen's ds ranged from .34 to 1.70). CONCLUSION: The virtual Diabetes Body Project appears to be a promising intervention worthy of more rigorous evaluation. A randomized controlled trial with at least a 1-year follow-up is warranted to determine its efficacy compared to a control condition.

Type 1 diabetes could begin with alterations in innate anti-viral immunity, which are already at this stage associated with HLA risk haplotypes.

AIMS: To investigate if HLA risk haplotypes and HbA1c levels are associated with the expression levels of innate anti-viral immune pathway genes in type 1 diabetes. MATERIALS AND METHODS: We investigated RNA expression levels of innate anti-viral immune pathway genes in laser-dissected islets from two to five tissue sections per donor from the Diabetes Virus Detection study and the network of Pancreatic Organ Donors in relation to HLA risk haplotypes (non-predisposed and predisposed) and HbA1c levels (normal, elevated, and high). RESULTS: The expression of innate anti-viral immune genes (TLR7, OAS1, OAS3 etc.) was significantly increased in individuals with predisposing vs non-predisposing HLA haplotypes. Also, the expression of several of the innate anti-viral immune genes from the HLA risk haplotype analysis was significantly increased in the group with high vs normal HbA1c. Furthermore, the gene expression of OAS2 was significantly increased in the group with high HbA1c vs elevated HbA1c. CONCLUSIONS: Expression of innate anti-viral immune pathway genes was increased in individuals with predisposing HLA risk haplotypes and those with high HbA1c. This indicates that type 1 diabetes might well begin with alterations in innate anti-viral immunity, and already at this stage be associated with HLA risk haplotypes.

Live enteroviruses, but not other viruses, detected in human pancreas at the onset of type 1 diabetes in the DiViD study.

AIMS/HYPOTHESIS: Enterovirus (EV) infection of pancreatic islet cells is one possible factor contributing to type 1 diabetes development. We have reported the presence of EV genome by PCR and of EV proteins by immunohistochemistry in pancreatic sections. Here we explore multiple human virus species in the Diabetes Virus Detection (DiViD) study cases using innovative methods, including virus passage in cell cultures. METHODS: Six recent-onset type 1 diabetes patients (age 24-35) were included in the DiViD study. Minimal pancreatic tail resection was performed under sterile conditions. Eleven live cases (age 43-83) of pancreatic carcinoma without diabetes served as control cases. In the present study, we used EV detection methods that combine virus growth in cell culture, gene amplification and detection of virus-coded proteins by immunofluorescence. Pancreas homogenates in cell culture medium were incubated with EV-susceptible cell lines for 3 days. Two to three blind passages were performed. DNA and RNA were extracted from both pancreas tissue and cell cultures. Real-time PCR was used for detecting 20 different viral agents other than EVs (six herpesviruses, human polyomavirus [BK virus and JC virus], parvovirus B19, hepatitis B virus, hepatitis C virus, hepatitis A virus, mumps, rubella, influenza A/B, parainfluenza 1-4, respiratory syncytial virus, astrovirus, norovirus, rotavirus). EV genomes were detected by endpoint PCR using five primer pairs targeting the partially conserved 5' untranslated region genome region of the A, B, C and D species. Amplicons were sequenced. The expression of EV capsid proteins was evaluated in cultured cells using a panel of EV antibodies. RESULTS: Samples from six of six individuals with type 1 diabetes (cases) and two of 11 individuals without diabetes (control cases) contained EV genomes (p<0.05). In contrast, genomes of 20 human viruses other than EVs could be detected only once in an individual with diabetes (Epstein-Barr virus) and once in an individual without diabetes (parvovirus B19). EV detection was confirmed by immunofluorescence of cultured cells incubated with pancreatic extracts: viral antigens were expressed in the cytoplasm of approximately 1% of cells. Notably, infection could be transmitted from EV-positive cell cultures to uninfected cell cultures using supernatants filtered through 100 nm membranes, indicating that infectious agents of less than 100 nm were present in pancreases. Due to the slow progression of infection in EV-carrying cell cultures, cytopathic effects were not observed by standard microscopy but were recognised by measuring cell viability. Sequences of 5' untranslated region amplicons were compatible with EVs of the B, A and C species. Compared with control cell cultures exposed to EV-negative pancreatic extracts, EV-carrying cell cultures produced significantly higher levels of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP1). CONCLUSIONS/INTERPRETATION: Sensitive assays confirm that the pancreases of all DiViD cases contain EVs but no other viruses. Analogous EV strains have been found in pancreases of two of 11 individuals without diabetes. The detected EV strains can be passaged in series from one cell culture to another in the form of poorly replicating live viruses encoding antigenic proteins recognised by multiple EV-specific antibodies. Thus, the early phase of type 1 diabetes is associated with a low-grade infection by EVs, but not by other viral agents.

Genetic predisposition in the 2'-5'A pathway in the development of type 1 diabetes: potential contribution to dysregulation of innate antiviral immunity.

AIMS/HYPOTHESIS: The incidence of type 1 diabetes is increasing more rapidly than can be explained by genetic drift. Viruses may play an important role in the disease, as they seem to activate the 2'-5'-linked oligoadenylate (2'-5'A) pathway of the innate antiviral immune system. Our aim was to investigate this possibility. METHODS: Innate antiviral immune pathways were searched for type 1 diabetes-associated polymorphisms using genome-wide association study data. SNPs within ±250kb flanking regions of the transcription start site of 64 genes were examined. These pathways were also investigated for type 1 diabetes-associated RNA expression profiles using laser-dissected islets from two to five tissue sections per donor from the Diabetes Virus Detection (DiViD) study and the network of Pancreatic Organ Donors (nPOD). RESULTS: We found 27 novel SNPs in genes nominally associated with type 1 diabetes. Three of those SNPs were located upstream of the 2'-5'A pathway, namely SNP rs4767000 (p = 1.03 × 10-9, OR 1.123), rs1034687 (p = 2.16 × 10-7, OR 0.869) and rs739744 (p = 1.03 × 10-9, OR 1.123). We also identified a large group of dysregulated islet genes in relation to type 1 diabetes, of which two were novel. The most aberrant genes were a group of IFN-stimulated genes. Of those, the following distinct pathways were targeted by the dysregulation (compared with the non-diabetic control group): OAS1 increased by 111% (p < 1.00 × 10-4, 95% CI -0.43, -0.15); MX1 increased by 142% (p < 1.00 × 10-4, 95% CI -0.52, -0.22); and ISG15 increased by 197% (p = 2.00 × 10-4, 95% CI -0.68, -0.18). CONCLUSIONS/INTERPRETATION: We identified a genetic predisposition in the 2'-5'A pathway that potentially contributes to dysregulation of the innate antiviral immune system in type 1 diabetes. This study describes a potential role for the 2'-5'A pathway and other components of the innate antiviral immune system in beta cell autoimmunity.

Characterisation of enterovirus RNA detected in the pancreas and other specimens of live patients with newly diagnosed type 1 diabetes in the DiViD study.

AIMS/HYPOTHESIS: The Diabetes Virus Detection (DiViD) study is the first study to laparoscopically collect pancreatic tissue and purified pancreatic islets together with duodenal mucosa, serum, peripheral blood mononuclear cells (PBMCs) and stools from six live adult patients (age 24-35 years) with newly diagnosed type 1 diabetes. The presence of enterovirus (EV) in the pancreatic islets of these patients has previously been reported. METHODS: In the present study we used reverse transcription quantitative real-time PCR (RT-qPCR) and sequencing to characterise EV genomes present in different tissues to understand the nature of infection in these individuals. RESULTS: All six patients were found to be EV-positive by RT-qPCR in at least one of the tested sample types. Four patients were EV-positive in purified islet culture medium, three in PBMCs, one in duodenal biopsy and two in stool, while serum was EV-negative in all individuals. Sequencing the 5' untranslated region of these EVs suggested that all but one belonged to enterovirus B species. One patient was EV-positive in all these sample types except for serum. Sequence analysis revealed that the virus strain present in the isolated islets of this patient was different from the strain found in other sample types. None of the islet-resident viruses could be isolated using EV-permissive cell lines. CONCLUSIONS/INTERPRETATION: EV RNA can be frequently detected in various tissues of patients with type 1 diabetes. At least in some patients, the EV strain in the pancreatic islets may represent a slowly replicating persisting virus.

Expression of immunoreactive inducible nitric oxide synthase in pancreatic islet cells from newly diagnosed and long-term type 1 diabetic donors is heterogeneous and not disease-associated.

Exposure of isolated human islets to proinflammatory cytokines leads to up-regulation of inducible nitric oxide synthase (iNOS), raised NO, and beta cell toxicity. These findings have led to increasing interest in the clinical utility of iNOS blockade to mitigate beta cell destruction in human type 1 diabetes (T1D). However, recent studies show that iNOS-derived NO may also confer beta cell protection. To investigate this dichotomy, we compared islet cell distributions and intensity of iNOS immunostaining in pancreatic sections, co-stained for insulin and glucagon, from new-onset T1D donors (group 1), with non-diabetic autoantibody-negative (group 2), non-diabetic autoantibody-positive (group 3) and long-term diabetic donors (group 4). The cellular origins of iNOS, its frequency and graded intensities in islets and number in peri-islet, intra-islet and exocrine regions were determined. All donors showed iNOS positivity, irrespective of disease and presence of beta cells, had variable labelling intensities, without significant differences in the frequency of iNOS-positive islets among study groups. iNOS was co-localised in selective beta, alpha and other endocrine cells, and in beta cell-negative islets of diabetic donors. The number of peri- and intra-islet iNOS cells was low, being significantly higher in the peri-islet area. Exocrine iNOS cells also remained low, but were much lower in group 1. We demonstrate that iNOS expression in islet cells is variable, heterogeneous and independent of co-existing beta cells. Its distribution and staining intensities in islets and extra-islet areas do not correlate with T1D or its duration. Interventions to inactivate the enzyme to alleviate disease are currently not justified.

Feasibility of a virtually delivered eating disorder prevention program for young females with type 1 diabetes.

OBJECTIVE: This study aimed to develop a virtual diabetes-specific version of the eating disorder (ED) prevention program the Body Project, and to assess feasibility and preliminary efficacy of this program for young females with type 1 diabetes. METHOD: Young females with type 1 diabetes aged 16-35 years were invited to participate in the study. A total of 35 participants were allocated to five Diabetes Body Project groups (six meetings over 6 weeks) and completed pretest assessments; 26 participants completed all sessions and posttest assessments (<7 days after last meeting). Primary measures included ED risk factors and symptoms, and secondary outcomes included diabetes-specific constructs previously found to be associated with ED psychopathology (e.g., diabetes distress and illness perceptions). RESULTS: The ease of recruitment, timely conduct of five groups, moderate drop-out rate and appreciation of the intervention by participants indicated that the Diabetes Body Project is feasible. Meaningful reductions occurred on the primary outcomes (i.e., ED psychopathology, body dissatisfaction, and thin ideal internalization) and on internalization of appearance ideals and appearance pressures at posttest (Cohen's d ranging from .63 to .83, which are medium to large effects). Small to medium effect sizes were found for diabetes illness perceptions and distress (.41 and .48, respectively). DISCUSSION: The virtual Diabetes Body Project is a promising and much-needed intervention, worthy of more rigorous evaluation. A randomized controlled trial is warranted to determine its effectiveness compared with a control condition.

IL-6 is present in beta and alpha cells in human pancreatic islets: Expression is reduced in subjects with type 1 diabetes.

IL-6 is a pro-inflammatory cytokine upregulated in some autoimmune diseases. The role of IL-6 in the development of type 1 diabetes (T1D) is unclear. Clinical studies are investigating whether tocilizumab (anti-IL-6 receptor) can help preserve beta cell function in patients recently diagnosed with T1D. However, in some rodent models and isolated human islets, IL-6 has been found to have a protective role for beta cells by reducing oxidative stress. Hence, we systematically investigated local tissue expression of IL-6 in human pancreas from non-diabetic, auto-antibody positive donors and donors with T1D and T2D. IL-6 was constitutively expressed by beta and alpha cells regardless of the disease state. However, expression of IL-6 was highly reduced in insulin-deficient islets of donors with T1D, and the expression was then mostly restricted to alpha cells. Our findings suggest that the implication of IL-6 in T1D pathogenesis might be more complex than previously assumed.

Procoagulant activity in children and adolescents on intensive insulin therapy.

BACKGROUND: Type 1 diabetes is associated with atherothrombosis, but limited data exist on procoagulant activity in the young. We investigated procoagulant activity in children/adolescents with type 1 diabetes using intensified insulin treatment compared with controls in a 5-year follow-up study, and further any associations with cardiovascular risk factors. METHODS: The study included 314 diabetes children/adolescents and 120 healthy controls. Prothrombin fragment 1+2 (F1+2), D-dimer, tissue-factor-procoagulant-activity (TF-PCA), and tissue-factor-pathway-inhibitor (TFPI) were analyzed with ELISAs. RESULTS: F1+2, D-dimer, and TF-PCA did not differ between the groups or correlate to HbA1c in the diabetes group at either time points. TFPI was significantly higher in the diabetes group compared with controls both at inclusion and follow-up (both P < .001). In the diabetes group, TFPI correlated significantly to HbA1c at both time points (r = 0.221 and 0.304, both P < .001). At follow-up, females using oral contraceptives had significantly elevated F1+2, D-dimer, and TF-PCA and lower TFPI compared to no-users (all P < .005), and females had lower TFPI (P = .017) and higher F1+2 compared with males (P = .052), also after adjusting for the use of oral contraceptives. CONCLUSIONS: The current results show similar procoagulant activity in children/adolescents with type 1 diabetes compared with controls over a 5-year period, indicating that these children using modern intensified insulin treatment are not at high thrombotic risk at younger age. The elevated levels of TFPI in the diabetes group, related to hyperglycaemia, are probably reflecting increased endothelial activation. These findings highlight the significance of optimal blood glucose control in children/adolescents with type 1 diabetes, to maintain a healthy endothelium.

International benchmarking in type 1 diabetes: Large difference in childhood HbA1c between eight high-income countries but similar rise during adolescence-A quality registry study.

OBJECTIVES: To identify differences and similarities in HbA1c levels and patterns regarding age and gender in eight high-income countries. SUBJECTS: 66 071 children and adolescents below18 years of age with type 1 diabetes for at least 3 months and at least one HbA1c measurement during the study period. METHODS: Pediatric Diabetes Quality Registry data from Austria, Denmark, England, Germany, Norway, Sweden, the United States, and Wales were collected between 2013 and 2014. HbA1c, gender, age, and duration were used in the analysis. RESULTS: Distribution of gender and age groups was similar in the eight participating countries. The mean HbA1c varied from 60 to 73 mmol/mol (7.6%-8.8%) between the countries. The increase in HbA1c between the youngest (0-9 years) to the oldest (15-17 years) age group was close to 8 mmol/mol (0.7%) in all countries (P < .001). Females had a 1 mmol/mol (0.1%) higher mean HbA1c than boys (P < .001) in seven out of eight countries. CONCLUSIONS: In spite of large differences in the mean HbA1c between countries, a remarkable similarity in the increase of HbA1c from childhood to adolescence was found.

Rationale for enteroviral vaccination and antiviral therapies in human type 1 diabetes.

In type 1 diabetes, pancreatic beta cells are destroyed by chronic autoimmune responses. The disease develops in genetically susceptible individuals, but a role for environmental factors has been postulated. Viral infections have long been considered as candidates for environmental triggers but, given the lack of evidence for an acute, widespread, cytopathic effect in the pancreas in type 1 diabetes or for a closely related temporal association of diabetes onset with such infections, a role for viruses in type 1 diabetes remains unproven. Moreover, viruses have rarely been isolated from the pancreas of individuals with type 1 diabetes, mainly (but not solely) due to the inaccessibility of the organ. Here, we review past and recent literature to evaluate the proposals that chronic, recurrent and, possibly, persistent enteroviral infections occur in pancreatic beta cells in type 1 diabetes. We also explore whether these infections may be sustained by different virus strains over time and whether multiple viral hits can occur during the natural history of type 1 diabetes. We emphasise that only a minority of beta cells appear to be infected at any given time and that enteroviruses may become replication defective, which could explain why they have been isolated from the pancreas only rarely. We argue that enteroviral infection of beta cells largely depends on the host innate and adaptive immune responses, including innate responses mounted by beta cells. Thus, we propose that viruses could play a role in type 1 diabetes on multiple levels, including in the triggering and chronic stimulation of autoimmunity and in the generation of inflammation and the promotion of beta cell dysfunction and stress, each of which might then contribute to autoimmunity, as part of a vicious circle. We conclude that studies into the effects of vaccinations and/or antiviral drugs (some of which are currently on-going) is the only means by which the role of viruses in type 1 diabetes can be finally proven or disproven.

No Evidence for Presence of Mucosal-Associated Invariant T Cells in the Insulitic Lesions in Patients Recently Diagnosed with Type 1 Diabetes.

Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize bacteria-infected cells and are thought to play a role in autoimmune diseases. Translocation of duodenal bacteria and viruses to the pancreas through the pancreatic duct has been hypothesized to initiate an innate inflammatory response that could contribute to the development of type 1 diabetes, a process that could involve MAIT cells. In this study, we used immunohistochemistry and quantitative PCR to search for evidence of MAIT cells in the insulitic lesions in the pancreas of human patients recently diagnosed with type 1 diabetes. Only a few scattered MAIT cells were found within the exocrine parenchyma in all pancreatic samples, but no MAIT cells were found in association to the islets. Also, only low gene expression levels of the MAIT T-cell receptor Vα7.2-Jα33 were found in the pancreas of patients with type 1 diabetes, in similar levels as that in nondiabetic organ donors used as control. The absence of MAIT cells shown in insulitic lesions in humans questions the direct cytotoxic role of these cells in ß-cell destruction.

Distribution of IL-1ß immunoreactive cells in pancreatic biopsies from living volunteers with new-onset type 1 diabetes: comparison with donors without diabetes and with longer duration of disease.

AIMS/HYPOTHESIS: Although IL-1ß is considered a key mediator of beta cell destruction, its cellular expression in islets during early type 1 diabetes remains unclear. We compared its expression in rare pancreatic biopsies from new-onset living volunteers with its expression in cadaveric pancreas sections from non-diabetic autoantibody-positive and -negative individuals and those with long-standing disease. METHODS: Pancreatic biopsy sections from six new-onset living volunteers (group 1) and cadaveric sections from 13 non-diabetic autoantibody-negative donors (group 2), four non-diabetic autoantibody-positive donors (group 3) and nine donors with diabetes of longer duration (0.25-12 years of disease; group 4) were triple-immunostained for IL-1ß, insulin and glucagon. Intra- and peri-islet IL-1ß-positive cells in insulin-positive and -negative islets and in random exocrine fields were enumerated. RESULTS: The mean number of IL-1ß-positive cells per islet from each donor in peri- and intra-islet regions was <1.25 and <0.5, respectively. In all study groups, the percentage of islets with IL-1ß cells in peri- and/or intra-islet regions was highly variable and ranged from 4.48% to 17.59% in group 1, 1.42% to 44.26% in group 2, 7.93% to 17.53% in group 3 and 3.85% to 42.86% in group 4, except in a single case where the value was 75%. In 25/32 donors, a higher percentage of islets showed IL-1ß-positive cells in peri-islet than in intra-islet regions. In sections from diabetic donors (groups 1 and 4), a higher mean number of IL-1ß-positive cells occurred in insulin-positive islets than in insulin-negative islets. In group 2, 70-90% of islets in 3/13 sections had weak-to-moderate IL-1ß staining in alpha cells but staining was virtually absent or substantially reduced in the remaining groups. The mean number of exocrine IL-1ß-positive cells in group 1 was lower than in the other groups. CONCLUSIONS/INTERPRETATION: At onset of type 1 diabetes, the low number of islet-associated IL-1ß-positive cells may be insufficient to elicit beta cell destruction. The variable expression in alpha cells in groups 2-4 suggests their cellular heterogeneity and probable physiological role. The significance of a higher but variable number of exocrine IL-1ß-positive cells seen in non-diabetic individuals and those with long-term type 1 diabetes remains unclear.

Abnormal islet sphingolipid metabolism in type 1 diabetes.

AIMS/HYPOTHESIS: Sphingolipids play important roles in beta cell physiology, by regulating proinsulin folding and insulin secretion and in controlling apoptosis, as studied in animal models and cell cultures. Here we investigate whether sphingolipid metabolism may contribute to the pathogenesis of human type 1 diabetes and whether increasing the levels of the sphingolipid sulfatide would prevent models of diabetes in NOD mice. METHODS: We examined the amount and distribution of sulfatide in human pancreatic islets by immunohistochemistry, immunofluorescence and electron microscopy. Transcriptional analysis was used to evaluate expression of sphingolipid-related genes in isolated human islets. Genome-wide association studies (GWAS) and a T cell proliferation assay were used to identify type 1 diabetes related polymorphisms and test how these affect cellular islet autoimmunity. Finally, we treated NOD mice with fenofibrate, a known activator of sulfatide biosynthesis, to evaluate the effect on experimental autoimmune diabetes development. RESULTS: We found reduced amounts of sulfatide, 23% of the levels in control participants, in pancreatic islets of individuals with newly diagnosed type 1 diabetes, which were associated with reduced expression of enzymes involved in sphingolipid metabolism. Next, we discovered eight gene polymorphisms (ORMDL3, SPHK2, B4GALNT1, SLC1A5, GALC, PPARD, PPARG and B4GALT1) involved in sphingolipid metabolism that contribute to the genetic predisposition to type 1 diabetes. These gene polymorphisms correlated with the degree of cellular islet autoimmunity in a cohort of individuals with type 1 diabetes. Finally, using fenofibrate, which activates sulfatide biosynthesis, we completely prevented diabetes in NOD mice and even reversed the disease in half of otherwise diabetic animals. CONCLUSIONS/INTERPRETATION: These results indicate that islet sphingolipid metabolism is abnormal in type 1 diabetes and suggest that modulation may represent a novel therapeutic approach. DATA AVAILABILITY: The RNA expression data is available online at https://www.dropbox.com/s/93mk5tzl5fdyo6b/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%2C%20RNA%20expression.xlsx?dl=0 . A list of SNPs identified is available at https://www.dropbox.com/s/yfojma9xanpp2ju/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%20SNP.xlsx?dl=0 .

Demonstration of Tissue Resident Memory CD8 T Cells in Insulitic Lesions in Adult Patients with Recent-Onset Type 1 Diabetes.

Subtypes of CD8+ T cells in insulitic lesions in biopsy specimens from six subjects with recent-onset type 1 diabetes (T1D) and six nondiabetic matched controls were analyzed using simultaneous multicolor immunofluorescence. Also, insulitic islets based on accumulation of CD3+ T cells were microdissected with laser-capture microscopy, and gene transcripts associated with inflammation and autoimmunity were analyzed. We found a substantial proportion, 43%, of the CD8+ T cells in the insulitic lesions to display a tissue resident memory T cell (TRM) (CD8+CD69+CD103+) phenotype in T1D subjects. Most TRM cells were located in the insulitic lesion in the endocrine-exocrine interface. TRM cells were also sporadically found in islets of control subjects. Moreover, gene expression analysis showed a lack of active transcription of genes associated with acute inflammatory or cytotoxic T-cell responses. We present evidence that a substantial proportion of T cells in insulitic lesions of recent-onset T1D patients are TRM cells and not classic cytotoxic CD8+ T cells. Our findings highlight the need for further analysis of the T cells involved in insulitis to elucidate their role in the etiology of T1D.

An immunohistochemical study of nitrotyrosine expression in pancreatic islets of cases with increasing duration of type 1 diabetes and without diabetes.

Peroxynitrite-induced nitration of cellular proteins has been shown to associate with various human pathologies. The expression of pancreatic nitrotyrosine and its cellular source relative to insulitis were analysed in cases with increasing duration of type 1 diabetes and compared with non-diabetic autoantibody-negative and -positive cases. Pancreatic tail sections from non-diabetic autoantibody-negative cases (Group 1; n = 7), non-diabetic autoantibody-positive cases (Group 2; n = 6), recently diagnosed cases (Group 3; n = 6), 0.25-5 years of diabetes (Group 4; n = 8) and 7-12 years of diabetes (Group 5; n = 6) were immunostained sequentially for nitrotyrosine, insulin and leucocytes. Nitrotyrosine expression was observed in selective beta cells only. In group 1, the percentage of insulin-positive islets with nitrotyrosine ranged from 7.6 to 58.8%. In group 2, it was minimally expressed in 2 cases and was present in 4.7-19.3% of insulin-positive islets in 3 cases and in all islets in 1 case. In group 3, it was absent in 1 case and in the remaining 5 cases, the values were 17.4-85.7%. In group 4, nitrotyrosine was absent in 6 cases and positive in 1.8 and 22.2% of insulin-positive islets in 2 cases. In group 5, the values were 60% (1 case) and 100% (2 cases), being absent in 3 cases, consistent with insulin-negativity. This case analysis shows that nitrotyrosine immunostaining is independent of the presence and severity of insulitis. Variable nitrotyrosine expression is present in some non-diabetic cases. Its increased expression in beta cells of recent-onset and long-standing disease requires further studies to determine whether beta cell nitration plays a pathogenic role during T1D.

Reduced HDL function in children and young adults with type 1 diabetes.

BACKGROUND: Patients with type 1 diabetes (T1D) are at increased risk of cardiovascular disease (CVD). Measures of high-density lipoprotein (HDL) function provide a better risk estimate for future CVD events than serum levels of HDL cholesterol. The objective of this study was to evaluate HDL function in T1D patients shortly after disease onset compared with healthy control subjects. METHODS: Participants in the atherosclerosis and childhood diabetes study were examined at baseline and after 5 years. At baseline, the cohort included 293 T1D patients with a mean age of 13.7 years and mean HbA1c of 8.4%, along with 111 healthy control subjects. Their HDL function, quantified by HDL-apoA-I exchange (HAE), was assessed at both time points. HAE is a measure of HDL's dynamic property, specifically its ability to release lipid-poor apolipoprotein A-I (apoA-I), an essential step in reverse cholesterol transport. RESULTS: The HAE-apoA-I ratio, reflecting the HDL function per concentration unit apoA-I, was significantly lower in the diabetes group both at baseline, 0.33 (SD = 0.06) versus 0.36 (SD = 0.06) %HAE/mg/dL, p < 0.001 and at follow-up, 0.34 (SD = 0.06) versus 0.36 (SD = 0.06)  %HAE/mg/dL, p = 0.003. HAE-apoA-I ratio was significantly and inversely correlated with HbA1c in the diabetes group. Over the 5 years of the study, the mean HAE-apoA-I ratio remained consistent in both groups. Individual changes were less than 15% for half of the study participants. CONCLUSIONS: This study shows reduced HDL function, quantified as HAE-apoA-I ratio, in children and young adults with T1D compared with healthy control subjects. The differences in HDL function appeared shortly after disease onset and persisted over time.

Advanced glycation end products in children with type 1 diabetes and early reduced diastolic heart function.

BACKGROUND: Reduced diastolic function is an early sign of diabetes cardiomyopathy in adults and is associated with elevated levels of HbA1c and advanced glycation end products (AGEs). OBJECTIVE: To assess the associations between early reduced diastolic function and elevated levels of HbA1c and AGEs in children and adolescents with type 1 diabetes (T1D). METHODS: One hundred fourty six T1D patients (age 8-18 years) without known diabetic complications were examined with tissue Doppler imaging and stratified into two groups according to diastolic function. A clinical examination and ultrasound of the common carotid arteries were performed. Methylglyoxal-derived hydroimidazolone-1 (MG-H1) was measured by immunoassay. RESULTS: At inclusion, 36 (25%) participants were stratified into a low diastolic function group (E'/A'-ratio < 2.0). Compared to the rest of the T1D children, these participants had higher body mass index (BMI), 22.8 (SD = 4.0) vs. 20.1 (SD = 3.4) kg/m2, p < 0.001, higher systolic blood pressure 104.2 (SD = 8.7) vs. 99.7 (SD = 9.3) mmHg, p = 0.010, and higher diastolic blood pressure, 63.6 (SD = 8.3) vs. 59.9 (SD = 7.9) mmHg, p = 0.016. The distensibility coefficient was lower, 0.035 (SD = 0.010) vs. 0.042 (SD = 0.02) kPa-1, p = 0.013, Young's modulus higher, 429 (SD = 106) vs. 365 (SD = 143), p = 0.009, and MG-H1 higher, 163.9 (SD = 39.2) vs. 150.3 (SD = 33.4) U/ml, p = 0.046. There was no difference in carotid intima-media thickness between the groups. There were no associations between reduced diastolic function and years from diagnosis, HBA1c, mean HBA1c, CRP or calculated glycemic burden. Logistic regression analysis showed that BMI was an independent risk factor for E'/A'-ratio as well as a non-significant, but relatively large effect size for MG-H1, indicating a possible role for AGEs. CONCLUSIONS: Early signs of reduced diastolic function in children and adolescents with T1D had higher BMI, but not higher HbA1c. They also had elevated serum levels of the advanced glycation end product MG-H1, higher blood pressure and increased stiffness of the common carotid artery, but these associations did not reach statistical significance when tested in a logistic regression model.

Eating patterns in adolescents with type 1 diabetes: Associations with metabolic control, insulin omission, and eating disorder pathology.

OBJECTIVE: The purpose of this study was to investigate eating patterns among male and female adolescents with type 1 diabetes (T1D), and the associations with age, zBMI, eating disorder (ED) pathology, intentional insulin omission, and metabolic control. METHOD: The sample consisted of 104 adolescents (58.6% females) with child-onset T1D, mean age of 15.7 years (SD 1.8) and mean zBMI of 0.4 (SD 0.8). The Child Eating Disorder Examination (ChEDE) assessed meal/snack frequency and ED pathology. T1D clinical data was obtained from the Norwegian Childhood Diabetes Registry. RESULTS: A significantly lower proportion of females than males (73.8% vs 97.7%) consumed breakfast on a daily basis. Approximately 50% of both genders ate lunch and 90% ate dinner daily. Among females, skipping breakfast was significantly associated with higher global ED psychopathology, shape concerns, self-induced vomiting, binge eating, insulin omission due to shape/weight concerns, and poorer metabolic control. Less frequent lunch consumption was significantly associated with poorer metabolic control. Skipping dinner was significantly associated with older age, higher dietary restraint, eating concerns, self-induced vomiting, and insulin omission. Among males, less frequent consumption of lunch and evening snacks was associated with attitudinal features of ED, including shape/weight concerns and dietary restraint. DISCUSSION: Among adolescents with T1D, irregular or infrequent meal consumption appears to signal potential ED pathology, as well as being associated with poorer metabolic control. These findings suggest the importance of routinely assessing eating patterns in adolescents with T1D to improve detection of ED pathology and to facilitate improved metabolic control and the associated risk of somatic complications.