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Background: SGLT2i proved efficacy in reducing cardiovascular morbidity and mortality in T2D patients at high cardiovascular risk. Aims: In real-word setting, we prospectively evaluated long-term effects of Dapagliflozin (DAPA) on left diastolic ventricular function (LDVF) and vascular reactivity in T2D patients without known cardiovascular disease. Population, Study and Methods: 13 patients (9 male, 57± 8 years, BMI 29.6± 3.6, HbA1c 8.9±1.2%, years of T2D 9±5, fasting glucose 185±51 mg/dl) initiated DAPA 10 mg daily as add-on to metformin. Following data were collected at baseline (T0), after 12 weeks (T1) and 52 weeks (T2) of treatment: blood pressure, anthropometrics, fasting glucose, HbA1c, eGFR and lipids. Ratio of the early (E) to late (A) ventricular filling velocities (E/A) was used to evaluate LDVF. Arterial stiffness was evaluated by Pulse Wave Velocity (PWV) and Augmentation Index (AIxHR75) with radial approach (SphygmoCor System). Endothelial function was assessed by non-invasive flow mediated dilatation (FMD). Results: We observed significant percent variation in HbA1c (T1 vs. T0 -17.9%, p=0.001; T2 vs. T0 -17.4%, p=0.01), fasting glucose (T1 vs. T0 -25.9%, p=0.002, T2 vs. T0 -31.8%, p=0.005) systolic blood pressure (T1 vs. T0 -6.7%, p=0.03; T2 vs. T0 -10.2% p=0.02) and BMI (T1 vs. T0 -3.3%, p=0.001; T2 vs. T0 -3%, p=0.02). Significant improvements from baseline of E/A ratio (T1 vs. T0 +15%, p=0.03; T2 vs. T0 +27.5%, p=0.003) and brachial FMD (T1 vs. T0 +50.7%, p=0.02; T2 vs. T0 +39.4%, p=0.04) were also seen. No significant changes in eGFR, lipids and arterial stiffness measures were seen. Conclusions: Intensification of treatment with DAPA 10 mg was accompanied by significant improvements in LVDF and endothelial-dependent vasodilation measures, which persist after 52 weeks of treatment. These preliminary observational results shed light on potential mechanisms responsible for cardiovascular protection with SGLT2i in T2D patients, even in absence of overt cardiovascular disease. Disclosure I. D'ippolito: None. E. De Carli: None. A. Andreadi: None. M. Romano: None. A. Galli: None. A. Capria: None. M. Massaro: None. P. Sbraccia: None. D. Della-Morte: None. A. Bellia: None. D. Lauro: None. Funding University of Rome

Diabetes mellitus is characterized by a state of hyperglycemia resulting from altered insulin secretion by pancreatic beta cell, insulin action or both. This pathological condition is frequently associated with muscle mass loss, a condition defined as sarcopenia, and diabetic myopathy, represented by an impairment of the regenerative power of muscle fiber and by an altered differentiation of progenitor cells. Oxidative stress has been identified as the main cause of muscular alterations typical of diabetic patients. We showed that Peroxiredoxin 6 (Prdx6), a relatively new antioxidant enzyme belonging from the Peroxiredoxin family, has a central role in glucose homeostasis by exerting a potent antioxidant role. Based on these results, in the present study we aimed to verify whether Prdx6 modulates the association between diabetes and the progression of myopathy and sarcopenia. Firstly, we evaluated the gene expression of the main factors involved in the differentiation of myogenic muscle cells such as MyoD and Myogenin in murine knockout models for Prdx6 (Prdx6-/-). We observed significant decreased levels of both genes in Prdx6-/- mice compared to controls, suggesting an impairment of the regenerative potential of muscle fibers. These data were, also, confirmed by using in vitro cell model of murine myoblasts (C2C7) knockdown for Prdx6. Moreover, in the murine models, the process of muscle atrophy was studied by evaluating the gene expression of MuRF1 and Atrogin-1 that finely regulate protein degradation at skeletal muscle level. According to our hypothesis, the expression levels of both enzymes were significantly increased confirming the presence of muscle atrophy. Our study, for the first time, highlights a fundamental role of Prdx6 in the preservation of muscle mass, suggesting how Prdx6 can be considered a potential therapeutic target for diabetic myopathy and sarcopenia. However, further studies are needed in order to understand the molecular mechanism underlying this phenomenon. Disclosure F. Pacifici: None. B. Capuani: None. F. Piermarini: None. D. Pastore: None. R. Arriga: None. A. Coppola: None. S. Rea: None. G. Donadel: None. A. Bellia: None. D. Della-Morte: None. D. Lauro: None.

OBJECTIVE In humans, it is unclear if insulin resistance at the monocyte level is associated with atherosclerosis in vivo. Here we have studied first-degree relatives of patients with type 2 diabetes to investigate whether a reduction in components of the insulin signal transduction pathways, such as the insulin receptor (InsR) or InsR substrate 1 or 2 (IRS1 or IRS2), or a reduction in genetic modifiers of insulin action, such as the TIMP3/ADAM17 (tissue inhibitor of metalloproteinase 3/A disintegrin and metalloprotease domain 17) pathway, is associated with evidence of atherosclerosis. RESEARCH DESIGN AND METHODS Insulin sensitivity was analyzed through euglycemic-hyperinsulinemic clamp, and subclinical atherosclerosis was analyzed through intimal medial thickness. Monocytes were isolated through magnetic cell sorting, and mRNA and proteins were extracted and analyzed by quantitative PCR and pathscan enzyme-linked immunosorbent assays, respectively. RESULTS In monocyte cells from human subjects with increased risk for diabetes and atherosclerosis, we found that gene expression, protein levels, and tyrosine phosphorylation of IRS2, but not InsR or IRS1, were decreased. TIMP3 was also reduced, along with insulin resistance, resulting in increased ectodomain shedding activity of the metalloprotease ADAM17. CONCLUSIONS Systemic insulin resistance and subclinical atherosclerosis are associated with decreased IRS2 and TIMP3 expression in circulating monocytes.

OBJECTIVE Insulin resistance (IR) and cardiovascular disease may share a common genetic background. We investigated the role of IR-associated ENPP1 K121Q polymorphism (rs1044498) on cardiovascular disease in high-risk individuals. RESEARCH DESIGN AND METHODS A prospective study (average follow-up, 37 months) was conducted for major cardiovascular events (myocardial infarction [MI], stroke, cardiovascular death) from the Gargano Heart Study (GHS; n = 330 with type 2 diabetes and coronary artery disease), the Tor Vergata Atherosclerosis Study (TVAS; n = 141 who had MI), and the Cardiovascular Risk Extended Evaluation in Dialysis (CREED) database (n = 266 with end-stage renal disease). Age at MI was investigated in cross-sectional studies of 339 type 2 diabetic patients (n = 169 from Italy, n = 170 from the U.S.). RESULTS Incidence of cardiovascular events per 100 person--years was 4.2 in GHS, 10.8 in TVAS, and 11.7 in CREED. Hazard ratios (HRs) for KQ+QQ versus individuals carrying the K121/K121 genotype (KK) individuals were 1.47 (95% CI 0.80–2.70) in GHS, 2.31 (95% CI 1.22–4.34) in TVAS, and 1.36 (95% CI 0.88–2.10) in CREED, and 1.56 (95% CI 1.15–2.12) in the three cohorts combined. In the 395 diabetic patients, the Q121 variant predicted cardiovascular events among obese but not among nonobese individuals (HR 5.94 vs. 0.62, P = 0.003 for interaction). A similar synergism was observed in cross-sectional studies, with age at MI being 3 years younger in Q121 carriers than in KK homozygotes among obese but not among nonobese patients (P = 0.035 for interaction). CONCLUSIONS The ENPP1 K121Q polymorphism is an independent predictor of major cardiovascular events in high-risk individuals. In type 2 diabetes, this effect is exacerbated by obesity. Future larger studies are needed to confirm our finding.

Enhanced oxidative stress contributes to the pathogenesis of diabetes and its complications. Peroxiredoxin 6 (PRDX6) is a key regulator of cellular redox balance, with the peculiar ability to neutralize peroxides, peroxynitrite, and phospholipid hydroperoxides. In the current study, we aimed to define the role of PRDX6 in the pathophysiology of type 2 diabetes (T2D) using PRDX6 knockout (−/−) mice. Glucose and insulin responses were evaluated respectively by intraperitoneal glucose and insulin tolerance tests. Peripheral insulin sensitivity was analyzed by euglycemic-hyperinsulinemic clamp, and molecular tools were used to investigate insulin signaling. Moreover, inflammatory and lipid parameters were evaluated. We demonstrated that PRDX6−/− mice developed a phenotype similar to early-stage T2D caused by both reduced glucose-dependent insulin secretion and increased insulin resistance. Impaired insulin signaling was present in PRDX6−/− mice, leading to reduction of muscle glucose uptake. Morphological and ultrastructural changes were observed in islets of Langerhans and livers of mutant animals, as well as altered plasma lipid profiles and inflammatory parameters. In conclusion, we demonstrated that PRDX6 is a key mediator of overt hyperglycemia in T2D glucose metabolism, opening new perspectives for targeted therapeutic strategies in diabetes care.

et al., Classically activated macrophages (M1) secrete proinflammatory cytokine and are predominant in obese adipose tissue. M2 macrophages, prevalent in lean adipose tissue, are induced by IL-13 and IL-4, mainly secreted by Th2 lymphocytes, and produce the anti-inflammatory cytokine IL-10. ITCH is a ubiquitously expressed E3 ubiquitin ligase involved in T-cell differentiation and in a wide range of inflammatory pathways. ITCH downregulation in lymphocytes causes aberrant Th2 differentiation. To investigate the role of Th2/M2 polarization in obesityrelated inflammation and insulin resistance, we compared wild-type and Itch2/2 mice in a context of diet-induced obesity (high-fat diet [HFD]). When subjected to HFD, Itch2/2 mice did not show an increase in body weight or insulin resistance; calorimetric analysis suggested an accelerated metabolism. The molecular analysis of metabolically active tissue revealed increased levels of M2 markers and genes involved in fatty acid oxidation. Histological examination of livers from Itch2/2 mice suggested that ITCH deficiency protects mice from obesity-related nonalcoholic fatty liver disease. We also found a negative correlation between ITCH and M2 marker expression in human adipose tissues. Taken together, our data indicate that ITCH E3 ubiquitin ligase deficiency protects from the metabolic disorder caused by obesity., This study was funded in part by Fondazione Roma 2008, ESFD/Lilly 2012, AIRC 2012 Project IG 13163, FP7-Health-241913 FLORINASH, FP-7 EURHYTHDIA, and PRIN 2009FATXW3_002 to M.Fe.; SAF-2012-33014 from Ministerio de Economía y Competitividad, Spain, to B.P.; and Medical Research Council, U.K., grants ACC12, MIUR/PRIN (20078P7T3K_001)/FIRB (RBIP06LCA9_0023, RBIP06LCA9_0C), AIRC (2011-IG11955), and AIRC 5xmille (MCO #9979), Telethon grant GGPO9133, Ministero della Salute, and IDI-IRCCS (RF08 c.15, RF07 c.57) to G.M.

Aspirin modestly influences cardiovascular events in patients with type 2 diabetes mellitus (T2DM), but the reason is unclear. The aim of the study was to determine whether in T2DM patients aspirin enhances platelet isoprostanes, which are eicosanoids with proaggregating properties derived from arachidonic acid oxidation by platelet NOX2, the catalytic subunit of reduced NAD phosphate oxidase. A cross-sectional study was performed comparing T2DM patients, treated (n = 50) or not treated (n = 50) with 100 mg/day aspirin, with 100 nondiabetic patients, matched for age, sex, atherosclerosis risk factors, and aspirin treatment. A short-term (7 days) treatment with 100 mg/day aspirin also was performed in 36 aspirin-free diabetic and nondiabetic patients. Higher platelet recruitment, platelet isoprostane, and NOX2 activation was found in diabetic versus nondiabetic patients and in aspirin-treated diabetic patients versus nontreated patients (P < 0.001). Platelet thromboxane (Tx) A2 (P < 0.001) was inhibited in all aspirin-treated patients. In the interventional study, aspirin similarly inhibited platelet TxA2 in diabetic and nondiabetic patients (P < 0.001). Platelet recruitment, isoprostane levels, and NOX2 activation showed a parallel increase in diabetic patients (P < 0.001) and no changes in nondiabetic patients. These findings suggest that in aspirin-treated diabetic patients, oxidative stress–mediated platelet isoprostane overproduction is associated with enhanced platelet recruitment, an effect that mitigates aspirin-mediated TxA2 inhibition.