Multiplexed MRM-based proteomics for identification of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) increases the risk of coronary heart disease (CHD) by 2-4 fold, and is associated with endothelial dysfunction, dyslipidaemia, insulin resistance, and chronic hyperglycaemia. The aim of this investigation was to assess, by a multimarker mass spectrometry approach, the predictive role of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus. METHODS: The study considered 34 patients with both T2DM and CHD, 31 patients with T2DM and without CHD, and 30 patients without diabetes with a diagnosis of CHD. Plasma samples of subjects were analysed through a multiplexed targeted liquid chromatography mass spectrometry (LC-MS)-based assay, namely Multiple Reaction Monitoring (MRM), allowing the simultaneous detection of peptides derived from a protein of interest. Gene Ontology (GO) Analysis was employed to identify enriched GO terms in the biological process, molecular function, or cellular component categories. Non-parametric multivariate methods were used to classify samples from patients and evaluate the relevance of the analysed proteins' panel. RESULTS: A total of 81 proteins were successfully quantified in the human plasma samples. Gene Ontology analysis assessed terms related to blood microparticles, extracellular exosomes and collagen-containing extracellular matrix. Preliminary evaluation using analysis of variance (ANOVA) of the differences in the proteomic profile among patient groups identified 13 out of the 81 proteins as significantly different. Multivariate analysis, including cluster analysis and principal component analysis, identified relevant grouping of the 13 proteins. The first main cluster comprises apolipoprotein C-III, apolipoprotein C-II, apolipoprotein A-IV, retinol-binding protein 4, lysozyme C and cystatin-C; the second one includes, albeit with sub-grouping, alpha 2 macroglobulin, afamin, kininogen 1, vitronectin, vitamin K-dependent protein S, complement factor B and mannan-binding lectin serine protease 2. Receiver operating characteristic (ROC) curves obtained with the 13 selected proteins using a nominal logistic regression indicated a significant overall distinction (p < 0.001) among the three groups of subjects, with area under the ROC curve (AUC) ranging 0.91-0.97, and sensitivity and specificity ranging from 85 to 100%. CONCLUSIONS: Targeted mass spectrometry approach indicated 13 multiple circulating proteins as possible biomarkers of cardiovascular damage progression associated with T2DM, with excellent classification results in terms of sensitivity and specificity.

The ankle-brachial index for assessing the prevalence of peripheral artery disease and cardiovascular risk in patients with type 2 diabetes mellitus.

BACKGROUND AND AIMS: Type 2 diabetes mellitus (T2DM) is an important risk factor for peripheral artery disease (PAD). Ankle-Brachial Index (ABI) was found associated with a higher cardiovascular (CV) risk and mortality. The main goals of this study were to establish the prevalence of PAD in a T2DM population, and assess the relationship between PAD and the CV risk calculated with the CUORE Project score (CPS) (https://www.cuore.iss.it/). The association between the ABI, the main risk factors for PAD and T2DM complications was also investigated. METHODS AND RESULTS: Two hundred patients were consecutively enrolled. The prevalence of PAD in this population was 17%. The CV risk tended to be higher (p = 0.0712) in the group with a pathological ABI than in the group with a normal ABI. Glycated hemoglobin (r = -0.1591; p = 0.0244), total cholesterol (r = -0.1958; p = 0.0054), LDL cholesterol (r = -0.1708; p = 0.0156) and systolic blood pressure (r = -0.1523; p = 0.0313) correlated significantly and inversely with the left ABI. The frequency of diabetic retinopathy was significantly higher in the group with a pathological ABI (p = 0.0316). CONCLUSIONS: The data reveal a high prevalence of PAD in patients with T2DM. The CPS confirmed that patients with a pathological ABI have tendency to a higher CV risk. The results point to the importance of an accurate CV assessment - also measuring individuals' ABI and calculating their CPS - to better pinpoint those at high risk of PAD, especially among patients with T2DM.

Mass Spectrometry Study about In Vitro and In Vivo Reaction between Metformin and Glucose: A Preliminary Investigation on Alternative Biological Behavior.

Metformin is the most prescribed glucose-lowering drug worldwide; globally, over 100 million patients are prescribed this drug annually. Some different action mechanisms have been proposed for this drug, but, surprisingly, no metabolite of metformin has ever been described. It was considered interesting to investigate the possible reaction of metformin with glucose following the Maillard reaction pattern. The reaction was first performed in in vitro conditions, showing the formation of two adducts that originated by the condensation of the two molecular species with the losses of one or two water molecules. Their structures were investigated by liquid chromatography coupled with mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS) and accurate mass measurements (HRMS). The species originated via the reaction of glucose and metformin and were called metformose and dehydrometformose, and some structural hypotheses were conducted. It is worth to emphasize that they were detected in urine samples from a diabetic patient treated with metformin and consequently they must be considered metabolites of the drug, which has never been identified before now. The glucose-related substructure of these compounds could reflect an improved transfer across cell membranes and, consequently, new hypotheses could be made about the biological targets of metformin.

Dietary inflammatory index score, glucose control and cardiovascular risk factors profile in people with type 2 diabetes.

We examined the relationships between the dietary inflammatory index (DII®), dietary habits and cardiovascular risk factor profiles in people with type 2 diabetes mellitus (T2DM). Energy-adjusted DII (E-DII™) scores were calculated from a Food Frequency Questionnaire in 2568 T2DM patients from different parts of Italy. Analyses were conducted according to quartiles of sex-specific E-DII scores. Higher, more pro-inflammatory, (quartile 4) E-DII scores were associated with overall poor quality of the diet characterised by higher content of refined carbohydrates, added sugars, saturated fat and cholesterol and lower unsaturated fat, fibre and polyphenols compared to quartile 1. Higher E-DII scores also were associated with higher waist circumference (105.4 vs. 103.5 cm; p = 0.002), triglycerides (154.6 vs. 146.1 mg/dL; p = 0.005), diastolic blood pressure (80.05 vs. 78.6 mmHg; p = 0.04) and lower HDL-cholesterol (45.3 vs. 47.4 mg/dL; p = 0.04). In conclusion, E-DII is a potent marker of overall quality of the diet and is associated with an unfavourable cardiovascular risk factor profile.

Possible role of fructosamine 3-kinase genotyping for the management of diabetic patients.

Diabetes mellitus is a global pandemic and continues to increase in numbers and significance. Several pathogenic processes are involved in the development of such disease and these mechanisms could be influenced by genetic, epigenetic and environmental factors. Non-enzymatic glycation reactions of proteins have been strongly related to pathogenesis of chronic diabetic complications. The identification of fructosamine 3-kinase (FN3K), an enzyme involved in protein deglycation, a new form of protein repair, is of great interest. FN3K phosphorylates fructosamines on the third carbon of their sugar moiety, making them unstable and causing them to detach from proteins, suggesting a protective role of this enzyme. Moreover, the variability in FN3K activity has been associated with some polymorphisms in the FN3K gene. Here we argue about genetic studies and evidence of FN3K involvement in diabetes, together with results of our analysis of the FN3K gene on a Caucasian cohort of diabetic patients. Present knowledge suggests that FN3K could act in concert with other molecular mechanisms and may impact on gene expression and activity of other enzymes involved in deglycation process.

Is eGFR Slope a Novel Predictor of Chronic Complications of Type 2 Diabetes Mellitus? A Systematic Review and Meta-Analysis.

Background: Diabetic kidney disease affects approximately 40% of patients with type 2 diabetes mellitus (T2DM) and is associated with an increased risk of end-stage kidney disease (ESKD) and cardiovascular (CV) events, as well as increased mortality. Among the indicators of decline in renal function, the eGFR slope is acquiring an increasing clinical interest. The aim of this study was to evaluate, through a systematic review of the literature and meta-analysis of the collected data, the association between the decline of the eGFR slope, chronic complications, and mortality of T2DM patients, in order to understand whether or not the eGFR slope can be defined as a predictive indicator of complications in T2DM. Methods: The review and meta-analysis were conducted according to PRISMA guidelines considering published studies on patients with T2DM. A scientific literature search was carried out on PubMed from January 2003 to April 2023 with subsequent selection of scientific papers according to the inclusion criteria. Results: Fifteen studies were selected for meta-analysis. Risk analysis as hazard ratio (HR) indicated a significant association between all events considered (all-cause mortality, CV events, ESKD, and microvascular events) for patients with steeper eGFR slope decline than subjects with stable eGFR. Calculated HRs (with 95% CI) were as follows: for all-cause mortality, 2.31 (1.70-3.15); for CV events, 1.73 (1.43-2.08); for ESKD, 1.54 (1.45-1.64); and for microvascular events, 2.07 (1.57-2.73). Overall HR was 1.82 (1.72-1.92). Conclusions: An association between rapid eGFR decline and chronic diabetes complications was demonstrated, suggesting that eGFR slope variability significantly impacts the course of T2DM and that eGFR slope should be considered as a predictor for chronic complications in patients with T2DM. According to the obtained results, the therapeutic management of the patient with diabetes should not focus exclusively on glycaemic control, and particular attention should be paid to preserve renal function.

Lipid profile in women of different ethnicity with gestational diabetes: Relationship with fetal growth.

AIMS/INTRODUCTION: Pregnancy complicated by gestational diabetes mellitus (GDM) is characterized by excessive insulin resistance that impairs the metabolism of glucose and lipids. the aim of the study was to examine lipid profiles during pregnancy of women with GDM, and its impact on fetal growth in a multiethnic population. MATERIALS AND METHODS: The study included 322 pregnant women of different ethnicity with GDM attending a clinical unit specializing in metabolic diseases. RESULTS: The area under the curve for the 75-g oral glucose tolerance test and glycated hemoglobin were significantly different among all groups. At the time of being diagnosed with GDM, Asian and African mothers had significantly lower levels of total and low-density liprotein cholesterol than European mothers (P < 0.001). The trend for high-density liprotein cholesterol was similar. Triglycerides levels in the Asian group (193.6 ± 65.5 mg/dL) were higher than in the African group (133.2 ± 49.6 mg/dL, P < 0.001), whereas the European group presented intermediate values (175.8 ± 58.8 mg/dL), which differed significantly only versus the African group (P < 0.001). Pre-partum lipid profiles showed a trend quite similar to that observed at diagnosis. The newborn's birthweight was significantly different, with that of African women (3,437 ± 503 g) being the highest, followed by that of European women (3,294 ± 455 g) and of Asian women (3,006 ± 513 g). The rates of macrosomia showed a trend with higher values in the African group (13.5%), followed by the European group (5.7%, P = 0.1162), whereas that of the Asian group was zero (P = 0.0023 vs African). CONCLUSIONS: Our data show that lipid profiles in women with GDM differ by ethnicity. The impact of lipid profile on fetal growth is limited and uninfluenced by ethnicity.

Telemedicine and its acceptance by patients with type 2 diabetes mellitus at a single care center during the COVID-19 emergency: A cross-sectional observational study.

INTRODUCTION: When Italy was placed under lockdown to contain the COVID-19 pandemic from 9 March to 18 May 2020, alternative approaches to delivering care-such as telemedicine-were promoted for patients with chronic diseases like diabetes mellitus (DM). The aim of this study was to analyze patients' perception of, and satisfaction with the telehealth services offered during the COVID-19 emergency at an outpatient diabetes care unit in Italy. METHODS: A cross-sectional survey was conducted on 250 patients with type 2 diabetes mellitus who regularly attended our diabetes care unit. Data were collected by means of telephone interviews, asking patients how they perceived the telehealth services, and their satisfaction with the televisit and computer-based care. A standardized questionnaire was administered: there were questions answered using a five-point Likert scale, and one open-ended question. Patients' demographic, anthropometric and biological data were collected from their medical records. Correlations between patients' characteristics, their perception of telemedicine, and their satisfaction with the televisit were examined. Spearman's rank-order correlation coefficient ρ (rho) and Kendall's rank correlation coefficient τ (tau) were used as nonparametric measures of the strength of the association between the scores obtained for the two ordinal variables, Perception and Satisfaction, and between other clinical parameters. Principal component analysis (PCA) was also used to assess overall links between the variables. RESULTS: Almost half of the interviewees expressed a strongly positive perception of the medical services received, and more than 60% were very satisfied with the telehealth service provided during the COVID-19 emergency. There was a strong correlation between patients' perception and satisfaction ratings (p<0.0001). Duration of disease showed a significant positive correlation with patients' satisfaction with their medical care. By means of PCA, it was found that BMI correlated inversely with both perception and satisfaction. Following a qualitative analysis of patients' answers to the open-ended question, contact with their specialist was important to them: it was reassuring and a source of scientifically correct information about their disease and the association between COVID-19 and diabetes. CONCLUSIONS: Based on our telephone interviews, patients appreciated the telehealth approach and were satisfied with it, regardless of the characteristics of their disease. Telemedicine proved essential to avoid interrupting the continuity of care, and therefore had not only clinical, but also psycho-social repercussions.

Long-term HbA1c variability and macro-/micro-vascular complications in type 2 diabetes mellitus: a meta-analysis update.

AIMS: The aim of the present study was to evaluate, by means of a meta-analysis approach, whether new available data, appeared on qualified literature, can support the effectiveness of an association of HbA1c variability with the risk of macro- and/or micro-vascular complications in type 2 diabetes mellitus (T2DM). METHODS: The meta-analysis was conducted according to PRISMA Statement guidelines and considered published studies on T2DM, presenting HbA1c variability as standard deviation (SD) or its derived coefficient of variation (CV). Literature search was performed on PubMed in the time range 2015-July 2022, with no restrictions of language. RESULTS: Twenty-three selected studies fulfilled the aims of the present investigation. Overall, the analysis of the risk as hazard ratios (HR) indicated a significant association between the HbA1c variability, expressed either as SD or CV, and the complications, except for neuropathy. Macro-vascular complications were all significantly associated with HbA1c variability, with HR 1.40 (95%CI 1.31-1.50, p < 0.0001) for stroke, 1.30 (95%CI 1.25-1.36, p < 0.0001) for transient ischaemic attack/coronary heart disease/myocardial infarction, and 1.32 (95%CI 1.13-1.56, p = 0.0007) for peripheral arterial disease. Micro-vascular complications yielded HR 1.29 (95%CI 1.22-1.36, p < 0.0001) for nephropathy, 1.03 (95%CI 0.99-1.08, p = 0.14) for neuropathy, and 1.15 (95%CI 1.08-1.24, p < 0.0001) for retinopathy. For all-cause mortality, HR was 1.33 (95%CI 1.27-1.39, p < 0.0001), and for cardiovascular mortality 1.25 (95%CI 1.17-1.34, p < 0.0001). CONCLUSIONS: Our meta-analysis on HbA1c variability performed on the most recent published data since 2015 indicates positive association between HbA1c variability and macro-/micro-vascular complications, as well as mortality events, in T2DM, suggesting that this long-term glycaemic parameter merits further attention as a predictive, independent risk factor for T2DM population.

Anti-diabetic combination therapy with pioglitazone or glimepiride added to metformin on the AGE-RAGE axis: a randomized prospective study.

Introduction: The ratio between advanced glycation end products (AGEs) and soluble form of receptor (s-RAGE) has been proposed as a risk marker for renal and cardiovascular diseases. The aim of this study was to evaluate in the diabetes condition the influence of two different oral anti-diabetic treatments on the AGE/s-RAGE ratio, during a 5-year observation period. Methods: Seventy-three patients with type 2 diabetes mellitus were randomly assigned to a drug therapy with pioglitazone or glimepiride, combined to metformin. Each subject was evaluated at baseline and after 5 years of treatment. Results: In both groups s-RAGE levels did not significantly vary, while the levels of AGE and AGE/s-RAGE were both significantly reduced, basal compared to 5-year values. Within pioglitazone group, as well within glimepiride group, significant variations (Δ, as difference between 5 years of treatment minus basal) were observed for AGE (Δ= -21.1±13.4 µg/ml, P<0.001 for pioglitazone; Δ= -14.4±11.4 µg/ml, P<0.001 for glimepiride) and in AGE/s-RAGE (Δ= -0.037±0.022 µg/pg, P<0.001 for pioglitazone; Δ= -0.024±0.020µg/pg, P<0.001 for glimepiride), suggesting an average decrease of the parameters by more than 50% in both treatments. Pioglitazone was more effective than glimepiride in reducing AGE/s-RAGE ratio after 5 years of therapy. Conclusion: These data can help to explain the benefits of oral anti-diabetic therapy in relation to the reduction of cardiovascular risk, as suggested by variations in AGE/s-RAGE ratio as biochemical marker of endothelial function; in particular, treatment with pioglitazone seems to offer greater long-term benefit on AGE-RAGE axis.

Omicron Variant of SARS-CoV-2 Virus: In Silico Evaluation of the Possible Impact on People Affected by Diabetes Mellitus.

The Omicron variant of SARS-CoV-2 (Spike mutant B.1.1.529) carrying more than 30-point mutations in its structure, of which 15 are localized in the receptor-binding domain (RBD), allows to hypothesize a relevant change in interactivity with ACE2. In previous reports we hypothesized that the worse outcome of the COVID-19 disease in diabetes mellitus condition could be related to the non-enzymatic glycation of ACE2 receptor and an in silico evaluation led to the demonstration that the number of interactions is decreased in comparison to the unmodified model, possibly shifting the virus attack through different, multiple alternative entry routes. Given the evidenced features of this variant, we aimed to investigate with a computational approach the characteristics of Omicron SARS-CoV-2 with respect to its binding to human ACE-2 receptor, in a particular population, namely people affected by diabetes mellitus, at risk for unfavorable outcomes of the COVID-19. The computational analysis, considering the case in which all the lysine residues in the system are subjected to non-enzymatic glycation, confirmed that lysine glycation causes a general loss of interactivity between wild-type (WT)-Spike-RBD and ACE2. In the Omicron variant, Lys417 mutates into an asparagine, preventing the possible non-enzymatic glycation of this residue. Therefore, if non-enzymatic glycation seemed to cause a shift in the way in which the virus enters the cell from the ACE2-mediated mechanism to other pathways, in the case of the Omicron variant the ACE2-mediated approach of the virus seems to remain an important event to take into account. Indeed, interaction profile analysis, together with molecular mechanics-generalized Born surface area (MM-GBSA) calculations, suggests that the Omicron-Spike-RBD maintains a higher affinity for ACE2 subsequently to non-enzymatic glycation with respect to WT-Spike-RBD. The finding of the present computational study may suggest a different clinical relevance of the Omicron variant for the diabetes mellitus field, also in the possible direction of a lower severity of the disease.

The Burden of Impaired Serum Albumin Antioxidant Properties and Glyco-Oxidation in Coronary Heart Disease Patients with and without Type 2 Diabetes Mellitus.

Human serum albumin (HSA) has an important antioxidant activity due to the presence of the reduced cysteine at position 34, which represents the most abundant free thiol in the plasma. In oxidative-based diseases, HSA undergoes S-thiolation (THIO-HSA) with changes in the antioxidant function of albumin that could contribute to the progression of the disease. The aim of this study was to verify, for the first time, the different burdens of THIO-HSA, glycated HSA (GLY-HSA), and advanced glycation end products (AGE) accumulation both in type 2 diabetes mellitus (T2DM) patients and in non-diabetic patients, with or without coronary heart disease (CHD). In this study, we assessed the presence of modified forms of HSA, THIO-HSA, and GLY-HSA by means of mass spectrometry in 33 patients with both T2DM and CHD, in 31 patients with T2DM and without CHD, in 30 patients without diabetes with a history of CHD, and 27 subjects without diabetes and CHD. All the patients' anthropometric and clinical data were recorded including age, sex, duration of diabetes, body mass index (BMI), blood pressure, and history of CHD defined with anamnestic data. Metabolic parameters, such as fasting plasma glucose (FPG), glycated hemoglobin (HbA1c), lipids, pentosidine, AGE, receptor for advanced glycation end-products (RAGE) and its soluble form (sRAGE), were measured. AGE and pentosidine are significantly higher in T2DM patients with and without CHD with respect to non-diabetic patients with CHD and control subjects. RAGE levels are significantly higher in T2DM patients with respect to non-diabetic patients, and among T2DM patients, the group with CHD showed significantly higher RAGE levels than those without CHD (217 ± 171 pg/mL and 140 ± 61 pg/mL, respectively). Albumin isoforms discriminate between non-diabetic patients with CHD and T2DM patients with and without CHD and control subjects, with GLY-HSA levels higher in T2DM with and without CHD, and THIO-HSA higher in CHD patients without T2DM. Finally, we demonstrated that the oxidized forms of HSA can increase the expression of the inflammatory cytokine Tumor Necrosis Factor-alpha (TNFα) in monocytic cells. In patients with CHD, GLY-HSA and THIO-HSA have a different prevalent distribution, the first one prevailing in patients with T2DM and the second one in patients without T2DM. These findings suggest that albumin quality and homeostasis balance between glyco-oxidation and thiolation might have an impact on the antioxidant defense system in cardiovascular diseases.

Genetic variability of the fructosamine 3-kinase gene in diabetic patients.

BACKGROUND: Nonenzymatic glycation appears to be an important factor in the pathogenesis of diabetic complications. Fructosamine 3-kinase (FN3K), initially identified in erythrocytes, appears to be responsible for the removal of fructosamine from proteins, suggesting a protective role in nonenzymatic glycation. Recently, genetic variants in the FN3K gene have been studied in diabetic patients. The aim of our study was the molecular characterization of the FN3K gene in a representative group of Italian patients with type 1 (T1DM) and 2 (T2DM) diabetes mellitus and in a cohort of healthy controls. METHODS: Seventy diabetic subjects (35 type 1 and 35 type 2) with stable glycemic control and 33 healthy control subjects were evaluated using PCR and direct sequencing of the FN3K gene. Denaturing high performance liquid chromatography (DHPLC) was used in controls for screening for the presence of the genetic variants previously found in diabetic patients. RESULTS: Seven different genetic variants were identified, five of them already reported and two new: the p.R187X and p.Y239C mutations identified in two females affected by T2DM. No significant association was found between certain polymorphisms and diabetes conditions. Preliminary haplotype studies are also reported. With respect to genotypes, we noted that some were not present in all the investigated cohort, and some were found related to higher glycated hemoglobin compared to others, although not at a significant level, probably because of the small number of subjects investigated. CONCLUSIONS: In conclusion, this study identified two new mutations and additional variants within the FN3K gene. This is the first study on FN3K in Italy. Future work is needed to achieve a better understanding of the FN3K enzyme and its possible clinical utility in the management of diabetic patients.

Effect of a New Formulation of Nutraceuticals as an Add-On to Metformin Monotherapy for Patients with Type 2 Diabetes and Suboptimal Glycemic Control: A Randomized Controlled Trial.

The aim of the study was to evaluate the overall biohumoral and metabolic effects of a 12-week add-on therapy consisting of a new nutraceutical formulation (BHC) based on berberine, hesperidin, and chromium picolinate in type 2 diabetes mellitus (T2D) patients with suboptimal glycemic compensation receiving metformin. After 12 weeks, participants in the group receiving metformin plus BHC, compared to the group receiving metformin only, saw a significant improvement in their glucose profile, in terms of both glycated hemoglobin (HbA1c) and fasting blood glucose (FBG). Their FBG dropped from 145 ± 20 mg/dL to 128 ± 23 mg/dL (p < 0.01), a decrease of 11.7% compared with the baseline. This decrease differed significantly from the situation in the control arm (p < 0.05). HbA1c decreased by 7.5% from the baseline, from 53.5 ± 4.3 mmol/mol to 49.5 ± 5.1 mmol/mol (p < 0.01), in the group given BHC, while no difference was seen in the control group. Advanced glycation end products (AGEs) and malondialdehyde (MDA) were found to be significantly reduced (p < 0.01) only in the BHC group, from 9.34 ± 7.61 µg/mL to 6.75 ± 6.13 µg/mL, and from 1.7 ± 0.15 µmol/L to 1.4 ± 0.25 µmol/L, respectively. In patients with T2D taking metformin with suboptimal glycemic compensation, adding BHC for 3 months significantly improved glucose control in terms of FBG and HbA1c, and had a positive effect on the lipid peroxidation profile, as indicated by a decrease in AGEs and MDA.

In silico evaluation of the interaction between ACE2 and SARS-CoV-2 Spike protein in a hyperglycemic environment.

The worse outcome of COVID-19 in people with diabetes mellitus could be related to the non-enzymatic glycation of human ACE2, leading to a more susceptible interaction with virus Spike protein. We aimed to evaluate, through a computational approach, the interaction between human ACE2 receptor and SARS-CoV-2 Spike protein under different conditions of hyperglycemic environment. A computational analysis was performed, based on the X-ray crystallographic structure of the Spike Receptor-Binding Domain (RBD)-ACE2 system. The possible scenarios of lysine aminoacid residues on surface transformed by glycation were considered: (1) on ACE2 receptor; (2) on Spike protein; (3) on both ACE2 receptor and Spike protein. In comparison to the native condition, the number of polar bonds (comprising both hydrogen bonds and salt bridges) in the poses considered are 10, 6, 6, and 4 for the states ACE2/Spike both native, ACE2 native/Spike glycated, ACE2 glycated/Spike native, ACE2/Spike both glycated, respectively. The analysis highlighted also how the number of non-polar contacts (in this case, van der Waals and aromatic interactions) significantly decreases when the lysine aminoacid residues undergo glycation. Following non-enzymatic glycation, the number of interactions between human ACE2 receptor and SARS-CoV-2 Spike protein is decreased in comparison to the unmodified model. The reduced affinity of the Spike protein for ACE2 receptor in case of non-enzymatic glycation may shift the virus to multiple alternative entry routes.

A role of glycation and methylation for SARS-CoV-2 infection in diabetes?

Type-2 diabetes (T2D) is a major comorbidity of COVID-19, and poorly controlled diabetes is associated with high mortality rate, emphasizing the necessity to improve glycemic control. Angiotensin-converting enzyme 2 (ACE2) is the receptor responsible for SARS-CoV-2 access to human cells, and ACE2 expression is increased in patients with diabetes and hypertension treated with ACE-inhibitors or angiotensin II receptor blockers. We hypothesize that an upregulation of ACE2 due to its non-enzymatic glycation could be considered, as well as a change of the protein tertiary structure in terms of amino acid (mostly lysine) available to be glycated. In fact, in a single ACE2 molecule, 34 lysine residues are present in the extracellular portion, and at least one of these is co-involved in a fundamental hydrogen-bond interaction with the SARS-CoV-2 receptor binding domain (RBD). The worse outcome of COVID-19 in people with diabetes could be related to the non-enzymatic glycation that triggers the activity of ACE2. Moreover, DNA methylation of genes regulating islet beta-cell function, as well as in insulin resistance of peripheral tissues such as liver, muscle, and adipose tissue may be involved, as already demonstrated for cancer conditions. DNA methylation, besides being considered as a biomarker to predict the risk of obesity and T2D, has been suggested also as a target for dietary and pharmacological treatments. The present observations may suggest further interventions in order to improve the outcome of COVID-19 in people affected by diabetes.

Role of fructosamine-3-kinase in protecting against the onset of microvascular and macrovascular complications in patients with T2DM.

INTRODUCTION: Microangiopathic and macroangiopathic complications are the main cause of morbidity and mortality in the diabetic population. Numerous publications have highlighted the role of glycation in the onset of complications of diabetes. In this context, the detection of fructosamine-3-kinase (FN3K)-an enzyme capable of counteracting the effect of hyperglycemia by intervening in protein glycation-has attracted great interest. Several studies have linked FN3K genetic variability to its enzymatic activity and glycated hemoglobin (HbA1c) levels. Here, we investigated the role of FN3K polymorphisms in the development of microvascular and macrovascular complications of diabetes. RESEARCH DESIGN AND METHODS: The anthropometric and biochemical parameters, and any medical history of microangiopathic and macroangiopathic complications, were documented in a sample of 80 subjects with type 2 diabetes. All subjects were screened for FN3K gene and analyzed for the combination of three polymorphisms known to be associated with its enzymatic activity (rs3859206 and rs2256339 in the promoter region and rs1056534 in exon 6). RESULTS: The combination of allelic variants of FN3K polymorphisms resulted in 13 distinct genotypic variants within the cohort. Comparison between genotypes showed no significant differences in terms of demographic, anthropometric and biochemical parameters, risk markers and long-term complications, except for a higher age and vitamin E levels associated with the genotype presenting GG at position -385, TT at position -232, and CC at c.900 A. Evaluating the microangiopathic and macroangiopathic complications as a whole, we found that they appeared significantly less present in this genotype compared with all other genotypes (p=0.0306). CONCLUSIONS: The group of patients carrying the favorable allele for the three polymorphisms of the FN3K gene revealed less severe microangiopathy and macroangiopathy, suggesting a protective role of this genotype against the onset of the complications of diabetes.

Patient-reported outcomes in elderly patients with type 2 diabetes mellitus treated with dual oral therapy: a multicenter, observational study from Italy.

Objective: To assess patient-reported outcomes after two years of use of dual oral anti-diabetes drug (OAD) therapy in elderly people (≥65 years) with type 2 diabetes mellitus (T2DM) from Italy under real-life settings.Methods: 3-AGE was a prospective, non-interventional study in elderly people with T2DM inadequately controlled on metformin monotherapy (defined as glycated hemoglobin [HbA1c] 7.0-9.0%), in whom a second OAD was prescribed. Primary endpoint was to assess the physical and psychological symptoms associated with T2DM from baseline to 24 months using the Diabetes Symptom Check List revised (DSC-R) questionnaire. Patient's quality of life and health status, treatment satisfaction, consumption of healthcare resources, and physician satisfaction with treatment were also assessed (secondary endpoints) using validated questionnaires. Additionally, safety and clinical characteristics were also evaluated.Results: The mean age of the study population (N = 860) was 71.5 ± 5.2 years. Addition of a second OAD significantly (p < .0001) reduced the DSC-R score from baseline (0.73 ± 0.68) to both Months 12 and 24 (0.63 ± 0.59 and 0.61 ± 0.56), and HbA1c from baseline (7.72% ± 0.54%) to Month 12 (6.95% ± 0.82%). Adding a second OAD improved quality of life and health status (baseline, 71.31 ± 15.16 to Month 12, 74.49 ± 13.64; p < .0001), patient's treatment satisfaction (p < .0001), and consumption of healthcare resources per patient. Physicians expressed good satisfaction with patients' treatment (across efficacy, tolerability and compliance domains) at Month 12. Overall, 32 adverse reactions (in 24 patients) and four hypoglycemic episodes were reported during the 24 months.Conclusion: Addition of a second OAD improved physical and psychological symptoms associated with T2DM and was well tolerated in elderly people under real-life settings.

Bariatric surgery improves atherogenic LDL profile by triglyceride reduction.

BACKGROUND: Small dense low-density lipoprotein (LDL) are atherogenic particles frequently observed in obese patients. Fatty acids modulate LDL. Objective of this study was to determine the relations between plasma phospholipid fatty acid composition and the presence of small dense LDL particles in morbidly obese patients treated with laparoscopic gastric banding (LAGB). METHODS: Small dense LDL, plasma lipids, lipoproteins, apoproteins, and phospholipid fatty acid composition (a marker of dietary fatty acid intake) were quantified before and 12 months after surgery in four men and 11 women who were morbidly obese and (BMI > 40 kg/m(2)) eligible for surgery, consecutively treated with LAGB at the Department of Medical and Surgical Sciences of the University of Padova. RESULTS: BMI was 48.3 +/- 4.8 kg/m(2) before and 36.1 +/- 5.5 kg/m(2) after LAGB. Plasma triglycerides and apoprotein E levels significantly decreased, while HDL cholesterol significantly increased after LAGB. A reduction of small dense LDL with an increase of LDL relative flotation (0.34 +/- 0.04 before vs 0.38 +/- 0.03 after LAGB, p < 0.001) was also observed. These modifications were neither related to weight reduction nor to changes in phospholipid fatty acid composition, but they were associated to triglyceride reduction, which explained 76.7% of the LDL relative flotation variation. CONCLUSION: Weight loss obtained by LAGB in morbidly obese subjects was accompanied by triglyceride reduction, high-density lipoprotein increase, and an improvement of the atherogenic LDL profile. Triglyceride reduction, but not the extent of weight loss or dietary fatty acid modifications, is the determinant of modifications of LDL physical properties in these patients.

Long-term effect of pioglitazone vs glimepiride on lipoprotein oxidation in patients with type 2 diabetes: a prospective randomized study.

AIMS: Type 2 diabetes (DM2) is associated to oxidative modifications of high-density lipoproteins (HDL), which can interfere with their function. Pioglitazone has proved effective in raising HDL cholesterol (HDL-C) and lowering small dense low-density lipoprotein (LDL), but no clinical studies have examined its effect on lipoprotein oxidation in patients with DM2. METHODS: We assessed the effect of pioglitazone vs glimepiride after 1 year on HDL oxidation, expressed as relative abundance of peptides containing Met112O in ApoA-I (oxApoA-I) estimated by mass spectrometry (MALDI/TOF/TOF), in 95 patients with DM2. The oxLDL and AGE were quantified by ELISA. RESULTS: Patients receiving pioglitazone showed a significant increase in the concentration of ApoA-I (Δ = 7.2 ± 14.8 mg/dL, p < 0.02) and a reduction in oxApoA-I (Δ = - 1.0 ± 2.6%, p < 0.02); this reduction was not significantly different from glimepiride. oxLDL showed a slight, but not significant increase in both treatment groups. Regression analysis showed a correlation between ΔoxApoA-I and ΔAGE (r = 0.30; p = 0.007) in all patients, while both of these parameters were unrelated to changes in HbA1c, HDL-C, duration of illness, or use of statins. CONCLUSIONS: Long-term treatment with pioglitazone was effective in reducing the oxidation of HDL, but not LDL in patients with DM2, while glimepiride didn't. This finding seems to be associated to the change of glyco-oxidation status, not to any improvement in glycemic control or lipid profile. TRIAL REGISTRATION: NCT00700856, ClinicalTrials.gov Registered June 18, 2008.