Prevenzano, Immacolata (2018) Effect of MGO accumulation on vascular function in mouse experimental models in vitro and in vivo. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: Effect of MGO accumulation on vascular function in mouse experimental models in vitro and in vivo.
Creators:
CreatorsEmail
Prevenzano, Immacolataimma.prevenzano@libero.it
Date: 10 December 2018
Number of Pages: 118
Institution: Università degli Studi di Napoli Federico II
Department: Medicina Veterinaria e Produzioni Animali
Dottorato: Scienze veterinarie
Ciclo di dottorato: 31
Coordinatore del Corso di dottorato:
nomeemail
Cringoli, Giuseppecringoli@unina.it
Tutor:
nomeemail
Beguinot, FrancescoUNSPECIFIED
Date: 10 December 2018
Number of Pages: 118
Uncontrolled Keywords: Glyoxalase1, Methylglyoxal, miRNAs
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Area 05 - Scienze biologiche > BIO/12 - Biochimica clinica e biologia molecolare clinica
Area 06 - Scienze mediche > MED/04 - Patologia generale
Area 06 - Scienze mediche > MED/05 - Patologia clinica
Additional Information: non rendo disponibile alla visualizzazione la presente tesi di dottorato
Date Deposited: 20 Dec 2018 09:04
Last Modified: 30 Jun 2020 08:26
URI: http://www.fedoa.unina.it/id/eprint/12545

Abstract

Methylglyoxal (MGO), a highly reactive dicarbonyl compound formed as by-product of glycolysis, is an ubiquitous metabolite of cellular metabolism. Therefore, it is produced in all cells, both under normal and pathological conditions. Under physiological circumstances, MGO is detoxified through the glyoxalase system, of which Glyoxalase 1 (Glo1) is the rate limiting enzyme. In pathological conditions, as in chronic hyperglycemia, high blood glucose levels lead to increased MGO accumulation. It is known that MGO plays a major role in endothelial cell damage and development of vascular disease. We have previously demonstrated that MGO induces endothelial insulin resistance both in vitro and in animal models. In the last few years, many evidence has provided a link between microRNAs (miRNAs) and diabetic complications. Indeed, miRNAs regulate cellular molecular pathways, including insulin signaling, thus controlling the pathophysiology of vascular bed. This study includes the investigation of two aspects of MGO effects on the pathophysiology of diabetes mellitus (DM) and its associated complications: 1. the evaluation of MGO accumulation on glucose homeostasis and vascular function in a mouse model knockdown for Glo1 (Glo1KD) and 2. the analysis of miRNAs contribution in MGO induced damaging effect on insulin responsiveness in mouse aortic endothelial cells (MAECs). The results obtained in vivo demonstrated that the endogenous accumulation of MGO in mice with a reduced expression of Glo1 leads to an age-dependent development of glucose intolerance, in absence of hyperglycemia. Indeed, despite the reduced glucose tolerance at 10 months of age, Glo1KD mice have no differences in body weight and in the glucose levels, compared to WT mice, neither at 5 months nor at 10 months of age. While no alterations in the whole-body insulin-sensitivity have been observed by insulin tolerance tests, Glo1KD mice show a basal hyperinsulinemia and impaired glucose-stimulated insulin-secretion, compared to WT mice. Moreover, an increased systolic blood pressure accompanied by impaired endothelium-dependent vasodilation are already shown starting from 5 months of age in Glo1KD mice. A deeper analysis of the molecular mechanisms involved in the endothelial dysfunction has been performed in vitro, in MAECs exposed to MGO, which we have previously demonstrated to display insulin resistance and an imbalanced production of vasoactive molecules: NO and ET-1. Our results demonstrate that MGO induces the down-regulation of 4 out of 84 diabetes-associated miRNAs. Among these, the reduced expression of miR-190a and miR-214 has been validated both in MAECs exposed to MGO and in aortae from Glo1KD mice. The inhibition of miR-190a and miR-214 impairs the insulin-induced activation of Akt1/eNOS pathway, whereas their overexpression prevents the MGO-induced insulin resistance in MAECs. In detail, we have identified the kinase KRAS and the phosphatase PHLPP2 as targets of miR-190a and miR-214, respectively. In MAECs increased KRAS levels result from the reduced expression of miR-190a and sustain the ERK hyperactivation, which is in turn responsible for the impairment of the insulin-stimulated IRS1/Akt/eNOS signal transduction in MAECs treated with MGO. Moreover, a reduced insulin-dependent activation of Akt in MGO-treated MAECs is fostered by higher protein levels of PHLPP2, which we validate here to be a direct target of miR-214. In conclusion, our results demonstrate that Glo1 silencing is enough to induce MGO accumulation in vivo in Glo1KD mice, causing glucose intolerance and β-cell dysfunction, which are characteristic of T2DM pathogenesis, together with the impairment of hemodynamic function (i.e blood pressure and endothelial-dependent vasodilation), in a context of normoglycemia. Moreover, miR-190a and miR-214 play a role in the endothelial insulin-resistance induced by MGO in MAECs. Thus, representing potential biomarkers of vascular dysfunction. Further efforts in the development of pharmacological intervention to interfere with these pathogenic events will be useful to provide new therapeutic options aimed at preventing the onset and progression of vascular complications in diabetes.

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