DISSERTATIONS | KAISA AIRAKSINEN | METABOLIC STRESS IN OBESITY | No 599
KAISA AIRAKSINEN
Metabolic stress in obesity
The impact of high-fat diet and food ingredients on adipose tissue inflammation and satiety
Dissertations in Health Sciences
THE UNIVERSITY OF EASTERN FINLAND
uef.fi
PUBLICATIONS OF
THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences
ISBN 978-952-61-3636-3 ISSN 1798-5706
Overweight and obesity are associated with several risk factors for type 2 diabetes
and cardiovascular diseases. Low-grade inflammation and perturbations in appetite
regulation are typically linked with the obese state. This thesis studied the effects of supplementary food ingredients, betaine,
polydextrose and lactitol, on adipose tissue inflammation and satiety in high-fat-diet- induced preclinical and clinical models and
offered dietary solutions to overcome the metabolic stress in obesity.
KAISA AIRAKSINEN
METABOLIC STRESS IN OBESITY
THE IMPACT OF HIGH-FAT DIET AND FOOD INGREDIENTS ON ADIPOSE TISSUE INFLAMMATION AND SATIETY
Kaisa Airaksinen
METABOLIC STRESS IN OBESITY
THE IMPACT OF HIGH-FAT DIET AND FOOD INGREDIENTS ON ADIPOSE TISSUE INFLAMMATION AND SATIETY
To be presented by permission of the
Faculty of Health Sciences, University of Eastern Finland for public examination in Mediteknia MD100 Auditorium, Kuopio
on December 18th, 2020, at 12 o’clock noon Publications of the University of Eastern Finland
Dissertations in Health Sciences No 599
University of Eastern Finland Kuopio
2020
Series Editors
Professor Tomi Laitinen, M.D., Ph.D.
Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences
Associate professor (Tenure Track) Tarja Kvist, Ph.D.
Department of Nursing Science Faculty of Health Sciences Professor Ville Leinonen, M.D., Ph.D.
Department of Neurosurgery Faculty of Health Sciences Professor Tarja Malm, Ph.D.
A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences
Lecturer Veli-Pekka Ranta, Ph.D.
School of Pharmacy Faculty of Health Sciences
Distributor:
University of Eastern Finland Kuopio Campus Library
P.O.Box 1627 FI-70211 Kuopio, Finland
www.uef.fi/kirjasto
Grano Oy Kuopio, 2020 ISBN: 978-952-61-3636-3 (print)
ISBN: 978-952-61-3637-0 (PDF) ISSNL: 1798-5706
ISSN: 1798-5706 ISSN: 1798-5714 (PDF)
Author’s Address: DuPont Nutrition & Biosciences Global Health and Nutrition Science KANTVIK
FINLAND
Supervisors: Docent Kirsti Tiihonen, Ph.D.
DuPont Nutrition & Biosciences Global Health and Nutrition Science KANTVIK
FINLAND
Professor Marjukka Kolehmainen, Ph.D.
Department of Clinical Nutrition Faculty of Health Sciences University of Eastern Finland KUOPIO
FINLAND
Reviewers: Professor Baukje de Roos, Ph.D.
Rowett Institute University of Aberdeen ABERDEEN
UK
Docent Anne-Maria Pajari, Ph.D.
Department of Food and Nutrition Nutrition Science
University of Helsinki HELSINKI
FINLAND
Opponent: Associate Professor Eriika Savontaus, M.D., Ph.D.
Institute of Biomedicine
Integrative Physiology and Pharmacology University of Turku
TURKU FINLAND
Airaksinen, Kaisa
Metabolic stress in obesity – The impact of high-fat diet and food ingredients on adipose tissue inflammation and satiety
Kuopio: University of Eastern Finland
Publications of the University of Eastern Finland Dissertations in Health Sciences 599. 2020, 130 p.
ISBN: 978-952-61-3636-3 (print) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3637-0 (PDF) ISSN: 1798-5714 (PDF)
ABSTRACT
A high-fat diet and the excess consumption of energy-dense foods are commonly regarded as unhealthy and will often lead to overweight and obesity. Low-grade adipose tissue inflammation and perturbations in appetite regulation are typically associated with the obese state. In order to study weight management and health-promoting food ingredients, there is a need to develop and apply relevant preclinical in vitro and in vivo models, as well as well-designed clinical studies with suitable study populations.
This PhD thesis aimed to study the effects of supplementary food ingredients—betaine, polydextrose and lactitol—on metabolic stress-related parameters in high-fat diet-induced models. Several methods were applied, such as in vitro human adipocyte model mimicking obese conditions, adipose tissue gene expression analysis and nontargeted adipose tissue metabolomics. These methods were utilized to study the metabolic changes following the consumption of selected food ingredients together with high-fat diets in preclinical and clinical settings.
This study revealed that the impact of high-fat diet and specific food ingredients was noticeable at the metabolic and genetic level. Tissue hypoxia—imitating the obese state within the visceral white adipose tissue—caused noticeable inflammation in human adipocytes cultivated under low-oxygen condition.
This was determined by real-time gene expression analyses and the concomitant secretion of selected adipokines into the cell culture medium. A high-fat diet significantly increased white adipose tissue inflammation also in mice, as measured by quantitative PCR and represented by IL-6 and leptin gene expression. Nontargeted metabolomics showed a significant decrease in the levels of certain metabolites, such as betaine derivatives and several carnitine species, due to the high-fat feeding. Then, a study with a rat model revealed that the high-fat diet had detrimental effects to glucose metabolism and body weight.
Furthermore, this study showed that the food ingredients, including betaine, polydextrose and lactitol, may assist in reversing the observed obesity-related metabolic changes. Betaine, which is a naturally occurring organic osmolyte, alleviated the inflammatory state in hypoxia-stressed human adipocytes in vitro. This was demonstrated with significantly decreased mRNA expression of inflammatory markers IL-6, leptin and TNF-α after betaine treatment. In a mouse study, betaine supplementation correlated with lower IL-6 expression in white adipose tissue depots and increased the carnitine production within the fat tissue. The supplementation of two indigestible carbohydrates (polydextrose and lactitol) led to a release of modified satiety hormone levels in humans and animals. Polydextrose supplementation significantly increased the plasma glucagon-like peptide 1 (GLP-1) levels in non-diabetic, obese participants when offered in conjunction with a high-fat meal. Lactitol increased the release of another gastrointestinal satiety hormone, peptide tyrosine tyrosine (PYY), in rats fed the high-fat diet. Both polydextrose and lactitol controlled weight gain and improved the metabolic profile in a rat model, especially through attenuated postprandial plasma triglycerides and insulin effect. In addition, polydextrose showed significant reduction in postprandial hunger sensation by 40% in an acute, postprandial, randomized, double-blind, and placebo-controlled clinical study.
In conclusion, this study suggested that specific food ingredients may provide additional means to regulate high-fat diet-induced changes in metabolism, attenuate post-prandial triglyceridemia and control weight-related outcomes. This thesis also showed that betaine had a potential to alleviate obesity-
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related adipose tissue inflammation and to improve the metabolic flexibility during high-fat feeding. In addition, polydextrose and lactitol influenced on the sensation of satiety and the overall postprandial health. This study addressed the metabolic consequences of high-fat eating in various models and offered dietary solutions to overcome the metabolic stress in obesity.
Key words: adipose tissue; gene expression analysis; lactitol; metabolic stress; polydextrose; postprandial; satiety
Medical Subject Headings: Adipocytes; Adipocytes, White; Adipose Tissue, White; Animal Experimentation;
Betaine; Diet, High-Fat; Food Ingredients; Inflammation; Metabolomics; Obesity; Appetite Regulation; Gene Expression; Postprandial Period; Satiation; Satiety Response
National Library of Medicine Classification: QS 532, QU 120, QZ 150, W 20.55.A5
Airaksinen, Kaisa
Metabolinen stressi lihavuudessa – Runsasrasvaisen ruokavalion ja ravintoaineiden vaikutus rasvakudoksen tulehdukseen ja kylläisyyteen
Kuopio: Itä-Suomen yliopisto
Publications of the University of Eastern Finland Dissertations in Health Sciences 599. 2020, 130 s.
ISBN: 978-952-61-3636-3 (nid.) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3637-0 (PDF) ISSN: 1798-5714 (PDF)
TIIVISTELMÄ
Runsasrasvainen ruokavalio nähdään yleisesti epäterveellisenä ja energiapitoisen ruuan ylenmääräisen kulutuksen tiedetään johtavan ylipainoon ja lihavuuteen. Rasvakudoksen matala-asteinen tulehdus ja häiriöt kylläisyyden säätelyssä ovat tyypillisiä piirteitä lihavuudessa. Ravitsemustutkimuksessa onkin tarve sopiville solu- ja eläinmalleille sekä hyvin suunnitelluille kliinisille tutkimuksille, joiden avulla voidaan selvittää energiapitoisten ruokavalioiden vaikutuksia aineenvaihduntaan ja jotka soveltuvat painonhallintaan suunnattujen elintarvikekomponenttien kehittämiseen.
Tämän väitöskirjan tavoitteena oli tutkia tiettyjen ravintoaineiden, kuten betaiinin, polydekstroosin ja laktitolin, käytön vaikutuksia ihmisten ja eläinten aineenvaihduntaan osana runsasrasvaista ruokavaliota. Tutkimuksessa keskityttiin etenkin lihavuuteen liittyvään metaboliseen stressiin ja rasvakudoksen tulehdustilaan. Tutkimuksessa hyödynnettiin monipuolisesti useita nykyaikaisia menetelmiä, esimerkiksi kudosten geenien ilmenemistä (geeniekspressioanalyysi) ja aineenvaihduntatuotteiden profilointia (metabolomiikka). Näitä menetelmiä käytettiin tutkittaessa aineenvaihdunnan muutoksia solu- ja eläinmalleissa sekä kliinisessä tutkimuksessa.
Tämä tutkimus osoitti, että runsasrasvainen ruokavalio ja tutkitut ravintoaineet vaikuttivat havaittavasti geenien ilmenemiseen ja aineenvaihduntaan sekä solu- että eläinmalleissa. Vähähappisia kasvatusolosuhteita käytettiin soluviljelyssä mallintamaan sisäelinten ympärille sijoittuvan rasvakudoksen hypoksiaa, jota tavataan lihavuudessa. Hypoksia aiheutti rasvasoluissa selkeästi havaittavan tulehdusreaktion, jonka tiedetään liittyvän oleellisesti lihavuuteen. Rasvakudoksen tulehdusreaktio osoitettiin reaaliaikaisella geeniekspressioanalyysillä (qPCR) sekä proteiininmääritys- menetelmällä. Rasvapitoinen ruokavalio puolestaan lisäsi hiirten rasvakudoksen tulehdustilaa. Tämä ilmeni qPCR:llä mitattujen tulehdusmarkkerien ekspressiotason nousuna. Hiirille syötetty rasvapitoinen ruokavalio vaikutti tiettyjen aineenvaihduntatuotteiden esiintyvyyteen rasvakudoksessa. Etenkin betaiinijohdannaiset sekä useat karnitiinien aineenvaihduntaan liittyvät yhdisteet vähenivät merkittävästi rasvaisen ruokavalion seurauksena. Toisessa eläinmallissa osoitettiin vastaavanlaisen rasvapitoisen ruokavalion vaikuttavan selvästi rottien painon lisääntymiseen ja glukoositason muutoksiin.
Tämä tutkimus osoitti myös, että betaiinin, polydekstroosin ja laktitolin käyttö voi lieventää lihavuuteen liittyviä aineenvaihdunnan muutoksia. Betaiini, joka tunnetaan luonnossa esiintyvänä osmolyyttinä, lievitti tulehduksellista tilaa ihmisen rasvasoluissa. Geeniekspressioanalyysi osoitti betaiinin laskevan useiden tulehdusmarkkerien tasoja rasvasolumallissa. Samansuuntainen vaikutus nähtiin myös hiiren valkoisessa rasvakudoksessa, jossa betaiini lisäsi myös karnitiinien tuotantoa.
Polydekstroosin ja laktitolin annostelu ihmisille ja eläimille muutti veren kylläisyyshormonien pitoisuuksia. Polydekstroosi ja laktitoli ovat elimistössä sulamattomia hiilihydraatteja, ja polydekstroosi luokitellaan myös liukoiseksi ravintokuiduksi. Polydekstroosi nosti plasman kylläisyyshormoni GLP-1:n tasoa lihavilla ei-diabeetikoilla, kun sitä annosteltiin yhdessä rasvaisen ruuan kanssa. Laktitoli puolestaan lisäsi kylläisyyshormoni PYY:n eritystä vereen rasvaisella ruokavaliolla ruokituissa rotissa. Sekä polydekstroosi että laktitoli hillitsivät painonnousua ja alensivat aterianjälkeistä veren triglyseriditason nousua rasvaisella ruokavaliolla rikastetussa rottamallissa. Väitöskirjan kliinisessä tutkimuksessa
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havaittiin, että polydekstroosi vähensi merkittävästi ruokailun jälkeistä nälän tunnetta, mikä on tyypillistä liukoisille kuiduille.
Tämä tutkimus osoitti, että tietyt ruokavalioon lisätyt ravintoaineet voivat tarjota apukeinoja rasvaisesta ruokavaliosta johtuvien aineenvaihdunnan muutosten ja suurentuneen veren triglyseridiarvon säätelyyn sekä painonhallintaan. Betaiini voi lievittää lihavuudesta johtuvaa rasvakudoksen tulehdustilaa ja täten auttaa kehon aineenvaihduntaa sopeutumaan muuttuviin oloihin.
Sulamattomat hiilihydraatit, polydekstroosi ja laktitoli, voivat vaikuttaa myönteisesti kylläisyyden tunteeseen ja veren triglyserideihin. Tämä tutkimus käsitteli rasvaisen ruokavalion aineenvaihdunnallisia seurauksia monipuolisesti useiden erilaisten mallien avulla ja tarjosi myös ratkaisuja metabolisen stressin lievittämiseen ruokavalioon lisättyjen ravintoaineiden avulla.
Avainsanat: geeniekspressioanalyysi; runsas-rasvainen ruokavalio; laktitoli; matala-asteinen tulehdus;
metabolinen stressi; polydekstroosi; rasvakudos
Yleinen suomalainen ontologia: aineenvaihdunta; aineenvaihduntatuotteet; betaiini; geeniekspressio; kylläisyys;
metabolinen oireyhtymä; rasvakudokset; ravintokuitu; ravintoaineet; painonhallinta; eläinkokeet Luokitus: QS 532, QU 120, QZ 150, W 20.55.A5
ACKNOWLEDGEMENTS
This PhD study was funded by DuPont Nutrition and Biosciences.
Firstly, I want to acknowledge all the co-authors of the articles included to my thesis: Docent Sampo Lahtinen (DuPont), Dr. Nina Rautonen (DuPont), Docent Kirsti Tiihonen (DuPont), Dr. Jenna Jokkala (University of Eastern Finland, UEF), Dr. Ilmari Ahonen (Vincit), Professor Seppo Auriola (UEF), Professor Marjukka Kolehmainen (UEF, VTT Technical Research Centre of Finland), Professor Kati Hanhineva (UEF), M.Sc. Markku Saarinen (DuPont), Docent Sofia Forssten (DuPont), M.Sc. Mari Madetoja (Made Consulting), Professor Karl-Heinz Herzig (University of Oulu), M.Sc. Krista Salli (DuPont), Docent Esa Alhoniemi (Vincit), Dr. Alvin Ibarra (DuPont) and Professor Tommi Vasankari (UKK Institute for Health Promotion Research). I also want to thank several persons for contributing to the work done for this thesis: Lauri Naski (DuPont) and Henri Ahokoski (DuPont) are thanked for their skillful support in the cell culture and ELISA analyses in Study I, respectively. Dr. Heli Anglenius (DuPont), John Cowasji and Docent Sofia Forssten (DuPont) are thanked for their help in image processing, proofreading and reference managing in Study I, respectively. Professor Hannu Mykkänen (UEF) and Professor Kaisa Poutanen (UEF) are thanked for participating in the design for Study II with Docent Sampo Lahtinen (DuPont). M.Sc. Anne Huotari (UEF) and Minna Eskola (DuPont) are thanked for their valuable assistance during the animal trial in Study II. M.Sc. Krista Salli (DuPont) is thanked for her kind contribution in the gene expression analysis in Study II, as is Miia Reponen (UEF) for performing the LC-QTOF-MS analysis at the LC-MS Metabolomics Center (Biocenter Kuopio). In addition, I want to thank Janne Kaskinoro (Toxis Ltd.) for performing the animal experiments and Docent Sampo Lahtinen (DuPont), Jaana Larsson-Leskelä (DuPont) and Brita Mäki (DuPont) for their help in microbial analyses during Study III. Lauri Naski (DuPont) and David Bishop (DuPont) are thanked for their skillful assistance in image processing and proofreading for Studies III and IV, respectively. M.Sc. Tarja Niskanen, M.Sc. Niina Tapola, Dr. Henna Karvonen and Dr. Essi Sarkkinen (Foodfiles CRO) are thanked for their contribution in the human clinical trial (Study IV) and for writing the clinical study report.
Secondly, I want to express my gratitude towards my supervisors, Docent Kirsti Tiihonen and Professor Marjukka Kolehmainen, for always supporting me and pushing me towards the end-goal. I value your comments and constructive feedback and sincerely feel that you have helped me to do my best with this thesis. I have learned a great deal from you and appreciate your patience and professionalism during all these years. I would also like to extend my gratitude to my thesis reviewers, Docent Anne-Maria Pajari (University of Helsinki) and Professor Baukje de Roos (University of Aberdeen), for your valuable comments and extensive reviews.
In addition, I want to recognize all my DuPont colleagues, past and present, for the support they have given me during this long journey. My special thanks go to M.Sc. Krista Salli, Dr. Liisa Lehtoranta and Dr. Lotta Stenman, who encouraged me to continue even when the journey seemed to never end, to M.Sc.
Nicolas Yeung, who helped me with the layout of this thesis and to Dr. Johanna Maukonen, Dr. Arja Laitila and Dr. Arthur Ouwehand for reviewing my thesis. Additional thanks go to Dr. Jenna Jokkala (UEF) for support and great team work while conducting the experiments in Kuopio and to Dr. Sean T.
Kim (Blue Pencil Science) for assistance in proofreading.
Finally, I want to thank my family, in-laws and dear friends for always believing in me and encouraging me to complete my doctoral studies. Mum, dad, grandpa and Susanna, you mean the world to me and I express my deepest gratitude for your endless love and support. My lovely friends, Anna- Maija, Henna, Siru, Tiina and Sanna – I am so grateful that you are in my life! Jyrki, my love, I could not have done this without your support. And Kaarina, my beloved daughter, thank you for bringing me great joy every day and for teaching me what really matters in life.
Espoo, October, 2020 Kaisa Airaksinen
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LIST OF ORIGINAL PUBLICATIONS
This dissertation is based on the following original publications:
I Olli K, Lahtinen S, Rautonen N, and Tiihonen K. Betaine reduces the expression of inflammatory adipokines caused by hypoxia in human adipocytes. British Journal of Nutrition, 109(1): 43-49, 2013.
II Airaksinen K, Jokkala J, Ahonen I, Auriola S, Kolehmainen M, Hanhineva K, and Tiihonen K. High- fat diet, betaine, and polydextrose induce changes in adipose tissue inflammation and metabolism in C57BL/6J mice. Molecular Nutrition & Food Research, 62(23): 1800455C, 2018.
III Olli K, Saarinen MT, Forssten SD, Madetoja M, Herzig K-H, and Tiihonen K. Independent and combined effects of lactitol, polydextrose, and Bacteroides thetaiotaomicron on postprandial metabolism and body weight in rats fed a high-fat diet. Frontiers in Nutrition, 3(15), 2016.
IV Olli K, Salli K, Alhoniemi E, Saarinen M, Ibarra A, Vasankari T, Rautonen N, and Tiihonen K.
Postprandial effects of polydextrose on satiety hormone responses and subjective feelings of appetite in obese participants. Nutrition Journal, 14(2): 2015.
The publications were adapted and reprinted with the kind permission of the copyright or license owners and original publishers: Cambridge Journals (Study I), John Wiley and Sons (Study II), Frontiers Media SA (Study III) and BioMed Central Ltd. (Study IV).
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CONTENTS
ABSTRACT ... 7
TIIVISTELMÄ ... 9
ACKNOWLEDGEMENTS ... 11
1 INTRODUCTION ... 21
2 REVIEW OF THE LITERATURE ... 23
2.1 Overview on obesity ... 23
2.1.1 Adipose tissue metabolism and inflammation in obesity ... 24
2.1.2 Obesity-related changes in the gut microbiota ... 26
2.2 Metabolic stress in obesity ... 28
2.2.1 Dietary modifications affecting metabolic stress in adipose tissue ... 28
2.2.2 Betaine and metabolic stress in different tissues ... 29
2.3 Hormonal control of satiety ... 31
2.3.1 Introduction to satiation and satiety ... 31
2.3.2 Satiety hormones ... 31
2.4 Dietary ingredients and satiety ... 33
2.4.1 The general effect of dietary fibers on health and satiety ... 33
2.4.2 Introducing polydextrose and lactitol – two indigestible carbohydrates ... 34
2.5 Summary of the literature review ... 35
3 AIMS OF THE STUDY ... 37
4 GENERAL EXPERIMENTAL PROCEDURES ... 39
4.1 Preclinical experiments ... 39
4.1.1 In vitro model ... 39
4.1.2 In vivo models ... 39
4.2 Clinical postprandial study ... 40
5 BETAINE REDUCES THE EXPRESSION OF INFLAMMATORY ADIPOKINES CAUSED BY HYPOXIA IN HUMAN ADIPOCYTES ... 41
5.1 Abstract ... 41
5.2 Introduction ... 41
5.3 Experimental methods ... 42
5.3.1 Cell culture ... 42
5.3.2 RNA extraction, cDNA synthesis and real-time PCR ... 42
5.3.3 Measurement of adipokines by ELISA ... 43
5.3.4 Data analysis and statistics... 43
5.4 Results ... 43
5.4.1 Effects of hypoxia on adipokine mRNA expression ... 43
5.4.2 Effects of betaine on adipocyte inflammation ... 44
5.4.3 Effects of hypoxia and betaine on adipokine secretion ... 45
5.5 Discussion ... 47
6 HIGH-FAT DIET, BETAINE, AND POLYDEXTROSE INDUCE CHANGES IN ADIPOSE TISSUE INFLAMMATION AND METABOLISM IN C57BL/6J MICE ... 49
6.1 Abstract ... 49
6.2 Introduction ... 49
6.3 Materials and methods ... 50
6.3.1 Experimental animals, diets, and tissue collection ... 50
6.3.2 Biochemical analyses ... 51
6.3.3 Gene expression analysis by qPCR ... 51
6.3.4 Sample preparation for nontargeted metabolomics ... 51
6.3.5 Liquid chromatography high-resolution mass spectrometry ... 51
6.3.6 Data collection and data preprocessing ... 52
6.3.7 Principal component analyses ... 52
6.3.8 Statistical analysis ... 52
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6.3.9 Identification of metabolites ... 53
6.4 Results ... 53
6.4.1 Biochemical analysis of plasma ... 53
6.4.2 Adipose tissue gene expression ... 53
6.4.3 Comparison of diet groups by PCA ... 54
6.4.4 The effect of HF diet on the identified metabolites ... 57
6.4.5 The effect of betaine and polydextrose on the identified metabolites ... 57
6.5 Discussion ... 66
7 INDEPENDENT AND COMBINED EFFECTS OF LACTITOL, POLYDEXTROSE, AND BACTEROIDES THETAIOTAOMICRON ON POSTPRANDIAL METABOLISM AND BODY WEIGHT IN RATS FED A HIGH-FAT DIET ... 69
7.1 Abstract ... 69
7.2 Introduction ... 69
7.3 Material and Methods ... 70
7.3.1 Animals and experimental design ... 70
7.3.2 Short-chain fatty acids... 72
7.3.3 Dry matter and heat value of feces ... 72
7.3.4 Quantification of Bacteroides spp. ... 72
7.3.5 Blood analyses ... 72
7.3.6 Statistical analysis ... 73
7.4 Results ... 73
7.4.1 Body weight gain and feed intake ... 73
7.4.2 SCFAs in cecum ... 75
7.4.3 Dry matter and heat value in feces ... 76
7.4.4 Bacteroides in cecal digesta ... 76
7.4.5 Blood glucose and triglyceride response ... 77
7.4.6 Plasma insulin response ... 79
7.4.7 PYY release ... 80
7.5 Discussion ... 81
8 POSTPRANDIAL EFFECTS OF POLYDEXTROSE ON SATIETY HORMONE RESPONSES AND SUBJECTIVE FEELINGS OF APPETITE IN OBESE PARTICIPANTS ... 85
8.1 Abstract ... 85
8.2 Background ... 85
8.3 Methods ... 87
8.3.1 Participants ... 87
8.3.2 Study design ... 87
8.3.3 Composition of the study meal ... 88
8.3.4 Blood sampling and analysis ... 88
8.3.5 Appetite ratings ... 89
8.3.6 Statistical analyses ... 89
8.4 Results ... 90
8.4.1 Characteristics of the study participants ... 90
8.4.2 Gastrointestinal peptides... 91
8.4.3 Plasma SCFAs and lactic acid ... 92
8.4.4 Appetite ratings ... 93
8.5 Discussion ... 95
8.6 Conclusion ... 96
9 SYNOPSIS OF THE RESULTS ... 97
9.1 Results to research question 1 ... 97
9.2 Results to research question 2 ... 97
9.3 Results to research question 3 ... 98
9.4 Results to research question 4 ... 99
10GENERAL DISCUSSION ... 101
10.1 Overview of the thesis and principal findings ... 101
10.2 Methodological considerations ... 102
10.3 Limitations and practical implications of this work and recommendations for further research .. 105
10.4 Conclusions ... 106
REFERENCES ... 109 APPENDICES ... 125
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ABBREVIATIONS
AUC Area under the curve ADIPOQ Adiponectin;
ANOVA Analysis of variance BCFA Branched-chain fatty acid
BET Betaine
BHMT Betaine–homocysteine S- methyltransferase
BIA Bioelectrical impedance analysis
BMI Body mass index
BWG Body weight gain
CCK Cholecystokinin CVD Cardiovascular disease DEXA/DXA Dual-energy X-ray
absorptiometry DIO Diet-induced obese ER Endoplasmic reticulum
FD Fold difference
FDR False discovery rate
FIAF Fasting-induced adipose factor GAPDH Glyceraldehyde-3-phosphate
dehydrogenase GI Gastrointestinal GLP-1 Glucagon-like peptide 1 HDL High density lipoprotein
HF High-fat diet
HIF-1α Hypoxia-inducible factor-1, α subunit
HILIC Hydrophilic interaction liquid chromatography
iAUC Incremental area under the curve
IL Interleukin
IL-6 Interleukin-6
ISAPP International Scientific Association for Probiotics and Prebiotics
LC-MS Liquid chromatography (LC) coupled to mass spectrometry LC-MS/MS LC-MS method with tandem
mass spectrometry LC-QTOF-MS Liquid chromatography
quadrupole time-of-flight mass spectrometry
LDL Low density lipoprotein
LF Low-fat diet
LPL Lipoprotein lipase LPS Lipopolysaccharide MCP-1 Monocyte chemoattractant
protein 1
MetS Metabolic syndrome MIF Macrophage migration
inhibitory factor
mRNA Messenger RNA
NAFLD Non-alcoholic fatty liver disease PAI-1 Plasminogen activator inhibitor-
1
PC Phosphatidylcholine
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PC1-5 Principal components 1-5 PCA Principal component analysis PDX Polydextrose
PE Phosphatidylethanolamine PG Glycerophosphoglycerol PI Phosphatidylinositole PPAR-γ Peroxisome proliferator-
activated receptor gamma PYY Peptide tyrosine tyrosine qPCR Quantitative polymerase chain
reaction
RP Reverse Phase (RP) chromatography
SAT Subcutaneous adipose tissue S-AM S-adenosylmethionine
SCFA Short chain fatty acid SD Standard deviation SEM Standard error of the mean SFA Saturated fatty acid
SM Sphingomyelin
TGF-β Transforming growth factor beta
TNF-α Tumor necrosis factor alpha T2D Type 2 diabetes mellitus UV Ultraviolet
VAS Visual analogue scale VAT Visceral adipose tissue WAT White adipose tissue WHO World Health Organization
1 INTRODUCTION
Obesity-related metabolic disturbances are a globally increasing health problem. Simultaneously, the interest for the low-glycemic and satiety-increasing food components is growing. The health problems related to the physical weight are traditionally avoided through body weight management and reduction of absolute fat mass, which can be achieved through balanced, nutritious diet and necessary physical exercise. However, these measures are not always enough, and there can be differences in individuals’
response to dietary modifications aiming to weight loss. Mechanistically, the obesity-related disturbances can be avoided by affecting the metabolism. For instance, ensuring attenuated postprandial lipid, glucose and insulin responses can decrease the risk of metabolic diseases and have a role in managing obesity- related incidents. The postprandial responses can be studied in both animal and human trials, and feeding trials allow us also to test the effects of additional food ingredients together with distinct diets.
White adipose tissue is a cofounder in the development of obesity, and the location and quantity of excess fat mass are key factors in defying the severity of the obese state. The distribution and morphology of adipose tissue is an important factor in the pathogenesis of obesity-related disturbances. Central or abdominal adiposity is considered more detrimental to the health status in obesity. The enlarged adipose tissue is metabolically active and individuals with hypertrophy (few large adipocytes) typically have a more adverse metabolic profile and body shape than those with hyperplasia, i.e., many small adipocytes (Arner et al., 2010). Adipocyte hypertrophy may weaken the adipose tissue functions by inducing local inflammation, mechanical stress and altered metabolism. In fact, the obese state is well characterized by a chronic, low-grade inflammation that can be concomitantly linked with increased prevalence of other obesity-related comorbidities, such as type 2 diabetes and cardiovascular diseases (Trayhurn et al., 2008, Holmer-Jensen et al., 2011). The enlarged adipose tissue is known to suffer from tissue hypoxia, and the effect of hypoxia on adipocyte gene and protein expression has been studied previously (Trayhurn et al., 2008). In this thesis, the potentially beneficial metabolic effect of betaine—a naturally occurring organic osmolyte—was examined in human adipocytes under hypoxia. White adipose tissue produces multiple adipokines and other cytokines that participate in the regulation of glucose, lipid and energy metabolism.
By altering its metabolism, adipose tissue can react readily and beneficially to different modifications in lifestyle, e.g., dietary changes. Therefore, the incidence of metabolic disorders could be affected not only by decreasing or limiting the amount of adipose tissue within the body but also by affecting the metabolism and the expression of adipokines within the tissue.
Clinical findings propose that the composition of a diet, especially low dietary fiber intake, can significantly contribute to the development of obesity (Davis et al., 2006). Specific food ingredients, such as fibers, may contain ability to regulate postprandial metabolism and thus potentially aid in weight management and alleviation of obesity-related stress. The kinds of dietary ingredients are already widely used in the food industry to reduce sugar and calorie load of food products, but their additional health effects need to be carefully validated. Several studies have recently investigated the satiety-promoting effect of polydextrose, a dietary fiber supplement, in different human populations (Ibarra et al., 2016, Ibarra et al., 2017). However, relevant postprandial studies to demonstrate that polydextrose can enhance satiety in obese population were lacking before this study. Eventually, the food industry has been interested in developing such food ingredients that can modulate the gut microbiota, since the biological relevance of the connection between gut microbiota and metabolic health has been widely acknowledged in the past decade (Davis, 2016).
This thesis aimed to investigate the impact of specific supplementary food ingredients on the metabolic stress-related parameters in vitro, in vivo and in human clinical study when challenged with the high-fat diet. The interactions between gene expression, protein release, metabolism and other attributes were studied with relevant models. It was studied in human adipocyte culture and in diet-induced obese mouse model whether the obesity-related adipose tissue inflammation can be positively affected by betaine supplementation and how the dietary modifications can alter the tissue metabolism. The effect of two indigestible carbohydrates—polydextrose and lactitol—on the satiety hormone responses and subjective feelings of appetite was further studied in animal models and in a postprandial clinical study
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with obese volunteers. Nutrigenomic and metabolomics provide useful tools for interpreting the health effects of food ingredients or modulated diets on the whole-body level. Nutrigenomics is defined as the study of the effects of food and food constituents on gene expression. Metabolomics is the quantitative measurement of metabolic responses of living systems to physiological stimuli. Together, these relevant methods help to provide a wider picture of the studied effects and can offer more accurate and detailed quantification. With the help of different omics, the nutrition-based effects to different physiological functions can be better understood and new biomarkers may even be found.
2 REVIEW OF THE LITERATURE
2.1 OVERVIEW ON OBESITY
Obesity is nowadays a global epidemic, and 39% of the world’s adult population was overweight and 13% was obese in 2016 (World Health Organization). The rise in the frequency of obesity over the last decades has led to an increase in obesity-related comorbidities, such as insulin resistance, hypertension, i.e., high blood pressure, and dyslipidemia. The complex cluster of these disorders is commonly called metabolic syndrome (MetS). Not only are obesity and overweightness associated with reduced life expectancy and limited quality of life, but also, they are associated with concomitantly increased blood lipid and glucose concentrations, which are real risk factors for the development of type 2 diabetes mellitus (T2D) and cardiovascular disease (CVD). Obese state is well characterized by a chronic, low- grade inflammation that is linked with increased prevalence of T2D, MetS, hypertension and CVD (Trayhurn et al., 2008, Holmer-Jensen et al., 2011). Increased concentrations of various inflammatory cytokines have been linked with obesity and insulin resistance (Dandona et al., 2004), which will further define the observed inflamed state within the obese. Additionally, an association between weight, body mass index (BMI), adiposity and the blood inflammatory status has also been demonstrated (Ellulu et al., 2017).
Overweight and obesity are defined as abnormal or excessive fat accumulation that presents a risk to the health of an individual or entire population. The occurrence of overweight or obesity can be defined by measuring the body fat and waist circumference and calculating the BMI (calculated by dividing weight in kilograms by height in square meters) (World Health Organization, 2000). However, the direct, large-scale measurement of the amount of body fat is often not part of everyday health care practices.
This is why BMI and waist circumference have become the main tools in estimating the amount of body fat, and they are used for nutritional evaluation and monitoring purposes. Nevertheless, the precise amount of fat can be measured with whole-body composition assessment tools, such as dual-energy X- ray absorptiometry (DEXA, or DXA) or bioelectrical impedance analysis (BIA) method. A large retrospective study has suggested that BIA and DXA methods are interchangeable at a population level (Achamrah et al., 2018). However, it seemed that the body composition values measured with BIA may overestimate fat-free mass and underestimate fat mass in individuals with BMI over 18,5 kg/m2 and less than 40 kg/m2, compared to the DXA method (Achamrah et al., 2018). According to the World Health Organization (WHO), BMI over or equal to 25 kg/m2 indicates overweight, and BMI over or equal to 30 kg/m2 indicates obesity (World Health Organization, 2000). However, it has been shown repeatedly that a given BMI may not correspond to the same degree of fatness across different populations (Carroll et al., 2008). It has been suggested that different waist circumference or BMI cut-off points may be necessary to sufficiently estimate the metabolic risk in different ethnicgroups due to their altered relationship between anthropometric measures and the quantity of visceral fat mass (Carroll et al., 2008). Effectively, WHO, the International Obesity Task Force and the International Association for the Study of Obesity (IASO) have proposed lower cut-off points for overweight (BMI = 23.0 kg/m2) and obesity (BMI = 25.0 kg/m2) in Asian and Pacific Island Populations (WHO Expert Consultation, 2004). Asian populations have indeed a higher risk of developing comorbidities, such as CVD and T2D, already at BMIs less than 25 kg/m2, and they seem to have different associations between BMI and body fat percentage than the Europeans (WHO Expert Consultation, 2004). However, BMI alone will not distinguish the origin of weight, i.e., if it originates mainly from fat depots or other dense tissues, such as muscle (World Health Organization, 2000). Therefore, additional measurements of waist circumference and waist-to-hip ratio are used when determining obesity. Waist circumference and waist-to-hip ratio are in fact more closely associated with the risk of CVD than BMI, since they are more connected to the fat distribution. In obesity, abnormal or excessive fat accumulation occurs in adipose tissue. Hence, the distribution of fat storages is relevant when considering the impact of obesity on health. Abdominal fat is considered more harmful to the health than peripheral fat depots, and it is linked to e.g., adipocyte hypertrophy, insulin resistance, T2D, hypertension and cardiovascular diseases (Wajchenberg, 2000, Calder et al., 2011).
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Lifestyle factors, such as diet and exercise, have an impact—beneficial or harmful—on the development of obesity-related comorbidities. However, the mechanisms by which different dietary nutrients and dietary patterns may be involved in the pathogenesis of these diseases or their risk factors are not yet fully understood. Several reviews imply that obesity can affect postprandial metabolic processes (Blaak et al., 2012, Chan et al., 2013, Klop et al., 2013), and the impaired ability of insulin to regulate glucose utilization in peripheral tissues could be an important link between obesity, MetS and dyslipidemia (Klop et al., 2013). In addition, the role of inflammation, especially inside the adipose tissue, has recently been studied with great attention for its probable role in mediating the development of T2D and CVD. According to one hypothesis, postprandial dysmetabolism, characterized as repeated high and long-term increases of glucose and lipid concentrations, may result in inflammation, endothelial dysfunction and other adverse cardiovascular events, even in non-diabetic subjects (O'Keefe & Bell, 2007).
One major factor in the development of inflammation is undoubtedly unhealthy diet, but the underlying genetic features seem to also influence the development of these obesity-related morbidities (O'Keefe &
Bell, 2007). Overall, obesity is considered to be the result of complex interaction of genetic, metabolic, environmental and behavioral factors.
Overweight and obesity are a multifactorial problem with several treatment options. Typically, therapies for overweightness and obesity include diet, exercise, behavioral therapy and drug therapy, and in extreme cases, the surgical options, such as gastric bypass surgery, can be used (Avenell et al., 2004).One of the main challenges in the treatment of obesity is the long-term weight loss maintenance, which often requires a complex and disciplined approach. The general goals of weight loss and weight management are to reduce body weight and to maintain a lower body weight over long term. Through successful weight loss—or weight control—it is possible to enhance the individual’s metabolic state, which can be demonstrated by e.g.,reduction in blood pressure, improvement in lipid profiles and improved glycemic control (Pi-Sunyer et al., 2007). The overall counseling for healthy lifestyle, including a balanced, healthy diet with decreased caloric intake and increased physical activity, should help to achieve a healthy weight and well-adjusted metabolism. However, not all individuals seem to benefit similarly from weight loss alone, and high individual variability in response to certain diets has been reported (de Roos & Brennan, 2017), hinting that additional strategies are needed for those individuals.
The energy value of the diet is a main factor affecting the positive energy balance, which can eventually lead to overweight or obesity and then cause more crucial complications to the metabolism. The nutritional composition of the diet naturally plays an important role in this, and there may be individuals or populations that would benefit from additional ‘bioactive’ nutrients as part of their diet. Consequently, it would be valuable to understand which populations would benefit the most from certain nutrients or supplementary ingredients, and this would further help us to develop new tools for weight management.
However, the impact of diet and exercise alone can not explain all the disadvantages observed in the health conditions of obese and overweight, and it has been postulated that the composition of the gut microbiota has an additional role to play in the metabolism of the host (Davis, 2016).
2.1.1 Adipose tissue metabolism and inflammation in obesity
According to the current view, the state of inflammation within adipose tissue plays a causal role in the initiation and development of the diseases linked to obesity, such as insulin resistance and T2D.
Originally white adipose tissue (WAT) has been seen as a simple energy storage reservoir; however, it is nowadays known to also function as a major endocrine and secretory organ which is interacting with various metabolic pathways and physiological tracks (Burhans et al., 2018). WAT produces multiple adipokines (adipocyte-derived factors) and other cytokines that participate in the regulation of glucose, lipid and energy metabolism and even immunity (Ahima, 2006). Many of the adipokines are linked to inflammation, and in total some 60 biologically active secretory factors have been recognized (Kershaw
& Flier, 2004, Trayhurn & Wood, 2004, Trayhurn et al., 2008). Positive energy balance causes WAT mass to expand due to increased storage of lipids and triglycerides and eventually leading to obesity. The expanding adipose tissue mass causes a major inflammatory response, and changes in the levels of adipokines released from the inflamed adipose tissue can cause insulin resistance and endothelial dysfunction (Trayhurn et al., 2008, Calder et al., 2011). Inflammatory response can be described as a
complex, self-limiting process that is coordinated by a timely release of mediators and requires expression of specific receptors, cytokines and chemokines. The main purpose of the inflammatory reaction is to maintain tissue and organ homeostasis and to function as a protective mechanism; however, it can be altered into a detrimental, chronic state of inflammation if its regulation is distorted. The long-lasting chronic inflammation is characterized by the presence of lymphocytes and macrophages and the proliferation of blood vessels and connective tissue (Ellulu et al., 2017).
Adipose tissue produces and secretes many pro-inflammatory and pro-thrombotic factors and also insulin-sensitive factors, such as adiponectin (Arner, 2005). At the state of obesity, especially abdominal obesity, adipose tissue affects actively in the development of insulin resistance and inflammation and, eventually, in the development of T2D (Arner, 2005). A short summary of changes observed in adipokine secretion from WAT in obesity is presented as Supplementary Table S1 (Appendix 1). The expression and release of inflammation-related adipokines, such as leptin, pro-inflammatory interleukins (IL-1β, IL-6, and IL-8) and tumor necrosis factor alpha (TNF-α), increase in obesity, while the level of adiponectin is decreased (Trayhurn & Wood, 2005). Increased serum IL-6 concentrations are reported to be associated with visceral adiposity, whereas increased concentration of circulating TNF-α has been shown to associate with the total amount of body fat (Dandona et al., 2004). A study by Tiihonen and co-workers has indicated a correlation between serum IL-6 concentration and the waist circumference in obese participants (Tiihonen et al., 2010). Besides being connected to high amount of fat, the increased levels of pro-inflammatory cytokines TNF-α and IL-6 are known to induce insulin resistance (Thomas et al., 2015).
Indeed, the increased expression of IL-6 observed in adipose tissue in obesity is positively correlated with impaired glucose tolerance and insulin resistance (Fernandez-Real & Ricart, 2003). This increased production of chemokines at the obese state is considered as a probable link between inflammation and insulin resistance (Hotamisligil, 2006). Also, by producing chemokines, e.g., monocyte chemoattractant protein-1 (MCP-1), the migration of macrophages to the adipose tissue is strongly reinforced (Sartipy &
Loskutoff, 2003).
There are also several other factors, such as fasting-induced adipose factor (FIAF) and circulating free fatty acids, that are involved in the maintenance of tissue homeostasis and linking MetS and adipose tissue dysfunction (Peredo-Escárcega et al., 2015). FIAF is a circulating lipoprotein lipase (LPL) inhibitor, which can modulate lipase activity in adipose tissue and thus cause an elevation of serum triglyceride levels (Kersten et al., 2000, Yoon et al., 2000, Sukonina et al., 2006). Free fatty acids, together with their by- products, are known to cause further metabolic reactions linked to MetS, such as adipogenesis and adipokine secretion (El Akoum et al., 2011). During obesity, the production and circulating levels of plasminogen activator inhibitor-1 (PAI-1)—an important adipose-derived factor in the homeostasis and an important acute-phase response protein—increase significantly (Alessi et al., 2000), which links the inflamed phenotype to MetS. On the other hand, a certain essential upstream component of the inflammatory cascade, macrophage migration inhibitory factor (MIF), is suggested to function as a potential therapeutic target for reducing the inflammatory status in metabolic and cardiovascular disorders (Verschuren et al., 2009). In addition to them, adipose tissue secretes also several hormones, such as leptin, adiponectin and resistin, which regulate long-term satiety, appetite and energy expenditure (Hall et al., 2009).
In severe obesity, the expanding adipose tissue suffers from low oxygen supply, causing hypoxic conditions to develop inside the tissue (Trayhurn et al., 2008). This kind of hypoxia has been demonstrated in adipose tissue in different obese mouse models (Ye et al., 2007). Hypoxia is known to inhibit the differentiation process of adipocytes (Zhou et al., 2005), and the expression of a central inducer of adipose differentiation, peroxisome proliferator-activated receptor-γ (PPAR-γ), is known to decrease under hypoxic conditions (Wang et al., 2008). Both obesity and low O2 tension measured in adipocytes are associated with increased leptin production and high plasma leptin concentrations (Considine et al., 1996, Wang et al., 2007). Cell culture studies on human adipocytes show that hypoxic conditions—induced by low oxygen supply—lead to stimulation of the expression and secretion of several inflammation-related markers, e.g., IL-6 and leptin (Wang et al., 2007). Normally, the net action of leptin is to inhibit appetite, promote energy expenditure, stimulate thermogenesis, enhance fatty acid oxidation, decrease glucose and reduce body weight and fat (Ahima, 2006). However, the rise in endogenous leptin, or exogenous leptin treatment, is unable to prevent weight gain in obese, which may eventually result in saturation of
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the leptin transport to the central nervous system, causing a so-called leptin resistance (Münzberg &
Myers, 2005). There are several indications to the leptin resistance in the literature demonstrating elevated plasma leptin levels in obese animal and human subjects (Considine et al., 1996, Ahima & Osei, 2004, Tiihonen et al., 2010). Leptin levels seem to also correlate positively with fat mass in normal-weight and obese humans (Considine et al., 1996). Leptin has profound effects also in the response to fasting, regulation of neuroendocrine and immune systems, hematopoiesis and bone and brain development (Ahima & Osei, 2004).
Together with skeletal muscle, WAT is considered to be the main site of lactate production and release (Rooney & Trayhurn, 2011). Previous studies postulate that the production of lactate in adipose tissue seems to increase together with the occurrence of obesity (DiGirolamo et al., 1992, Rooney & Trayhurn, 2011). This suggests a switch-like function towards anaerobic metabolism when hypoxic conditions occur within the adipose tissue (Rooney & Trayhurn, 2011). Moreover, the circulating lactate concentration correlates positively with obesity and obesity-related insulin resistance (DiGirolamo et al., 1992, Lovejoy et al., 1992). Lactate functions in tissues as a signal of anaerobic metabolism, and therefore, it may induce an inflammatory response in adipose tissues (Tiihonen et al., 2010).
Adipose tissue may contain few large adipocytes (hypertrophy) or many small adipocytes (hyperplasia). Subcutaneous adipose tissue hypertrophy and hyperplasia are strongly related to the total adipocyte number in adults, but they occur independently of sex and body fat content (Arner et al., 2010).
Individuals with hypertrophy have a more adverse metabolic profile and body shape than those with hyperplasia (Arner et al., 2010). Adipocyte hypertrophy may weaken the adipose tissue functions by inducing local inflammation, mechanical stress and altered metabolism (Jernås et al., 2006, Monteiro et al., 2006, Skurk et al., 2007). Large fat cells in the visceral region are linked to dyslipidemia, whereas large subcutaneous adipocytes are important for glucose and insulin abnormalities (Hoffstedt et al., 2010, Eriksson-Hogling et al., 2015). Increased adipocyte size, i.e., hypertrophy, correlates with serum insulin concentrations and insulin resistance (Lundgren et al., 2007) and predicts the incidence of T2D (Weyer et al., 2000, Lönn et al., 2010, Eriksson-Hogling et al., 2015). There are also studies relating increased mRNA concentrations of inflammation-related genes, e.g., macrophage migration inhibitory factor (MIF), to adipocyte size and in vivo insulin action in obese individuals (Koska et al., 2009). Obese subjects with hypertrophy are more glucose-intolerant and hyperinsulinemic than those having the same degree of obesity but with hyperplasia (Arner et al., 2010). Furthermore, lipolysis is enhanced in the large adipocytes (Berger & Barnard, 1999). The increased release of free fatty acids in the visceral fat produces a great problem, since the released fatty acids can reach the liver through portal circulation strongly affecting the hepatic metabolism. An accumulation of free fatty acids in adipocytes induces also endoplasmic reticulum (ER) stress and oxidative stress at the mitochondrial level, both of which can impair the adipocyte function (de Ferranti & Mozaffarian, 2008).
2.1.2 Obesity-related changes in the gut microbiota
Diets can alter the microbial composition of the gut, which will have long-lasting effects on the overall health status of the individuals. The impact of microbiota on various health conditions and diseases is nowadays largely investigated, and there is evidence that the microbial composition of the gastrointestinal (GI) tract contributes to the altered metabolism related to obesity (Bäckhed et al., 2004, Davis, 2016). Certain changes in the microbial composition of the gut have been shown to lead towards increased adiposity, as demonstrated initially in mice (Turnbaugh et al., 2006). In humans, there are also distinct differences in the gut microbial community reported between the lean and obese phenotypes (Ley et al., 2006).
Obesity has been associated with changes in the relative abundance of the two dominant bacterial divisions, Firmicutes and Bacteroidetes, with reduced amount of Bacteroides. An increased abundance of Firmicutes over Bacteroidetes has been reported initially in obese (ob/ob) mice (Ley et al., 2005). According to further studies in mice, the Western diet rich in fat was able to provide a competitive advantage to the Firmicutes division (Turnbaugh et al., 2008). However, it has been shown in other animal studies that prebiotic supplementations could normalize the reduced Bacteroidetes count (Parnell & Reimer, 2012).
Evidence from human clinical studies has shown that fecal microbial composition differs between obese
and normal-weight subjects, and especially Akkermansia muciniphila seems to be more abundant in the lean phenotype (Dao et al., 2016). In addition, a higher relative amount of gut Bacteroidetes has been reported with lean subjects (Ley et al., 2006, Turnbaugh et al., 2006); however, there are also contradictory results published (Duncan et al., 2008). In a study by Tiihonen and co-workers, overweight participants showed less sulphate-reducing bacteria, as well as a trend towards less Bacteroides (Tiihonen et al., 2010).
Furthermore, an inverse correlation was reported between fecal Bacteroides levels and waist circumference (Tiihonen et al., 2010). Nevertheless, the effect of Bacteroidetes on satiety remains unclear. There is also evidence suggesting that the amount of beneficial bifidobacteria in the gut may differ according to the state of obesity. Decreased bifidobacteria counts have been detected in obese mice and higher counts in lean subjects (Cani et al., 2007). The study by Cani et al. (2007) indicates that increased abundance of bifidobacteria in the gut microbiota may in fact improve high-fat diet-induced diabetes in mice. Thus, the gut microbiota could affect the pathophysiological regulation of metabolic endotoxemia. This refers to an imbalanced state during which the gut microbes—or microbial fragments, such as lipopolysachharide (LPS)—can enter the bloodstream and end up in different tissues, causing exaggerated lipolysis and low- grade inflammation (Cani et al., 2007, Amar et al., 2011).
It is currently hypothesized that gut dysbiosis, i.e., microbial imbalance in the gut, can lead to a disbalanced inflammatory processes and loss of epithelial integrity. As bacterial components, like endotoxins, are leaking out of the gut. this can summon a low-grade, chronic and systemic inflammation, which is typical for the obese state. The gut microbiota composition can affect the intestinal barrier function and thus regulate the translocation of inflammatory gut microbes and their components, which can cause concomitant tissue inflammation by affecting immunomodulatory metabolic pathways (Burcelin, 2012). Increased gut permeability has been linked with obesity both in animal (Brun et al., 2007) as well as in human studies (Damms-Machado et al., 2016). As demonstrated in mice, the translocation of commensal intestinal bacteria into circulation and adipose tissue is increased during the onset of high-fat diet-induced diabetes (Amar et al., 2011). This translocation of bacteria can result into low-grade bacteremia, which is associated with components of innate immune system. However, it has been demonstrated that the consumption of a prebiotic fiber (oligofructose) may lower metabolic endotoxemia and systemic inflammatory tone and that bifidobacteria are the main gut bacteria involved in the positive effects observed after such prebiotic supplementation (Cani et al., 2007). Recently, the association between gut microbiota, inflammation and obesity has been an important research subject, especially in the pursuit of strategies against high-fat-induced metabolic disorders.
There are three recognized concepts of functional dietary supplements that are able to modify the gut microbiota composition and/or activity, and by doing so, these ingredients can affect the health of the host. These are ‘probiotics’, ‘prebiotics’ and ‘synbiotics’, and they have been studied extensively in the past decades. According to the current definition articulated by the International Scientific Association for Probiotics and Prebiotics (ISAPP), probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host (Hill et al., 2014). The interpretation of the definition of probiotics has been clarified over the years (Binda et al., 2020), and according to the current view, the adequate dose is the dose demonstrated to be efficacious in well-established clinical trials. Probiotics containing products should deliver this tested dose at the end of the shelf life. The administration of probiotics happens traditionally through ingestion, but it can also be administered, for example, via the surface of the skin. The proven health benefits should always be tested in the target host in properly controlled studies; however, the acceptance of the term ‘probiotic’ differs between different regulatory regions worldwide. The definition of prebiotics was first released in 1995 by Gibson and Roberfroid (Gibson & Roberfroid, 1995), and ever since, the potential health effects of prebiotics have been investigated with great interest. The current consensus states that a prebiotic is ‘a substrate that is selectively utilized by host microorganisms conferring a health benefit’ (Gibson et al., 2017). When Gibson and Roberfroid first introduced the prebiotic concept, they also suggested that prebiotics could be combined with probiotics to form synbiotics. In 2011, Gibson, together with Kolida, described additional criteria for synbiotics, as they proposed that synbiotics could have either complementary or synergistic activities (Kolida & Gibson, 2011). According to the most recent definition by ISAPP, a synbiotic is a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host (Swanson et al., 2020).
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The GI microbiota is able to produce short-chain fatty acids (SCFAs) from otherwise indigestible foods via fermentation processes. It has been noted that obesity and changes in the concentrations of these volatile SCFAs seem to be interconnected and that SCFAs can exert multiple beneficial effects on mammalian energy metabolism (den Besten et al., 2013). SCFAs can be transported systemically from the intestinal lumen and be taken up by different organs in order to act as substrates for cell growth or as signal molecules (den Besten et al., 2013). These processes involve a complex interplay between diet, gut microbiota and host energy metabolism. Several GI microbes are able to produce lactic acid, which can be further utilized by other intestinal microbes (Duncan et al., 2004). Normally, lactic acid is not accumulated to the feces of healthy subjects. However, fecal lactic acid concentrations have been found in obese subjects, and they were associated with increased blood inflammatory biomarkers, such as C- reactive protein (CRP) and IL-6 (Tiihonen et al., 2010). Thus, it has been suggested that the increased postprandial blood lactate level may be partly explained by the intestinal lactic acid production. In addition, elevated lactic acid concentrations generated by the intestinal bacterial fermentation may have a larger systemic effect also in other tissues, such as muscle (Lombardi et al., 1999).
It is now well known that the microbiota of the obese is different than that of the lean subjects (Ley et al., 2006, Turnbaugh et al., 2006, Turnbaugh et al., 2009). However, there are indications that the quality of diet, not obesity itself, is responsible for the changes within microbiota (Hildebrandt et al., 2009).
Especially, the high-energy containing Western diet has been shown to alter the gut microbial diversity in mouse models (Turnbaugh et al., 2008). Gut microbiota has an important role also in fat deposition (Bäckhed et al., 2004), and several microbiological studies (Turnbaugh et al., 2008, Turnbaugh et al., 2009) are supporting this view. Gut microbes in obese subjects can also be more efficient in caloric extraction (Turnbaugh et al., 2006). Therefore, via such dietary modifications, which can also alter the gut microbiota composition towards a more beneficial direction, it may be possible to help obese subjects to lose weight and maintain healthier metabolic condition. This is one reason why weight management and the pursuit of new ways to reduce caloric intake are a driving force also for the food industry. In the future, not only exercise and various pharmacological agents but also the diet and ingested food ingredients may open new means to alleviate the obesity-related adipose tissue dysfunction and postprandial dysmetabolism.
2.2 METABOLIC STRESS IN OBESITY
Stress can be explained as a state of threatened or imbalanced homeostasis. Metabolic stress occurs in both obesity and T2D, and it can be explained as a group of stress responses that are dysregulated in metabolically relevant sites. Obesity is considered as one causative factor in the development of MetS, which is known to cover several interlinked detrimental conditions involving metabolic stress. Chronic, metabolically triggered inflammation—often referred to as chronic low-grade inflammation—has been recognized as one of the potential causes to MetS, and it is considered as a risk factor for a cluster of metabolic diseases (Hotamisligil, 2006, Esser et al., 2014). The chronic low-grade inflammation, together with an activation of the immune system, is involved in the pathogenesis of obesity-related insulin resistance and T2D (Esser et al., 2014). However, not all obese individuals develop MetS, and even lean individuals can be insulin-resistant (Kotronen & Yki-Järvinen, 2008). Regardless of their BMI, insulin- resistant individuals tend to develop excess, non-alcohol related fat in the liver—a condition defined as non-alcoholic fatty liver disease (NAFLD) (Kotronen & Yki-Järvinen, 2008). Independent of the obesity class, this insulin-resistant fatty liver correlates strongly with MetS, and the prevalence of both MetS and NAFLD is known to increase with obesity (Kotronen & Yki-Järvinen, 2008, Yki-Järvinen, 2014). In addition, the obese individuals with a dysfunctional fatty liver are more likely to have inflammatory changes in adipose tissue, especially in the intra-abdominal area, where surplus of fat is accumulated (Kotronen & Yki-Järvinen, 2008).
2.2.1 Dietary modifications affecting metabolic stress in adipose tissue
The high abundance of fat in the diet can drastically alter the health and is considered the main cause of obesity-related metabolic stress. The development of metabolic diseases is closely associated with weight gain and Western diet, which contains excess of simple carbohydrates and dietary fat, especially saturated
fat. These unhealthy diets are also known to modify intestinal microbiota and induce low-grade inflammation and insulin resistance.
Adipose tissue can react readily and beneficially to different modifications in lifestyle, especially to dietary changes. Beneficial dietary changes, such as increasing the consumption of whole-grain cereals, foods with a low glycemic index and dietary resistant starch in the diet, can improve the clinical outcome and eventually associate with changes in gene expression profiles in adipose tissue (Dahlman et al., 2005, Kallio et al., 2007, Kolehmainen et al., 2008). Adipocytes, and adipose tissue in general, can sense and react to the dietary changes by altering their metabolism. The effect of dietary changes on adipose tissue function has been greatly studied on genetic level and in the light of weight reduction. Kolehmainen and co-workers showed e.g., reduced expression of genes regulating the extracellular matrix and cell death after long-term weight reduction, while another group reported downregulation of genes regulating the production of polyunsaturated fatty acids. (Dahlman et al., 2005, Kolehmainen et al., 2008).
Nordic diet—following Nordic Nutrition recommendations—is generally considered to be beneficial for health. It has been suggested as an alternative to the Mediterranean diet, which is commonly known to improve health and to support the prevention of CVD, certain cancers and T2D (Uusitupa et al., 2013).
The typical Nordic diet is composed of whole grains, rapeseed oil, vegetable oil–based margarines, low- fat or fat-free milk products and local berries, fruits, vegetables and fish of Nordic origin. The foundation of the Mediterranean diet is very similar to Nordic diet, favoring the use of vegetables, fruits, herbs, nuts, beans and whole grains with moderate amounts of dairy, poultry, eggs and seafood. Nordic diet has reportedly reduced inflammatory gene expression in subcutaneous adipose tissue (SAT), compared with an isoenergic control diet, independent of body weight change in individuals with features of MetS (Kolehmainen et al., 2015). Especially, the genes expressed in immune-related pathways were differentially expressed in individuals consuming Nordic diet and those on control diet for 18 to 24 weeks (Kolehmainen et al., 2015). Healthy Nordic diet can also have beneficial effect on lipid metabolism and reduce the signs of systemic inflammation, as reported in the same randomized dietary intervention study [Systems Biology in Controlled Dietary Interventions and Cohort Studies (SYSDIET)] (Uusitupa et al., 2013, Brader et al., 2014).
2.2.2 Betaine and metabolic stress in different tissues
Betaine (glycine betaine, trimethylglycine) is a naturally occurring organic osmolyte that can be found in living organisms, such as various plant, animal and microbial species. Important sources of betaine include marine invertebrates (≈1%), wheat germ or bran (≈1%) and spinach (≈0.7%) and sugar beets (Craig, 2004). Dietary intake of betaine is estimated to range from an average of 1 g/d to 2.5 g/d, the latter measured from individuals consuming a diet high in whole wheat and seafood (Craig, 2004). Sugar beet (Beta vulgaris) is especially known for accumulating betaine and the mechanistic extraction of betaine from sugar beet molasses can be executed by a patented chromatographic separation method (Heikkilä et al., 1982).
By the chemical structure, betaine molecule is small (117.2 Da) N-methylated amino acid with three chemically reactive methyl groups attached to the nitrogen atom of a glycine molecule. It is a stable, non- toxic, highly water-soluble (160 g/100 g) and very hygroscopic molecule. In fact, betaine has a tendency to attach to several water molecules (Mathlouthi, 1997). Since betaine can carry water molecules without immobilizing them, it can assist other biological reactions that utilize water.
The diet must provide adequate amount of methyl groups, since they cannot be synthesized in the body. Methyl groups are essential for many metabolic processes, including the synthesis of phospholipids, adrenal hormones, RNA and DNA. Choline, methionine and betaine can donate methyl groups for these reactions using varying biochemical routes (Craig, 2004). The primary methyl donor in most metabolic reactions in the body is activated methionine, S-adenosylmethionine (S-AM). S- adenosylmethionine is converted to S-adenosylhomocysteine after it donates the methyl group and S- adenosylhomocysteine is then further converted to homocysteine (Craig, 2004). If genetic or nutritional defects occur in the metabolic processes of homocysteine, this can lead to elevated plasma homocysteine levels which are known to be associated with increased risk of CVD (Craig, 2004). However, betaine can re-methylate homocysteine back to methionine via the activity of betaine–homocysteine S-
