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Project’s title: Molecular mechanisms of hyperlipidemia-induced insulin resistance; metabolic connections between the intestine, liver steatosis and atherosclerosis

Titlu proiect: Mecanisme moleculare ale rezistentei la insulina induse de hiperlipidemie; conexiuni metabolice intre intestin, steatoza hepatica si ateroscleroza

Acronym: MECLIPINSMETAB

Abstract: Hyperlipidemia plays a critical role in lipid metabolism disorders, such as non-alcoholic fatty liver disease (NAFLD), insulin resistance (IR), diabetes and atherosclerosis, but the mechanisms involved are not elucidated. We aim to investigate the molecular mechanisms linking lipid accumulation, oxidative stress and endoplasmic reticulum (ER) stress with IR in the intestine and liver of hyperlipidemic (HL) hamsters. Our preliminary data show that paraoxonase-1, an essential anti-oxidant enzyme associated with high density lipoproteins (HDL), has a decreased specific activity in the intestine and liver of HL hamsters with IR versus HL hamsters. Our hypothesis is that HDL production in the intestine and liver is a key mechanism affected by ER stress and involved in IR development, NAFLD and atherosclerotic plaques progression, with life-threatening consequences. The novelty of the present project is the uncovery of relations between ER stress intensity and the levels of accumulated lipids, their oxidation products (oxysterols), the appearance of dysfunctional plasma HDL, having as consequence the alteration of the insulin signalling pathway. The originality of the project consists in the interconnected investigation of the intestine, liver and myocardium in HL conditions, the comparative analysis of in vivo and in vitro sources of HDL (intestine vs. cultured enterocytes) and the use of omega-3 fatty acids to increase functional HDL production for arresting or reversing the IR.

Rezumat: Hiperlipidemia are un rol critic in aparitia bolilor metabolismului lipidic, precum boala ficatului gras non-alcoolic (NAFLD), rezistenta la insulina (RI), diabetul si ateroscleroza, insa mecanismele implicate nu sunt elucidate. Scopul prezentului proiect este de a investiga mecanismele moleculare care leaga acumularea de lipide, stresul oxidativ si stresul reticolului endoplasmic (RE) cu RI in intestinul si ficatul hamsterilor hiperlipidemici (HL). Datele noastre preliminare arata ca paraoxonaza-1, enzima anti-oxidanta esentiala asociata cu lipoproteinele de densitate inalta (HDL), prezinta o activitate specifica scazuta in intestinul si ficatul hamsterilor HL cu RI vs hamsterii HL. Ipoteza noastra este ca producerea HDL in intestin si ficat este un mecanism cheie afectat de stresul RE si implicat in dezvoltarea RI, progresia NAFLD si a placilor aterosclerotice care afecteaza grav sanatatea. Noutatea prezentului proiect este descoperirea unor relatii intre intensitatea stresului RE, nivelele de lipide si produsii lor de oxidare (oxisteroli) si aparitia HDL disfunctional plasmatic avand drept consecinta alterarea caii de semnalizare a insulinei. Originalitatea proiectului consta in investigarea interconectata a intestinului, ficatului si miocardului in conditii HL, analiza comparativa a surselor de HDL in vivo si in vitro (intestin vs enterocite in cultura) si utilizarea acizilor grasi omega-3 pentru a induce producerea HDL functional cu scopul de a stopa sau reversa RI.

Proiect’s team:

Project’s manager: Camelia Sorina Stancu, Ph.D.

Mihaela G. Carnuta, Ph.D. student

Mariana Deleanu, Ph.D. student

Gabriela M. Sanda, PhD

Anca V. Sima, Ph.D., Associate member of the Romanian Academy

Loredan Stefan Niculescu, PhD

Laura Toma, Ph.D.

Teodora Barbălată, Master student

Emanuel Dragan, Vet. M.D.

Daniela Rogoz, Assistant

Cristina Dobre, Assistant

Duration: 01.10.2015-30.11.2017 (26 months, updated 2017)

Total funds: 550.000 lei (2015-2017)

Funds for 2015: 90.000 lei

Funds for 2016: 197.494 lei

Funds for 2017: 262.506 lei

General objectives

Objective 1: To analyze the impact of hyperlipidemia (HL) on the intestine of hamsters with HL or HL with insulin resistance (HL-IR) by investigating the gene and protein regulation of the molecules involved in the Lp synthesis and secretion.

Objective 2: To assess the stage of hepatic steatosis (NAFLD) in association with the alteration of Lp secreted by the intestine of HL-IR versus HL hamsters.

Objective 3: To analyze the effect of the Lp secreted by the intestine and liver on the development of atherosclerotic lesions in HL-IR compared to HL hamsters vasculature.

Objective 4: To evaluate the potential of PON1 and apoAI synthesized by the enterocytes overexpressing PON1/apoAI or stimulated with omega-3 FA to reverse endothelial cells dysfunction.

2015 Workplan

Task 1.1. Establishing the Golden Syrian hamster groups (N, HL) and starting the saturated fat diet administration (for 16 weeks)

Task 1.2. Characterization of the HL animals that spontaneously develop IR (HL-IR) compared to HL hamsters by determination of the HOMA-IR, plasma Lp profile, Lp biochemical composition and associated enzymes and their lipid oxidation products every 4 weeks during the diet.

Objective 2: To disseminate the results of the project.

Task 2.1. Design and build of a website dedicated to the project’s results.

Objective 3: Management of the project.

Task 3.1. Monitoring the project’s progress, defining the working protocol and the specific role of each team member.

Task 3.2. Organizing periodic meetings between the team members to discuss the results and the future strategy; integration of the PhD and postdoctoral program of the team members in the project’s workplan.

Task 3.3. Preparation of periodic deliverables and reports; delivery of financial and scientific reports to the Contracting Authority.

Deliverables for 2015

Technical-scientific report (month 3).

The plasma lipid profile on hamsters before and after two months of hyperlipidemic diet administration.

Project’s website.

2016 Workplan

Objective 1: To analyse the impact of hyperlipidemia (HL) on the intestine of hamsters with HL or HL with insulin resistance (HL-IR) by investigating the gene and protein regulation of the molecules involved in the Lp synthesis and secretion – months 4-11

Task 1.1 Blood collection and sampling of the organs of interest (intestine, liver and myocardium) after 16 weeks of fat-diet administration; echocardiographic evaluation of the animals.

Task 1.2 Measurement of the ER stress in association with the accumulated lipid levels (FA, free and esterified cholesterol) and their specific oxidation products in the intestine.

Task 1.3 Quantification of the gene and protein expression of the key players involved in HDL secretion (ApoAI, PON1, ABCA1) from the intestine;

Task 1.4 Measurement of the gene and protein expression of the lipid transporters (MTTP, NPC1L1) involved in chylomicron production and of the glucose transporters (GLUT-1/2) from the intestine;

Task 1.5 Quantification of the gene and protein expressions of the transporters involved in TICE process (ABCG5/8) and their correlation with the feces cholesterol levels.

Task 1.6 Assessment of the protein expression of the transcription regulation factors (LXRs, PPARs) involved in the regulation of HDL and chylomicrons production by the intestine.

Objective 2: To assess the stage of hepatic steatosis (NAFLD) in association with the alteration of Lp secreted by the intestine of HL-IR versus HL hamsters – months 12-15

Task 2.1 Determination of the histological scoring system for NAFLD (lipid deposits, inflammation markers, fibrosis) in the liver of HL-IR versus HL hamsters

Task 2.2 Measurement of the ER stress in association with the lipid levels and their specific oxidation products accumulated in the liver.

Task 2.3 Quantification of the gene and protein expression of the key players of HDL secretion (ApoAI, PON1, ABCA1 and SRBI) in the liver.

Objective 3: To disseminate the results of the project - months 4-15

Task 3.1. Communication of the obtained results at the national/international scientific events.

Task 3.2. Preparing and publishing the results in ISI-Thomson ranked journals.

Task 3.3. Periodic update of a website dedicated to the project.

Objective 4: To manage the overall project objectives, tasks, milestones and potential risks - months 4-15

Task 4.1. Monitoring the project’s progress and the working protocol of each team member.

Task 4.2. Organizing periodic meetings between the team members to discuss the results and the future strategy.

Task 4.3. Preparation of periodic reports, delivery of the financial and scientific reports to the Contracting Authority.

Deliverables for 2016

Technical-scientific report (month 15)

Impact of hyperlipidemia (HL) on the lipoprotein synthesis and secretion in the small intestine of hamsters with HL or HL with insulin resistance

Effect of the lipoproteins synthetized and secreted by the small intestine on the liver of hamsters with HL or HL with insulin resistance

Communications at national/international scientific meetings

Article in ISI-Thomson and/or BDI journal

Project’s website update

2017 Workplan

Objective 1: To assess the stage of hepatic steatosis (NAFLD) in association with the alteration of Lp secreted by the intestine of HL-IR versus HL hamsters - months 16-22

Task 1.1 Measurement of the gene expression of proteins involved in de novo lipogenesis (Fasn, Acaca, Scd1) and lipolysis (hepatic lipase, PLA₂) and of the glucose transporters (GLUT-1/2) from different areas of the hepatic tissue using laser pressure catapulting dissection microscopy.

Task 1.2 Assessment of the protein expression of the transcription regulation factors (LXRs, PPARs, pMAPK, pJNK) involved in regulation of the Lp secretion by the liver.

Objective 2: To analyze the effect of the Lp secreted by the intestine and liver on the development of atherosclerotic lesion in HL-IR compared to HL hamsters vasculature - months 16-22

Task 2.1 Determination of the lipid levels and their specific oxidation products in the myocardium.

Task 2.2 Assessment of the atherosclerotic plaques formed in aortic valves, coronary arteries and aortic arch in association with the metabolic status of myocardium (FA oxidation, ATP production).

Task 2.3 Measurement of the ER stress in association with the lipid levels and their oxidation products.

Task 2.4 Quantification of the protein expression of the key factors representing the insulin receptor signaling pathway in the myocardium (IRbeta, IRS1/2, p85, p110, pAkt, GLUT-4).

Objective 3: To evaluate the potential of PON1 and apoAI synthesized by the enterocytes overexpressing PON1/apoAI or stimulated with omega-3 FA to reverse EC dysfunction - months 20-26

Task 3.1 Obtaining transfected Caco-2 cells (tCaco-2) overexpressing human PON1 and/or apoAI.

Task 3.2 Determination of the PON1, apoAI, ABCA1/G5/8 and SRBI gene and protein expression in tCaco-2 cells compared to Caco-2 cells exposed to omega-3 FA.

Task 3.3 Quantification of the PON1 and apoAI levels secreted by tCaco-2 cells versus Caco-2 cells exposed to omega-3 FA.

Task 3.4 Measurement of the gene and protein expression of lipids and glucose transporters (MTTP, NPC1L1, GLUT-1/2) in Caco-2 cells exposed to omega-3 FA.

Task 3.5 Determination of the transcription regulation factors (LXRs, PPARs, pMAPK, pJNK) gene and protein expression in Caco-2 cells incubated with omega 3 FA versus Caco-2 cells.

Task 3.6 Determination of the potential of the culture medium from the tCaco-2 cells or Caco-2 cells exposed to omega 3 FA to exert lipid lowering, antioxidant or anti-inflammatory effects on EC pre-exposed to atherogenic Lp.

Objective 4: To disseminate the results of the project - months 16-26

Task 4.1. Communication of the obtained results at the national/international scientific events.

Task 4.2. Preparing and publishing the results in ISI-Thomson ranked journals.

Task 4.3. Periodic update of a website dedicated to the project.

Objective 5: To manage the overall project objectives, tasks, milestones and potential risks - months16-26

Task 5.1. Monitoring the project’s progress and the working protocol of each team member.

Task 5.2. Organizing periodic meetings between the team members to discuss the results and the future strategy.

Task 5.3. Preparation of periodic reports, delivery of the financial and scientific reports to the Contracting Authority.

Deliverables for 2017

Technical-scientific report (month 26)

Gene and protein expression of key molecules involved in de novo lipids synthesis in the steatotic livers from HL and HL-RI hamsters

Correlation between oxidized lipids content and the altered insulin signaling pathway in myocardium; aortic valves lesions severity in HL-RI compared to HL hamsters

Gene and protein expression of key molecules involved in HDL secretion in enterocytes stimulated with omega-3 fatty acids or overexpressing ApoA-I/PON1

Communications at national/international scientific meetings

Article in ISI-Thomson and/or BDI journal

Project’s website update

Articles in ISI-ranked journals:

2016

Toma L, Sanda GM, Deleanu M, Stancu CS, Sima AV. Glycated LDL increase VCAM-1 expression and secretion in endothelial cells and promote monocyte adhesion through mechanisms involving endoplasmic reticulum stress. Mol Cell Biochem. 417(1-2): 169-79 (2016)

Sanda GM, Deleanu M, Toma L, Stancu CS, Simionescu M, Sima AV. Oxidized LDL-Exposed Human Macrophages Display Increased MMP-9 Expression and Secretion Mediated by Endoplasmic Reticulum Stress. J Cell Biochem. accepted: doi: 10.1002/jcb.25637 (2016)

2017

Carnuta MG, Stancu CS, Toma L, Sanda GM, Niculescu LS, Deleanu M, Popescu AC, Popescu MR, Vlad A, Dimulescu DR, Simionescu M, Sima AV.Dysfunctional high-density lipoproteins have distinct composition, diminished anti-inflammatory potential and discriminate acute coronary syndrome from stable coronary artery disease patients. Scientific Reports 7(1): 7295. doi: 10.1038/s41598-017-07821-5 (2017)

Toma L, Sanda GM, Niculescu LS, Deleanu M, Stancu CS, Sima AV. Caffeic acid attenuates the inflammatory stress induced by glycated LDL in human endothelial cells by mechanisms involving inhibition of AGE-receptor, oxidative, and endoplasmic reticulum stress. Biofactors 43(5): 685-697. doi: 10.1002/biof.1373 (2017)

Sanda GM, Deleanu M, Toma L, Stancu CS, Simionescu M, Sima AV. Oxidized LDL-Exposed Human Macrophages Display Increased MMP-9 Expression and Secretion Mediated by Endoplasmic Reticulum Stress. Journal of Cellular Biochemistry 118(4): 661-669. doi: 10.1002/jcb.25637 (2017)

Communications at scientific events:

2016

Posters

Stancu C.S., Carnuta M.G., Sanda G.M., Toma L., Deleanu M., Niculescu L.S., Sima A.V. Fat-activated ER stress induces the reduction of ABCG5/8 expression in the small intestine and liver of hyperlipidemic hamsters and the appearance of dysfunctional HDL. 84^th Congress of European Atherosclerosis Society, 29 May 29 – 2 June 2016, Innsbruck, Austria

Toma L., Sanda G.M., Deleanu M., Stancu C.S., Sima A.V. Glycated LDL induce endoplasmic reticulum stress along with the stimulation of VCAM-1 Expression in human endothelial cells, and the ensuing monocyte adhesion. 84^th Congress of European Atherosclerosis Society, 29 May 29 – 2 June 2016, Innsbruck, Austria

Sanda G.M., Deleanu M., Stancu C.S., Simionescu M., Sima A.V. Human macrophages exposed to oxidized LDL or tunicamycin exhibit increased MMP-9 expression, secretion and activity by a mechanism involving ER stress. 84^th Congress of European Atherosclerosis Society, 29 May 29 – 2 June 2016, Innsbruck, Austria

Stancu C.S., Carnuta M.G., Sanda G.M., Toma L., Deleanu M., Niculescu L.S., Sima A.V. The hyperlipidemic diet inhibits ABC transporters in the intestine and liver of hamsters and induces HDL dysfunction. 5^th National Symposium of ARSAL, 10-11 November 2016, Bucharest, Romania

2017

Oral communications

Cărnuță MG, Stancu CS, Toma L, Sanda MG, Niculescu LS, Deleanu M, Popescu AC, Popescu MR, Vlad A, Doina RD, Simionescu M, Sima AV. Dysfunctional high density lipoproteins with diminished anti-inflammatory potential discriminate between acute coronary syndrome and stable coronary artery disease. International Symposium Acad. Nicolae Cajal of the Academy for Medical Sciences: Translational research - Actualities in virusology, Bucuresti, 30 Martie - 1 April 2017.

Toma L, Deleanu M, Sanda GM, Niculescu LS, Stancu CS, Raileanu M, Sima AV. Caffeic acid attenuates the inflammation in glycated LDL-exposed endothelial cells by reducing the oxidative and endoplasmic reticulum stress. The 9^th National Congress of the Romanian Society for Cell Biology with International Participation and The 35^th RSCB Annual scientific Session, Iasi, Romania, 7-11 Iunie 2017, Buletinul RSCB nr. 45, pag. 15, 2017.

Posters

Cărnuță MG, Camelia CS, Sanda GM, Toma L, Niculescu LS, Deleanu M, Popescu AC, Popescu MR, Vlad A, Dimulescu DR, Simionescu M, Sima AV. Dysfunctional HDL characterized by decreased paraoxonase 1 and increased apolipoproteins AII, CIII and myeloperoxidase levels discriminate between acute coronary syndrome and stable CAD. The 85^th Congress of European Atherosclerosis Society (EAS), Prague, Czech Republic, 23 – 26 April 2017, Atherosclerosis, vol 263, p. 219, August 2017, doi.org/10.1016/j.atherosclerosis.2017.06.713.

Deleanu M, Cărnuță MG, Stancu CS, Toma L, Barbalata T, Raileanu M, Popa ME, Sima AV. Ginger extract reduces SCD1 activity and gene expression in the liver of hyperlipidemic hamsters, as opposed to high fat-diet interruption. NUTRICON 2017 Congress dedicated to Food Quality and Safety, Health and Nutrition, Skopje, Republica Macedonia, 5-7 October 2017.

Deleanu M, Cărnuță MG, Stancu CS, Barbalata B, Raileanu M, Popa ME, Sima AV. Treatment with ginger extract ameliorates SCD activity in the liver during a controlled high fat dietary intervention on syrian hamsters. The 9^th National Congress of the Romanian Society for Cell Biology with International Participation and The 35^th RSCB Annual scientific Session, Iasi, Romania, 7-11 June 2017, Buletinul RSCB nr. 45, pag. 92, June 2017.

Cărnuță MG, Stancu CS, Toma L, Sanda GM, Niculescu LS, Deleanu M, Popescu AC, Popescu MP, Vlad A, Dimulescu DR, Simionescu M, Sima AV. Dysfunctional HDL from coronary artery disease patients contain oxidized lipids, altered apolipoproteins and enzymes activity. The 9^th National Congress of the Romanian Society for Cell Biology with International Participation and The 35^th RSCB Annual scientific Session, Iasi, Romania, 7-11 June 2017, Buletinul RSCB nr. 45, pag. 59, June 2017.

Awards obtained by the team members

EAS Young Investigator Fellowship for Mihaela Cărnuță to attend The 85^th Congress of European Atherosclerosis Society, Prague, Czech Republic, 23 – 26 April 2017 with the communication Dysfunctional HDL characterized by decreased paraoxonase 1 and increased apolipoproteins AII, CIII and myeloperoxidase levels discriminate between acute coronary syndrome and stable CAD, authors Cărnuță MG, Camelia CS, Sanda GM, Toma L, Niculescu LS, Deleanu M, Popescu AC, Popescu MR, Vlad A, Dimulescu DR, Simionescu M, Sima AV.