NANČA ČEBRON LIPOVEC METABOLIČNI PROFILI BOLNIKOV S KRONIČNO OBSTRUKTIVNO PLJUČNO BOLEZNIJO PRED IN PO REHABILITACIJI DOKTORSKA DISERTACIJA

Transcription

1 NANČA ČEBRON LIPOVEC METABOLIČNI PROFILI BOLNIKOV S KRONIČNO OBSTRUKTIVNO PLJUČNO BOLEZNIJO PRED IN PO REHABILITACIJI DOKTORSKA DISERTACIJA LJUBLJANA, 2016 I

2 NANČA ČEBRON LIPOVEC METABOLIC PROFILES OF PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE BEFORE AND AFTER REHABILITATION DOCTORAL DISSERTATION LJUBLJANA, 2016 II

3 Avtor: Nanča Čebron Lipovec Naslov: Metabolični profili bolnikov s kronično obstruktivno pljučno boleznijo pred in po rehabilitaciji Imenovanje mentorja na seji senata dne 13. januar Imenovanje somentorja na seji senata dne 13. januar Komisija za oceno in zagovor imenovana na seji senata dne 27. junij Datum zagovora: 15. november Mentor: Somentorica: Predsednica komisije: Član: Članica: Članica: izr. prof. dr. Mitja Lainščak, dr. med. prof. dr. Annemie Schols izr. prof. dr. Marjeta Terčelj, dr. med. izr. prof. dr. Tomaž Marš, dr. med. doc. dr. Barbara Artnik, dr. dent. med. prof. dr. Ellen Blaak III

4 ZAHVALA / ACKNOWLEDGEMENT Zahvaljujem se vsem, ki so prispevali k nastanku doktorske naloge in me podpirali na poti: mentorju prof. Lainščaku, somentorici prof. Schols, vsem sodelavcem s Klinike Golnik in iz Univerze v Maastrichtu, prijateljem in družini. I would like to thank everyone who contributed to my doctoral dissertation and supported me on the way: my supervisor prof. Lainščak, my co-supervisor prof. Schols, all colleagues from the Clinic Golnik as well as Maastricht University, my friends and family. IV

5 VSEBINA / TABLE OF CONTENTS SEZNAM SLIK / LIST OF FIGURES... VIII SEZNAM PREGLEDNIC / LIST OF TABLES... IX SEZNAM OKRAJŠAV / LIST OF ABBREVIATIONS... X RAZŠIRJEN POVZETEK... 1 ABSTRACT UVOD Kronična obstruktivna pljučna bolezen Presnova pri kroničnih boleznih in spremembe telesne sestave Inzulinska rezistenca in metabolični sindrom Izguba telesne mase in mišične mase Ocena telesne sestave KOPB kot sistemska bolezen Kardiovaskularno in metabolično tveganje pri KOPB Dejavniki kardiovaskularnega in metaboličnega tveganja pri KOPB Inzulinska rezistenca in metabolični sindrom Izguba mišične mase in spremembe presnove mišic Kronično sistemsko vnetje Življenjski slog Pljučna rehabilitacija INTRODUCTION Chronic obstructive pulmonary disease Metabolism in chronic diseases and changes in body composition Insulin resistance and metabolic syndrome Whole body and muscle wasting Body composition assessment COPD as a systemic disease Cardiovascular and metabolic risk in COPD V

6 2.5 Drivers of cardiovascular and metabolic risk in COPD Insulin resistance and metabolic syndrome Muscle wasting and muscle metabolism abnormalities Low grade systemic inflammation Lifestyle Pulmonary rehabilitation NAMENI IN CILJI AIMS AND OBJECTIVES MATERIALS AND METHODS RESULTS General characteristics of included patients Insulin resistance and Metabolic syndrome Sarcopenia Overlap of sarcopenia and metabolic risk factors Pulmonary rehabilitation outcomes Characteristics of patients who did not complete the rehabilitation program Effect of rehabilitation on physical performance and quality of life Whole group Sarcopenia vs. no sarcopenia Effect of pulmonary rehabilitation on metabolic risk Whole group Sarcopenia vs no sarcopenia Effect of pulmonary rehabilitation on inflammatory profile CRP IL Relation between changes in metabolic risk factors and changes in physical performance Relation between changes in HOMA-IR indices and changes in body composition Protocol for intervention study of COPD patients on rehabilitation VI

7 7 DISCUSSION CONCLUSIONS ZAKLJUČKI PUBLICATIONS AND ACHIEVEMENTS BIBLIOGRAPHY VII

8 SEZNAM SLIK / LIST OF FIGURES Slika 1 Kombinirana ocean KOPB klasifikacija po GOLD smernicah Slika 2 Poročilo o telesni sestavi, DEXA...14 Slika 3 Pospeševalci kardiovaskularnega in metaboličnega tveganja pri KOPB...16 Slika 4 Spekter podpore bolnikoom s KOPB, ki prikazuje pljučno rehabilitacijo kot pomemben del celostne obravnave Figure 1 Combined assessment of COPD GOLD 2011 classification...25 Figure 2 Body composition analysis report by DEXA...30 Figure 3: Drivers of cardiovascular and metabolic risk in COPD Figure 4 Spectrum of support for COPD patients showing pulmonary rehabilitation as an important part of integrated care (74)...38 Figure 5 Study design outline...43 Figure 6 Pulmonary rehabilitation program...44 Figure 7 Examples of muscle biopsies prepared for fiber typing Figure 8 Association between percentage muscle type I fibers and MyHCI expression in COPD and controls Figure 9 Prevalence of MetS across BMI groups...55 Figure 10 Prevalence of sarcopenia across BMI groups...56 Figure 11 MyHC1 mrna expression in sarcopenic and non-sarcopenic patients...60 Figure 12 Changes in physical performance and HRQoL for whole group Figure 13 Response to pulmonary rehabilitation Figure 14 Change in HOMA-IR index Figure 15 Change in body composition in sarcopenic and non-sarcopenic patients Figure 16 Change in lipids...68 Figure 17 Correlation between changes in FFMI and changes in fasting insulin in sarcopenic patients Figure 18 Study design and intervention...74 VIII

9 SEZNAM PREGLEDNIC / LIST OF TABLES Preglednica 1 Klasifikacija stopenj plju-čne obstrukcije po GOLD-u (na osnovi postbronhodilatatornega FEV1).. 9 Preglednica 2 Najpogostejše definicije metaboličnega sindroma Table 1 GOLD Classification of airflow limitation in COPD (based on post bronchodilator FEV1)...25 Table 2 Most common definitions of MetS...28 Table 3 General characteristics of included patients...53 Table 4 Medication use...54 Table 5 Prevalence of metabolic risk factors at baseline...54 Table 6 General characteristics of sarcopenic and non-sarcopenic patients...57 Table 7 Medication use, sarcopenic and non-sarcopenic patients...58 Table 8 Vitamin D and amino acids levels in sarcopenic and non-sarcopenic patients...59 Table 9 Prevalence of metabolic risk factors in sarcopenic and non-sarcopenic patients...61 Table 10 Characteristics of patients who finished vs. patients who did not finish pulmonary rehabilitation...62 Table 11 Change in metabolic risk factors before and after rehabilitation Table 12 Subgroup analysis of fasting glucose, fasting insulin and HOMA-IR before and after rehabilitation...67 Table 13 Serum IL-6 and serum CRP levels pre and post rehabilitation...69 Table 14 Correlations between changes in physical performance and changes in selected metabolic risk factors Table 15 Correlations between changes in body composition and changes in HOMA-IR indices...71 Table 16 Eligibility criteria for intervention study...73 IX

10 SEZNAM OKRAJŠAV / LIST OF ABBREVIATIONS 6MWT ASMI ATS BCAA BIA BMI CHF COPD CPET CRP CVD DEXA ESC ERS FEV1 FFMI FMI FVC GOLD HDL HOMA-IR HRQoL 6-minute Walk Test Appendicular Skeletal Mass Index American Thoracic Society Branched-chain Amino Acids Bioelectrical impedance analysis Body Mass Index Chronic Heart Failure Chronic Obstructive Pulmonary Disease Maximum cycling ergometry C-reactive protein Cardiovascular disease Dual X-ray absorptiometry European Society of Cardiology European Respiratory Society Forced Expiratory Volume in 1 second Fat-free Mass Index Fat Mass Index Forced Vital Capacity Global Initiative for Chronic Obstructive Lung Disease High-density Lipoprotein Homeostatic Model Assessment of Insulin Resistance Health-related Quality of Life X

11 IDF International Diabetes Federation IL-6 Interleukin 6 IR ITM ISWT LDL LOD MCID MetS MNA Insulin Resistance / Inzulinska Rezistenca Indeks telesne mase Incremental Shuttle Walk Test Low-density Lipoprotein Limit of Detection Minimal clinically important difference Metabolic Syndrome Mini Nutritional Assessment MyHC1 Myosin Heavy-chain 1 RNA SGRQ T2DM TG WHR Ribonucleic Acid St. George s Respiratory Questionnaire Type 2 Diabetes Mellitus Triglycerides Waist to Hip Ratio XI

12 RAZŠIRJEN POVZETEK Uvod: Kronična obstruktivna pljučna bolezen (KOPB) je eden glavnih vzrokov obolevnosti in umrljivosti. Po napovedih Svetovne zdravstvene organizacije naj bi bila leta 2020 tretji najpogostejši vzrok smrti na svetu in peti najpogostejši vzrok invalidnosti in bremena bolezni. Vedno več je podatkov, da KOPB ni omejena na pljuča in se kaže tudi sistemsko (Vestbo et al. 2013). Najpogostejša spremljajoča stanja in bolezni vključujejo kronično srčno popuščanje in druge bolezni srca in ožilja, sladkorno bolezen tipa 2, depresijo, slabokrvnost, metabolični sindrom, zmanjšano moč skeletnih mišic, kaheksijo in osteoporozo (Vanfleteren et al. 2013). Večina bolnikov s KOPB umre zaradi spremljajočih bolezni, vključno z boleznimi srca in ožilja, raka in drugih vzrokov. Kronične bolezni, tudi KOPB, so pogosto povezane s spremembami v presnovi in posledičnimi spremembami telesne sestave, ki lahko skupno vodijo v višje kardiometabolično tveganje (van den Borst, Gosker in Schols 2013). Metabolični sindrom je skupek dejavnikov tveganja, ki povečujejo tveganje za bolezni srca in ožilja. Inzulinska rezistenca (IR) je temelj metaboličnega sindroma in osnovna patofiziologija sladkorne bolezni tipa 2 (Alberti, Zimmet, in Shaw 2006). Podatkov je malo o prevalenci inzulinske rezistence in metaboličnega sindroma pri KOPB. Drugi metabolični fenotipi, ki prevladujejo v KOPB, so podhranjenost, kaheksija in sarkopenija (Vanfleteren et al. 2013). Zmanjšanje telesne mase in kaheksija sta povezani z višjo umrljivostjo. Indeks telesne mase (ITM) pa je neodvisen napovedni dejavnik smrti pri bolnikih s KOPB (Schols et al. 2005). Prevalenca različnih metaboličnih profilov pri bolnikih s KOPB ni dobro raziskava, malo je tudi podatkov o povezavi teh profilov s kardiometaboličnim tveganjem in izidi zdravljenja. Pljučna rehabilitacija je pomembna intervencija v sklopu celostne obravnave bolnika s KOPB, saj izboljša fizično zmogljivost in kakovost življenja (Spruit et al. 2013). Vpliv rehabilitacije na kardiometabolično tveganje pri bolnikih s KOPB pa je prav tako slabo raziskan. Namen in cilji: Oceniti metabolični profil bolnikov s KOPB pred rehabilitacijo in po njej; oceniti prevalenco metaboličnih dejavnikov tveganja glede na različne fenotipe telesne sestave in spremembe po rehabilitaciji; oceniti povezave med metaboličnimi spremembami in kliničnimi izidi ob rehabilitaciji; na podlagi rezultatov opazovalne raziskave pripraviti protokol prospektivne intervencijske raziskave za optimizacijo pljučne rehabilitacije. Hipoteze: Prevalenca metaboličnega sindroma je visoka pri bolnikih s KOPB, vendar ni povezana z indeksom telesne mase. 1

13 Prevalenca spremenjene presnove glukoze je pri bolnikih s KOPB visoka in je povezana z nizko vrednostjo indeksa puste telesne mase. Štiritedenska visoko intenzivna rehabilitacija izboljša občutljivost na inzulin pri bolnikih s KOPB. Štiritedenska visoko intenzivna rehabilitacija izboljša vnetni profil bolnikov s KOPB. Metode: Izvedli smo prospektivno opazovalno študijo. Vključili smo bolnike s KOPB, ki so bili napoteni na pljučno rehabilitacijo in se jim v zadnjih štirih tednih bolezen ni poslabšala. Iz študije so bili izključeni bolniki, ki so imeli srčni spodbujevalnik, ki so zavrnili sodelovanje ali niso zmogli slediti programu rehabilitacije. Štiritedenski program rehabilitacije je vključeval naslednje intervencije: individualiziran vadbeni trening moči in trening vzdržljivosti, transkutano električno stimulacijo mišic, dihalne vaje, socialno in psihološko podporo glede na potrebe bolnika in prehransko podporo z individualizirano dieto, svetovanjem in po potrebi predpisom prehranskih dopolnil s strani dietetika. Bolniki so bili pod dnevnim zdravniškim nadzorom. Demografski podatki, zdravljenje in kadilski status so bili ocenjeni pred pričetkom rehabilitacije. Rutinske meritve, prehranski status (Mini Nutritional Assessment Test - MNA vprašalnik), ocena telesne sestave (bioelektrična impedanca - BIA, dvoenergijska rentgenska absorpciometrija - DEXA) so bile izvedene pred začetkom rehabilitacije. Vzorci seruma in mišičnih biopsij so bili odvzeti pred rehabilitacijio in po njej in so bili nato zmrznjeni ter shranjeni na -80 o C za nadaljnje analize vnetnih parametrov (IL-6), inzulinske občutljivosti (HOMA-IR) in metabolitov (aminokisline, vitamin D), v mišičnih biopsijah pa smo analizirali porazdelitev mišičnih vlaken. Prevalenca metaboličnega sindroma je bila ocenjena na podlagi kriterijev Svetovne zveze za diabetes (IDF), sarkopenija pa je bila ocenjena na podlagi indeksa skeletnih mišic (ASMI). Inzulinska rezistenca je bila definirana kot HOMA-IR indeks nad 2,5. Klinični izidi so zajemali rezultate šestminutnega testa hoje, testa Shuttle, vprašalnika St. George's ter maksimalno obremenitev pri cikloergometriji. Zbrane podatke smo statistično analizirali s pomočjo programa SPSS Statistics Rezultati: V študijo smo vključili 112 bolnikov. Bolniki so bili starejši (66 ± 9 let), pretežno moškega spola (67 %), v veliki meri kadilci (82 %) in so po stopnji pljučne obstrukcije večinoma spadali v skupini GOLD 3 in 4 (83 %). Več kot 90 % bolnikov je imelo vrednosti vitamina D nižje od priporočenih. Inzulinska rezistenca, metabolični sindrom in sarkopenija so bili prisotni pri 59 %, 47 % and 55 % bolnikov. Najpogostejše komponente metaboličnega sindroma so bile hipertenzija (86 %), povečan obseg pasu (72 %) in hiperglikemija (69 %). Bolniki s sarkopenijo so imeli v primerjavi z nesarkopeničnimi slabšo fizično zmogljivostjo in nižjo kakovostjo življenja ob začetku rehabilitacije. Opazili smo tudi pomembno nižje vrednosti razvejanih aminokislin v serumu teh bolnikov (399 nmol/ml proti 445 nmol/ml, p = 0,018). Sarkopenija je bila pogostejša pri bolnikih z nižjim indeksom telesne mase, vendar je bila 2

14 pogosta tudi pri bolnikih z indeksom telesne mase nad 25 kg/m 2. Inzulinska rezistenca je bila prisotna pri 54 % sarkopeničnih bolnikov, 37 % pa jih je izpolnjevalo kriterije za metabolični sindrom. Sarkopenija je bila tudi pogostejša pri bolnikih, ki se jim je med programom rehabilitacije bolezen poslabšala ali programa niso uspešno dokončali. Po rehabilitaciji se je izrazito izboljšala telesna zmogljivost in kakovost življenja tako na ravni celotne skupine kot podskupin (sarkopenični, nesarkopenični). Nesarkopeničnim bolnikom se je pomembno znižal HOMA-IR indeks (iz 2,8 na 1,9, p = 0,031), indeks maščobne mase (iz 10,1 na 9,7 kg/m 2, p = 0,013), obseg pasu (iz 103 na 101 cm, p = 0,002) in LDL holesterol (iz 3,2 na 3,0 mmol/l, p = 0,034). Pri sarkopeničnih bolnikih nismo opazili spremembe HOMA-IR ali drugih metabolnih parametrov; pri sarkopeničnih bolnikih brez inzulinske rezistence je HOMA-IR težil k povišanju (iz 1,5 na 1,9, p = 0,058) ob povišanju inzulina (iz 5,6 na 8,4 miu/l, p = 0,031). Sprememba HOMA-IR je bila pri sarkopeničnih bolnikih povezana s spremembo v indeksu puste telesne mase (R = 0,471, p < 0,05). HDL-holesterol se je po rehabilitaciji pomembno znižal na ravni celotne skupine (iz 1,7 na 1,4 mmol/l, p < 0,001). Vrednosti CRP se po rehabilitaciji niso pomembno spremenile, spremembe IL-6 pa nismo mogli oceniti zaradi številnih vzorcev z vrednostmi pod mejo zaznavanja. Razprava: Naša študija je ena prvih, ki ocenjuje prevalenco kardiometabolnega tveganja pri bolnikih z napredovano KOPB in ocenjuje učinek pljučne rehabilitacije na kardiometabolno tveganje v tej skupini bolnikov. Metabolični sindrom je bil opazen pri približno polovici vključenih bolnikov, najpogostejše komponente metaboličnega sindroma pa so bile hipertenzija, abdominalna debelost in hiperglikemija, kar sovpada z objavljeno literaturo (Cebron Lipovec et al. 2016; Vanfleteren et al. 2013). Slednje kaže na visoko kardiometabolno tveganje bolnikov z napredovalo KOPB. Sarkopenija je bila prisotna pri polovici bolnikov; v literaturi variira prevalenca sarkopenije pri KOPB od 4 do 35 % oz. do 50 % v KOPB stopnje 4 (Maltais et al. 2014). Metabolični sindrom in sarkopenija sta pogosto soobstajala, kar nakazuje obstoj t. i. sarkopenične debelosti, ki je pri bolnikih s KOPB pogosta in negativno vpliva na izide rehabilitacije (van de Bool et al. 2015). Sarkopenični bolniki so bili karakterizirani ne samo z znižano mišično maso, temveč tudi s prehranskimi deficiti in spremembami v mišičnem metabolizmu. Rezultati študije tako kažejo, da je sarkopenija večdimenzionalen metabolni profil, ki bistveno vpliva na fizično zmogljivost, kakovost življenja, poslabšanja bolezni ter adherenco pri rehabilitaciji in zato zahteva pozornost v klinični praksi. Rehabilitacija je izboljšala kakovost življenja in fizično zmogljivost bolnikov, kar je v skladu s pričakovanji (Spruit et al. 2013). Učinek pljučne rehabilitacije na kardiometabolno tveganje pa se razlikuje glede na profil telesne sestave. Pri nesarkopeničnih bolnikih smo opazili izboljšanje 3

15 lipidograma, indeksa maščobne mase in obsega pasu. Inzulinska rezistenca je bila pogosta tako pri sarkopeničnih kot nesarkopeničnih bolnikih, vendar se je njena prevalenca znižala le pri nesarkopeničnih. Slednje je lahko posledica nižjihi vrednostmi inzulina pri sarkopeničnih bolnikih pred začetkom rehabilitacije, o čemer literatura že poroča (Franssen et al. 2004). Zvišanje inzulina in s tem HOMA-IR pri sarkopeničnih bolnikih je lahko tudi odraz anabolnega učinka treninga. Nadaljnje študije so potrebne za razjasnitev povezave med sarkopenijo in vplivom rehabilitacije na kardiometabolno tveganje pri bolnikih s KOPB. Tako mi kot Gale in sodelavci (Gale et al. 2011) smo pokazali, da rehabilitacija zniža vrednosti HDL-holesterola, ki pa so pri bolnikih s KOPB v osnovi višje kot pri kontrolnih skupinah (Reed et al. 2011), zato klinični pomen tega znižanja ni jasen. Po rehabilitaciji tudi nismo opazili večjega vpliva na markerje vnetja (CRP, IL-6), kar je v skladu z objavljeno literaturo (Bolton et al. 2007; Vanfleteren et al. 2014). Na podlagi rezultatov študije predlagamo, da se nadaljnje intervencijske študije osredotočijo na bolnike s sarkopenijo. Pri teh naj ocenijo, kako prehranska dopolnila, bogata z razvejanimi aminoksilinami in vitaminom D v kombinaciji s treningom vplivajo na izide rehabilitacije, telesno sestavo in kardiometabolno tveganje pri tej skupini bolnikov. Zaključki: Z višjim kardiometabolnim tveganjem povezani metabolični profili, kot sta npr. metabolični sindrom in sarkopenija, so pogosti pri bolnikih s KOPB, vključenih v program pljučne rehabilitacije. Ti profili pogosto soobstajajo. Rehabilitacija ima pozitiven vpliv na fizično zmogljivost in kakovost življenja, na učinek na kardiometabolno tveganje pa vpliva prisotnost sarkopenije. Alberti KG, Zimmet P, Shaw J. Metabolic syndrome - a new world-wide definition. A Consensus Statement from the International Diabetes Federation. Diabet Med. 2006;23(5): Bolton CE, Evans M, Ionescu AA, Edwards SM, Morris RH, Dunseath G, et al. Insulin resistance and inflammation - A further systemic complication of COPD. COPD. 2007;4(2): Cebron Lipovec N, Beijers RJ, van den Borst B, Doehner W, Lainscak M, Schols AM. The Prevalence of Metabolic Syndrome In Chronic Obstructive Pulmonary Disease: A Systematic Review. COPD. 2016;13(3): Franssen FM, Broekhuizen R, Janssen PP, Wouters EF, Schols AM. Effects of whole-body exercise training on body composition and functional capacity in normal-weight patients with COPD. Chest. 2004;125(6): Gale NS, Duckers JM, Enright S, Cockcroft JR, Shale DJ, Bolton CE. Does pulmonary rehabilitation address cardiovascular risk factors in patients with COPD? BMC Pul Med. 2011;11:20. 4

16 Reed RM, Iacono A, DeFilippis A, Eberlein M, Girgis RE, Jones S. Advanced chronic obstructive pulmonary disease is associated with high levels of high-density lipoprotein cholesterol. J Heart Lung Transplant. 2011;30(6): Schols AM, Broekhuizen R, Weling-Scheepers CA, Wouters EF. Body composition and mortality in chronic obstructive pulmonary disease. The American journal of clinical nutrition. 2005;82(1):53-9. Spruit MA, Singh SJ, Garvey C, ZuWallack R, Nici L, Rochester C, et al. An official American Thoracic Society/European Respiratory Society statement: key concepts and advances in pulmonary rehabilitation. Am J Respir Crit Care Med. 2013;188(8):e van de Bool C, Rutten EP, Franssen FM, Wouters EF, Schols AM. Antagonistic implications of sarcopenia and abdominal obesity on physical performance in COPD. Eur Respir J. 2015;46(2): van den Borst B, Gosker HR, Schols AM. Central fat and peripheral muscle: partners in crime in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2013;187(1):8-13. Vanfleteren LE, Spruit MA, Groenen M, Gaffron S, van Empel VP, Bruijnzeel PL, et al. Clusters of comorbidities based on validated objective measurements and systemic inflammation in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2013;187(7): Vanfleteren LE, Spruit MA, Groenen MT, Bruijnzeel PL, Taib Z, Rutten EP, et al. Arterial stiffness in patients with COPD: the role of systemic inflammation and the effects of pulmonary rehabilitation. Eur Respir J. 2014;43(5): Vestbo J, Hurd SS, Agusti AG, Jones PW, Vogelmeier C, Anzueto A, et al. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease GOLD executive summary. Am J Respir Crit Care Med. 2013;187(4):

17 ABSTRACT Introduction: Metabolic abnormalities such as metabolic syndrome (MetS), body wasting and sarcopenia are frequent in patients with COPD, but the prevalence and correlation with cardiometabolic risk in those eligible for pulmonary rehabilitation is not well studied. Exercise training generally modifies cardiometabolic risk but whether and to what extent this is present in patients with COPD is largely unknown. The impact of various metabolic profiles on rehabilitation outcomes is also poorly investigated. Aim: We aimed to investigate the prevalence of metabolic profiles (insulin resistance (IR), MetS, sarcopenia) in COPD patients eligible for pulmonary rehabilitation. We also aimed to assess the cardiometabolic effects of a short-term high-intensity rehabilitation program in sarcopenic and non-sarcopenic patients.and the change in inflammatory profile. Based on the findings, we suggest a nutritional intervention study protocol to tailor pulmonary rehabilitation program for sarcopenic patients. Methods: We have conducted a prospective observational study. Patients were recruited from an in-patient 4-week short term high-intensity pulmonary rehabilitation programme; their characteristics, laboratory biomarkers and muscle biopsies were analysed at baseline and/or after rehabilitation. Sarcopenia was assessed at baseline (skeletal muscle index <7.23 kg/m 2 for men and <5.67 kg/m 2 for women, as measured by whole-body dual energy X-ray absorptiometry). International Diabetes Federation definition of MetS was applied. IR was defined as homeostasis model assessment of insulin resistance (HOMA-IR) >2.5. At baseline, levels of vitamin D and amino acids from serum were also evaluated. Effects of rehabilitation on change in physical performance, quality of life, cardiometabolic risk, inflammatory profile, and body composition were studied. Results: IR, MetS, and sarcopenia were present in 59%, 47% and 55% of patients, respectively, with a significant overlap. Sarcopenic patients were characterized by low branched-chain amino acids (BCAA), namely valine and leucine, and low vitamin D levels. A higher number of sarcopenic patients also experienced exacerbations or dropped-out of rehabilitation. In non-sarcopenic patients, rehabilitation decreased HOMA-IR (2.8 to 1.9, p=0.031), fat mass index (10.1 to 9.7 kg/m 2, p=0.013), waist circumference (103 to 101 cm, p=0.002) and LDL-cholesterol (3.2 to 3.0 mmol/l, p=0.034). Sarcopenic patients showed no significant change in HOMA-IR. A decrease in total cholesterol levels and HDL-cholesterol was observed in both groups. No change was observed in inflammatory profile. 6

18 Conclusions: The prevalence of altered metabolic profiles in COPD patients eligible for rehabilitation is high. Rehabilitation decreased IR and other cardiometabolic risk markers in non-sarcopenic patients, but this effect was not seen in sarcopenic patients. Sarcopenic patients also tend to experience more exacerbations and program drop-out. Our findings suggest that in pulmonary rehabilitation, body composition assessment is essential to better characterize patients. Sarcopenic patients may profit from a personalized rehabilitation program including nutritional interventions; we suggest to focus on supplementation with a leucine or it s active metabolite HMB and vitamin D. 7

19 1 UVOD 1.1 KRONIČNA OBSTRUKTIVNA PLJUČNA BOLEZEN Kronična obstruktivna pljučna bolezen (KOPB) je eden glavnih vzrokov obolevnosti in umrljivosti. Po napovedih SZO naj bi bila leta 2020 tretji najpogostejši vzrok smrtnosti na svetu in peti najpogostejši vzrok invalidnosti. KOPB je karakterizirana s perzistentno obstrukcijo dihalnih poti, ki je navadno progresivna in je povezana s povečanim vnetnim odzivom dihalnih poti na (cigaretni) dim in toksične delce. Obstrukcija dihalnih poti je posledica obolenja malih dihalnih poti in uničenja parenhima, ki sta pri vsakem bolnikom prisotni v različnem obsegu in določata dva glavna fenotipa KOPB emfizem in kronični bronhitis. Pljučni emfizem je definiran kot nenormalno trajno povečanje distalnih dihalnih poti, ki ga spremlja uničenje alveolarnih sten brez očitne fibroze. S tem ko se stene med alveoli uničijo, se izrazito zmanjša tudi površina za izmenjavo plinov. Motnje izmenjave plinov vodijo v hipoksemijo, hiperkapnijo, ali oboje. Izguba elastičnosti in strukturne podpore vodi v kolaps malih dihalnih poti med izdihom in progresivno, ne povsem reverzibilno obstrukcijo dihanja. Kronični bronhitis je posledica kroničnega vnetja. Tipična je hipersekrecija bronhialne sluzi in tanšanje sten dihalnih poti, ki vodita v kronični nastajanje izmečka in kronični kašelj. To povzroči hiperplazijo in hipertrofijo podsluzničnih žlez in celic, ki izločajo sluz. Hipersekrecija sluzi in zmanjšno delovanje cilijev povečata tveganje za kolonizacijo bakterij, kar vodi v ponavljajoče se okužbe. Diagnoza kroničnega bronhitisa se postavi, ko je kroničen kašelj prisoten več kot 3 mesece dve zaporedni leti (1). Najpogostejši dejavnik tveganja za nastanek KOPB je kajenje oz. izpostavljenost kajenju, v manjšem obsegu prispevajo k tveganju tudi drugi dejavniki, kot je na primer onesnaženost zraka, izpostavljenost toksičnim plinom in genetski faktorji (2). Vdihovanje teh delcev povzroči kronični vnetni odziv v pljučih (3). Glavni simptomi KOPB so kronični kašelj, dispneja in kronično preobilno nastajanje bronhialne sluzi, kar bolnike pogosto ovira pri opravljanju dejavnosti v vsakdanjem življenju, pogosto zbolevajo za okužbanmi dihal in nenamerno izgubljajo telesno težo. Poleg kroničnih 8

20 simptomov pri bolnikih s KOPB pride pogosto tudi do poslabšanj osnovne bolezni, kar zahteva hospitalizacije in povzroči upad v splošnem zdravstvenem stanju bolnika. Na diagnozo KOPB posumimo ob značilnih simptomih in izpostavljenih dejavnikih tveganja. Obstrukcijo v dihalnih poteh ugotovimo s preiskavo pljučne funkcije, njeno reverzibilnost pa preverjamo z bronhodilatatornim testom. Obstruktivna bolezen dihal je definirana kot razmerje FEV1/FVC, nižje od 0,7 po uporabi bronhodilatatorja. Na podlagi rezultata testa pljučne funkcije razdelimo KOPB na 4 stopnje (Tabela 1). Preglednica 1: Klasifikacija stopenj pljučne obstrukcije po GOLD-u (na osnovi postbronhodilatatornega FEV1) Stopnja I KOPB blage stopnje FEV1/FVC<0,70 FEV1 80 % normale Stopnja II KOPB zmerne stopnje FEV1/FVC<0,70 FEV % normale Stopnja III KOPB hude stopnje FEV1/FVC<0,70 FEV % normale Stopnja IV KOPB zelo hude stopnje FEV1/FVC<0,70 FEV1 < 30 % normale Pri bolnikih s KOPB testi pljučne funkcje ne odražajo vedno stopnje simptomov in števila poslabšanj. Leta 2011 je bila zato predlagana nova lestvica ocene KOPB. Slednja vključuje tako stopnjo obstrukcije v dihalnih poteh, stopnjo dispneje in pogostost poslabšanj osnovne bolezni, na osnovi katere razdelimo bolnike v skupino A, B C in D (Slika 1). Slika 1: Kombinirana ocena KOPB klasifikacija po GOLD smernicah

21 Opustitev kajenja je pri bolniku s KOPB osrednji ukrep, saj prepreči nadaljnje napredovanje upada pljučne funkcije in je priporočena vsem bolnikom. Farmakološko zdravljenje omili simptome, zmanjša število poslabšanj, pripomore z izboljšanju fizične zmogljivosti in splošnega zdravstvenega stanja. K farmakološkemu zdravljenju štejemo bronhodilatatorje, inhalacijske kortikosteroide, antiholinergike, inhibitorje 4-fosfodiesteraze in metilksantine. Poleg farmakoloških ukrepov so vedno bolj poudarjeni tudi nefarmakološki ukrepi. Rehabilitacija temelji na telesni vadbi in ostalih podpornih ukrepih in je indicirana pri bolnikih vseh stopenj KOPB, saj se izboljša telesna zmogljivost, zmanjšata se občutek dispneje in utrujenosti (glej razdelek Pljučna rehabilitacija) (4). 1.2 PRESNOVA PRI KRONIČNIH BOLEZNIH IN SPREMEMBE TELESNE SESTAVE Za kronične bolezni so značilne patofiziološke spremembe presnove na nivoju organizma. Spremembe se pojavljajo pri različnih kroničnih boleznih od kroničnega srčnega popuščanja, kronične ledvične bolezni in tudi KOPB (5-7). Bolezenski dejavniki kot tudi dejavniki, vezani na način življenja (npr. kajenje, fizična neaktivnost, nezdrave prehanjevalne navade), spodbujajo razvoj inzulinske rezistence, metaboličnega sindroma, izgube mišične mase in izgube telesne mase INZULINSKA REZISTENCA IN METABOLIČNI SINDROM Inzulinska rezistenca je osnovni patofiziološki mehanizem sladkorne bolezni tipa 2 in hkrati tudi temelj metaboličnega sindroma. Definirana je kot stanje, pri katerem normalne ali povišane koncentracije inzulina ne dosegajo kliničnih učinkov prenosa glukoze v tarčna tkiva. Primarno mesto inzulinske rezistence so skeletne mišice (8). Inzulinsko rezistenco telo sprva premaguje s povečanim delovanjem beta celic in s tem povišanim izločanjem inzulina. Hiperinzulinemija in IR sta zato lahko prisotni leta ali celo desetletja pred klinično manifestacijo sladkorne bolezni tipa 2 (9). IR naj bi bila del skupne poti različnih dejavnikov tveganja, vezanih predvsem na način življenja in genetske predispozicije, ki s hkrati prisotnim vnetnim odzivom povzročijo prenosne in hemodinamske sprembe (10, 11). IR je tako hkrati dejavnik tveganja za razvoj številnih kroničnih bolezni kot tudi posledica teh kroničnih bolezni (12, 13). Metabolični sindrom je konstelacija različnih presnovnih dejavnikov tveganja, ki povečajo tveganje za nastanek sladkorne bolezni tipa 2 in kardiovaskularnih obolenj. 10

22 Sindrom je prvi poimenoval Reaven leta 1988 kot sindrom X in ga označil kot skupek različnih dejavnikov tveganja, ki izhajajo iz IR (14). Danes diagnozo metaboličnega sindroma postavimo na osnovi več kriterijev, ki vključujejo centralno debelost, hipertenzijo, dislipidemijo in hiperglikemijo. V uporabi so različne definicije metaboličnega sindroma, ki se delno razlikujejo v kombinaciji teh komponent in uporabljenih mejnih vrednostih (Tabela 1). Prevalenca metaboličnega sindroma v splošni populaciji je od 21% do 31 % v Aziji (15, 16) do 34 % v ZDA (17) in je višja pri starejših osebah in osebah z višjim ITM (18). Predispozicijski dejavniki za nastanek metaboličnega sindroma so kajenje (19, 20) in sedeči življenjski slog (21, 22), kar je hkrati značilnost številnih kroničnih bolezni (23). Prevalenca metaboličnega sindroma je pogosto višja pri bolnikih s kroničnimi obolenji v primerjavi s kontrolnimi skupinami (24, 25). Patofiziologija IR in metaboličnega sindroma sta v številnih pogledih podobni. Visceralno maščobno tkivo velja za osrednjo komponento razvoja metaboličnega sindroma. Znano je, da maščobno tkivo prispeva k sistemskemu vnetju. Adipokini, kot npr. leptin, adiponektin in rezistin, so del regulatornih poti presnove glukoze in energijskega ravnotežja. Adipozno tkivo proizvaja in sprošča tudi druge vnetne parametre kot npr. TNF-α, IL-6 and IL-1 (26). Debelost je centralna komponenta metaboličnega sindroma in pomemben dejavnik tveganja za razvoj kardiovaskularnih obolenj. Debelost ne vpliva vedno negativno na napredovanje in izide bolezni. V nasprotju z epidemiološkimi podatki iz splošne populacije naj bi debelost pri bolnikih s kroničnimi boleznimi imela protektiven efekt in je pravzaprav povezana z boljšim izidom bolezni. Ta fenomen, imenovan paradoks debelosti, je bil zaznan pri različnih kroničnih boleznih, kot je npr. kronično srčno popuščanje, kronična ledvična odpoved kot tudi KOPB (27-29). Ozadje tega fenomena, ki debelost ocenjuje preko ITM, še ni popolnoma razjasnjeno, bi pa lahko bilo povezano z zvišano mišično maso, ne pa tudi količino maščevja (27). Čeprav je IR navadno povezana z debelostjo in metaboličnim sindromom, ne smemo pozabiti, da ima inzulin tudi anabolni efekt. Pri bolnikih s kroničnim srčnim popuščanjem na primer je bila IR povezana z rezistenco na anaboličen učinek in s pomanjkanjem testosterona ter s povečanim katabolizmom (6). Če so torej spremembe presnove pri kroničnih obolenjih primarno povezane s povišano telesno maso in zvišanim maščevjem, lahko sčasoma te motnje vodijo tudi v izgubo telesne mase in posebno mišične mase. 11

23 after rehabilitation. Doctoral dissertation. University of Ljubljana, Faculty of Medicine, 2016 Preglednica 2 Najpogostejše definicije metaboličnega sindroma Centralna debelost Hiperglikemija Dislipidemija WHO (1998) (30) EGIR (31) Obseg pasu > 30 kg/m² in/ali WHR > 0,90 pri moških ali WHR > 0,85 pri ženskah IGT, IFG, ali T2DM ali znižana inzulinska občutljivost TG 150 mg/dl in/ali HDL < 35 mg/dl pri moških ali < 39 mg/dl pri ženskah Obseg pasu > 94 cm pri moških in > 80 cm pri ženskah NCEP ATP III (2001 and 2005) (32, 33) Obseg pasu > 102 cm pri moških in > 88 cm pri ženskah AACE (2003) (34) IDF (35) Alberti (2009) (36) ITM 25 kg/m² Obseg pasu 94 cm prih moške in 80 cm pri ženskah; etnično prilagojene vrednosti Obseg pasu > 102 cm pri moških in > 88 cm pri ženskah; etnično prilagojene vrednosti IGT or IFG 110 mg/dl IGT or IFG 100 mg/dl 100 mg/dl TG 150 mg/dl in/ali HDL < 40 mg/dl za moške ali < 50 mg/dl pri ženskah ali specifično zdravljenje TG 150 mg/dl c HDL < 40 mg/dl pri moških ali < 50 mg/dl pri ženskah TG 150 mg/dl HDL < 40 mg/dl pri moških ali < 50 mg/dl pri ženskah TG 150 mg/dl ali specifično zdravljenje HDL < 40 mg/dl pri moških ali < 50 mg/dl pri ženskah ali specifično zdravljenje TG 150 mg/dl HDL < 40 mg/dl pri moških ali < 50 mg/dl pri ženskah 130/85 mmhg 130/85 mmhg ali Hipertenzija 140/90 mmhg 140/90 mmhg 130/85 130/85 mmhg mmhg zdravljenje že diagnosticirane hipertenzije Ostalo Mikroalbuminurija - - Drugi znaki inzulinske rezistence e - - Diagnoza MetS Hiperglikemija plus dva druga kriterija Inzulin > 75 percentilom plus dva druga kriterija Trije ali več kriterijev Hiperglikemija plus dva druga kriterija Centralna debelost plus dva druga kriterija Trije ali več kriterijev 12

24 1.2.2 IZGUBA TELESNE MASE IN MIŠIČNE MASE Izguba telesne mase je navadno posledica negativnega energijskega ravnotežja, pri čemer je vnos energije nižji od njene porabe. Normalen odziv na tako znižan energetski vnos (stradanje) je znižanje bazalnega metabolizma. Pri mnogih kroničnih boleznih pa je bazalni metabolizem povišan (37). Poleg tega se pri mnogih kroničnih boleznih, kot npr SP, zmanjša apetit in je zmanjšana gastrointestinalna absorpcija (6). Te značilnosti še poglobijo neravnotežje med vnosom in porabo energije. Klinična manifestacija tega neravnotežja je izguba telesne mase na račun izgube mase različnih tkiv maščobnega tkiva, mišičnega tkiva kot tudi kostnine. Izguba mišične mase je posledica katabolnega stanja, ki je rezultat neravnovesja med sintezo in razgradnjo proteinov (38). Inzulinska rezistenca in kronično vnetje sta pogosto značinli za kronične bolezni in poglabljata katabolno stanje. Sarkopenija je definirana kot proces izgube mišične mase, ki spremlja staranje in ob čemer je delež maščobnega tkiva zvišan ali nespremenjen (39). Točno prevalenco sarkopenije je težko določiti, saj se zanjo uporabljajo različne mejne vrednosti. Za zdrave osebe se uporablja deseti percentile indeksa puste telesne mase (< 17 kg/m 2 za moške, < 15 kg/m 2 za ženske pri Kavkazijcih) (40). S pomočjo napredkov v oceni telesne sestave lahko danes ocenimo regionalne spremembe telesne sestave. Na podlagi tega je bil razvit t. indeks skeletne mišične mase (SMI; skeletna mišična masa na meter kvadrat, (kg/m 2 )), za mejno vrednost pa se uporablja vrednost 2 standardnih deviacij pod povprečjem splošne populacije (< 7,23 kg/m 2 za moške in 5,67 kg/m 2 za ženske). Te mejne vrednosti se najpogosteje uporablja za oceno prevalence sarkopenije pri starostnikih. (39) Sarkopenija je med starostniki pogost pojav in je povezana z oviranim gibanjem, ovirano možnostjo opravljanja dnevnih aktivnosti, izgubo samostojnosti in povečano smrtnostjo (39). Poleg sprememb v količini mišične mase, ki se kaže kot izguba mišične mase, opazimo pri kroničnih boleznih tudi spremembe mišične sestave. Te spremembe lahko vodijo v nadaljnje slabšanje IR, zmanjšano energijsko učinkovitost in prikrito kopičenje maščob (6). Posledično se lahko izguba mišične mase uravnoteži s povečanjem maščobne mase brez spremembe v telesni masi. Zaradi teh prikritih sprememb je bistveno oceniti telesno sestavo pri kroničnih bolnikih in ne zgolj njihovo telesno težo ali ITM. 13

25 1.2.3 OCENA TELESNE SESTAVE Za oceno telesne sestave obstajajo različne metode. V klinični praksi sta najpogostejši bioelektrična impedanca (BIA) in dvoenergijske rentgenske absorpciometrije (DEXA). BIA je neinvazivna, enostavna metoda, ki ocenjuje električno impedanco različnih telesnih tkiv in izračuna vsebnost vode v telesu. Iz teh vrednosti se nato preračuna vsebnost suhe puste mase in posredno tudi maščobno maso. BIA je torej dvoprostorni model za oceno telesne sestave. Kot metoda je enostavna in dostopna, ima pa tudi nekatere omejitve; ker ocenjuje vsebnost vode v telesu, stanje hidracije bolnika ali prisotnost edemov, kar lahko vpliva na pridobljeni rezultat (41, 42). Druga metoda je DEXA. Rentgenski žarek presvetli telo in na osnovi različnih absorpcij se preračuna vsebnost maščobnega, mišičnega in kostnega tkiva. DEXA je torej triprostorni model ocene telesne sestave, saj razkrije informacije o vsebnosti kostnega tkiva, kar uporaba metode BIA ne razkrije. DEXA tudi omogoča oceno telesne sestave posameznih predelov telesa, npr. skeletnega mišičnega tkiva in tako ocene SMI. Kot metoda je torej DEXA bolj informativna, vendar je hkrati tudi bolj invazivna zaradi (sicer nizke) stopnje radioaktivnosti. Aparat tudi ni prenosen, meritve pa lahko izvaja le izkušeno osebje (43). Slika 2: Poročilo o telesni sestavi, DEXA 14

26 1.3 KOPB KOT SISTEMSKA BOLEZEN V zadnjem desetletju vedno več podatkov kaže, da učinki KOPB niso omejeni na pljuča, temveč se izražajo sistemsko (44). Danes je KOPB definirana kot heterogena in multikomponentna bolezen (45), vpliv komorbidnosti na breme bolezni pa je bil poudarjen tudi v zadnji definiciji KOPB v smernicah GOLD (1). Komorbidnosti ne vplivajo samo na breme bolezni, invalidnost in splošno zdravstveno stanje bolnika (46), temveč višajo število hospitalizacij in smrtnost (47). Bolniki s KOPB pravzaprav pogosteje umirajo zaradi nerespiratornih zapletov, npr. kardiovaskularnih bolezni, raka in drugih vzrokov (3). Najpogostejše sistemske značilnosti in komorbidnosti vključujejo kronično srčno popuščanje, sladkorno bolezen tipa 2, depresijo, anemijo, metabolični sindrom, zmanjšano mišično moč, kaheksijo in osteoporozo (48). 1.4 KARDIOVASKULARNO IN METABOLIČNO TVEGANJE PRI KOPB Bolniki s KOPB imajo približno 2- do 3-krat višje kardiovaskularno tveganje kot bolniki brez KOPB, ne glede na stopnjo bolezni ali kadilski status (49). Najpogostejše kardiovaskularne komorbidnosti so aritmija, kronično srčno popuščanje, akutni miokardni infarkt, periferna vaskularna bolezen in možganska kap; vse naštete bolezni so bolj pogoste pri bolnikih s KOPB kot pri kontrolnih skupinah (49). Visoka prevalenca je posledica intrinzičnih faktorjev, kot so npr. sistemsko vnetje, oksidativni stres in hipoksemija pa tudi ekstrinzičnih faktorjev, npr. nezdravega življenjskega sloga. 15

27 Slika 3: Pospeševalci kardiovaskularnega in metaboličnega tveganja pri KOPB. Povzeto po Van den Borst, AJRCCM 2013 (7). 1.5 DEJAVNIKI KARDIOVASKULARNEGA IN METABOLIČNEGA TVEGANJA PRI KOPB INZULINSKA REZISTENCA IN METABOLIČNI SINDROM Prevalenca in dejavniki, ki k njej prispevajo Sladkorna bolezen tipa 2 je pogosta komorbidnost pri bolnikih s KOPB (50), prevalenca IR pa je slabo raziskana. Študije nakazujejo, da je IR bolj pogosta pri bolnikih s KOPB kot pri zdravih kontrolah (51, 52). Bolton in sodelavci so pokazali, da je IR (ocenjena s HOMA-IR indeksom) pri nehipoksemičnih bolnikih s KOPB bolj pogosta kot pri zdravih posameznikih. Pokazali so tudi, da je IR vezana na sistemsko vnetje (51). V nedavni študiji Van den Borsta so opazili višje vrednosti HOMA-IR in CRP tudi pri bolnikih z blago do zmerno KOPB v primerjavi z zdravimi posamezniki. Poročali so tudi o šibki korelaciji med HOMA-IR in CRP (52). Skyba in sodelavci so poročali tudi o višji prevalenci IR pri KOPB bolnikih s povišanim ITM, ki je bila asocirana z zvišano ekspresijo vnetnih genov (53). Prevalenca metaboličnega sindroma pri bolnikih s KOPB je bila natančno predstavljena v preglednem članku (24). Povprečna prevalenca je znašala 34 % (21 % do 58 %). Bolniki z 16

28 metaboličnim sindromom so bili pogosteje ženskega spola, imeli so višji ITM in višji FEV1 v primerjavi z bolniki s KOPB brez metaboličnega sindroma. Manjše število študij je poročalo o višjih vrednostih CRP in IL-6 pri bolnikih s KOPB z metaboličnim sindromom v primerjavi s KOPB brez metaboličnega sindroma. Najbolj pogoste komponente metaboličnega sindroma so bile hipertenzija (56 %), hiperglikemija (44 %) in abdominalna debelost (39 %). Nizek HDLholesterol je bil bistveno manj prevalenten pri bolnikih s KOPB v primerjavi s kontrolnimi skupinami. Abdominalna debelost, merjena z obsegom pasu, je pogosta komponenta metaboličnega sindroma pri bolnikih s KOPB. Dve pred kratkim objavljeni študiji sta pokazali, da imajo tudi bolniki s KOPB z normalnim ali nizkim ITM več visceralnega maščevja v primerjavi s kontrolami. Ti bolniki niso bili kategorizirani kot debeli s pomočjo ITM in niso imeli povišanega obsega pasu, kar nakazuje obstoj prikritega kopičenja (visceralnega) maščevja (54, 55). Adipokini, ki se sproščajo iz maščobnega tkiva, delujejo tako lokalno kot sistemsko in spodbujajo sistemsko vnetje in inzulinsko rezistenco pri bolnikih s KOPB. Maščobno tkivo in abdominalna debelost močno vplivata na prevalenco MetS pri KOPB, vendar nanjo vplivajo tudi drugi faktorji kot na primer genetska predispozicija (56), zdravila (glukokortikoidi) in fizična neaktivnost (24). Vpliv na izid bolezni Podatki o vplivu metaboličnega sindroma na izide zdravljenja pri bolnikih s KOPB so slabo raziskani in deloma tudi kontradiktorni. Študije kažejo, da je metabolični sindrom pri KOPB povezan z višjim številom komorbidnosti in bolj pogostim občutkom dispneje (57). Podatki tudi kažejo na povezavo med prisotnostjo metaboličnega sindroma in številom in trajanjem poslabšanj KOPB (58). Abdominalna debelost naj bi prispevala k obstrukciji pljučne funkcije (59). Hkrati pa so študije tudi pokazale prisotnost t. i. paradoksa debelosti, saj imajo bolniki s KOPB z višjim ITM boljše preživetje (29). Poleg tega je fizična zmogljivost (ocenjena z maksimalno porabo kisika) splošno ohranjena pri zmerno debelih bolnikih s KOPB. Pri teh bolnikih naj bi povečana inspiratorna kapaciteta in nižji volumni pljuč zmanjšali dodatno delo dihalnih mišic in s tem zmanjšali občutek dispneje med vadbo (60). Tako sklepanje nasprotuje splošnim prepričanjem, da ima debelost negativen učinek na fizično zmogljivost in dipsnejo pri bolnikih s KOPB (61). Obstoječi podatki tako kažejo, da vpliva metaboličnega sindroma in debelosti na fizično zmogljivost in prognozo bolnikov s KOPB ne moremo posploševati, temveč ga moramo razumeti v kontekstu tako stopnje bolezni kot tudi prisotnosti metabolnih in mišičnoskeletnih motenj. 17

29 1.5.2 IZGUBA MIŠIČNE MASE IN SPREMEMBE PRESNOVE MIŠIC Čeprav sta pri bolnikih s KOPB pogosta tako metabolični sindrom kot IR presnovne motnje, tipične za debelost, je prav tako pogosto tudi hujšanje in slabo stanje prehranjenosti. Ta dva fenotipa telesne sestave spominjata na klasično razdelitev bolnikov s KOPB na t. i. rožnate pihalce (ang. pink puffers ) in modre pihalce (ang. blue bloaters ). Rožnati pihalci emfizemski tip bolnikov, so bili karakterizirani z nizkim ITM in izgubo telesne teže, modri pihalci pa so imeli pretežno prekomerno težo in bolj izražen kronični bronhitis (62). Prevalenca in dejavniki, ki nanjo vplivajo Nizek ITM (< 20 kg/m2) je prisoten pri približno petini bolnikov s KOPB, vendar je nizka mišična masa (definirana kot indeks puste telesne mase < 16 kg/m 2 za moške in < 15 kg/m 2 za ženske) dvakrat bolj pogosta (25). Poročana prevalenca mišične atrofije je odvisna od uporabljene mejne vrednosti in variira od 4 % do 35 % (29). Nizka mišična masa je bolj pogosta pri bolnikih z višjo stopnjo bolezni in doseže prevalence do 50 % pri bonikih s KOPB stopnje 4. Pogostejša je tudi pri ženskah (29). Nizek indeks puste telesne mase je opaziti tudi pri četrtini bolnikov s KOPB z normalno telesno težo (29). Izguba mišične mase pri bolnikih s KOPB je posledica tako bolezenskih dejavnikov kot dejavnikov, povezanih z življenjskim slogom. Fizična neaktivnost, kronična hipoksija, slaba prehranjenost, kronično vnetje in uporaba glukokortikoidov zmanjšujejo mišično maso. Osrednji razlog za izgubo mišične mase je neravnovesje med sintezo in razgradnjo mišičnih proteinov. Natančen pregled molekularnih mehanizmov v ozadju presega namen te naloge; za nadaljnjo razlago je na voljo pregledni članek Langena in sodelavcev (38). Poleg sprememb v mišični masi so pri bolnikih s KOPB opazne tudi spremembe v metabolizmu mišic. V mišičnem tkivu pride do prehoda od oksidativnih na glikolitična vlakna in zmanjšane encimske oksidativne kapacitete (63). Tipično je tudi zmanjšanje deleža vlaken tipa I in povečanje deleža vlaken tipa II, kar je v nasprotju s spremembami pri normalnem staranju. Sprememba je izrazitejša pri bolnikih višjih stopenj bolezni, nižjega indeksa telesne mase in je tipična za mišice spodnjih okončin. Zmanjšani sta tudi količina in funkcija mitohondrijev v mišicah (64). Pri bolnikih s KOPB, posebno z nizkim ITM, je tako izguba mišične mase tesno povezana z izgubo mišičnega oksidativnega fenotipa. Sprememba mišičnih vlaken zmanjša kapaciteto za oksidacijo maščob ob povečani razpoložljivosti maščobnih kislin; to posledično spremeni preferenco izrabe maščobe na izrabo glukoze (65). Glede na to, da so skeletne mišice glavno mesto porabe glukoze, lahko sarkopenija tako povzroča inzulinsko rezistenco neposredno in neodvisno od KOPB (66). Metabolne spremembe, opažene v skeletnih mišicah bolnikov s KOPB, kažejo izjemne 18

30 podobnosti s spremembami, opaženimi pri bolnikih s sladkorno boleznijo. Zmanjšana mitohondrijska respiracija in mitohondrijska biogeneza sta primerljivi med bolniki s KOPB nizke/normalne telesne teže in debelimi bolniki s sladkorno boleznijo (67-69). Sladkorni bolniki imajo tudi podobno zmanjšan delež vlaken tipa I v primerjavi s kontrolami (70). Sarkopenija torej predstavlja ranljiv KOPB fenotip, kar ne vpliva zgolj na fizično zmogljivost, temveč lahko tudi poveča kardiometabolno tveganje. Vpliv na izide bolezni Izguba mišične mase močno vpliva na klinične izide bolnikov s KOPB. Zmanjšuje fizično zmogljivost, dodatno poveča utrujenost in zmanjša zmožnost opravljanja vsakodnevnih aktivnosti. Hkrati vpliva na znižanje kakovosti življenja in poveča verjetnost za hospitalizacije (71). Najpomembnejši vpliv pa ima mišična masa na smrtnost, saj je izguba mišične mase povezana s krajšo življenjsko dobo, neodvisno od stopnje bolezni, mase maščevja in indeksa telesne mase (29, 72, 73) KRONIČNO SISTEMSKO VNETJE Sistemski znaki KOPB so tesno povezani s sistemskim vnetjem. Podatki kažejo, da je tovrstno vnetje prisotno pri bolnikih s KOPB, tudi če se bolezen ne poslabša. V primerjavi z zdravimi kontrolami imajo bolniki s KOPB povišane ravni CRP, fibrinogena, leukocitov in TNF-alfa, kar kaže na kronični vnetni proces (74). Vir vnetja še ni popolnoma jasen. Sprva so kronično vnetje razlagali kot posledico prenosa vnetja iz pljuč, vendar raziskave nakazujejo, da tudi drugi organi, npr. maščobno tkivo in tudi drugi vplivi, npr. hipoksija, doprinesejo k sistemskemu vnetju (75). Sistemsko vnetje pri bolnikih s KOPB je vezano na različne zunajpljučne manifestacije, kot je na primer izguba telesne teže, kaheksija in kardiovaskularna obolenja. Novejši podatki kažejo, da so različni vnetni mediatorji vezani na različne profile komorbidnosti. Vanfleteren in sodelavci so predstavili t. i. skupine komorbidnosti. Bolniki v kardiovaskularni skupini, v kateri sta bili najpogostejši komorbidnosti hipertenzija in ateroskleroza, so imeli močno zvišane ravni IL-6. V metabolni skupini, kjer je bila pogosta debelost, dislipidemija in hiperglikemija, pa so opazili povišane ravni TNF receptorjev (44). Vzroki in povezave med sistemskim vnetjem in izvenpljučnimi znaki pri bolnikih s KOPB še niso povsem jasni in so predmet intenzivnega raziskovanja. 19

31 1.5.4 ŽIVLJENJSKI SLOG Kajenje Kajenje je neodvisen dejavnik tveganja tako za KOPB kot za kardiovaskularna obolenja. Raziskave kažejo, da kajenje doprinaša k inzulinski rezistenci v skeletnih mišicah in povečani abdominalni debelosti, ki posledično zvišujejo kardiovaskularno in metabolno tveganje (76, 77). Bolniki s KOPB kot tudi kadilci brez KOPB imajo zmanjšano mišično maso v primerjavi z nekadilci (78). Kadilci brez KOPB kažejo tudi znake izgube oksidativnega fenotipa v skeletnih mišicah (79). Kronična izpostavljenost kajenju zvišuje sistemsko vnetje in s tem vpliva na vse prej opisane pospeševalce povečanega metaboličnega tveganja (80). Fizična (ne)aktivnost Za bolnike s KOPB je značilen sedeči življenjski slog z manj pogostimi in krajšimi obdobji fizične aktivnosti v primerjavi s kontrolami (63, 67). Simptomi, kot so dispneja in utrujenost, bistveno vplivajo na neaktivnost. Študije nakazujejo, da zmanjšana fizična aktivnost lahko pospeši razvoj KOPB, saj imajo fizično aktivni kadilci in nekadilci zmanjšan upad pljučne funckije. Fizična neaktivnost povzroča tudi številne metabolne motnje kot npr. inzulinsko rezistenco mišic, povečano vsebnost maščob v mišicah, mišično atrofijo in spremembe v tipu mišičnih vlaken (81). Fizična neaktivnost verjetno pomembno zmanjšuje metabolno zdravje bolnikov s KOPB. Prehrana Čeprav je debelost pogost pojav pri bolnikih s KOPB, so mnogi bolniki tudi slabo prehranjeni. Dispneja, utrujenost in depresija so pogosti simptomi KOPB in vplivajo na znižan apetit (82). Zaradi zmanjšanega apetita in slabšega prehranjevanja, ki ni uravnoteženo s povečanimi energijskimi potrebami, bolniki izgubljajo telesno težo (37, 82). Študije so tudi pokazale, da bolniki s KOPB pogosto uživajo nezdravo hrano, bogato z nasičenimi mašobami, uživanje nenasičenih maščobnih kislin in vitaminov, npr vitamina D, pa je pod priporočili (83). Znana je povezava med nezdravim načinom prehranjevanja (Western diet) in povečanim kardiovaskularnim tveganjem, torej prehranjevalne navade tudi prispevajo k povečanemu kardiometaboličnemu tveganju bolnikov s KOPB. 20

32 1.6 PLJUČNA REHABILITACIJA Farmakološko zdravljenje KOPB je zelo razširjeno, vendar je omejeno na simptomatsko lajšanje obstrukcije dihanja. Hkrati stopajo v ospredje izvenpljučni znaki KOPB in z njimi povezani negativni vplivi na izide zdravljenja, zaradi česar se povečuje zanimanje za nefarmakološke ukrepe kot npr. pljučno rehabilitacijo. Pljučna rehabilitacija je definirana kot celovita intervencija, osnovana na celostni oceni bolnika, kateri sledijo bolniku prilagojene terapije, ki vključujejo, vendar niso omejene na vadbeni trening, izobraževanje in spremembe vedenjskega sloga in ki imajo za cilj izboljšanje fizičnega in psihološkega stanja bolnikov s kroničnimi pljučnimi obolenji ter promoviranje dolgoročne adherence življenjskemu slogu, ki izboljšuje zdravje (84). Pljučna rehabilitacija je osnovni del celovite obravnave bolnikov s KOPB in je vključena v smernice GOLD zdravljenja KOPB (3). Po smernicah GOLD je izboljšanje fizične zmogljivosti, kakovosti življenja in zmanjšanje stroškov zdravljenja po pljučni rehabilitaciji ocenjeno kot dokaz stopnje A. Čeprav so dobrobiti pljučne rehabilitacije dobro znane, kažejo raziskave, da je ta intervencija močno podcenjena in premalo uporabljena (85). 21

33 Slika 4: Spekter podpore bolnikom s KOPB, ki prikazuje pljučno rehabilitacijo kot pomemben del celostne obravnave (74) Vadbeni trening je osnova pljučne rehabilitacije. Tipi treninga vključujejo intervalni trening, trening moči, trening zgornjih okončin in transkutano nevromuskularno električno stimulacijo. Intenzivnost treninga je simptomatsko omejena in prilagojena zmogljivosti posameznega bolnika. Trajanje pljučne rehabilitacije ni natančno definirano, vendar naj bi največji doprinos za bolnika imeli programi z minimalno 20 treningi (84). Vadbeni trening povzroči strukturne in funkcionalne spremembe v mišicah zdravih. Spremembe vključujejo spremembe aktivnosti oksidativnih encimov, zvišano število mitohondrijev in povečano kapilarizacijo mišic (86). Pri bolnikih s KOPB aerobni trening izboljša oksidativno kapaciteto mišic in zmanjša mišično utrujenost, anaerobni trening pa povzroči hipertrofijo mišic (87-89). Čeprav je znano, da pljučna rehabilitacija izboljša telesno zmogljivosti, zmanjša simptome in izboljša kakovost življenja, (90) študije kažejo, da program ne pripomore vsem bolnikom 22

34 enako. V času omejenih virov financiranja se pojavlja vedno več pozivov k boljši karakterizaciji bolnikov s KOPB, pri katerih ima rehabilitacija največ učinka, in k iskanju novih intervencij pri tistih skupinah bolnikov, kjer s tem program ne dosežemo želenih ciljev. Močno je poudarjena potreba po personaliziranem program pljučne rehabilitacije (91, 92). Hkrati je ob visoki prevalenci komorbidnosti in posebno visokem kardiovaskularnem tveganju nujno ovrednotiti tudi učinek pljučne rehabilitacije na te parametre pri bolnikih s KOPB. 23

35 2 INTRODUCTION 2.1 CHRONIC OBSTRUCTIVE PULMONARY DISEASE Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. It is predicted to be the 3 rd leading cause of death by 2020 according to the WHO (1). Moreover, COPD is predicted to rank fifth as chronic disability and burden of disease by COPD is characterized by persistent airflow limitation that is usually progressive and associated with an enhanced inflammatory response of the airway and lung to noxious particles or gases. The chronic airflow limitation is attributable to the disease of small airways and parenchymail destruction. The latter varies from patient to patient and is related to the two main patients phenotypes emphysema and chronic bronchitis. Emphysema is defined as an abnormal permanent enlargement of the airspaces distal to the terminal bronchioles, accompanied by destruction of their walls and without obvious fibrosis. As the walls between alveoli are destroyed, the surfacee area for gas exchange becomes significantly reduced. Abnormalities in gas exchange ultimately leads to hypoxemia, hypercarbia, or both. Loss of lung recoil and structural support promotes collapse of small airways during expiration and progressive, not fully reversible airflow limitation. In chronic bronchitis, mucus hypersecretion and thickening of the airway walls occur and result in chronic cough and sputum production. The large airways, which include the trachea and first generations of the bronchi, are a major site of inflammation and mucus hypersecretion. This results from hyperplasia and hypertrophy of the submucosal glands and mucus-producing goblet cells within the surface epithelium. Mucus hypersecretion coupled with impaired ciliary function, reduces mucociliary clearance, increases the accumulation of secretions, and enhances the risk of bacteria colonization. As a result, recurrent infections often occur owing to the inability to clear pathogens. Chronic bronchitis is diagnosed based on the presence of chronic productive cough for 3 months in 2 consecutive years (1). The most common risk factor for COPD is cigarette smoking, although other risk factors have been identified, such as air pollution, toxic gases exposure and genetics. (2) The inhalation of these particles is believed to trigger the persistent pulmonary inflammatory response (3). The main symptoms of COPD are chronic cough, dyspnea and chronic sputum productions; patients also frequently experience limitations in activities of daily living, persistent respiratory 24

36 infections and unintended weight loss. Besides the chronic symptoms, COPD patients also suffer of frequent disease exacerbations, which may lead to hospitalizations and a general decline in health status. COPD is evaluated based on patients symptoms and is confirmed using pulmonary function tests and arterial blood gas analysis. Airflow obstruction is defined as a reduced postbronchodilator FEV1/FVC ratio of <0.7. Based on the results, the severity of airflow limitation is staged into 4 different groups: Table 1 GOLD Classification of airflow limitation in COPD (based on post bronchodilator FEV1) (1) Stage I Mild COPD FEV1/FVC<0.70 FEV1 80% normal Stage II Moderate COPD FEV1/FVC<0.70 FEV % normal Stage III Severe COPD FEV1/FVC<0.70 FEV % normal Stage IV Very Severe COPD FEV1/FVC<0.70 FEV1 <30% normal As the degree of airflow limitation does not necessarily reflect the severity of symptoms and number of exacerbations, in 2011, a new GOLD classification was proposed. It includes assessment of airflow limitation severity, degree of dyspnea and frequency of exacerbations to categorize patients into groups A, B, C, and D (Figure 1) (1). Figure 1 Combined assessment of COPD GOLD 2011 classification 25

37 Smoking cessation has the greatest impact on the natural course of COPD and should be encouraged in all patients. The pharmacological treatment is used to reduce symptoms, reduce the frequency of exacerbations, improve exercise limitation and patients health status. It comprises inhalation therapy with bronchodilators (beta-agonists, anticholinergics), inhaled corticosteroids (mostly in a combination), phosphodiesterase-4-inhibitors and methylxanthines. In stage IV disease (or with chronic hypoxia), long-term oxygen therapy is also used. Besides pharmacological treatment, non-pharmacological interventions have been gaining interest and are indicated in patients of all stages as this reduces exercise limitation and improves dyspnea and fatigue (see section Pulmonary rehabilitation) (4). 2.2 METABOLISM IN CHRONIC DISEASES AND CHANGES IN BODY COMPOSITION Chronic diseases are marked with pathophysiological changes in whole body metabolism. These changes are shared across various chronic diseases, such as chronic heart failure, COPD, chronic kidney disease (5-7). Disease-related as well as lifestyle-related factors, such as smoking, physical inactivity, malnutrition, lead to the development of IR, MetS, muscle and whole body wasting INSULIN RESISTANCE AND METABOLIC SYNDROME IR is the underlying pathophysiological mechanism of type II diabetes mellitus (T2DM) and the cornerstone of the MetS. It is defined as a state where normal or elevated insulin levels produce an impaired sensitivity to insulin mediated glucose disposal. The primary site of IR is skeletal muscle (8). IR may initially be overcome by increased beta cell activity to increase endogenous insulin levels. Accordingly, hyper-insulinaemia and IR precede the clinical manifestation of T2DM by years or even decades (9). IR has been suggested to be part of a common pathway of various lifestyle factors, which combined with genetic predispositions cause metabolic and hemodynamic derangements in association with inflammation (10, 11). As such, IR is both a risk factor for numerous chronic diseases as well as a consequence of them (12, 13). MetS is a constellation of metabolic risk factors that increase the risk of T2DM and CVD development. The syndrome was originally named Syndrome x by Reaven in 1988 and was termed as a clustering of various risk factors, stemming from IR (14). Nowadays, the diagnosis 26

38 of MetS is based on the presence of central obesity, hypertension, dyslipidemia, and hyperglycemia. Various MetS definitions are available and differ in specific cut points and combination of the components (Table 1). The prevalence of MetS in the general population varies from 21%-31% in Asia (15, 16) to 34% in the USA (17), and increases with age and body mass index (BMI) (18). Predisposing factors associated with MetS development are smoking (19, 20) and a sedentary lifestyle (21, 22), which are well-described features in numerous chronic diseases (23). Indeed, in numerous chronic diseases, MetS is reported to be more prevalent compared to healthy controls (24, 25). From a pathophysiological perspective, Mets and IR are intertwined. Visceral adipose tissue is believed to be central to the development of MetS. Adipose tissue is by now known to be a potent site of inflammation. Adipocytokines such as leptin, adiponectin and resistin are involved in the regulation of glucose metabolism and energy balance. Adipose tissue also produces and secretes inflammatory mediators, such as TNF-α, IL-6 and IL-1 (26). Obesity is a central feature of MetS and an important risk factor for CVD. However, this does not seem to be the case for disease progression and outcome. In contrast to epidemiological data from the general population, obesity in specific chronic diseases seems to have a protective effect and contributes to a longer life expectancy. This phenomenon, termed the obesity paradox, has been observed in other chronic conditions, such as chronic heart failure, chronic kidney disease and COPD (27-29). The reasons behind this phenomenon, which evaluated obesity with BMI, are not yet fully elucidated, but might be also related to higher lean mass reserves rather than increased fat mass (27). Although IR is usually related to obesity and MetS, it is important to acknowledge that insulin also has anabolic effects. In patients with CHF, IR has been related to anabolic failure with blunted anabolic capacity and increased catabolism (6). Hence, although the metabolic changes typical for chronic conditions are related to increased body size and fat mass, through time these derangements may lead to body wasting and especially muscle wasting. 27

39 after rehabilitation. Doctoral dissertation. University of Ljubljana, Faculty of Medicine, 2016 Table 2 Most common definitions of MetS Central obesity Hyperglycemia Dyslipidemia WHO (1998) (30) EGIR (31) BMI >30 kg/m² and/or WHR >0.90 in men or WHR>0.85 in women IGT, IFG, or T2DM or low insulin sensitivity TG 150 mg/dl and/or HDL <35 mg/dl in men or <39 mg/dl in women c WC >94 cm in men and >80 cm in women NCEP ATP III (2001 and 2005) (32, 33) WC >102 cm in men and >88 cm in women AACE (2003) (34) IDF (35) BMI 25 kg/m² WC 94 cm for Europid men and 80 cm for Europid women, with ethnicity specific values for other groups a Alberti (2009) (36) WC >102 cm in men and >88 cm in women, with ethnicity specific values IGT or IFG 110 mg/dl b c IGT or IFG 100 mg/dl 100 mg/dl c TG 150 mg/dl and/or HDL <40 mg/dl in men or <50 mg/dl in women or specific treatment for this lipid abnormality TG 150 mg/dl c HDL <40 mg/dl in men or <50 mg/dl in women c TG 150 mg/dl and HDL <40 mg/dl in men or <50 mg/dl in women TG 150 mg/dl or specific treatment for this lipid abnormality HDL <40 mg/dl in men or <50 mg/dl in women or specific treatment for this lipid abnormality 130/85 mmhg 130/85 mmhg or Hypertension 140/90 mmhg 140/90 mmhg 130/85 mmhg c treatment of previously diagnosed hypertension TG 150 mg/dl c HDL <40 mg/dl in men or <50 mg/dl in women c 130/85 mmhg c Other Microalbuminuria d - - Other features of IR e - - MetS diagnose Hyperglycemia plus any two of the other criteria Plasma insulin >75 th percentile plus any two of the other criteria Three or more of the five criteria Hyperglycemia plus any of the other criteria Central obesity plus any two of the other criteria Three or more of the five criteria a Ethnic specific cutoffs are: Europids, Sub-Saharan Africans, Mediterranean and Middle East: male 94cm, female 80cm; South Asians, Chinese and South and Central Americans: male 90cm, female 80cm; Japanese: male 85cm, female 90cm. b In the revised NCEP ATP III definition of 2005 criterion for hyperglycemia was glucose level of 100 mg/dl. c In the revised NCEP ATP III definition of 2005 and in the definition of Alberti, treatment for elevated glucose, elevated triglycerides, reduced HDL-C and antihypertensive drug treatment in a patient with a history of hypertension were also positive criteria. d Urinary albumin excretion of 20 µg/min or albumin-to-creatinine of 30 g/g. e Includes family history of type 2 diabetes mellitus, hypertension and cardiovascular disease, polycystic ovary syndrome, sedentary lifestyle, advancing age, and ethnic groups susceptible to type 2 Diabetes Mellitus 28

40 2.2.2 WHOLE BODY AND MUSCLE WASTING Body wasting is usually a result of a negative energy balance, in which the energy input does not cover the energy expenditure. A normal response to such decreased energy input (starvation) is a decreased resting energy metabolism. In numerous chronic diseases however, the resting energy metabolism is increased (37). Decreased appetite as well as impaired gastrointestinal absorption is common for example in patients with CHF (6). These features further contribute to the imbalance between energy needs and input. The clinical manifestation of such imbalance is inevitable weight loss and wasting of different tissue compartments fat, muscle and bone. Wasting of skeletal muscle is due to a net catabolic state, which may result from an imbalance in muscle protein breakdown and synthesis (38). IR and chronic inflammation, frequent features of chronic diseases, can contribute to this catabolism. Sarcopenia is defined as the process of decreasing muscle mass that occurs with advancing age and can be accompanied with an increase or no change in fat mass (39). The exact prevalence of muscle wasting is difficult to assess due to the different cut-offs in use. Traditionally, fat-free mass index (FFMI) below the 10 th percentile for healthy subjects was used (<17 kg/m 2 for males, <15 kg/m 2 for females in Caucasians) (40). Nowadays, with the advances of body composition imaging, regional changes in body composition are also possible to evaluate. Hence, the appendicular skeletal muscle mass index (ASMI; skeletal muscle mass per height squared (kg/m 2 )) has been developed, based on the cutoff of 2 standard deviations below the mean of healthy persons. This cut-off of ASMI - <7.23 kg/m 2 for males, 5.67kg/m 2 for females are now most commonly used for defining sarcopenia in the elderly (39). In aging adults, sarcopenia is common and is associated with mobility disorders, impaired ability to perform activities of daily living, loss of independence and increased mortality (39). Besides the changes in muscle quantity, which results in muscle mass loss, alterations in the quality of the muscle are also observed in chronic diseases. These can lead to further worsening of IR, impaired energy efficiency and hidden fat accumulation (6). In turn, skeletal muscle wasting can be balanced by increased fat mass and thus no net change in total body weight is observed. This is why assessment of body composition is essential to unravel these metabolic abnormalities. 29

41 2.2.3 BODY COMPOSITION ASSESSMENT Various methods exist for assessing body composition. In clinical practice, two most common techniques are bioelectrical impedance analysis (BIA) and dual x-ray absorptiometry (DEXA). BIA is a non-invasive, simple method which evaluates the electrical impedance of different body tissues and calculates total body water. This value is then used to calculate fat-free mass in the body and indirectly fat mass. BIA is therefore a two-compartment model for body composition evaluation. Although simple and readily available, BIA also has some limitations; for example, as it evaluates the water content in the body, the hydration state of the patient or presence of edema can affect its results (41, 42). A second method is DEXA. An x-ray beam passes through the body and based on different absorption, the content of fat mass, lean mass and bone mass is evaluated. DEXA is therefore a three-compartment model for evaluating body composition, as it also offers information on bone mass, which is not available with BIA measurements It also allows analysis of specific body part, for example skeletal muscle mass alone and hence evaluation of SMI. Although more informative, DEXA is a more invasive measurement due to the radiation exposure, although very low. The apparatus is not portable and it requires a higher expertise of the technician (43). Figure 2 Body composition analysis report by DEXA 30

42 2.3 COPD AS A SYSTEMIC DISEASE In the last decade, evidence is accumulating that the effects of COPD reach beyond the lungs and are manifested systemically (44). COPD has been defined as a heterogeneous and multicomponent disease (45) and the impact of its comorbidities to the disease burden as has been recently recognized also in the GOLD definition of COPD (1). Comorbidities affect not only patient disease burden, disability and overall health status (46), but contribute also to the rates of hospitalizations as well as mortality (47). In fact, COPD patients mainly die because of non-respiratory complications, including cardiovascular diseases, cancer and other causes (3). The most common systemic features and comorbidities include chronic heart failure and other cardiovascular disease, type 2 diabetes, depression, anemia, MetS, skeletal muscle weakness, cachexia and osteoporosis (48). 2.4 CARDIOVASCULAR AND METABOLIC RISK IN COPD COPD patients have an approximate 2 to 3 fold increased cardiovascular risk compared to patients without COPD irrespective of disease severity and smoking status (49). The most common cardiovascular comorbidities are arrhythmia, chronic heart failure, acute myocardial infarction, peripheral vascular disease and stroke, all of which are more prevalent in COPD patients compared to controls (49). This higher occurrence has been related to intrinsic factors, such as low-grade systemic inflammation, oxidative stress and hypoxemia as well as extrinsic factors, such as an unhealthy lifestyle. 31

43 Figure 3: Drivers of cardiovascular and metabolic risk in COPD. Adopted from Van den Borst, AJRCCM 2013 (7) 2.5 DRIVERS OF CARDIOVASCULAR AND METABOLIC RISK IN COPD INSULIN RESISTANCE AND METABOLIC SYNDROME Prevalence and contributing factors T2DM is a well-known and frequent comorbidity in COPD patients (50), however data on the prevalence of IR in COPD is scarce. Available studies suggest that IR is more common in COPD patients compared to healthy controls. Bolton and colleagues showed that IR (assessed by HOMA index) in non-hypoxemic patients with COPD is greater than in healthy subjects (51, 52). They also showed that IR was related to systemic inflammation (51). In a recent study by Van den Borst, a higher HOMA-IR and CRP was also observed in patients with mild to moderate COPD compared to healthy controls. A weak association between HOMA-IR and CRP was found (52). Skyba and colleagues also reported a higher prevalence of IR in patients 32

44 COPD with increased BMI, which was associated with higher expression of pro-inflammatory genes (53). Our group has recently published a systematic review on the prevalence of MetS in COPD patients. The overall mean prevalence of MetS in COPD was 34% (21% to 58%). COPD patients with MetS were more frequently females, have higher BMI, higher FEV 1 compared to COPD patients without MetS. A hand-full of studies reported higher serum CRP and IL-6 levels in COPD patients with MEtS compared to patients without MetS. The most prevalent MetS components were hypertension (56%), hyperglycemia (44%) and abdominal obesity (39%). Low HDL-cholesterol was significantly less prevalent in COPD patients compared to controls (24). Abdominal obesity is a prevalent component of Mets in COPD patients. However, two studies confirmed that visceral fat mass is higher in non-obese COPD patients compared to controls (54, 55). It is important to note that these patients were not obese nor had increased waist circumference, which implies the presence of hidden (visceral) fat accumulation. Adipocytokines excreted from fat tissue function both locally and systemically and contribute to the low-grade systemic inflammation as well as IR in COPD patients. Fat tissue and abdominal obesity therefore probably have a significant impact on the prevalence of MetS in COPD. However, other factors, including genetics (56), medications (especially glucocorticoids) and physical inactivity may also play a role (24). Effect on outcome Data on the effect of MetS on outcome are somehow contradicting and not well-studied. Some studies have shown that MetS in COPD is associated with more dyspnea and more comorbidities (57). Associations between exacerbations of COPD and its duration with MetS were also observed (58). Abdominal obesity was suggested to be a strong predictor of lung function impairment (59). On the other hand, the so called obesity paradox has been observed in COPD patients, suggesting that patients with higher BMI have better survival (29). Studies have also concluded that exercise capacity (assessed by peak symptom-limited VO2) is generally preserved in COPD patients with mild to moderate obesity. It has been argued that increased inspiratory capacity and lower operating lung volumes may minimize the extra work of pulmonary muscles and consequently decrease the intensity of dyspnea during exercise (60). This conclusion challenges the common belief that obesity has a deleterious effect on exercise capacity and dyspnea in COPD patients (61). It thus seems that the impact of MetS and obesity on the functional capacity and prognosis of COPD patients cannot be generalised, but should be viewed in relation to disease severity and metabolic and muscoskeletal abnormalities. 33

45 2.5.2 MUSCLE WASTING AND MUSCLE METABOLISM ABNORMALITIES Although MetS and IR metabolic abnormalities typically linked to obesity are common in COPD, tissue wasting and malnutrition are also highly prevalent. These two body composition phenotypes resemble the classical division of COPD patients into pink puffers and blue bloaters. Pink puffers, the emphysematous type, were characterized by low body mass index and weight loss, while in the blue bloaters, usually overweight, chronic bronchitis prevailed (62). Prevalence and contributing factors Very low BMI (<20 kg/m2) is seen in approximately one fifth of patients, but lean body mass depletion (defined as fat free mass index <16 kg/m 2 for males and <15 kg/m 2 for females) is twice more common in these patients (25). The reported prevalence of muscle atrophy depends on the cut-off used and is reported in 4 to 35% of COPD patients (29). Applying the 10 th percentile cut-off, low FFMI is more common in higher disease stages, reaching a prevalence of 50% in patient with GOLD 4, and is more common in females (29). Furthermore, low FFMI has been reported in one quarter of normal-weight COPD patients (29). Loss of muscle mass in COPD is a consequence of disease-related and lifestyle-related factors. Disuse, chronic hypoxia, malnutrition, chronic inflammation and glucocorticoids use all contribute to a decrease in muscle mass. The underlying cause for muscle wasting is an imbalance in muscle protein synthesis and results in a general catabolic state. A presentation of the molecular mechanisms involved is beyond the scope of this thesis; for details see the review by Langen et al (38). Besides the catabolic state, a shift from oxidative towards glycolytic fibers and decreased oxidative enzyme capacity is also observed in sarcopenic COPD patients (63). A shift from type I fibers to type II fibers is typical for COPD and in contrast to normal ageing. The shift is more pronounced in higher disease stages and was associated with lower BMI (64). The mitochondrial mass and function may also be decreased. Altogether, in COPD patients, especially those with lower BMI, the loss of muscle mass is closely related to loss of muscle oxidative phenotype. The fibre type switch reduces the capacity for fat oxidation in response to increased fatty acid availability; this in turn alters energy substrate preference from primarily fat to glucose utilisation (65). Given that skeletal muscle is the primary site of glucose utilisation (65), sarcopenia can promote IR directly and independently of COPD (66). In fact, the metabolic changes observed in skeletal muscle of COPD patients show striking similarities with changes observed in diabetic patients. Decreased mitochondrial respiration and reduced mitochondrial 34

46 biogenesis is comparable between under/normal weight COPD patients and obese diabetic patients (67-69). Similarly, diabetic patients show a reduced proportion of type I muscle fibers compared to controls (70). Therefore, sarcopenia represents a vulnerable COPD phenotype as it not only affects physical functioning, but may also increase cardiometabolic risk. Effect on outcomes Muscle wasting significantly affects outcomes of COPD patients. Muscle wasting reduces physical activity, contributes to exercise intolerance and reduced the capacity to perform daily activities. It also contributes to a lowered health-related quality of life (HRQoL) in COPD patients. Decreased muscle mass also increases the probability of hospitalizations (71). Most importantly, muscle atrophy is associated with a shorter life expectancy independent of airflow impairment, fat mass or BMI (29, 72, 73) LOW GRADE SYSTEMIC INFLAMMATION The systemic effects of COPD have been closely linked to low grade systemic inflammation. Increasing evidence suggests that systemic inflammation is present also in stable COPD patients. Compared to healthy controls, stable COPD patients show higher levels of CRP, fibrinogen, leucocytes and TNF-alpha, which indicates a persistent inflammatory process (74). It is not yet clear what is the source of this inflammation. At first it was believed it is a spill-out of the inflammatory mediators from the lung department, however it is also possible that other inflammatory organs, such as adipose tissue, and COPD-related factors such as hypoxia contribute to the systemic inflammation (75). Low-grade systemic inflammation in COPD has been linked to various extra-pulmonary manifestations, such as weight loss, cachexia and cardiovascular diseases (44). Recent data suggest that different inflammatory mediators are more common in different comorbidities profiles. From the so called comorbidity clusters recently presented by Vanfleteren et al, the cardiovascular cluster, characterized by hypertension and atherosclerosis, showed elevated IL-6 levels, while the metabolic cluster with more obesity, dyslipidemia and hyperglycemia, showed elevated TNF receptors (44). The causes of and relations between low grade systemic inflammation and extra-pulmonary COPD manifestations are not yet clear, but are a topic of intense research. 35

47 2.5.4 LIFESTYLE Smoking Smoking is an independent risk factor for both COPD and cardiovascular disease. Recent finding suggest that smoking contributes to more insulin sensitivity in the skeletal muscles, increased abdominal obesity, which in turn increases cardiovascular and cardiometabolic risk (76, 77). COPD patients as well as age-matched smokers without COPD have a lower muscle and fat mass compared to non-smoking controls (78). Smokers without COPD also show a loss of muscle oxidative phenotype (79). Chronic exposure to smoking also induces systemic inflammation, hence contributing to all previously presented drivers of increased risk (80). Physical (in)activity COPD patients have a predominantly sedentary lifestyle with fewer and shorter periods of physical activity compared to controls (63, 67). Symptoms such as dyspnea and exercise intolerance contribute to the inactivity. Studies suggest that decreased physical activity might be implicated in the development of COPD, as physically active smokers and non-smokers have an attenuated FEV1 decline. Physical inactivity also induces numerous metabolic abnormalities, such as IR of the muscle, increase in intramyocellular lipid content, muscle atrophy and muscle fiber shift, etc (81). The effect of physical inactivity on metabolic health in COPD patients is likely to be considerable. Diet and nutrition Although obesity is frequent in COPD patients as discussed earlier, many patients are also malnourished. Dyspnea, fatigue and depression are common symptoms and comorbidities in COPD that contribute to low appetite (82). The latter together with low dietary intake which does not balance the increased energy requirements contributes to weight loss (37, 82). On the other hand, studies have shown that COPD patients consume a poor and rather unhealthy diet, rich in saturated fats, while the intake of vitamins, eg. vitamin D and poly-unsaturated faty acids is below recommended (83). The relation between a poor, so called Western diet and increased cardiometabolic risk is well known, hence dietary habit of COPD patients can contribute to the elevated cardiometabolic risk observed in this patient population. 36

48 2.6 PULMONARY REHABILITATION Treatment of COPD has been, for a long time, limited to symptomatic pharmacological treatment of airflow obstruction. With the increasing awareness of the multisystem manifestations of COPD and its negative influences on patient outcomes, nonpharmacological treatments such as pulmonary rehabilitation have been gaining interest. Pulmonary rehabilitation is defined as a comprehensive intervention based on a thorough patient assessment followed by patient-tailored therapies that include, but are not limited to, exercise training, education, and behavior change, designed to improve the physical and psychological condition of people with chronic respiratory disease and to promote the longterm adherence to health-enhancing behaviors (84). Pulmonary rehabilitation is a fundamental part of the integrated care for COPD patients and is included in the GOLD guidelines for COPD management (3). The GOLD guidelines assessed the improvement in exercise performance, HRQoL and reduction in health care utilization by pulmonary rehabilitation as evidence grade A. Although the benefits of pulmonary rehabilitation are well-recognized, studies have shown that rehabilitation as an intervention is largely undervalued and underused (85). 37

49 Figure 4 Spectrum of support for COPD patients showing pulmonary rehabilitation as an important part of integrated care (74) Exercise training is the cornerstone of pulmonary rehabilitation. Training modalities include interval training, strength training, upper limb training, and transcutaneous neuromuscular electrical stimulation. Training intensity is recommended to be symptom-limited and adopted to patient s physical capacity. The optimal duration of pulmonary rehabilitation has not been strictly defined, however programs including at least 20 training sessions have shown to benefit patients the most (84). Exercise training, including resistance and endurance training, has been shown to cause structural and functional alterations in muscles in healthy subjects. The observed alterations included changes in muscle oxidative enzyme activity, increased number of mitochondria and muscle capillarization (86). In COPD, aerobic training improves muscle oxidative capacity and reduced fatigue, while resistance training induces muscle hypertrophy (87-89). Although it has been shown that pulmonary rehabilitation improved exercise intolerance, decreases symptom burden and improves HRQoL (90), it has also been observed that not all 38

50 patients benefit equally from PR. In the current time of limited resources, calls are being made to better characterize COPD patients who benefit the most from pulmonary rehabilitation and find new targets and additional interventions for those subgroups of patients that don t reach the desired goals. The need for a more personalized pulmonary rehabilitation programs is being highly emphasized (91, 92). In parallel, the increasingly high prevalence of comorbidities and especially cardiovascular risk in COPD patients calls also for studies focusing on the effect of pulmonary rehabilitation on cardiometabolic risk in COPD patients. 39

51 3 NAMENI IN CILJI NAMEN Oceniti metabolični profil bolnikov s KOPB pred rehabilitacijo in po njej CILJI Oceniti prevalenco metaboličnih dejavnikov tveganja glede na različne fenotipe telesne sestave Oceniti spremembe metaboličnih dejavnikov pred rehabilitacijo in po njej, Oceniti povezave med metaboličnimi spremembami in kliničnimi izidi po rehabilitaciji; Pripraviti protokol prospektivne intervencijske raziskave za optimizacijo pljučne rehabilitacije. HIPOTEZE Prevalenca metaboličnega sindroma je visoka pri bolnikih s KOPB, vendar ni povezana z indeksom telesne mase. Prevalenca spremenjene presnove glukoze je pri bolnikih s KOPB visoka in je povezana z nizko vrednostjo indeksa puste telesne mase. Štiritedenska visoko intenzivna rehabilitacija izboljša občutljivost na inzulin pri bolnikih s KOPB. Štiritedenska visoko intenzivna rehabilitacija izboljša vnetni profil bolnikov s KOPB. 40

52 4 AIMS AND OBJECTIVES AIM To provide a comprehensive picture of the metabolic profile of COPD patients prior to and after rehabilitation OBJECTIVES To estimate the prevalence of metabolic disorders in COPD patients referred for pulmonary rehablitation in relation to different body composition phenotypes To evaluate the responsiveness of metabolic parameters to rehabilitation To investigate the associations between metabolic changes with clinical outcomes in COPD patients To prepare a protocol for a prospective interventional study to optimize metabolic modulation during rehabilitation HYPOTHESES The prevalence of MetS is high in patients with COPD but is not associated with BMI. MetS The prevalence of altered glucose metabolism is high in patients with COPD and is associated with low fat-free mass index. High intensity four-week rehabilitation improves insulin sensitivity in patients with COPD. High intensity four-week rehabilitation improves the inflammatory profile in patients with COPD. 41

53 5 MATERIALS AND METHODS The patients The pulmonary rehabilitation program is developed for patients with pulmonary diseases and mostly hosts (advanced) COPD patients. Patients can be referred to the rehabilitation unit by their GP or by a specialist, eg. after being hospitalized for an exacerbation. Study inclusion criteria Confirmed diagnosis of COPD, Stable disease, Eligible for PR program. Study exclusion criteria Patients were excluded from the study if the following criteria applied: Hospitalization due to a COPD exacerbation within the last 4 weeks, implanted pace maker (contraindication for BIA measurement), no data available on baseline body composition or metabolic risk factor prevalence, refused the collaboration or if they were not able to follow the rehabilitation program due to any cause. Patients experiencing an exacerbation during the rehabilitation program and patients who did not complete the rehabilitation program were excluded from follow-up analysis. Study design In this prospective observational study we recruited COPD patients from the in-patient pulmonary rehabilitation unit between July 2012 and August Patients were required to be in a stable disease state, free from exacerbation in the previous 4 weeks prior to start of the programme. Patients were excluded from the study if their metabolic risk factor data was unavailable for baseline analysis. Patients experiencing an exacerbation during the rehabilitation program were excluded from follow-up analysis as they did not attend the whole rehabilitation program (Figure 5). The study protocol was reviewed and approved by the Slovenian National Ethics Committee. Informed consent was obtained from all patients. The study is registered at clinicaltrials.gov (NCT ). 42

54 138 patients invited to attend pulmonary rehabilitation 6 patients did not respond to the invite: - 3 personal reasons - 2 exacerbation - 1 logistic reasons 7 patients excluded during baseline testing: - 4 not interested - 2 psychiatirc comorbidities - 1 rehabilitation duration too long 125 patients included in pulmonary rehabilitation 13 patients without data on metabolic risk factors 112 included in baseline analysis 21 exacerbations 6 unable to follow program 85 successfully finished PR 15 patients without body composition results 17 patients not fasted at sample withdrawal 70 patients included in body composition analysis 68 patients included in HOMA-IR index analysis Figure 5 Study design outline 43

55 Pulmonary rehabilitation program The 5-week program consisted of an extensive baseline assessment, 4 weeks of intervention and a post-rehabilitation assessment. In total, patients received 20 training days that included at least one session of combined endurance and resistance training with the following modalities: a) interval training on a cycle-ergometer with alternating 40-50% maximal capacity and 80% maximal capacity, 30 minutes per day, b) treadmill training with alternating slope, 20 to 30 minutes per day, c) transcutaneous electrostimulation of thigh muscles, d) upper-limb and trunk muscles training, e) respiratory muscle training. Exercise intensity was adjusted weekly during patient supervision visits according to patient performance. In addition to exercise training, patients also received nutritional assessment with counseling and support through diets tailored to patient needs and preferences as well as counseling to quit smoking. Furthermore patients also received social and psychological support in line with the latest ERS/ATS recommendations on pulmonary rehabilitation (84). At baseline, all patients had a cardiac examination which included a non-invasive assessment with echocardiography and exercise testing in order to rule out any relevant cardiovascular contraindications for exercise training and rehabilitation. All training programs were adjusted to the cardiovascular and pulmonary performance levels of the individual patients in order to meet the patients training objectives. During the rehabilitation, patients were under daily medical supervision. Figure 6 Pulmonary rehabilitation program 44

56 2.2 Baseline clinical assessment The baseline clinical assessment included lung function testing, body composition analysis, physical performance testing, cardiovascular system evaluation and nutritional assessment. Furthermore, multiple laboratory biomarkers were routinely assessed from fasting blood samples at before and after rehabilitation. Lung function Spirometry was performed to determine forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC) and their ratio (FEV1/FVC) according to European Respiratory Society guidelines (93). Based on these measurements, the patients disease severity was classified according to the Global Initiative for Chronic Obstructive lung Disease (GOLD) criteria (3). Metabolic and cardiac comorbidities At baseline, participants underwent diagnostic work-up for selected metabolic and cardiac comorbidities. Diabetes Mellitus Type II (T2DM) was diagnosed using IDF guidelines (35). Arterial hypertension, heart failure, ischemic heart disease, and valve disease were diagnosed per relevant ESC guidelines (94). Body composition Body height, and waist and hip circumference were measured to the nearest cm. Body weight was measured to the nearest 0.1 kg using a standard balance beam scale. Based on the height and weight, body mass index (BMI) was calculated as weight/height squared (kg/m2). Patients were categorised as underweight (BMI below 20 kg/m2), normal weight (BMI between 20 and 25 kg/m2), overweight (BMI between 25 and 30 kg/m2) and obese (BMI above 30 kg/m2). Body composition was assessed using dual-energy X-ray absorptiometry (DXA) and bioelectrical impedance (BIA). DXA was assessed baseline only using Hologic QDR Series Explorer bone densitometer (Hologic Inc, MA, USA). BIA was performed using a tetra polar RJL System Quantum II (RJL Systems, Clinton TWP, MI, USA) with the emission of a low electrical current (500 to 800 ma and 50 khz). The patient was studied in the supine position without shoes and stockings and after 10 minutes of rest. The patient s upper and lower-limbs were arranged at approximately 30 from the midline. Electrodes were connected to the hands (wrist and middle finger) and feet (ankle and above the knuckle of the toe) after the areas were cleaned with alcohol. The patient s right side was standardized for the test. The instructions for use of the equipment were obtained from the manufacturer. We evaluated body fat, lean mass and body water. BIA was performed at 45

57 baseline and at the end of the rehabilitation program using a tetra polar RJL System Quantum II (RJL Systems, Clinton TWP, MI, USA) with the emission of a low electrical current (500 to 800 ma and 50 khz). Fat free mass index (FFMI) was calculated by dividing fat free mass by body height squared. DEXA was performed in supine position (QDR Series Explorer). This technique is based on the principle of different attenuation of ionizing radiation (x-rays) upon passing through different body tissues. The x-ray source releases a beam of two different energies, therefore allows assessment of different body compartments and differentiation between the mineralized tissue (bone), fat and fat free mass. Appendicular skeletal muscle index (ASMI) was calculated as the lean mass of the extremities divided by body height squared. Nutritional status Nutritional status has been assessed using the Mini Nutritional Assessment Test (Appendix 1). This is a questionnaire aiming at defining the patient s eating habits and recent body weight changes and assessing the risk of malnutrition. The questionnaire results categorize patients into malnourished (MNA below 17 point), at risk of malnourishment (MNA between 17.5 to 23.5 points) and well nourished (MNA points 24 and above). Laboratory biomarkers Blood samples were collected after an 8 hour overnight fast. Samples were separated by centrifugation at 4 C at 3,000 rpm for 10 minutes and if not analyzed straightaway, subsequently frozen and stored at -80 C until assayed. Metabolic markers including fasting plasma glucose, insulin, cholesterol and triglycerides were evaluated. Routine biochemistry was performed in the University Clinic Golnik laboratory according to standard procedures. Insulin resistance Fasting insulin levels were determined from frozen serum samples using Immunochemiluminescention-assay (ILMA). The analysis was performed in the Department of Clinical Chemistry, Reinier de Graaf Hospital in Delft, The Netherlands. HOMA-IR index was calculated with the formula proposed by Matthews (95): fasting glucose (mmol HOMA IR = L ) fasting insulin (miu L )

58 Amino acids and vitamin D analysis Amino acid levels and vitamin D levels were assessed from frozen serum samples. After precipitation of proteins and polypeptides with perchloric acid, the sample was centrifuged. In the supernatant the content of the individual amino acids was determined by UFLC (Shimadzu Corporation) using a pre-column derivatization with o-phtaldialdehyde and fluorimetry as detection. The analysis was performed in the Department of Clinical Chemistry, Reinier de Graaf Hospital in Delft, The Netherlands. Due to lack of samples, these analysis were assessed at baseline only. Inflammatory profile The inflammatory profile was assessed by measuring CRP levels and interleukin-6 (IL-6) levels. CRP was evaluated from serum samples as part of the routine biochemistry at the Clinic Golnik at start and end of the rehabilitation program. Measurement of IL-6 was performed in a well-characterized subgroup of patients (significant improvement in 6MWT after rehabilitation, CRP levels below 10 at start and end of program). IL-6 levels were measured using enzyme-linked immunosorbent assay (R&D Systems). The analysis was performed at the Department of Pharmacology, Maastricht University, the Netherlands. Muscle biopsy analysis Biopsies were obtained from quadriceps muscle (vastus lateralis) of the dominant leg by needle biopsy, at least 20 hours after the last exercise test. The biopsy site was cleaned and draped, to ensure for sterile conditions. After anesthetizing the skin and underlying tissues with lidocaine, a 1cm incision allowed for introduction of a Bergström needle to harvest the skeletal muscle tissue. Obtained samples were snap frozen in liquid nitrogen, and stored in aliquots at -80 C for further analyses. Histological analysis of muscle samples was planned. This, however, was abandoned due to artifacts in biopsy samples that prevented reliable assessment of fiber typing. (Figure 7) As an alternative, myosin heavy chain 1 (MyHC1) expression in RNA was used. Muscle fiber type I content in COPD correlates with MyHC1 expression in RNA. (Figure 8, unpublished data from Maastricht Laboratory) We therefore used MyHC1 levels, which we were able to extract, as a surrogate for muscle fiber typing. 47

59 Figure 7 Examples of muscle biopsies prepared for fiber typing. A: High-quality muscle biopsy, fiber typing possible. B: Muscle biopsy with visible artefacts, fiber typing not possible. Figure 8 Association between percentage muscle type I fibers and MyHCI expression in COPD and controls. Unpublished data from Maastricht Laboratory. qrt-pcr Tissue was homogenized and RNA was isolated by TRI-reagentl/Chloroform extraction and subsequently precipitated from the aqueous phase using glycogen containing isopropanol. RNA was reconstituted in 30 μl RNA storage solution and stored at -80 C. RNA concentrations were measured spectrophotometrically. 400 ng RNA was diluted in nuclease free H 2O and reverse transcribed to cdna using the Tetro cdna synthesis kit (Bioline) according to the manufacturer s instructions. qrt-pcr primers were designed based on Ensembl transcript 48

60 sequences and ordered from Sigma Genosys. qrt-pcr reactions contained Sensimix SYBR & ROX and primermix, and were run in a 384 well white opaque plate on a LightCycler 480 system. Melting curves were analyzed to verify specificity of the amplification, and relative quantity of the targets was assessed by LinRegPCR software. Three reference genes (RPLP0, B2M, Clyclophilin A) were used to calculate a GeNorm correction factor, which was used to normalize expression levels of the target genes. Western Blot Tissue was homogenized in 600 μl of IP lysis buffer (50mM Tris, 150 mm NaCl, 10% glycerol, 0,5% Nonidet P40, protease and phosphatase inhibitors) with a Micro Tissue Homogenizer. After homogenization, samples were incubated on a tube rotator at 4 C for 30 minutes and centrifuged at 14000g at 4 C for 30 minutes. The supernatant was stored at -80 C until analysis. Total protein concentration was determined using BCA Protein Assay kit according to the manufacturer s instructions. For Western Blot analyses, 4x Laemmli sample buffer (0.25M Tris-HCL ph6.8; 8% (w/v) SDS; 40% (v/v) glycerol; 0.4M DTT and 0.02% (w/v) Bromophenol Blue) was added and samples were heated to 100 C for 5 minutes. 10 μg protein was separated on a Criterion XT Precast 4-12% Bis-Tris gel with XT MOPS running buffer by gel electrophoresis. Proteins were transferred to a nitrocellulose membrane by electroblotting at 100V for 60 minutes in transfer buffer (25 mm Tris, 192 mm Glycine, 20% (vol/vol) methanol). For total protein detection, the membrane was stained with PonceauS solution (0.2% Ponceau S in 1% acetic acid; Sigma-Aldrich Chemie) and imaged using the Amersham imager 600RGB. The membrane was blocked for 1 hour at room temperature in Tris-buffered saline with Tween20 (TBST; 20 mm Tris, 137 mm NaCl, 0.1% (vol/vol) Tween20, ph 7.6) containing 3% (w/v) nonfat dry milk. The membranes were washed in TBST, followed by overnight incubation at 4 C with primary antibody diluted in TBST with 3% BSA or nonfat dry milk. Membranes were incubated with a peroxidase conjugated secondary antibody solution for 1 hour at RT, visualized by chemiluminescence using Supersignal West PICO or FEMTO Chemiluminescent Substrate according to the manufacturer s instructions, and detected using the using the Amersham imager 600RGB. Signals were quantified with Image Quant software. For analyses, samples from COPD patients were randomly distributed within and between blots, and reference samples were loaded onto all blots to correct for between blot variations. 49

61 Physical performance and HRQoL Physical performance was evaluated with the 6-minute walking test (6MWT), Incremental Shuttle Walk Test (ISWT) and maximum cycling ergometry (CPET) according to standardised protocol. (96) HRQoL was determined through the Saint George s Respiratory Questionnaire (SGRQ) scores (Appendix 2). 2.3 Rehabilitation outcome evaluation The effectiveness of the rehabilitation program was evaluated by measures of physical performance and HRQoL. The improvement in physical performance and HRQoL was calculated as the absolute value as well as the percentage of participants attaining the minimal clinically important difference (MCID; 25 m for 6MWT(97), 47.5 m for ISWT (98), 4 Wats for maximum CPET (99), 4 points for SGRQ (100). 2.4 Body composition profiles and metabolic risk factors Sarcopenia and non-sarcopenia was the main body composition profile analysed in our study. Sarcopenia was defined by the appendicular skeletal muscle index cut-offs (<7.23 kg/m2 for males and <5.67 kg/m2 for females) (39). Metabolic risk factors included MetS and its components, IR, lipids levels, amino acids and vitamin D levels. MetS was defined using the International Diabetes Federation definition (35). IR was defined based on the HOMA-IR values. IR was defined as an HOMA-IR higher than 2.5 (95). Study endpoints and statistical analysis The primary outcome is the change in IR (assessed using HOMA-IR index) from baseline to end of rehabilitation. Secondary outcomes include: prevalence of other metabolic disorders (fasting glucose, lipids), prevalence of other metabolic disorders (fasting glucose, lipids) in relation to body composition, changes in other metabolic markers (fasting glucose, lipids), inflammatory markers and metabolomics before and after the rehabilitation program, correlation of metabolic disorders with clinical outcomes. 50

62 Continuous variables are presented as mean values ± standard deviation. Categorical variables are presented as absolute numbers. To compare values between the two groups, independent samples t-test was used for continuous variables and Chi-square for categorical variables. For comparison of data before and after rehabilitation, paired samples t-test was used to compare normally distributed data and Wilcoxon sign-rank test for non-normally distributed data. In correlation analysis, Pearson s correlation coefficient was reported for normally distributed data and Spearman s rank correlation coefficient for non-normally distributed data. All analyses were performed with SPSS 21.0 (SPSS Inc., 2007, Chicago, IL, USA) and a p-value of <0.05 was considered statistically significant. 51

63 6 RESULTS 6.1 GENERAL CHARACTERISTICS OF INCLUDED PATIENTS During the study inclusion, 138 COPD patients were invited to attend the pulmonary rehabilitation program at the Clinic Golnik and 125 patients actually started the program (Figure 5). Of these, 13 patients were excluded from data analysis because of lack of data on body composition or metabolic risk factors. In total, 112 were included in the study analysis. (Table 3) Patients were older (mean age 66 years) and predominantly male (66%). The majority were in GOLD stages III (52%) and IV (31%) and most of them reported to be current or ex-smokers (82%). Approximately half of the patients were overweight or obese, while a minority was underweight. Based on the mini-nutritional assessment test, almost half of all the patients were malnourished or at risk of malnourishment. Diabetes as a comorbidity was present in 23% of patients, of which half were newly diagnosed at start of the rehabilitation program. Heart failure was present in one quarter of all patients. On average, patients managed to walk 335m during the 6-minute walk test and achieved an average of 40 wats during maximum cycling tests. The average score for HRQoL evaluation was 40 points out of 100. The most frequent medications used by the included patients are presented in Table 4. 52

64 Table 3 General characteristics of included patients All patients (N=112) Sex (male, %) 66% Age (years, mean) 66±8 Smokers, current or ex (%) 82% FVC (% pred) 81% FEV1 (% pred) 38±14 FEV1/FVC 36±13 GOLD stage 2 17% 3 52% 4 31% MNA (points) 23±4 MNA category Malnourished 13% At risk of malnourishment 34% Well-nourished 53% BMI (kg/m 2 ) 25±5 BMI category (%) Underweight 14% Normal weight 39% Overweight 23% Obese 24% Total body mass (kg) 71±16 FMI (kg/m 2 ) 8.6 ±3.1 FFMI (kg/m 2 ) 16.7±3.1 ASMI (kg/m 2 ) 6.6±1.2 Bone mass (kg) 2.1±0.6 Waist circumference (cm) 97±13 WHR 0.98±0.08 CRP (mg/l) 3.0 ( ) Diabetes Mellitus Type II 23% Heart failure 25% Atrial fibrillation 7% Ischemic heart disease 10% 6MWT (m) 335±105 ISWT (m) 253±113 Maximum CPET (W) 58±22 HRQoL (SGRQ points) 40±13 53

65 Table 4 Medication use All patients (N=112) Inhaled corticosteroids 70% Long-acting beta agonists 80% Short-acting beta agonists 71% Long-acting muscarinic agonists 71% Theophylline 15% Angiotensin converting enzyme (ACE) inhibitors 32% Angiotensin receptor blockers 7% Calcium channel blockers 10% Diuretics 27% Beta-blockers 14% Statins 21% Oral antidiabetics 9% Long-term oxygen therapy 34% 6.2 INSULIN RESISTANCE AND METABOLIC SYNDROME The prevalence of metabolic risk factors in the whole group level is presented in table 5. IR and MetS was present in 59% and 47% of patients, respectively. The most prevalent MetS components were abdominal obesity (72%), hypertension (86%) and hyperglycemia (69%). Hypertriglyceridemia and low HDL-cholesterol levels were present in approximately 10% of patient. Table 5 Prevalence of metabolic risk factors at baseline All patients (N=112) MetS 47% Abdominal obesity 72% Arterial hypertension 86% Hyperglycemia 69% Hypertriglyceridemia 12% Low HDL-cholesterol 10% IR 59% HOMA-IR 3.2 ( ) Fasting glucose (mmol/l) 5.9 ( ) Fasting insulin (miu/l) 11.1 ( ) Total cholesterol (mmol/l) 5.1±1.1 Increased total cholesterol 48% LDL-cholesterol (mmol/l) 3.1±1.0 Increased LDL-cholesterol 66% HDL-cholesterol (mmol/l) 1.7 ( ) Triglycerides (mmol/l) 1.2±0.5 54

66 Patients with MetS were characterized by a higher BMI (26.9 vs 23.8 kg/m2, p=0.001); MetS was more prevalent in higher BMI categories (Figure 9, p=0.015). Patients with MetS were characterized also by higher fat mass (BFMI: 9.4 vs 7.9 kg/m2, p=0.010) and higher fat-free mass (FFMI: 17.5 vs 16.1, p=0.021), lower GOLD stages (FEV1 %pred: 41 vs 35, p=0.023) and a higher prevalence of comorbidities (81% vs 54%, p=0.002), including IR (77% vs 42%, p<0.001). There was no difference in age, sex, baseline physical performance or HRQoL between patients with and without MetS. Insulin resistant patients also had higher FFMI compared to non-ir patients (17.3 kg/m2 vs kg/m2, p=0.014). 60 NUMBER OF PATIENTS Underweight Normal weight Overweight Obese METABOLIC SYNDROME NO METABOLIC SYNDROME Figure 9 Prevalence of MetS across BMI groups 6.3 SARCOPENIA Sarcopenia was present in 61 (55%) patients. Sarcopenia was most prevalent in underweight patients, but also present in more than half of normal and overweight patients. (Figure 10). 55

67 Figure 10 Prevalence of sarcopenia across BMI groups Sarcopenic patients had lower fat-free mass, lower skeletal muscle mass, lower fat mass and waist circumference and were less well-nourished compared to non-sarcopenic patients. Bone mass was also lower in sarcopenic patients. 56

68 Table 6 General characteristics of sarcopenic and non-sarcopenic patients Sarcopenia (N=61) No sarcopenia (N=51) P-value Sex (male, %) 72% 59% Age (years, mean) 66±9 66± Smokers, current or ex (%) 80% 83% FVC (% pred) 79% 83% FEV1 (% pred) 36±13 40± FEV1/FVC 35±13 37± GOLD stage % 18% 3 50% 52% 4 33% 28% MNA points 22±4 24± MNA category Malnourished 21% 6% At risk of malnourishment 43% 22% Well-nourished 36% 72% BMI (kg/m 2 ) 23±4 29±5 <0.001 BMI category (%) <0.001 Underweight 23% 2% Normal weight 51% 25.5 % Overweight 13% 25.5% Obese 3% 47% Total body mass (kg) 65±13 79±15 <0.001 FMI (kg/m 2 ) 7.4± ±3.7 <0.001 FFMI (kg/m 2 ) 15.7± ±2.8 <0.001 ASMI (kg/m 2 ) 5.9± ±1.0 <0.001 Bone mass (kg) 2.0± ± Waist circumference (cm) 93±11 103±13 <0.001 WHR 0.97± ± CRP (mg/l) 3.3 ( ) 2.8 ( ) Diabetes Mellitus Type II 22% 24% Heart failure 20% 31% Atrial fibrillation 9% 6% Ischemic heart disease 12% 8% MWT (m) 316± ± ISWT (m) 230±16 285± Max CPET (Ws) 22±3 20± HRQoL (SGRQ points) 43±13 36± Sarcopenic patients reported worse results in all three measures of physical performance (6MWT, ISWT, maximum cycling tests). The reported HRQoL was also significantly lower in sarcopenic patients compared to non-sarcopenic patients. 57

69 However, sarcopenic patients did not differ from non-sarcopenic patients in gender, age, degree of airflow obstruction, prevalence of CVD or received drug therapy. No difference was observed in CRP levels between sarcopenic and non-sarcopenic patients. (Table 6, Table 7) Table 7 Medication use, sarcopenic and non-sarcopenic patients Sarcopenia No sarcopenia (N=61) (N=51) P-value Inhaled corticosteroids 66% 75% Long-acting beta agonists 75% 86% Short-acting beta agonists 64% 78% Long-acting muscarinic agonists 67% 75% Theophylline 16% 14% Angiotensin converting enzyme (ACE) inhibitors 26% 37% Angiotensin receptor blockers 7% 8% Calcium channel blockers 8% 12% Diuretics 27% 28% Beta-blockers 12% 18% Statins 13% 31% Oral antidiabetics 10% 8% Long-term oxygen therapy 30% 39% Sarcopenic patients showed significantly lower levels of BCAA, specifically leucine and valine. Essential amino acids also showed a trend towards a significant decrease in the sarcopenic group. No difference was observed in other amino acid levels. Both the sarcopenic and non-sarcopenic group had substantially low vitamin D levels; almost 90% of patients in both groups were reported to have vitamin D insufficiency (serum levels between75 and 50 nmol/l) or deficiency (serum levels below 50 nmol/l). (Table 8) 58

70 Table 8 Vitamin D and amino acids levels in sarcopenic and non-sarcopenic patients Sarcopenia (N=61) No sarcopenia (N=51) P-value Amino acids (nmol/ml) 3164± ± Prolin 175±67 174± Cysteine 210±34 220± Aspartic acid 26±13 26± Glutamic acid 75±28 81± Asparagine 47±10 49± Serine 133±44 126± Glutamine 594±85 584± Histidine 72±13 76± Glycine 258±74 242± Threonine 122±35 123± Citruline 36±11 37± Arginine 114±20 109± Alanine 374±78 372± Taurine 118±34 122± Tyrosine 70±17 73± Lysine 188±37 197± Methionine 23±6 25± Tryptophan 55±11 55± Phenylalanine 72±15 76± Isoleucine 60±17 68± Leucine 123±28 139± Valine 216±41 238± BCAA 399±80 445± EAA 931± ± Vitamin D (nmol/l) 43±25 43± Vitamin D insufficiency 19% 33% Vitamin D deficiency 69% 61% Muscle characteristics The MyHC1 mrna expression was significantly lower in sarcopenic patients compared to non-sarcopenic (2.7*10-6 ±2.4*10-6 in sarcopenic patients vs. 4.8*10-6 ± in nonsarcopenic patients, p=0,035). (Figure 11) 59

71 Figure 11 MyHC1 mrna expression in sarcopenic and non-sarcopenic patients 6.4 OVERLAP OF SARCOPENIA AND METABOLIC RISK FACTORS The prevalence of metabolic risk factors in sarcopenia is presented in table 9. IR was present in more than half of sarcopenic patients compared to two thirds in the non-sarcopenic group. Fasting glucose levels were comparable between sarcopenic and non-sarcopenic patients, while fasting insulin levels tended to be lower in the sarcopenic group. The prevalence of MetS was lower in the sarcopenic group, but still present in approximately one third of patients. In terms of MetS components, sarcopenic patients had a lower prevalence of abdominal obesity, however all other components were comparable with non-sarcopenic patients. Non-sarcopenic patients had a higher prevalence of MetS, abdominal obesity and higher levels of triglycerides. 60

72 Table 9 Prevalence of metabolic risk factors in sarcopenic and non-sarcopenic patients Sarcopenia (N=61) No sarcopenia (N=51) P-value IR 54% 65% HOMA-IR 2.7 ( ) 3.4 ( ) Fasting glucose (mmol/l) 5.9 ( ) 5.9 ( ) Fasting insulin (miu/l) 9.5 ( ) 11.9 ( ) MetS 37% 57% Abdominal obesity 63% 83% Arterial hypertension 82% 92% Hyperglycemia 68% 69% Hypertriglyceridemia 6% 19% Low HDL-cholesterol 8% 13% Total cholesterol (mmol/l) 5.0± ± LDL-cholesterol (mmol/l) 3.0± ± HDL-cholesterol (mmol/l) 1.8 ( ) 1.7 ( ) Triglycerides (mmol/l) 1.1± ± PULMONARY REHABILITATION OUTCOMES Eighty-five patients successfully completed the four-week high-intensity training. The reasons for not completing the program are presented in Figure 2 in the Methods section CHARACTERISTICS OF PATIENTS WHO DID NOT COMPLETE THE REHABILITATION PROGRAM The characteristics of patients who dropped-out of the pulmonary rehabilitation program are presented in Table 10. At baseline, these patients were characterised by a significantly lower BMI, lower waist circumference, lower physical performance and HRQoL, and showed a trend towards higher prevalence of sarcopenia. No difference was observed in age, disease severity or prevalence of comorbidities. 61

73 Table 10 Characteristics of patients who finished vs. patients who did not finish pulmonary rehabilitation Dropped out (N=27) Completed (N=85) P-value Sex (male, %) 70% 65% Age (years, mean) 66±9 66± Smokers, current or ex (%) 87% 79% FVC (% pred) 83±21 80± FEV1 (% pred) 38±14 38± FEV1/FVC 37±15 36± GOLD stage % 16% 3 46% 53% 4 33% 30% MNA points 23±4 23± MNA category Malnourished 26% 11% At risk of malnourishment 26% 36% Well-nourished 48% 54% BMI (kg/m 2 ) 24±4 26± BMI category (%) Underweight 19% 12% Normal weight 44% 38% Overweight 26% 22% Obese 11% 28% FMI (kg/m 2 ) 7.8± ± FFMI (kg/m 2 ) 16.1± ± ASMI (kg/m 2 ) 6.2± ± Sarcopenia (%) 70% 49% Waist circumference (cm) 92±9 99± WHR 0.96± ± CRP (mg/l) 3.5 ( ) 2.8 ( ) Diabetes Mellitus Type II 32% 20% Heart failure 37% 21% Atrial fibrillation 12% 6% Ischemic heart disease 20% 7% MWT (m) 284±97 349± ISWT (m) 202± ± Max CPET (Ws) 54±19 62± HRQoL (SGRQ points) 48±13 38±

74 6.6 EFFECT OF REHABILITATION ON PHYSICAL PERFORMANCE AND QUALITY OF LIFE WHOLE GROUP From the patients who successfully completed the program, the majority improved both physical functioning and HRQoL. More than 50% patients improved 6MWT above the minimal clinically important difference. A similar effect was observed in changes in ISWT. The improvement was even more significant in HRQoL as 75% of patients improved above the MCID. (Figure 12) Figure 12 Changes in physical performance and HRQoL for whole group SARCOPENIA VS. NO SARCOPENIA Both sarcopenic and non-sarcopenic patients significantly improved HRQoL and physical performance measured via the ISWT and maximum cycling power. The improvement in physical performance measured with 6MWT did not reach significance in the sarcopenic group. (Figure 13) 63

75 Figure 13 Response to pulmonary rehabilitation. A: Non-sarcopenic patients. B: Sarcopenic patients. 6.7 EFFECT OF PULMONARY REHABILITATION ON METABOLIC RISK WHOLE GROUP On the whole group level, selected changes have been observed in the metabolic risk after 4 weeks of high-intensity pulmonary rehabilitation. Fasting glucose significantly decreased (5.9 to 5.3 mmol/l, p=0.001), however no change was observed in fasting insulin levels (9.9 to 9.5 miu/l, p=0.594). No significant difference was observed in HOMA-IR (2.6 to 2.2, p=0.215). Patients significantly decreased in waist circumference and a trend toward decreased body fat was also seen. No change was observed in lean mass on the whole group level. All patients significantly decreased total cholesterol levels, LDL-cholesterol levels and, surprisingly, also HDL-cholesterol levels. No change was observed in triglyceride levels. (Table 11) 64

76 after rehabilitation. Doctoral dissertation. University of Ljubljana, Faculty of Medicine, 2016 Table 11 Change in metabolic risk factors before and after rehabilitation. All patients who completed pulmonary rehabilitation (N=85) A. Sarcopenic (N=42) B. Non-sarcopenic (N=43) Post-pre, A vs B Pre Post p-value Pre Post p-value Pre Post p-value p-value Fasting glucose (mmol/l) 5.9 ( ) 5.3 ( ) ( ) 5.2 ( ) ( ) 5.3 ( ) Fasting insulin (miu/l) 9.9 ( ) 9.5 ( ) ( ) 10.0 ( ) ( ) 9.1 ( ) HOMA-IR 2.6 ( ) 2.2 ( ) ( ) 2.4 ( ) ( ) 1.9 ( ) Total cholesterol (mmol/l) 5.1± ±1.1 < ± ± ± ± LDL-cholesterol (mmol/l) 3.0± ± ± ± ± ± HDL-cholesterol (mmol/l) 1.7 ( ) 1.4 ( ) < ( ) 1.51 ( ) ( ) 1.44 ( ) < Triglycerides (mmol/l) 1.2± ± ± ± ± ± Waist circumference (cm) 98±13 96±13 < ±11 93± ±13 101± WHR 0.98± ± ± ± ± ± Fat mass (kg) 21.1 ( ) 20.9 ( ) ( ) 20.3 ( ) ( ) 23.2 ( ) Lean mass (kg) 47.2± ± ± ± ± ±

77 6.7.2 SARCOPENIA VS NO SARCOPENIA In non-sarcopenic patients, the IR prevalence decreased by 10% and was accompanied by a significant decrease in HOMA-IR and fasting glucose. In sarcopenic patients the opposite was observed, the prevalence of IR slightly increased, along with an increase in fasting insulin levels and HOMA-IR (Figure 14). The difference in the change of HOMA-IR values between sarcopenic and non-sarcopenic patients was statistically significant (median, IQR: 0.06 ( ) in sarcopenic patients versus ( ) in non-sarcopenic patients, p=0.031). Figure 14 Change in HOMA-IR index. A: All patients. B: Sarcopenic patients. C: Nonsarcopenic patients Further stratifying patients per sarcopenia and IR revealed that sarcopenic non-ir patients did not show a change in fasting glucose levels, but significantly increased fasting insulin and showed a trend towards increased HOMA-IR. Non-sarcopenic IR patients on the other hand significantly decreased both fasting glucose and HOMA-IR, but showed no difference in fasting insulin levels. (Table 12) 66

78 Table 12 Subgroup analysis of fasting glucose, fasting insulin and HOMA-IR before and after rehabilitation Sarcopenic Insulin resistant Non-sarcopenic Pre Post p-value Pre Post p-value Fasting glucose (mmol/l) Fasting insulin (miu/l) 6.4 ( ) 5.9 ( ) ( ) 6.0( ) ( ) 11.8 ( ) (15,3-38,0) 18.5( ) HOMA-IR 5.8 ( ) 3.5 ( ) ( ) 5.0( ) Sarcopenic Non-insulin resistant Non-sarcopenic Pre Post p-value Pre Post p-value Fasting glucose (mmol/l) Fasting insulin (miu/l) 5.4 ( ) 5.2 ( ) ( ) 5.1( ) ( ) 8.4( ) ( ) 4.6( ) HOMA-IR 1.5( ) 1.9 ( ) ( ) 1.0( ) Neither group showed a significant change in lean mass, while the non-sarcopenic group decreased in body fat and waist circumference. (Figure 15) Figure 15 Change in body composition in sarcopenic and non-sarcopenic patients. A: FFMI Fat-free mass index. B: FMI Fat mass index. C: Waist circumference. D: Waist to hip ratio A significant decrease in total cholesterol levels as well as HDL-cholesterol levels was observed in both groups. Non-sarcopenic patients also decreased LDL-cholesterol levels. No change was observed in triglyceride levels. (Figure 16) 67

79 Figure 16 Change in lipids. A: Total cholesterol. B: LCL-cholesterol. C: HDL-cholesterol. D: Triglycerides. 6.8 EFFECT OF PULMONARY REHABILITATION ON INFLAMMATORY PROFILE CRP No changes have been observed in patients' inflammatory profile assessed with CRP levels after rehabilitation (median with IQR: 2.8 ( ) mg/l at baseline, 3.0 ( ) mg/l after rehabilitation; p= 0.317). No signifcant change was observed also in sarcopenic or nonsarcopenic patients (median, sarcopenic: 3.1 ( ) mg/l at baseline, 3.8 ( ) mg/l after rehabilitation, p=0.437; median, non-sarcopenic: 2.6 ( ) mg/l at baseline, 3.0 ( ) mg/l after rehabilitation, p=0.856) IL-6 In total, 19 patients were included in the IL-6 analysis: 12 patients had IL-6 serum levels below limit of detection both before and after rehabilitation. Of the remaining 7 patients, only two had detectable IL-6 levels both at start and end of the program. (Table 13). The change in IL-6 was not significant (mean: 0,35 pg/ml at baseline, 0,84 pg/ml after rehabilitation, p=0,140). We also analyzed the change in CRP serum levels in this subgroup of patients. The change in CRP was also not significant (median: 2,0 mg/l at baseline, 2.5 mg/l after rehabilitation, p= 0,984). 68

80 Table 13 Serum IL-6 and serum CRP levels pre and post rehabilitation Serum IL-6 (pg/ml)* Serum CRP (mg/l) Pre Post Pre Post *Serum values below LOD are presented as

81 6.9 RELATION BETWEEN CHANGES IN METABOLIC RISK FACTORS AND CHANGES IN PHYSICAL PERFORMANCE Besides the effect of the overall rehabilitation program on the changes in metabolic risk factors, we were also interested in the relation between the change in physical performance and the change in metabolic risk factors. No association has been observed between changes in body composition or changes in insulin sensitivity and rehabilitation outcomes on the whole group level. In non-sarcopenic patients, an improvement in ISWT was positively associated with a decrease in fat mass index. (Table 14) Table 14 Correlations between changes in physical performance and changes in selected metabolic risk factors. Δ 6MWT Δ ISWT Δ Max CPET All patients Δ HOMA-IR Δ Fasting insulin Δ Fasting glucose Δ FFMI Δ FMI Sarcopenic patients Δ HOMA-IR Δ Fasting insulin Δ Fasting glucose Δ FFMI Δ FMI Non-sarcopenic patients Δ HOMA-IR Δ Fasting insulin Δ Fasting glucose Δ FFMI Δ FMI *

82 6.10 RELATION BETWEEN CHANGES IN HOMA-IR INDICES AND CHANGES IN BODY COMPOSITION On the whole group level, no associations were found between changes in body composition and changes in HOMA-IR indices. Stratifying patients per sarcopenia revealed that this association is present in sarcopenic patients. A significant positive correlation was observed between change in fasting insulin and change in fat-free mass index. (Table 15, Figure 17) Table 15 Correlations between changes in body composition and changes in HOMA-IR indices Δ FFMI Δ FMI All patients Δ HOMA-IR Δ Fasting insulin Δ Fasting glucose Sarcopenic patients Δ HOMA-IR Δ Fasting insulin 0.477* Δ Fasting glucose Non-sarcopenic patients Δ HOMA-IR Δ Fasting insulin Δ Fasting glucose

83 Figure 17 Correlation between changes in FFMI and changes in fasting insulin in sarcopenic patients. 72

84 6.11 PROTOCOL FOR INTERVENTION STUDY OF COPD PATIENTS ON REHABILITATION Study design: Randomized double-blind interventional study on sarcopenic COPD patients included to pulmonary rehabilitation program Eligibility criteria: Study inclusion and exclusion criteria are given in Table 16. Table 16 Eligibility criteria for intervention study Study inclusion criteria Confirmed diagnosis of COPD, Stable disease, Sarcopenia as defined by the appendicular skeletal muscle index cutoffs (<7.23 kg/m2 for males and <5.67 kg/m2 for females), Eligible for pulmonary rehabilitation program. Study exclusion criteria Hospitalization due to a COPD exacerbation within the last 4 weeks, Refusal of collaboration or if patients are not able to follow the rehabilitation program due to any cause, Patients experiencing an exacerbation during the rehabilitation program, Patients who do not complete the rehabilitation program. Study intervention: Group A: Supplementation with nutritonal supplement (1.5 kcal/ml, 200 ml) rich in protein, HMB (leucine metabolite) and vitamin D, Group B: Supplementation with normocaloric normoproteinic supplement (1.5 kcal/ml, 200 ml). 73

85 Figure 18 Study design and intervention The supplement/placebo will be given to the patients between breakfast and lunch. Rehabilitation program: The program will be performed as presented in the Methods section. Both groups will receive the same training and educational program and psychological and social support. Body composition will be evaluated at start and end of the rehabilitation program using DEXA. Study duration: 4 weeks Primary outcome: Improvement in physical performance (6MWT, ISWT, max CPET) Secondary outcomes: Improvement in PR program adherence (number of exacerbations and program dropouts), Improvements in metabolic risk factors (IR, body composition), Improvement in HRQoL. 74