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Effects of glycated insulin on adipogenic differentiation. A. Piccoli1, T. Bisogno1,2, M. Maccarrone1,3, P. Pozzilli1,4, N. Napoli1,5, R. Strollo1. 1Universita Campus Bio-Medico di Roma, Rome, Italy; 2National Research Council, Pozzuoli, Italy; 3European Center for Brain Research (CERC)/Santa Lucia Foundation IRCCS, Rome, Italy; 4Queen Mary, University of London, London, UK; 5Washington University in St Louis, St Louis, MO, USA Hyperglycaemia and chronic low-grade inflammation associated with obesity and diabetes are a main source of reactive oxidants (ROS). High levels of ROS may generate oxidative post-translational modification (oxPTMs) that may impair proteins biological functions. We have shown that insulin could also be a target of these modifications that include glycation, oxidation and chlorination. Adipose tissue is a primary target of this hormone and a key player in insulin resistance (IR): evidence from animal studies show that the glycation of insulin or other proteins result in a significant impairment of biological function of adipose tissue, affecting adipokines expression and insulin sensitivity. However, how oxPTMs such as glycation interfere with insulin function and their involvement in IR and type 2 diabetes (T2D) is still unknown. We hypothesize that insulin glycation may be involved in insulin resistance and in the pathogenesis of T2D. Therefore, the aim of this study was to assess the possible effects of glycated vs. native insulin on adipocyte differentiation. Human recombinant insulin was glycated in vitro following our established protocol. Insulin glycation was monitored through PAGE. Human preadipocytes (HPAd), a primary cell line from subcutaneous adipose tissue, and adipose-derived stem cells (ASCs), isolated from tissues of lean and obese subjects (BMI30), were differentiated with standard adipogenic medium in presence of native or glycated insulin. Cellular differentiation was assessed by gene and protein expression analyses of adipogenic markers (adiponectin, peroxisome proliferator-activated receptor gamma -Ppar?-, fatty acid binding protein 4 -Fabp4-, glucose transporter 4 -Glut4-) and of receptors for adiponectin (AdipoR1) and for insulin (InsR), through real time RT-PCR and immunoblot respectively. Our preliminary results showed that preadipocytes (both HPAd and ASCs) exposed to glycated insulin had impaired differentiation capacities: Oil Red O-staining showed that these cells had lower number of lipid droplets compared to those exposed to native insulin, suggesting an impaired adipocyte differentiation. Considering gene expression, we observed an overall downregulation of adipogenic markers, with a significant decrease of adiponectin both in leans- and in obese-derived cells (p=0.007 and p=0.017 respectively), lower levels of Glut4 and a significant lower expression of Fabp4 (p=0.002) in cells from leans differentiated with glycated insulin. Immunoblotting showed a similar pattern to that observed with gene expression analysis but, regarding insulin receptor, protein levels of the receptor presented a significant down regulation in cells treated with glycated insulin, with a p value of 0.0266 in leans and p=0.011 in obese subjects. Conversely, gene expression of insulin receptor did not present any difference between the native and glycated insulin. In conclusion, our data suggest that glycated insulin could affect adipocytes differentiation and impair insulin receptor expression, acting at translational/post-translational level or increasing its degradation. Its role and mechanisms that lead to these effects should be investigated as a possible mediator in the pathogenesis of T2D.

Charcot neuroarthropathy (CN) is a disabling complication, culminating in bone destruction and involving joints and articular cartilage with high inflammatory environment. Its real pathogenesis is as yet unknown. In autoinflammatory diseases, such as rheumatoid arthritis, characterized by inflammation and joint involvement, autoantibodies against oxidative post-translationally modified (oxPTM) collagen type I (CI) and type II (CII) were detected. Therefore, the aim of our study was to assess the potential involvement of autoimmunity in charcot neuroarthropathy, investigating the presence of autoantibodies oxPTM-CI and oxPTM-CII, in participants with charcot neuroarthropathy.In this case-control study, we enrolled 124 participants with type 2 diabetes mellitus (47 with charcot neuroarthropathy, 37 with diabetic peripheral neuropathy without charcot neuroarthropathy, and 40 with uncomplicated diabetes), and 32 healthy controls. The CI and CII were modified with ribose and other oxidant species, and the modifications were evaluated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding of sera from the participants was analyzed with enzyme-linked immunosorbent assay.Age, body mass index, waist and hip circumferences, and lipid profile were similar across the 4 groups, as well as glycated hemoglobin and duration of diabetes among people with diabetes. An increased binding to both native and all oxidation-modified forms of CII was found in participants with CN and diabetic neuropathy. Conversely, for CI, an aspecific increased reactivity was noted.Our results detected the presence of autoantibodies against oxidative post-translational modified collagen, particularly type 2 collagen, in participants with charcot neuroarthropathy and diabetic neuropathy, suggesting the possible involvement of autoimmunity. Further studies are required to understand the role of autoimmunity in the pathogenesis of charcot neuroarthropathy.

Contains fulltext : 125353.pdf (Publisher’s version ) (Open Access) BACKGROUND: Vitamin D supplementation in childhood improves the achievement of peak bone mass. We investigated the effect of supplementation with calcitriol on bone turnover in recent-onset type 1 diabetes (T1D). Moreover, the association between osteocalcin and parameters of beta-cell function and metabolic control was examined. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a post-hoc analysis of a double-blind, placebo-controlled study of calcitriol supplementation to preserve beta-cell function. 27 recent-onset T1D subjects, mean age 22 years, were randomized to 0.25 microg calcitriol per day or placebo (1:1) and followed up for one year. Changes in bone formation (osteoclacin) and resorption (beta-CrossLaps) markers, and differences between placebo and calcitriol-treated group were evaluated. At baseline, osteocalcin levels were significantly lower in female than in male patients (P

Acute lymphoblastic leukemia (ALL) is the most common subtype of leukemia in children. Although ALL and type 1 diabetes appear to be biologically unrelated, there are common threads in both epidemiology and etiology. Rising incidence rates of both ALL (1) and type 1 diabetes (2) observed over recent decades in many Western countries seem to support common etiological factors (3). In the current study, we report on diabetes-related autoantibodies (Abs) in a group of patients with ALL. Thirty-four consecutive children (19 males and 15 females, mean age 6.2 ± 4.6 years) were referred to our institution in 2004 for newly diagnosed ALL. Patients were tested for Abs to islet and thyroid antigens. After the initial investigation and treatment, 31/34 (91%) patients (3 died in the mean time) were followedup for 6 years to evaluate the evolution of the autoimmune markers and progression toward type 1 diabetes. Glutamic acid decarboxylase (GAD) Abs by direct …

Aims/hypothesis Antibodies specific to oxidative post-translational modifications (oxPTM) of insulin (oxPTM-INS) are present in most individuals with type 1 diabetes, even before the clinical onset. However, the antigenic determinants of such response are still unknown. In this study, we investigated the antibody response to oxPTM-INS neoepitope peptides (oxPTM-INSPs) and evaluated their ability to stimulate humoral and T cell responses in type 1 diabetes. We also assessed the concordance between antibody and T cell responses to the oxPTM-INS neoantigenic peptides. Methods oxPTM-INS was generated by exposing insulin to various reactive oxidants. The insulin fragments resulting from oxPTM were fractionated by size-exclusion chromatography further to ELISA and LC-MS/MS analysis to identify the oxidised peptide neoepitopes. Immunogenic peptide candidates were produced and then modified in house or designed to incorporate in silico-oxidised amino acids during synthesis. Autoantibodies to the oxPTM-INSPs were tested by ELISA using sera from 63 participants with new-onset type 1 diabetes and 30 control participants. An additional 18 fresh blood samples from participants with recently diagnosed type 1 diabetes, five with established disease, and from 11 control participants were used to evaluate, in parallel, CD4+ and CD8+ T cell activation by oxPTM-INSPs. Results We observed antibody and T cell responses to three out of six LC-MS/MS-identified insulin peptide candidates: A:12–21 (SLYQLENYCN, native insulin peptide 3 [Nt-INSP-3]), B:11–30 (LVEALYLVCGERGFFYTPKT, Nt-INSP-4) and B:21–30 (ERGFFYTPKT, Nt-INSP-6). For Nt-INSP-4 and Nt-INSP-6, serum antibody binding was stronger in type 1 diabetes compared with healthy control participants (p≤0.02), with oxidised forms of ERGFFYTPKT, oxPTM-INSP-6 conferring the highest antibody binding (83% binders to peptide modified in house by hydroxyl radical [●OH] and >88% to in silico-oxidised peptide; p≤0.001 vs control participants). Nt-INSP-4 induced the strongest T cell stimulation in type 1 diabetes compared with control participants for both CD4+ (p+ (p=0.049). CD4+ response to oxPTM-INSP-6 was also commoner in type 1 diabetes than in control participants (66.7% vs 27.3%; p=0.039). Among individuals with type 1 diabetes, the CD4+ response to oxPTM-INSP-6 was more frequent than to Nt-INSP-6 (66.7% vs 27.8%; p=0.045). Overall, 44.4% of patients showed a concordant autoimmune response to oxPTM-INSP involving simultaneously CD4+ and CD8+ T cells and autoantibodies. Conclusions/interpretation Our findings support the concept that oxidative stress, and neoantigenic epitopes of insulin, may be involved in the immunopathogenesis of type 1 diabetes. Graphical abstract

Aims/hypothesis: We assessed the levels of blood circulating immune checkpoint molecules (ICMs) at diagnosis of type 1 diabetes, and determined their association with the risk of developing an additional autoimmune disorder over time. Methods: Children with new-onset type 1 diabetes (n = 143), without biological and/or clinical signs of additional autoimmune disorders, and healthy children (n = 75) were enrolled, and blood circulating levels of 14 ICMs were measured. The children with type 1 diabetes were divided into two groups on the basis of the development of an additional autoimmune disease in the 5 years after diabetes onset. Differences in soluble ICM levels between the groups were assessed, and a Cox regression analysis was used to evaluate their association with the risk of development of an additional autoimmune disease over time. To validate the data, circulating ICMs were measured in an independent cohort of 60 children with new-onset type 1 diabetes stratified into two groups. Results: We found that the levels of circulating ICMs were significantly higher in children with new-onset diabetes compared with healthy children. Further, we observed that children with type 1 diabetes who developed a second autoimmune disease over time (T1D-AAD+ children) had higher levels of soluble ICMs than children with type 1 diabetes who did not (T1D-AAD− children). Cox regression models revealed that high circulating levels of CD137/4-1BB and PD-1 molecules at diabetes diagnosis were associated with the risk of developing an additional autoimmune disease in both type 1 diabetes cohorts. Conclusions/interpretation: Our findings suggest that soluble CD137/4-1BB and PD-1 molecules may be used as prognostic biomarkers in children with type 1 diabetes, and may pave the way for novel immunological screening at diabetes onset, allowing early identification of children at higher risk of developing other autoimmune conditions over time. Graphical abstract: [Figure not available: see fulltext.]

Currently, there is no effective therapy for COVID-19, and several approaches are under investigation. Nevertheless, some drugs are used off-label despite the absence of clear data on their effectiveness. Among these, hydroxychloroquine suppresses SARS-CoV-2 replication in vitro,1 and clinical trials are ongoing to evaluate its use as an anti-COVID-19 agent. To date, the FDA and EMA allow its use only in hospitalized patients with severe COVID-19 or in those at high risk, in cases where other trials are not feasible. According to a small non-randomized study, hydroxychloroquine’s efficacy might be enhanced by azithromycin, as the combination of these two drugs appeared to accelerate viral clearance.2 However, these findings were not substantiated by another study performed in severe COVID-19 cases.3 In addition to the lack of consistent or conclusive efficacy data of these drugs, whether they are used individually or in combination, concern is increasing about potential safety issues. Both hydroxychloroquine and azithromycin may lead to QT prolongation-related life-threatening arrhythmias through the blockage of the rapid delayed rectifier potassium current (IKr). Their combination seems to be more harmful than their single use. Importantly, the risk of azithromycin-mediated QT prolongation is higher in males and in elderly groups,4 that represent a large proportion of the COVID-19 population. Data from a recent Chinese report showed that 16.7% of hospitalized COVID-19 patients and 44.4% of those in intensive care units had arrhythmias.5 The two main mechanisms involved could be hypoxia and electrolyte abnormalities, common features in the acute phase of severe illness. These factors may further enhance the risk of arrhythmic toxicity associated with hydroxychloroquine or azithromycin. Therefore, strategies to mitigate this potential risk have been proposed. The American College of Cardiology suggested QT monitoring in order to discontinue azithromycin (if used) and/or reduce the hydroxychloroquine dose in the case of QT prolongation. While drugs currently used against COVID-19 may seem crucial to treat the infection (in the absence of better options), it is imperative to mitigate toxicity to guarantee the maximum possible safety. According to 2019 international guidelines by the American Thoracic Society and Infectious Diseases Society of America, in pneumonia patients with QT prolongation, macrolides should be replaced by tetracyclines. While providing a broad-spectrum antibiotic activity, compared with other antibiotics tetracyclines also display additional properties that can be advantageous in COVID-19. In particular, the tetracycline derivative minocycline exerts anti-inflammatory effects by suppressing proinflammatory cytokines, including interleukin-6 (IL-6), which plays a key role in COVID-19, and may inhibit poly(ADP-ribose) polymerase-1 (PARP-1) which seems to be implicated in SARS-CoV-2 replication.6,7 Moreover, minocycline displays a synergistic effect when added to hydroxychloroquine.8 Compared with azithromycin, minocycline does not prolong the QT interval, and experimental evidence suggests that it may even be beneficial in conditions associated with increased risk of QT prolongation or arrhythmia. In an experimental model of epilepsy, intrathecal administration of minocycline, by antagonizing the action of microglia, reduced autonomic nervous system activity and seizure-induced QT prolongation.9 In other murine models, treatment with minocycline attenuated ischaemia-induced arrhythmias by blocking the mitochondrial K-ATP channel and inhibiting the PI3K/Akt signalling pathway mediated by L-type calcium channels both involved in myocardial ischaemia-induced arrhythmias.10 Although not proven in humans yet, it is reasonable to believe that minocycline may prevent (or at least mitigate) the increased risk of QT prolongation, and potentially fatal arrhythmias, due to hydroxychloroquine/azithromycin combination therapy, while having the same efficacy as azithromycin but a lower arrhythmogenic effect. For these reasons, until clinical trials prove the efficacy of azithromycin alone or in combination with hydroxychloroquine, we suggest that, whenever antibiotic prophylaxis is needed, azithromycin should be replaced by minocycline.

Aims/hypothesis: We aimed to analyse the association between plasma circulating microRNAs (miRNAs) and the immunometabolic profile in children with type 1 diabetes and to identify a composite signature of miRNAs/immunometabolic factors able to predict type 1 diabetes progression. Methods: Plasma samples were obtained from children at diagnosis of type 1 diabetes (n = 88) and at 12 (n = 32) and 24 (n = 30) months after disease onset and from healthy control children with similar sex and age distribution (n = 47). We quantified 60 robustly expressed plasma circulating miRNAs by quantitative RT-PCR and nine plasma immunometabolic factors with a recognised role at the interface of metabolic and immune alterations in type 1 diabetes. Based on fasting C-peptide loss over time, children with type 1 diabetes were stratified into the following groups: those who had lost >90% of C-peptide compared with diagnosis level; those who had lost

Objective: To determine clinical diabetes-related risk factors for fragility fractures in type 1 diabetes (T1D). Research design and methods: History of bone fragility fractures occurring after T1D diagnosis was assessed by questionnaire in this cross-sectional study in 600 T1D subjects. Glycated hemoglobin A1c (HbA1c)over the previous 5 years was used as an index of long-term glycemic control; complications were adjudicated by physician assessment. Multinomial logistic regression models were used to assess the associations between diabetes-related risk factors and fracture history. Results: One-hundred-eleven patients (18.5%)reported at least one fracture; of these 73.8% had only one and 26.2% had more than one fracture. Average age was 41.9 ± 12.8 years, with even gender distribution; disease duration was 19.9 ± 12.0 years; and BMI was 24.4 ± 3.7 kg/m2. The 5-year average HbA1c was 7.6 ± 1.0% (60 mmol/mol). In adjusted models, reduced risk for 1 fracture was found in those with higher creatinine clearance rate (CCr)(RRR 0.22 [95% CI: 0.06–0.83]for 1 unit increase in lnCCr, p = 0.03)and increased risk in those with neuropathy (RRR 2.57 [1.21–5.46], p = 0.01). Increased risk for ≥2 fractures was found in subjects in the highest tertile of HbA1c (≥7.9%)compared with the lowest tertile (≤7.17%)(RRR 3.50 [1.04–11.7], p = 0.04)and of disease duration (≥26 years versus

Ambient noise measurements were performed at the island of Ischia (southern Italy) along alignments of 2.4–7 km by using two three-component seismic stations. Synchronous noise recordings of 2–20 h were cross-correlated over 20–30 s windows, stacked and iteratively band-pass filtered to enhance the dispersive wave trains. Frequency time analysis was performed on the vertical and radial components of cross-correlations and the fundamental-mode Rayleigh wave group velocity was obtained. Validation of the dispersion data was possible with those obtained from an earthquake recording along a close path. The non-linear inversion of average Rayleigh wave group velocity dispersion curves along 13 paths (receiver inter-distances) allowed the definition of shear wave velocity models in the uppermost 1–2 km of the crust. The correlation of VS profiles vs. depth and drilling stratigraphy allowed to attribute VS lower than 1 km/s to tuffs and VS of 1.41 km/s to very fractured lavas. Higher VS are found in the central area of the island, in correspondence of the resurgent area. The top of the trachytic lava basement, with VS of 2.2–2.4 km/s and density of 2.3 g/cm3 is about 0.6–0.7 km deep b.s.l. in the centre of Ischia, below altered, very fractured lava or thermally altered tuff.