Beneficial effects of SIRT1 activation in skeletal muscle and adipose tissue

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DISSERTATIONS | SHALEM RAJU MODI | BENEFICIAL EFFECTS OF SIRT1 ACTIVATION IN SKELETAL... | No 476

Dissertations in Health Sciences

THE UNIVERSITY OF EASTERN FINLAND

SHALEM RAJU MODI

BENEFICIAL EFFECTS OF SIRT1 ACTIVATION IN SKELETAL MUSCLE AND ADIPOSE TISSUE uef.fi

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences

ISBN 978-952-61-2845-0 ISSN 1798-5706

This thesis investigates the role of SIRT1 and SIRT1 activators in glucose, energy and fat metabolism in the key insulin-sensitive tissues,

skeletal muscle and adipose tissue. The study reports the association of SIRT1 expression

with energy expenditure, insulin sensitivity and mitochondrial function in humans and introduces strigolactone GR24 and pinosylvin

as SIRT1 activators, having beneficial effects on metabolism of skeletal muscle cells and

adipocytes.

SHALEM RAJU MODI

Shalem_Modi_Thesis_kannet_18_07_17.indd 1 17.7.2018 8:55:56

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Beneficial effects of SIRT1 activation in

skeletal muscle and adipose tissue

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SHALEM RAJU MODI

Beneficial effects of SIRT1 activation in skeletal muscle and adipose tissue

To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Auditorium MD 100, Mediteknia building, University of Eastern Finland,

Kuopio, on Friday, August 10^th 2018, at 12 noon

Publications of the University of Eastern Finland Dissertations in Health Sciences

476

Department of Medicine, Institute of Clinical Medicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland

Kuopio 2018

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Grano Oy Jyväskylä, 2018

Series Editors:

Professor Tomi Laitinen, M.D., Ph.D.

Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences

Professor Tarja Kvist, Ph.D.

Department of Nursing Science Faculty of Health Sciences

Professor Kai Kaarniranta, M.D., Ph.D.

Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences

Associate Professor (Tenure Track) Tarja Malm, Ph.D.

A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy

Faculty of Health Sciences

Distributor:

University of Eastern Finland Kuopio Campus Library

P.O. Box 1627 FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto ISBN (print): 978-952-61-2845-0

ISBN (pdf): 978-952-61-2846-7 ISSN (print):1798-5706

ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

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Author’s address: Institute of Clinical Medicine, Internal Medicine, School of Medicine, Faculty of Health Sciences

University of Eastern Finland P.O. Box 1627

70210 KUOPIO, FINLAND Email: shalem.modi@uef.fi

Supervisors: Professor Markku Laakso, M.D., Ph.D.

Institute of Clinical Medicine, Internal Medicine, School of Medicine, Faculty of Health Sciences

University of Eastern Finland and Kuopio University Hospital KUOPIO

FINLAND

Docent Tarja Kokkola, Ph.D.

University of Eastern Finland KUOPIO

FINLAND

Professor Johanna Kuusisto, M.D., Ph.D.

University of Eastern Finland and Kuopio University Hospital KUOPIO

FINLAND

Reviewers: Professor Zsolt Radak, D.Sc.

Research Institute of Sport Sciences, University of Physical Education, BUDAPEST

HUNGARY

Professor Olavi Ukkola, Ph.D.

Research Unit of Internal Medicine, University of Oulu

OULU FINLAND

Opponent: Professor Kirsi Pietiläinen, M.D., Ph.D.

Department of Medicine University of Helsinki HELSINKI

FINLAND

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Modi, Shalem Raju, Beneficial effects of SIRT1 activation in skeletal muscle and adipose tissue University of Eastern Finland, Faculty of Health Sciences

Publications of the University of Eastern Finland. Dissertations in Health Sciences Number. 476. 2018. 77 p.

ISBN (print):978-952-61-2845-0 ISBN (pdf):978-952-61-2846-7 ISSN (print):1798-5706 ISSN (pdf):1798-5714 ISSN-L:1798-5706

ABSTRACT

Insulin resistance is a characteristic finding in hyperglycemia and type 2 diabetes. Sirtuin 1 (SIRT1) is a NAD⁺-dependent deacetylase involved in the regulation of insulin sensitivity, mitochondrial function, energy metabolism and adipocyte metabolism. Skeletal muscle and adipose tissue are the important insulin-sensitive tissues responsible for maintaining whole body glucose homeostasis. We investigated the association of SIRT1 expression with energy expenditure and insulin sensitivity in these tissues in humans. We showed that energy expenditure associates with insulin sensitivity during the hyperinsulinemic euglycemic clamp, and SIRT1 mRNA expression significantly correlated with the expression of genes regulating energy metabolism and mitochondrial function. We demonstrated that SIRT1 expression in muscle and adipose tissue is associated with energy expenditure and insulin sensitivity.

SIRT1 regulates energy metabolism by interacting with AMPK, a master cellular energy sensor, and PGC-1α, a central regulator of mitochondrial biogenesis. SIRT1 activation has been a proven strategy of extending the life span in animal models by delaying the development of age-related diseases and preventing metabolic syndrome. SIRT1 activators, including plant polyphenols such as resveratrol, have beneficial effects on glucose homeostasis, insulin sensitivity and energy metabolism. We aimed to elucidate the potential effects of the novel plant-derived compounds strigolactone analogue GR24 and pinosylvin on SIRT1 function, glucose uptake, mitochondrial biogenesis and gene expression in skeletal muscle cells. We demonstrated in L6 skeletal muscle myotubes with resveratrol as a reference compound that strigolactone GR24 upregulated and activated SIRT1 without activating AMPK, enhanced the production of NAD⁺, which is an essential SIRT1 substrate, and increased glucose uptake, insulin signaling, GLUT4 translocation and mitochondrial biogenesis. Pinosylvin activated SIRT1 in vitro and stimulated glucose uptake through the activation of AMPK.

Obesity is characterized by excess fat accumulation in white adipose tissue, which triggers chronic low-grade inflammation through secretion of pro-inflammatory factors by the enlarged adipocytes and infiltrated macrophages. This affects glucose and lipid metabolism in adipose tissue, inducing type 2 diabetes. SIRT1 activation inhibits adipogenesis by regulating the key adipogenic transcription factors, PPARγ and C/EBPα. We aimed to investigate the role of strigolactone GR24 and pinosylvin in adipogenesis and inflammation of 3T3-L1 adipocytes with resveratrol as a reference compound. GR24 upregulated SIRT1, enhanced NAD⁺ production and inhibited the activation of NF-κB. GR24 and pinosylvin attenuated adipogenesis via downregulating PPARγ and C/EBPα and protected against inflammation by inhibiting TNF-α- stimulated IL-6 secretion.

In conclusion, our studies demonstrated promising novel therapies via SIRT1 activation in the treatment of insulin resistance and obesity-associated diseases.

National Library of Medicine Classification:QU 125; QU 465; QU 475; WD 210; WK 810; WK 815

Medical Subject Headings: Adipose Tissue; Diabetes Mellitus, Type 2; Inflammation; Insulin Resistance;

Mitochondria; Muscle Cells; Phytochemicals; Pinosylvin; Sirtuin 1

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Modi, Shalem Raju, SIRT1-aktivaation hyödylliset vaikutukset lihas- ja rasvakudoksessa Itä-Suomen yliopisto, terveystieteiden tiedekunta

Publications of the University of Eastern Finland. Dissertations in Health Sciences 476. 2018. 77 s.

ISBN (print): 978-952-61-2845-0 ISBN (pdf):978-952-61-2846-7 ISSN (print):1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

ABSTRAKTI

Insuliiniresistenssi on tyypillinen löydös tyypin 2 diabeteksessa. Sirtuiini 1 (SIRT1) on NAD⁺ -riippuvainen deasetylaasientsyymi, joka osallistuu insuliiniherkkyyden, mitokondrioiden toiminnan, energia-aineenvaihdunnan sekä rasvasolujen aineenvaihdunnan säätelyyn.

Luurankolihakset ja rasvakudos ovat tärkeitä kudoksia insuliiniherkkyyden ja glukoositasapainon kannalta. Tutkimme SIRT1:n ilmentymisen yhteyttä energiankulutukseen ja insuliiniherkkyyteen vapaaehtoisilla tutkittavilla, joilla ei ollut diabetesta. Osoitimme, että energian kulutuksen ja insuliiniherkkyyden välillä oli positiivinen korrelaatio. SIRT1:n lähetti- RNA:n määrä korreloi merkitsevästi energia-aineenvaihdunnan ja mitokondrioiden toimintaa säätelevien geenien kanssa. Tutkimus osoitti, että SIRT1-geenin ilmentyminen lihaksessa ja rasvakudoksessa liittyi energiankulutukseen ja insuliiniherkkyyteen.

SIRT1 säätelee energia-aineenvaihduntaa vuorovaikutuksessa AMPK:n (tärkein solujen energiatasoja säätelevä proteiini) ja PGC-1α:n (mitokondrioiden biogeneesin keskeisin säätelijä) kanssa. SIRT1-aktivaation on eläinmalleissa osoitettu pidentävän elinikää, viivästyttävän ikään liittyvien sairauksien kehittymistä ja ehkäisevän metabolista oireyhtymää. SIRT1- aktivaattoreilla, joihin kuuluvat mm. tietyt kasviperäiset polyfenolit, kuten resveratroli, on edullisia vaikutuksia glukoositasapainoon, insuliiniherkkyyteen ja energia-aineenvaihduntaan.

Selvitimme uusien kasviperäisten yhdisteiden, strigolaktoni-analogi GR24:n ja pinosylviinin, vaikutuksia SIRT1:n toimintaan, glukoosin soluunottoon, mitokondrioiden biogeneesiin ja geenien ilmentymiseen luurankolihassoluissa käyttäen resveratrolia vertailuyhdisteenä.

Strigolaktoni GR24 lisäsi SIRT1:n määrää ja aktiivisuutta ilman AMPK-aktivaatiota sekä SIRT1- substraatti NAD⁺-tuotantoa. Lisäksi GR24 paransi glukoosin soluunottoa, insuliinin signalointia, GLUT4-translokaatiota ja mitokondrioiden biogeneesiä. Pinosylviini aktivoi SIRT1:n in vitro ja lisäsi glukoosin soluunottoa AMPK:n aktivoitumisen kautta.

Lihavuus johtuu ylimääräisen rasvan kerääntymisestä rasvakudokseen ja se aiheuttaa kroonisen matala-asteisen tulehduksen suurentuneiden rasvasolujen ja kudokseen kerääntyvien makrofaagien erittämien tulehdusta lisäävien tekijöiden välityksellä. Tämä vaikuttaa epäedullisesti rasvakudoksen glukoosi- ja rasva-aineenvaihduntaan, mikä voi johtaa tyypin 2 diabetekseen. SIRT1-aktivaatio estää adipogeneesiä eli rasvakudoksen muodostumista vaikuttamalla tärkeisiin adipogeneesin transkriptiotekijöihin (PPARy ja C/EBPα). Tutkimme strigolaktoni GR24:n ja pinosylviinin vaikutuksia 3T3-L1-rasvasolujen adipogeneesiin ja tulehdusreaktioon käyttäen resveratrolia vertailuyhdisteenä. GR24 lisäsi SIRT1:n määrää, tehosti NAD⁺-tuotantoa ja vähensi NF-κB:n aktivaatiota. GR24 ja pinosylviini estivät lisäksi adipogeneesia vähentämällä PPARy:n ja C/EBPα:n määrää sekä suojelivat soluja tulehdukselta estämällä TNF-α:lla stimuloitua IL-6-eritystä.

Tutkimuksemme osoittaa, että SIRT1-aktivaatio vaikuttaa edullisesti aineenvaihduntaan.

Tämän vuoksi SIRT1-aktivaatiota voidaan käyttää hyväksi uusien lääkkeiden kehittämiseen sairauksissa, joihin liittyy insuliiniresistenssi ja ylipaino.

Luokitus: QU 125; QU 465; QU 475; WD 210; WK 810; WK 815

Yleinen suomalainen asiasanasto: diabetes; insuliiniresistenssi; kasvituotteet; lihassolut; mitokondriot; polyfenolit;

rasvakudokset; sirtuiinit; tulehdus

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“Be joyful in hope, patient in affliction, faithful in prayer.” – Romans 12:12

To my beloved father and in the eternal memory of my mother

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Acknowledgements

This study was performed in the Institute of Clinical Medicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland.

I express my deepest gratitude and immensely thank my principal supervisor Professor Markku Laakso for providing me the privilege of pursuing my Ph.D. studies in his laboratory under his esteemed supervision. I am indebted to his incessant support and constant encouragement during the entire period of my research. His brilliant ideas and constructive comments have been instrumental in improving my abilities and conducting my thesis. I feel honored to share the invaluable knowledge and time with a visionary scientist having a persistent passion to contribute to scientific research. His intelligence, dedication, enthusiasm, accomplishments and his phenomenal career will inspire me forever.

I am extremely grateful to my second supervisor Docent Tarja Kokkola for directing my work with her appreciable expertise. I am ever thankful for her impeccable guidance and significant contribution. A perfect colleague on all accounts, she is always willing to dispense her extensive knowledge with a charismatic smile. I have learnt a lot from her cordial and affirmative discussions and working with her has been a remarkable experience. I would like to acknowledge my third supervisor Professor Johanna Kuusisto for her generous support and guidance in pursuing my research.

I offer my sincere thanks to Docent Antero Salminen for providing me space to perform my experiments in his laboratory. His invariable humility and his receptive personality have always been alluring. His intellectual instructions during our meetings have been highly beneficial. Many thanks to Tapio Nuutinen for sharing his thoughts and for helping me with the experiments initially. I thank Tiina Suuronen for her suggestions during my research in Antero’s lab. Thanks to Associate Professor Anu Kauppinen for her genial nature and in providing their cell culture facility whenever needed. I thank Professor Mikko Hiltunen, Associate Professor Annakaisa Haapasalo, Petra Mäkinen and Eveliina Korhonen for helping with the reagents in critical situations. Big thanks to Anne Seppänen for her nice concern and goodwill.

My considerable thanks to Jarno Rutanen for his significant contribution in the first article included in this thesis. Many thanks to the pre-examiners Professor Olavi Ukkola and Professor Zsolt Radak for graciously evaluating my thesis; their valid comments enabled to revise the text and improve the content of this thesis. I would like to thank Doctor David Laaksonen for the linguistic revision of my thesis and Professor Kai Kaarniranta for kindly revising my thesis.

I strongly admire my association with long-time friend, Nagendra Yaluri, who helped me prior to and after my arrival in Finland. I appreciate his contribution in all the research projects that we worked together. We shared substantial responsibilities and valid information for a long duration and our journey taught me important practical aspects. I am thankful to Alena Stancakova, my colleague and friend for her valuable advices and pleasant composure, providing an encouraging momentum. I express my substantial thanks to Jagadish Vangipurapu, for his great friendship during my entire stay in Kuopio and perhaps in future.

Our regular conversations over coffee and lunch were essential stress-relievers. I gladly thank him for being a confidant and I cherish plentiful memories of our camaraderie.

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My heartfelt thanks to Niyas K Saleem for his selfless continuous help and for accompanying me in exploring Finland, giving me a multitude of memorable moments. I thank Yuvaraj Mahendran for all the fun we shared and for his cooperation in plenty of activities that we participated together that kept me in high spirits. I appreciate Maykel Lopez Rodriquez for his collaboration in various projects and applaud his affable behavior. I acknowledge Martin Javorsky, Milka Hakkarainen, Diana Kujala, Jackson Woo and Marinko Rade for many noteworthy moments. I thank Docent Eija Pirinen, Docent Henna Cederberg, Maija Tuusa and Marc Cerrada-Giminez for their scholarly discussions and my thanks to Jemal Adem, Dorota Kaminska, Johanna Viiri and Maria Fizelova for being amiable.

I am very much thankful to Seija Laitinen & Reijo, Leena & Jukka Uschanoff for introducing and showing the Finnish way of leisure and joy by inviting me to many of their gatherings giving unforgettable memories. It has always been a gratifying eventful experience to spend my precious time with their families. I sincerely thank the responsive approach of Raija Räisänen, Teemu Kuulasmaa, Maritta Siloaho, Katja Kostinen-Kokko, Arja Afflekt and all the members of our department in creating a congenial ambience. I warmly thank my fellow Indians, Lakshman Puli, my enduring friend in assisting me on various occasions, Sireesha and Nesna especially for their elegant hospitality and charming nature. My absolute thanks extend to Narasinha &

Varsha Shurpali, Heramb & Jayashree, Rolls John, Mohan Babu & Durga, Bhima & Smitha, Sudipta & Jani Paukkonen, Suresh, Ashik, Uma, Ashok, Bala, Rammohan, Feroze and all the members of Indian Association of Kuopio for their tremendous contribution in making my social life in Kuopio delightfully awesome. I earnestly thank John Mills and Revd. Panu Pohjolainen for their spiritual guidance and for including me in the services of the Lutheran church. Considerable thanks to all my friends and relatives in India who stood by me regardless of the situations with their endearing supportive nature.

My gratitude will never suffice the eternal love of my beloved parents, Satyanandam Modi and Grace Vimala (Late). Their practice of hard work, patience, optimism to overcome the adversities of life has greatly influenced my disposition. With tremendous pride, I bear the testimony of their incredible love, support, courage and infinite sacrifices that constantly motivates me. I credit all my achievements to their enduring support. Special thanks to my beloved parents-in-law, Veda Ratnam & Anita Kiran for being a marvelous strength to me with their overwhelming affection. My hearty thanks to my brother-in-law Mervin for being genuinely supportive and adorable. I duly bestow my success to my lovely wife Merlin for showering me with happiness, kindness, patience, care and for filling my life with endless love.

I thank all the academic and administrative staff of the University of Eastern Finland in providing excellent resources for teaching and learning, sophisticated lab equipment for research, appropriate facilities for sports and ample freedom necessary for enriching my knowledge. Kuopio has been truly hospitable in all the possible ways. Finland taught me the essence of enjoying life with humility and peace and the happiness of dwelling with nature. The spectacular scenic beauty of Finland with its distinct colorful seasons and all its serenity has always been captivating for me. I wholeheartedly thank Finland and the welcoming nature of its beautiful honest people for gifting me perpetual treasures of wonderful life.

I gratefully acknowledge strategic research funding from the University of Eastern Finland, Academy of Finland, Sigrid Jusélius Foundation and Diabetes Wellness Finland for the generous financial support of my studies.

Above all, I praise The Almighty GOD for blessing me abundantly.

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List of the original publications

This dissertation is based on the following original publications:

I Rutanen J, Yaluri N, Modi S, Pihlajamäki J, Vänttinen M, Itkonen P, Kainulainen S, Yamamoto H, Lagouge M, Sinclair DA, Elliott P, Westphal C, Auwerx J, Laakso M:

SIRT1 mRNA expression may be associated with energy expenditure and insulin sensitivity. Diabetes. 59:829-835, 2010

II Modi S, Yaluri N, Kokkola T, Laakso M: Plant-derived compounds strigolactone GR24 and pinosylvin activate SIRT1 and enhance glucose uptake in rat skeletal muscle cells. Sci Rep. 7:17606-017-17840-x, 2017

III Modi S, Yaluri N, Kokkola T: Strigolactone GR24 and pinosylvin attenuate adipogenesis and inflammation of white adipocytes. Biochem Biophys Res Commun.

499:164-169, 2018

The publications were adapted with the permission of the copyright owners.

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Abbreviations

3T3-L1 Murine preadipocyte cell line ADP Adenosine diphosphate AMPK AMP-activated protein kinase ATP Adenosine triphosphate BAT Brown adipose tissue BMI Body mass index

C/EBPα CCAAT/enhancer binding protein α CR Calorie restriction

DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide

EE Energy expenditure FBS Fetal bovine serum FFA Free fatty acids

FOXO1 Forkhead box protein O1 GLUT4 Glucose transporter type 4 IR Insulin resistance

L6 L6 rat myoblast cell line LBM Lean body mass

NAD Nicotinamide adenine dinucleotide NADH Reduced form of NAD

NAMPT Nicotinamide phosphoribosyltransferase NF-κB Nuclear factor kappa B

NRF1 Nuclear respiratory factor 1 OGTT Oral glucose tolerance test

PGC-1α Peroxisome proliferator-activated receptor gamma coactivator 1-alpha PI3K Phosphatidylinositol 3-kinase

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PPARγ Peroxisome proliferator-activated receptor gamma SIRT1 Sirtuin 1

SIRT2 Sirtuin 2 SIRT3 Sirtuin 3 SIRT4 Sirtuin 4 SIRT5 Sirtuin 5 SIRT6 Sirtuin 6 SIRT7 Sirtuin 7

T2D Type 2 diabetes TBS Tris buffered saline

TNF-α Tumor necrosis factor-alpha WAT White adipose tissue WBGU Whole body glucose uptake

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1. Introduction

Type 2 diabetes (T2D) is a progressive heterogeneous disease and a major global health problem, attributable to two pathophysiological disturbances, impaired insulin secretion and insulin resistance (Kahn et al. 2014). Currently there are 415 million people worldwide diagnosed with T2D, and this number is estimated to climb to 642 million in 2040 (Singh et al.

2016). Insulin resistance (IR) is a pathological condition where the key insulin-sensitive tissues such as skeletal muscle, adipose tissue and liver become insensitive to insulin action. Skeletal muscle is the predominant tissue of insulin-stimulated glucose uptake. IR in skeletal muscle, due to impaired glucose and lipid metabolism, mitochondrial dysfunction and reduced glycogen synthesis, contributes to the development of T2D (Abdul-Ghani and DeFronzo 2010, Hesselink et al. 2016, Szendroedi et al. 2011).

Skeletal muscle IR is associated with obesity and increased levels of circulating fatty acids.

Adipocyte metabolism plays a key role in regulating insulin sensitivity and links obesity with T2D (Guilherme et al. 2008). Adipose tissue stores excess nutrients as triacylglycerols and releases free fatty acids (FFA) to meet the energy demands of the body during fasting. Obesity, a multifactorial disorder due to excess fat accumulation in the body, is the the most significant risk factor for T2D. Obesity-associated chronic low-grade inflammation, a pathological condition where the resident lymphocytes and macrophages enhance the production of proinflammatory cytokines, induces IR in adipocytes (Makki et al. 2013, Greenberg and Obin 2006).

SIRT1 is a NAD⁺ dependent deacetylase controlling glucose metabolism, insulin sensitivity in muscle tissue (Schenk et al. 2011), and lipid metabolism in adipose tissue (Qiang et al. 2012, Picard et al. 2004), stimulates mitochondrial biogenesis (Menzies et al. 2013, Price et al. 2012) among other vital functions in the pancreas, liver, heart, hypothalamus and macrophages.

SIRT1 regulates energy metabolism by interacting with AMP-activated protein kinase (AMPK) (Price et al. 2012, Canto et al. 2010), a master sensor of cellular energy status, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a major mitochondrial biogenesis regulator. Skeletal muscle IR has been associated with impaired mitochondrial function and down-regulation of mitochondrial genes including PGC-1α (Petersen et al. 2004, Mootha et al. 2003). SIRT1 activation prevents the development of metabolic syndrome through the deacetylation and activation of PGC-1α (Canto and Auwerx 2009, Lagouge et al. 2006).

SIRT1 activators, including plant polyphenols such as resveratrol, have been shown to have beneficial effects on glucose homeostasis, energy metabolism and insulin sensitivity in muscle and adipose tissues in animal models (Szkudelski and Szkudelska 2015, Milne et al. 2007).

Strigolactones are plant hormones having endogenous roles in regulating shoot and root branching, and exogenous roles in enhancing beneficial symbiosis of the plant with mycorrhizal fungi. In mycorrhiza, strigolactones have been shown to promote mitochondrial biogenesis, hyphal branching and adenosine triphosphate (ATP) production (Al-Babili and Bouwmeester 2015, Besserer et al. 2008, Besserer et al. 2006). Strigolactones have only been investigated in mammalian cells in the context of their beneficial effects in cancer (Pollock et al. 2014, Croglio et al. 2016). Pinosylvin is a natural stilbenoid polyphenol found in high concentrations in the Pinus species with structural resemblance to resveratrol. Pinosylvin has been found to possess anti- bacterial, anti-fungal and anti-cancer properties (Lee et al. 2005, Park et al. 2013), but its effects in metabolic tissues are largely unknown (van Summeren-Wesenhagen and Marienhagen 2015).

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Plant bioactive compounds improve insulin sensitivity and metabolic health, but the effects of strigolactones and pinosylvin on these parameters have not been previously studied. We investigated the association of energy expenditure (EE), insulin sensitivity with SIRT1 and PGC- 1α mRNA expression in human skeletal muscle and adipose tissue. We further elucidated the effects of the novel plant compounds strigolactone analogue GR24 and pinosylvin on SIRT1 function, glucose uptake, mitochondrial biogenesis and gene expression in L6 myotubes, and the effects of GR24 and pinosylvin on SIRT1 function, adipogenesis and inflammation in 3T3-L1 adipocytes.

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2. Review of the Literature

2.1 TYPE 2 DIABETES

According to the recent estimate of the World Health Organization (WHO), the prevalence of diabetes is 9% in the world, accounting for 422 million adults in 2014 (WHO, 2016), and by 2030 it is predicted to become the 7th leading cause of death (Mathers and Loncar 2006). The number of diabetic patients is expected to increase up to 642 million over the next decade (Jaacks et al.

2016).

T2D is a chronic state of hyperglycemia caused by two pathophysiological conditions, namely IR, a condition where insulin does not have its normal effects in insulin sensitive tissues, and impaired insulin secretion, resulting in elevated blood glucose levels (Kahn et al. 2014). T2D is diagnosed by fasting plasma glucose ≥ 7.0 mmol/l, 2-hour blood glucose ≥ 11.1 mmol/l in an oral glucose tolerance test (OGTT), and HbA1c levels ≥ 48 mmol/mol, according to the criteria of the American Diabetes Association (ADA).

The radical changes in lifestyle and environment are responsible for an extensive increase in the prevalence and incidence of T2D. Genetic and environmental factors, such as high calorific diet and sedentary lifestyle, play a prominent role in the predisposition to T2D.

2.1.1 Pathophysiology of type 2 diabetes

Insulin is an endocrine hormone secreted from the pancreatic β-cells that plays a significant role in the regulation of blood glucose. Elevated glucose levels after a meal stimulate the release of insulin from the β-cells. Insulin stimulates glucose uptake into insulin sensitive tissues, such as skeletal muscle, liver and adipose tissue. Ultimately, glucose is either used as energy or stored as glycogen.

The pancreas, skeletal muscle, liver, adipose tissue, kidney, gastrointestinal tract and brain play a vital role in the pathophysiology of T2D (Figure 1). Impaired insulin secretion from the pancreatic β-cells, decreased muscle glucose uptake indicating IR, and liver IR leading to increased hepatic glucose production or reduced glucose uptake by the liver, are the major contributors for hyperglycemia. Chronic low-grade inflammation in the adipose tissue and enhanced lipolysis due to impaired insulin’s antilipolytic effect, deficient incretin hormone release in the gastrointestinal tract, increased glucagon secretion from the pancreatic alpha cells, enhanced glucose reabsorption by the kidneys, and brain IR are other important factors contributing to hyperglycemia (DeFronzo 2009).

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Figure 1.Tissues and organs in the pathophysiology of T2D (Cornell 2015).

2.1.2 Insulin secretion

Pancreatic β-cells secrete insulin in response to elevated blood glucose levels to maintain normoglycemia. Elevated insulin secretion compensates for IR, but when the β-cell compensatory response to IR fails, hyperglycemia develops. Pancreatic β-cell dysfunction and β-cell failure are pathological defects observed in patients with T2D (Prentki and Nolan 2006).

Chronic hyperglycemia-induced glucotoxicity and lipotoxicity due to elevated circulating FFA are responsible for β-cell apoptosis, eventually resulting in increased β-cell loss (Butler et al.

2003, Cnop et al. 2005, Karaca et al. 2009).

Glucose is transported into the β-cells with the glucose transporter 2 (GLUT2). Intracellular glucose is metabolized to glucose-6-phosphate and generates ATP in mitochondria. An increased ATP/ADP ratio induces the closure of the ATP-sensitive potassium channels (K^ATP) and leads to the depolarization of cell membrane resulting in membrane depolarization, opening of voltage-dependent Ca²⁺ channels (VDCC), and extracellular Ca²⁺ influx into the cell.

The rise in intracellular calcium concentrations triggers the exocytosis of insulin from insulin granules (MacDonald and Wheeler 2003, Henquin et al. 2003).

Transcription and translation of the insulin gene is dependent on the stimulation of glucose (Poitout et al. 2006). A biologically inactive 110-amino acid precursor, known as preproinsulin, is encoded by the insulin gene. Preproinsulin is converted to proinsulin in the endoplasmic reticulum by the cleavage of the signal peptide from its N-terminus by signal peptidase.

Proinsulin consists of 86 amino acids, and the A and B chains are bridged together by two

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disulfide bonds and joined by C-peptide. The folded proinsulin is then transported to the Golgi apparatus where C-peptide is removed by prohormone convertases, PC1 and PC3 (Fu et al.

2013, Weiss 2009). The mature insulin has a molecular weight of 5.8 kDa containing 51 amino acids and stored within the secretory granules as Zn²⁺-stabilized hexamers. Plasma proinsulin levels and C-peptide are used as markers of insulin secretion and β-cell dysfunction in patients with diabetes (Breuer et al. 2010, Jones and Hattersley 2013).

Insulin secretion is biphasic. The rapid first phase of insulin release happens in response to elevated blood glucose levels within two minutes and lasts for 10 minutes. The second phase of insulin secretion extends for 2-3 hours until normoglycemia is achieved. Insulin granules are recruited to the plasma membrane from intracellular storage pools during the second phase. If the first phase of insulin secretion is decreased, this may lead to a compensatory increase in hyperglycemia in the second phase of insulin secretion. Impairment of the first phase insulin secretion is an indicator of impaired glucose tolerance or early stage of T2D (Del Prato and Tiengo 2001). The second phase insulin release also declines during the progression of T2D (Jenssen and Hartmann 2015).

2.1.3 Insulin resistance

IR is a pathological condition where the body becomes insensitive to insulin action. As a result, insulin-stimulated glucose uptake in muscle, liver and adipose tissue decreases. Obesity and lack of physical exercise increase IR, an important risk factor for the development of T2D (Hishinuma et al. 2008).

In normal insulin signaling, insulin binds to the α subunit of the insulin receptor, which consists of two α and two β subunits linked by disulphide bonds. This results in activation of tyrosine kinase of the β subunits and autophosphorylation of the β subunit and leads to tyrosine phosphorylation of insulin receptor substrates (IRSs). These phosphorylated IRSs then associate with the regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and activate the catalytic subunit of PI3K, which phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) generating phosphatidylinositol 3,4,5-trisphosphate (PIP3). Membrane-bound PIP3 recruits 3- phosphoinositide-dependent kinase 1 (PDK1) and serine/threonine protein kinase Akt to the membrane and activates PDK1. PDK1 then phosphorylates and activates Akt mediating the translocation of the insulin sensitive glucose transporter type 4 (GLUT4) to the plasma membrane (Saini 2010). Perturbations in the insulin signaling pathway play a significant role in the development of IR (Boucher et al. 2014). Hyperglycemia (Jellinger 2009), lipotoxicity (DeFronzo 2010), low-grade inflammation (Osborn and Olefsky 2012), mitochondrial dysfunction (Montgomery and Turner 2015), endoplasmic reticulum stress (Flamment et al.

2012), and genetic factors (Watanabe 2010) worsen IR.

2.1.4 Insulin resistance in different tissues 2.1.4.1 Skeletal muscle

Skeletal muscle is the predominant insulin-sensitive tissue, accounting for about 75% of insulin- stimulated glucose uptake in the whole body (Björnholm and Zierath 2005). IR in skeletal muscle contributes to the development of T2D. IR is also associated with defects in insulin receptor signaling, such as reduced insulin receptor substrate 1 (IRS-1) phosphorylation, decreased PI3K activation, reduced Akt activation and decreased GLUT4 translocation to the

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plasma membrane, resulting in impaired glucose uptake in skeletal muscle (Krook et al. 2000, DeFronzo and Tripathy 2009, Deshmukh 2016).

The translocation of glucose transporter GLUT4 to the plasma membrane is an important event in insulin-stimulated glucose uptake in skeletal muscle (Björnholm et al. 2005). Impairment of GLUT4 trafficking and translocation contributes to muscle IR (Garvey et al. 1998, Zisman et al.

2000, Leto and Saltiel 2012). Additionally, impairment in insulin-stimulated glycogen synthesis is another significant finding in IR (Abdul-Ghani and DeFronzo 2010).

Mitochondrial dysfunction has been associated with IR and T2D in skeletal muscle (Kelley et al.

2002, Di Meo et al. 2017). Patients with IR and T2D showed decreased expression of genes involved in oxidative phosphorylation (Patti et al. 2003). Several studies in humans have implicated the role of mitochondria and the genes involved in oxidative phosphorylation and mitochondrial function in T2D (Mootha et al. 2003, Petersen et al. 2004, Morino et al. 2005, Montgomery and Turner 2015).

2.1.4.2 Adipose tissue

Adipose tissue is a key metabolic tissue for storing excess energy in the form of lipids during abundant caloric intake. White adipose tissue (WAT) stores FFAs as triglycerides and provides energy to the body during starvation and fasting by releasing FFAs. Brown adipose tissue (BAT) stores lipids for regulating body temperature via adaptive thermogenesis (Saely et al.

2012). When the capacity of adipose tissue to store triglycerides is exceeded, triglycerides accumulate in other tissues such as liver and skeletal muscle, leading to IR in these tissues (Ruan and Lodish, 2003). In genetically predisposed individuals, lipotoxicity due to overproduction of FFAs impairs insulin secretion and induces IR in muscle and liver (Bays et al.

2004).

Obesity is associated with chronic low-grade systemic inflammation and increased production of proinflammatory cytokines and macrophages in the adipose tissue (Gregor and Hotamisligil 2011, Makki et al. 2013). In lean individuals, adipose tissue secretes anti-inflammatory adipokines such as adiponectin, transforming growth factor beta (TGFβ), interleukin (IL)-10, IL- 4, IL-13, IL-1 receptor antagonist (IL-1Ra), and apelin. In obese individuals adipose tissue secretes proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) (Miyazaki et al.

2003), IL-6, leptin, visfatin, resistin, angiotensin II, and plasminogen activator inhibitor 1.

Additionally, adiponectin concentration is reduced (Kern et al. 2003). Macrophages in the pro- inflammatory state may be found in almost 50% of adipose tissue mass (Makki et al. 2013, Sears and Perry 2015). Expression of GLUT4 in adipose tissue is markedly reduced in insulin-resistant subjects (Shepherd and Kahn 1999).

2.1.4.3 Liver

Liver maintains systemic glucose homeostasis by producing glucose in the postabsorptive state by glycogenolysis, during fasting by gluconeogenesis, and by glycogenesis for storage of glucose. During feeding conditions, glycolysis serves as a major pathway to produce energy as ATP by the catabolism of glucose. Under conditions of fasting, liver acts as a fuel reserve for brain, muscle and red blood cells. Hepatic glucose production (HGP) primarily determines the fasting plasma glucose concentration, regulated by plasma insulin (DeFronzo et al. 1989). In T2D, plasma insulin concentration fails to suppress HGP.

Glycogen is a key energy reserve in the body stored in the liver and muscle. Glucose produced by the liver of healthy subjects in the fasting state normally meets the demands of the brain, and glucose uptake by brain is independent of insulin. Under conditions of starvation or short term

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fasting or during intense exercise, glycogen stored in the liver and muscle is hydrolyzed to glucose by glycogen phosphorylase. This process is largely stimulated by glucagon and adrenaline. During prolonged fasting where glycogen is depleted, glucose is synthesized by gluconeogenesis through non-carbohydrate sources such as pyruvate, lactate, glycerol and amino acids (Rui 2014, Han et al. 2016). Glucose or ingestion of meal induces the release of insulin into the portal vein, inhibits glucagon release and suppresses HGP. If the liver fails to recognize this signal, it leads to elevated glucose production by the liver (Bugianesi et al. 2005, Cersosimo et al. 2015). Enhanced gluconeogenesis, elevated plasma FFA, and triglyceride synthesis contribute to hepatic IR (Cerosimo et al. 2015, Ferris and Kahn 2016).

2.2 SIRTUINS

Sirtuins form a family of NAD⁺-dependent deacetylases highly conserved from bacteria and lower eukaryotes to humans (Fyre 1999, 2000). Sir2 name originates from the gene involved in cellular regulation in yeast. Sirtuins affect energy metabolism and stress responses. Some sirtuins extend longevity in many organisms via their sensitivity towards NAD⁺. Mammalian sirtuins (SIRT1-7) play a vital role in aging, energy metabolism and cell survival in many tissues (Morris 2013). The sirtuins belong to the class III family of histone deacetylases and utilize NAD⁺ as a cofactor for their enzymatic activity, yielding deacetylated O-acetyl-ADP-ribose and nicotinamide (Landry et al. 2000). In addition to deacetylase activity, some sirtuins also have other enzymatic activities. Sirtuins are expressed in all metabolically active tissues.

2.2.1 Types of sirtuins

Sirtuins are divided into five classes based on their conserved amino acid core domain (Fyre, 2000). There are seven sirtuins in mammals (SIRT1-7), and all of them use NAD⁺ as a co- substrate (Figure 2). SIRT1, sirtuin 2 (SIRT2) and sirtuin 3 (SIRT3) are the mammalian sirtuins belonging to class I sirtuins. SIRT1 shares the highest sequence similarity with yeast Sir2 and Hst1, and SIRT2 and SIRT3 with Hst2 (North et al. 2003). Sirtuin 4 (SIRT4) belongs to class II, sirtuin 5 (SIRT5) to class III, and sirtuin 6 (SIRT6) and sirtuin 7 (SIRT7) to class V sirtuins.

Mammalian sirtuins have a conserved sirtuin core domain but have different functions.

SIRT1 is predominantly localized in the nucleus (Michishita et al. 2005), but it can also be found in the cytoplasm (Byles et al. 2010) and in some conditions in mitochondria (Aquilano et al.

2010). The localization of SIRT1 varies in different tissues under specific conditions depending on nutrient availability and sensing of the metabolic status. SIRT1 shuttles between the nucleus and cytoplasm, and this mechanism regulates its activity (Tanno et al. 2007). For example, calorie restriction (CR) influences the nuclear localization of SIRT1 (Shinmura et al. 2008). SIRT2 is primarily located in the cytoplasm but translocates to the nucleus under certain conditions (Vaquero et al. 2006). SIRT3, SIRT4 and SIRT5 are located in the mitochondria, and SIRT6 in the nucleus. SIRT7 is a nuclear protein specifically localized in the nucleolus (Vassilopoulus et al.

2011, Kupis et al. 2016).

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Figure 2. Types of sirtuins, their primary structures and their subcellular localization. The figure shows mammalian sirtuins (SIRT1-7) in alignment with yeast Sir2. The conserved, catalytic domains of sirtuins are in yellow. Numbers represent the amino acids in the proteins. NLS- Nuclear localization sequence; MTS- Mitochondrial targeting sequence (Guarente 2013).

All sirtuins exhibit deacetylase activity. In this enzymatic reaction, sirtuins utilize NAD⁺ as a substrate to remove the acetyl groups from ε-acetyl lysine residues of target proteins. The lysine-bound acetyl group is transferred to the 2’-OH position of ADP-ribose, eventually generating a deacetylated protein and producing nicotinamide and 2’-O-acetyl-ADP-ribose (Cantó and Auwerx 2012) (Figure 3). SIRT2 also acts as demyristoylase (Teng et al. 2015), SIRT3 as decrotonylase (Tan et al. 2010), SIRT4 and SIRT6 act as mono-ADP-ribosyl transferases in reactions which involve the transfer of ADP-ribosyl moiety of NAD⁺ to a substrate protein.

SIRT4 acts as a lipoamidase and SIRT5 acts as demalonylase, desuccinylase and deglutarylase (Kupis et al. 2016). In addition to their deacetylase activity, mammalian sirtuins are also capable of catalyzing long-chain deacylation, demonstrated by the decrotonylase activity of SIRT1 and SIRT2 and delipoylation activity of SIRT1, SIRT2, SIRT3, and SIRT4 (Feldman et al. 2013).

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Figure 3. The NAD⁺-dependent SIRT1 deacetylase reaction (Canto and Auwerx 2012).

2.2.2 Sir2, the prototypic sirtuin

The SIR2 gene was originally identified and characterized as one of the genes that regulates selected loci in Saccharomyces cerevisiae (Klar et al. 1979). Sir2 overexpression was initially demonstrated to promote histone deacetylation (Braunstein et al. 1993). The role of Sir2 in the regulation of transcriptional silencing at mating-type loci, telomeres and extrachromosomal rDNA circles suggested the involvement of Sir2 in extending the life span of yeast (Sinclair and Guarente 1997). The significant step in elevating Sir2 to the core of aging research was the discovery that overexpression of Sir2 alone can promote longevity in yeast (Kaeberlein et al.

1999). Imai et al. first studied the dependence of yeast and mouse Sir2 proteins on NAD⁺ for their histone deacetylase activity (Imai et al. 2000). Other studies showed the NAD⁺-dependent histone deacetylase activity of bacterial and human Sir2 proteins (Smith et al. 2000, Landry J et al. 2000). SIR2 gene is evolutionarily conserved from bacteria to humans (Brachmann et al. 1995).

The overexpression of Sir2 homologs in flies, nematodes and mice was found to increase the lifespan of the organisms (Tissenbaum and Guarente 2001, Rogina and Helfand 2004, Viswanathan and Guarente 2011; Banerjee et al. 2012). SIRT1 is the mammalian homolog of yeast Sir2 and the most extensively studied and characterized sirtuin.

2.2.3. The roles of sirtuins in type 2 diabetes

The numerous functions of sirtuins in glucose, lipid metabolism, energy homeostasis, insulin secretion and insulin sensitivity have projected them as promising therapeutic targets for treating T2D.

SIRT1 is well documented to mediate the beneficial effects of CR, and its role in metabolism and protection against T2D has been studied in detail. The role of SIRT1 in insulin signaling and nutrient sensing has been demonstrated in several animal models and in humans. SIRT1 enhances glucose-stimulated insulin secretion, regulates the insulin signaling pathway, protects

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pancreatic β-cells from apoptosis and is vital for glucose metabolism (Kitada and Koya 2013).

SIRT1 enhances insulin sensitivity in metabolic tissues such as liver, skeletal muscle and adipose tissue by regulating several proteins and transcription factors through its deacetylase activity (Cao et al. 2016).

SIRT2 has been implicated in several metabolic functions, such as adipocyte differentiation through regulation of forkhead box protein O1 (FOXO1) (Jing et al. 2007) and fatty acid synthesis (Lin et al. 2013). It also promotes fatty acid oxidation and mitochondrial biogenesis via PGC-1α deacetylation in adipocytes with the inactivation of hypoxia-inducible factor 1α (HIF1α) (Krishnan et al. 2012).

SIRT3 is highly expressed in tissues rich in mitochondria such as brain, heart liver and BAT and functions as a mitochondrial deacetylase (Lombard et al. 2007). Increased triglycerides and low levels of FFA oxidation in the liver in the fasted state have been observed in SIRT3-deficient mice (Hirschey et al. 2010). SIRT3 regulates mitochondrial function, plays a crucial role in adaptive thermogenesis in BAT and is down-regulated in BAT of genetically obese mice.

Overexpression of SIRT3 enhances respiration and reduces membrane potential and production of reactive oxygen species (Shi et al. 2005). SIRT3 knock-out mice have increased oxidative stress and impaired insulin signaling in skeletal muscle (Jing E et al. 2011). Furthermore, SIRT3 protects high-fat-fed mice against IR and defects in skeletal muscle glucose uptake (Lantier et al.

2015).

SIRT4 is a mitochondrial sirtuin that mainly acts as an ADP-ribosyl transferase. SIRT4 interacts with glutamate dehydrogenase (GDH) and downregulates it in mitochondria of pancreatic β- cells. Loss of SIRT4 activates GDH, eventually enhancing insulin secretion stimulated by amino acids (Hagis et al. 2006). Overexpression of SIRT4 leads to a decrease in glucose-stimulated insulin secretion (Ahuja et al. 2007), in contrast to SIRT1.

SIRT5 is also a mitochondrial sirtuin with several enzymatic activities. SIRT5 has a potent lysine desuccinylase activity and its loss causes hyper-succinylation of mitochondrial proteins and impaired fatty acid β-oxidation (Rardin et al. 2013). SIRT5 regulates glycolysis via lysine malonylation; glycolysis is suppressed in the absence of SIRT5 due to increased malonylation of glycolytic enzymes (Nishida et al. 2015). In a study on adipose tissue of BMI-discordant monozygotic twins, SIRT5 expression was found to correlate positively with insulin sensitivity and negatively with inflammation (Jukarainen et al. 2016).

SIRT6 overexpression in mice protects against the accumulation of fat and impaired glucose tolerance induced by high fat diet and regulates lipid homeostasis by down-regulating key genes involved in lipid metabolism (Kanfi et al. 2010). SIRT6 deficiency is associated with severe hypoglycemia, enhanced insulin signaling, and increased basal and insulin-stimulated glucose uptake (Xiao et al. 2010). SIRT6 also functions as ADP-ribosyltransferase (Liszt et al. 2005), shows anti-inflammatory properties, and is downregulated in diabetic atherosclerotic plaques (Balestrieri et al. 2015). The deficiency of SIRT6 blocks adipocyte differentiation (Chen et al.

2017).

SIRT7 is a nuclear sirtuin that also regulates mitochondrial function. SIRT7 expression correlates positively with nuclear DNA encoded mitochondrial genes, and SIRT7 deficient mice have multi-systemic mitochondrial dysfunction (Ryu et al. 2014). SIRT7 has been found to promote obesity in mice (Cioffi et al. 2015), but in another study SIRT7 expression was decreased in obesity and increased after weight loss (Rappou et al. 2016).

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2.3 SIRT1 – METABOLIC FUNCTIONS AND MOLECULAR MECHANISMS

SIRT1 is a vital regulator of many functions of cellular metabolism such as aging, glucose homeostasis, energy metabolism, lipid metabolism, cell cycle, inflammation, apoptosis, oxidative stress, mitochondrial function, modulation of circadian rhythms, and DNA repair. It protects from several diseases such as diabetes, obesity, atherosclerosis, cardiovascular diseases and neurodegenerative diseases (Kitada and Koya 2013, Schug and Li 2011, Kitada et al. 2016, Herskovits and Guarente 2014) (Figure 4⁾.

Figure 4. Multiple cellular functions of SIRT1 (Adapted from Rahman and Islam 2011).

SIRT1 regulates several physiological functions by sensing the variations in intracellular NAD⁺ levels and adapting to the energy requirements of the cell. NAD⁺ acts as a substrate for NAD⁺- dependent enzymes and is essential for the redox reactions of the cell. The activity of SIRT1 is dependent on dynamic changes in cellular NAD⁺ concentration, which facilitates the function of SIRT1 as a nutritional and metabolic sensor. (Imai et al. 2000). SIRT1 expression is upregulated during conditions of energy deprivation and repressed during excess energy. Sir2/ SIRT1 distinctly deacetylates histones H1 (K26), H3 (K9, K14) and H4 (K16) in vitro which promotes

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the formation of heterochromatin (Imai et al. 2000, Vaquero et al. 2004, Vaquero et al. 2007). In addition to histone substrates, SIRT1 deacetylates a wide range of predominantly nuclear substrates and regulates transcription factors such as p53, nuclear factor kappa B (NF-κB), FOXO1, PGC-1α and many co-factors as well as nuclear receptors, thus linking the regulation of gene expression with the metabolic status of the cell (Zhai et al. 2017).

SIRT1 activity is affected by several posttranslational modifications such as phosphorylation, methylation, SUMOylation and nitrosylation (Revollo and Li 2013). SIRT1 activity is also regulated by microRNAs (miRNAs). miRNAs are short noncoding RNAs that are about 22 nucleotides long and function in RNA silencing and in controlling the post-transcriptional gene expression by suppressing translation. SIRT1 is regulated by a multitude of miRNAs in different tissues (Yamakuchi 2012, Buler et al. 2016). SIRT1 deacetylates multiple transcription factors and other proteins and modulates multiple fundamental cellular functions. SIRT1 is ubiquitously expressed also in metabolic tissues such as muscle (Rathbone et al. 2009), liver (Kemper et al. 2013), adipose tissue (Picard et al. 2004), pancreas (Bordone et al. 2006), heart (Sakamoto et al. 2004) and brain (Zachary et al. 2010).

2.3.1 Aging and calorie restriction

CR, defined as the lowering of nutritional intake without causing malnutrition, has been found to be the most effective intervention for slowing aging and its associated diseases in several animal models and humans (Fontana et al. 2010, Fontana and Partridge 2015). Lin et al. first reported that enhancement of longevity induced by CR depends on the activation of Sir2 by NAD⁺ in Saccharomyces cerevisiae (Lin et al. 2000). The studies on the role of Sir2 in aging and CR have been further confirmed in Caenorhabditis elegans (Tissenbaum et al. 2001) and Drosophila melanogaster (Rogina et al. 2004). Cohen et al. demonstrated that the effects of CR are due to the induction of SIRT1 in mammalian cells (Cohen et al. 2004).

SIRT1 activation mediates several beneficial effects induced by CR. Data from several recent randomized clinical trials in humans also indicate the health benefits that CR offers and supports the implementation of this strategy to protect from aging-associated disorders in the promotion of healthy aging (Most et al. 2016). The onset of age-related diseases such as atherosclerosis, diabetes and neurodegenerative diseases could be delayed by the activation of SIRT1 (Kitada et al. 2016, Chang and Guarente 2014, Kitada and Koya 2013, Herskovits and Guarente 2014).

2.3.2 Energy metabolism and mitochondrial function

Nutrient deprivation by fasting or exercise induces a shift from glucose to fatty acid oxidation for energy production in the skeletal muscle. The activation of SIRT1 during low energy status contributes to this metabolic switch and promotes fatty acid oxidation (Gerhart- Hines et al.

2007). The activity of SIRT1 is controlled by the levels of NAD⁺, which is an essential co-enzyme involved in redox reactions. As NAD⁺ is produced by oxidation and NADH in electron transport, the concentration of NAD⁺ reflects the energy status of the cell. CR, fasting and exercise enhance cellular NAD⁺levels and SIRT1 activity, whereas energy abundance and inflammation decrease both NAD⁺levels and SIRT1 activity (Li 2013). Thus, SIRT1 acts as a key component of energy metabolism and sensing.

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Mitochondria are key cellular organelles responsible for maintaining energy homeostasis, cell metabolism, protecting the cells from oxidative stress and apoptosis and regulating cell viability. Oxidative phosphorylation in the mitochondrial respiratory chain is the main cellular source of energy. Nuclear respiratory factor 1 (NRF1) is a transcription factor playing a crucial role in mitochondrial biogenesis (Zamora et al. 2015). The importance of NRF1 in IR has been shown in human gene expression studies, where IR was found to be associated with a decrease in the expression of NRF1 along with genes involved in oxidative metabolism and mitochondrial function (Patti et al. 2003).

PGC-1α controls energy homeostasis and is a major regulator of mitochondrial biogenesis (Puigserver and Spiegelman, 2003). PGC-1α-responsive genes that are involved in oxidative phosphorylation were coordinately downregulated in human diabetic muscle (Mootha et al.

2003). SIRT1 interacts with and deacetylates PGC-1α and activates it in vitro and in vivo (Nemoto et al. 2005). SIRT1 controls glucose homeostasis by functioning together with PGC-1α in adapting to nutrient deprivation by regulating genes involved in gluconeogenesis and glycolysis in the liver (Rodgers et al. 2005). The SIRT1 activator resveratrol improves mitochondrial function and insulin sensitivity via induction of PGC-1α activity through SIRT1 in the skeletal muscle (Lagouge et al. 2006). The deacetylation of PGC-1α by SIRT1 and the necessity of SIRT1 in influencing the regulation of mitochondrial and fatty acid metabolism has been demonstrated in skeletal muscle (Gerhart-Hines et al. 2007, Amat et al. 2009). Furthermore, Aquilano et al. showed the localization of SIRT1 and PGC-1α in mitochondria and their involvement in mitochondrial biogenesis (Aquilano et al. 2010).

AMPK is a metabolic master regulator that is activated in response to changes in the cellular AMP/ATP ratio. AMPK activation reflects energy levels of the cell, and activated AMPK stimulates processes that produces ATP by turning on catabolic pathways and shutting down energy-consuming anabolic pathways. During energy deficiency conditions, AMPK activates genes regulating glycolysis and mitochondrial respiration and represses genes involved in lipid synthesis (Canto and Auwerx 2009). Hou et al. reported that SIRT1 regulated hepatic lipid metabolism by acting upstream of LKB1/AMPK signaling pathway (Hou et al. 2008). AMPK modulates SIRT1 activity by increasing intracellular NAD⁺ levels (Fulco et al. 2008, Canto et al.

2009). SIRT1 activators enhance the phosphorylation of AMPK, whereas nicotinamide, an inhibitor of SIRT1 suppresses the activity of SIRT1 and subsequent phosphorylation of AMPK and enhances acetylation of PGC-1α (Suchankova et al. 2009).

AMPK activation also enhances the expression of PGC-1α (Terada et al. 2002). AMPK directly phosphorylates PGC-1α in skeletal muscle, and this interaction plays an essential role in GLUT4 gene expression and mitochondrial function (Jäger et al. 2007). Canto et al. reported the well- coordinated interaction of SIRT1, AMPK and PGC-1α in controlling EE and mitochondrial function to adapt to different energy demands (Canto et al. 2010). In another report, resveratrol was found to improve mitochondrial function through stimulation of AMPK, and SIRT1 played an essential role in mediating these effects (Price et al. 2012). Hence, SIRT1 interacts with AMPK and PGC-1α in sensing energy requirements of the cell and regulates the genes essential for cell survival and metabolism.

2.3.3 Insulin secretion

SIRT1 regulates insulin secretion and maintains glucose homeostasis (Moynihan et al. 2005;

Banks et al. 2008). Uncoupling protein 2 (UCP2) is expressed in pancreatic islets and influences mitochondrial uncoupling and insulin secretion. UCP2 is a critical component of glucose

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sensing in β-cells. It mediates the proton leak in mitochondria, reducing the production of ATP and acts as a negative regulator of insulin secretion (Zhang et al. 2001).

Figure 5. SIRT1 and insulin secretion (Adapted from Liang et al. 2009).

SIRT1 enhances glucose-stimulated insulin secretion via transcriptional repression of UCP2, which uncouples ATP production in mitochondria (Bordone et al. 2006) (Figure 5)

.

^SIRT1

deficiency in β-cells impairs insulin secretion (Luu et al. 2013, do Amaral et al. 2011, Moynihan et al. 2005). Research on pancreatic INS-1E cells and human islets showed a marked SIRT1- mediated enhancement of glucose response and glucose-stimulated insulin secretion (Vetterli et al. 2011). Enhancement of SIRT1 expression and activity protects pancreatic β-cells from apoptosis (Prud’homme et al. 2014). Hence, SIRT1 modulates ATP production, promotes glucose sensing and enhances insulin secretion of pancreatic β-cells.

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2.3.4 Insulin sensitivity

Muscle, liver and adipose tissue form the key tissues that play prominent roles in accounting for whole body insulin sensitivity and glucose homeostasis. SIRT1 plays a significant role in enhancing insulin sensitivity by regulating insulin signaling and interacting with several proteins and transcription factors (Kitada and Koya 2013, Cao et al. 2016) in metabolic tissues (Figure 6). SIRT1 activators, including resveratrol, have been demonstrated to enhance insulin sensitivity in different tissues (Milne et al. 2007, Timmers et al. 2012).

Figure 6. Effects of SIRT1 activation in metabolic tissues (Adapted from Lavu et al. 2008).

2.3.4.1 Skeletal muscle

CR results in increased insulin-stimulated glucose uptake due to enhanced cell surface recruitment of GLUT4 (Dean et al. 1998). CR also stimulates insulin signaling in the skeletal muscle, leading to enhanced insulin sensitivity (Sharma et al. 2011, Wang et al. 2009, McCurdy et al. 2005, Dean et al. 2000). SIRT1 regulates skeletal muscle glucose and lipid metabolism, and increasing evidence suggests that the decreased expression or activity of SIRT1 contributes to skeletal muscle IR. Enhanced expression of SIRT1 improves insulin sensitivity by repressing

Beneficial effects of SIRT1 activation in skeletal muscle and adipose tissue

Dissertations in Health Sciences

SHALEM RAJU MODI

BENEFICIAL EFFECTS OF SIRT1 ACTIVATION IN SKELETAL MUSCLE AND ADIPOSE TISSUE uef.fi

SHALEM RAJU MODI

Beneficial effects of SIRT1 activation in

skeletal muscle and adipose tissue

Beneficial effects of SIRT1 activation in skeletal muscle and adipose tissue

Acknowledgements

List of the original publications

Contents

Abbreviations

1. Introduction

2. Review of the Literature

.