Mitokondriometabolia ja oksidatiivinen stressi fyysisen harjoittelun ja ravintoneuvonnan funktiona T2DM:ssä (MITOX)

Ei väliintuloa: ND-INAKT

Osallistujat, jotka eivät saaneet kumpaakaan ravitsemusinterventiota tai liikuntaohjelmaa. Heidän ohjeistettiin ylläpitämän normaaleja elämäntapojaan liittyen fyysiseen aktiivisuuteen ja ruokavalioon.

Active Comparator: Keskitehoinen jatkuva harjoittelu (ND-MICT)

Osallistujat, jotka eivät saaneet ravitsemusinterventiota, mutta osallistuivat kohtalaisen intensiteetin jatkuvaan harjoitusohjelmaan.

Muut: Kohtalaisen intensiteetin jatkuva harjoittelu (MICT)

MICT-ryhmään sijoitetut osallistujat suorittivat 3 viikoittaista istuntoa, joissa oli 1–2 päivän lepojakso istuntojen välillä, polkupyöräergometrillä 12 viikon ajan.

MICT koostui 50 minuutin jatkuvasta polkemisesta noin 10 % laktaattikynnyksen yläpuolella olevalla intensiteetillä, polkemistiheydellä 60–80 rpm. Harjoituskuormaa (TL) lisättiin 10 %, jos nämä kaksi ehtoa täyttyivät: 1) sydämen sykkeen nousu vähintään 2 lyöntiä minuutista 20 minuuttiin 40 harjoituksen aikana ei tapahtunut, ja 2) laktaattitasot olivat alle 2 mmol/L istunnon 48. minuutilla. Ensimmäisen istunnon TL määritettiin säätötestillä, joka koostui asteittaisesta testistä viidestä peräkkäisestä 10 minuutin vaiheesta vakiopolkemistiheydellä (60–80 rpm) 90–130 % hengityskynnyksen tehosta, laskettuna aiemmin suoritetun FatOx-testin datasta, 10 %:n lisäyksin jokaisessa vaiheessa. TL määritettiin vaiheesta, jossa sydämen syke nousi ≥ 3 lyöntiä vaiheiden välillä ja veren laktaattitasot nousivat ≥ 1 mmol/L edellisestä vaiheesta.

Active Comparator: Korkean intensiteetin intervalliharjoittelu (ND-HIIT)

Osallistujat, jotka eivät saaneet ravitsemusinterventiota, mutta osallistuivat korkean intensiteetin intervalliharjoitusohjelmaan.

Muut: The High-Intensity Interval Training (HIIT)

Participants assigned to the HIIT completed 3 weekly sessions, with 1-2 days of rest between sessions, on a cycle ergometer for 12 weeks.

The training load (TL) was determined from an incremental test until volitional exhaustion with one-minute work intervals (from 85% to 165% of maximal power output from a previously performed VO2max test [Wmax] and with 10% increments) separated by 1.5-minute rest periods (at 20% Wmax).

The program consisted of a 3 minutes warm-up with the first 2 minutes at 20% and last minute at 40% of the TL, followed by 10 series of 1 min duration at 90% of TL, with 1 minute of rest between sets at 20% of TL and ending with 2 minutes of cool-down at 20% of TL (estimated total time of the session: 25 minutes). A 5% increase in workload was applied when, for two consecutive sessions, the first three intervals failed to exceed 85%, and at least five did not reach 90% of maximal HR. Participants were asked to maintain a cadence above 80RPM.

Active Comparator: Ravitsemusinterventio (D-INACT)

Osallistujat, jotka saivat ravitsemusinterventiota mutta eivät liikuntaohjelmaa.

Käyttäytyminen: Nutritional intervention

The nutritional intervention was conducted through biweekly (± 3 days) in-person consultations with a nutritionist or dietitian. Firstly, the patient's diet was analyzed. Then, participants were guided to follow a mildly calorie-restricted diet (-250-500 kcal/day deficit), with the primary goal of achieving a 5 % reduction in body weight and reducing blood glucose levels. Meal plans were created using Nutrium® software , personalized to each participant's lifestyle and food preferences, and reviewed at each biweekly consultation. The dietary plan included at least 4 or 5 meals per day, ensuring a well-distributed intake of carbohydrates, a balanced distribution of macro- and micronutrients, and the inclusion of foods rich in prebiotics and probiotics. Moreover, some suggestions about the combination of foods and culinary techniques were provided. Only one session through the intervention was allowed to be attended telephonically.

Kokeellinen: Ravitsemusinterventio Kohtalauhteinen jatkuva harjoittelu (D-MICT)

Osallistujat, jotka saivat ravitsemusinterventiota ja osallistuivat kohtalaisen intensiteetin jatkuvan harjoittelun liikuntaohjelmaan.

Muut: Kohtalaisen intensiteetin jatkuva harjoittelu (MICT)

MICT-ryhmään sijoitetut osallistujat suorittivat 3 viikoittaista istuntoa, joissa oli 1–2 päivän lepojakso istuntojen välillä, polkupyöräergometrillä 12 viikon ajan.

MICT koostui 50 minuutin jatkuvasta polkemisesta noin 10 % laktaattikynnyksen yläpuolella olevalla intensiteetillä, polkemistiheydellä 60–80 rpm. Harjoituskuormaa (TL) lisättiin 10 %, jos nämä kaksi ehtoa täyttyivät: 1) sydämen sykkeen nousu vähintään 2 lyöntiä minuutista 20 minuuttiin 40 harjoituksen aikana ei tapahtunut, ja 2) laktaattitasot olivat alle 2 mmol/L istunnon 48. minuutilla. Ensimmäisen istunnon TL määritettiin säätötestillä, joka koostui asteittaisesta testistä viidestä peräkkäisestä 10 minuutin vaiheesta vakiopolkemistiheydellä (60–80 rpm) 90–130 % hengityskynnyksen tehosta, laskettuna aiemmin suoritetun FatOx-testin datasta, 10 %:n lisäyksin jokaisessa vaiheessa. TL määritettiin vaiheesta, jossa sydämen syke nousi ≥ 3 lyöntiä vaiheiden välillä ja veren laktaattitasot nousivat ≥ 1 mmol/L edellisestä vaiheesta.

Käyttäytyminen: Nutritional intervention

The nutritional intervention was conducted through biweekly (± 3 days) in-person consultations with a nutritionist or dietitian. Firstly, the patient's diet was analyzed. Then, participants were guided to follow a mildly calorie-restricted diet (-250-500 kcal/day deficit), with the primary goal of achieving a 5 % reduction in body weight and reducing blood glucose levels. Meal plans were created using Nutrium® software , personalized to each participant's lifestyle and food preferences, and reviewed at each biweekly consultation. The dietary plan included at least 4 or 5 meals per day, ensuring a well-distributed intake of carbohydrates, a balanced distribution of macro- and micronutrients, and the inclusion of foods rich in prebiotics and probiotics. Moreover, some suggestions about the combination of foods and culinary techniques were provided. Only one session through the intervention was allowed to be attended telephonically.

Kokeellinen: Ravitsemuksellinen interventio Korkeatehoinen intervalliharjoittelu (D-HIIT)

Osallistujat, jotka saivat ravitsemusinterventiota ja osallistuivat korkean intensiteetin intervalliharjoitusohjelmaan.

Käyttäytyminen: Nutritional intervention

The nutritional intervention was conducted through biweekly (± 3 days) in-person consultations with a nutritionist or dietitian. Firstly, the patient's diet was analyzed. Then, participants were guided to follow a mildly calorie-restricted diet (-250-500 kcal/day deficit), with the primary goal of achieving a 5 % reduction in body weight and reducing blood glucose levels. Meal plans were created using Nutrium® software , personalized to each participant's lifestyle and food preferences, and reviewed at each biweekly consultation. The dietary plan included at least 4 or 5 meals per day, ensuring a well-distributed intake of carbohydrates, a balanced distribution of macro- and micronutrients, and the inclusion of foods rich in prebiotics and probiotics. Moreover, some suggestions about the combination of foods and culinary techniques were provided. Only one session through the intervention was allowed to be attended telephonically.

Muut: The High-Intensity Interval Training (HIIT)

Participants assigned to the HIIT completed 3 weekly sessions, with 1-2 days of rest between sessions, on a cycle ergometer for 12 weeks.

The training load (TL) was determined from an incremental test until volitional exhaustion with one-minute work intervals (from 85% to 165% of maximal power output from a previously performed VO2max test [Wmax] and with 10% increments) separated by 1.5-minute rest periods (at 20% Wmax).

The program consisted of a 3 minutes warm-up with the first 2 minutes at 20% and last minute at 40% of the TL, followed by 10 series of 1 min duration at 90% of TL, with 1 minute of rest between sets at 20% of TL and ending with 2 minutes of cool-down at 20% of TL (estimated total time of the session: 25 minutes). A 5% increase in workload was applied when, for two consecutive sessions, the first three intervals failed to exceed 85%, and at least five did not reach 90% of maximal HR. Participants were asked to maintain a cadence above 80RPM.

Arvioidut muutokset oksidatiivisessa stressissä

Hapen haitallisen stressin tasoa arvioitiin 8-OHdG:n pitoisuudella DNA:n hapettumisen biomarkkerina. Seruminäytteissä käytettiin kilpailevaa ELISA-tunnistusmenetelmää. Yksinkertaisesti sanottuna, standardi ja näyte lisättiin esipäällystettyyn mikrolaatikkoon yhdessä biotinyylatun tunnistusvastaisen kanssa. Pesun jälkeen lisättiin HRP-Streptavidinia. Sitten lisättiin TMB-substraatti, ja reaktio pysäytettiin happoliuoksella. Värin tuottoa mitattiin spektrofotometrisesti 450 nm:n aallonpituudella. Näytteiden optisen tiheyden arvoja verrattiin standardikäyrän arvoihin 8-OHdG:n pitoisuuden (ng/ml) määrittämiseksi.

Lihasnäytteen biopsia: Sitraattisyntaasin aktiivisuus

2–5 mg jäähdytettyä lihasta homogenisoitiin 1:20 laimennuksessa CelLytic™ MT Tissue Lysis -liuotinta (C3228, Sigma-Aldrich, Dorset, UK), johon oli lisätty 1 % proteaasien estäjäkoktailia (P8340, Sigma-Aldrich, Dorset, UK) 4 °C:ssa. Lysaatti sentrifugoitiin 12 000 × g 10 minuutin ajan 4 °C:ssa saostamaan jäämökset, ja ylämaine varastoitiin −80 °C:ssa. CS-aktiivisuus määritettiin kaksinkertaisina näytteinä käyttäen testisarjaa valmistajan ohjeiden mukaisesti (CS0720: Sigma-Aldrich, Dorset, UK). Lyhyesti sanottuna endogeeninen aktiivisuus rekisteröitiin DTNB:n aiheuttaman värinän emittoinnin kautta, ja kokonaisaktiivisuus laskettiin oksaloasetaatin lisäyksen jälkeen. Proteiinipitoisuus määritettiin kaksinkertaisina näytteinä käyttäen bikinikonihappotestisarjaa (BCA1: Sigma-Aldrich, Dorset, UK), mikä mahdollisti CS-aktiivisuusarvojen normalisoinnin kokonaisproteiinipitoisuuteen (µmol/g/min). CS-aktiivisuus ja proteiinipitoisuus mitattiin 96-kuoppalevyllä mikrolevylukijalla aallonpituuksilla 412 nm ja 562 nm (MQX200R2, Biotek instruments, Burlington, USA).

Muscle biopsy samples: mitochondrial respiratory capacity

Skin was locally anesthetized, followed by an incision of approximately 6-7mm on the dermal surface. Using sterilized Bergstrom needles, approximately 3-6 mg of skeletal muscle from the lateral vastus were extracted and placed in a BIOPS buffer for immediate analysis of mitochondrial respiration (MR) through high-resolution respirometry. Before, the fibers were mechanically separated in BIOPS buffer using sterilized needles and chemically permeabilized with a 50 μg/ml saponin solution.

MR was measured in a specific buffer (Mir05) in duplicate in hyperoxygenated chambers (250-450 nmol/mL of oxygen) in an Oxygraph-2k at 37ºC (Oroboros, Innsbruck, Austria). A substrate-uncoupler titration protocol was employed by adding (final concentration): malate (2mM), pyruvate (5mM), glutamate (2mM), MgCl2 (3mM), and ADP (5mM), cytochrome C (10μM), succinate (10mM), and p-trifluoromethoxyphenylhydrazone. Oxygen flux was presented as mass-specific (pmol·s/mg wet weight) and normalized to CS activity.

Arvioitu insuliiniresistenssin muutokset: HOMA-IR

Insuliiniresistenssi arvioitiin käyttäen Homeostaattista Mallia Insuliiniresistenssin Arviointiin (HOMA-IR), joka laskettiin kertomalla paastovaiheen insuliini (µU/mL) paastovaiheen glukoosilla (mg/dL) ja jakamalla tulos luvulla 405.

Arvioidut muutokset insuliiniherkkyydessä: Matsuda-indeksi

Koko kehon insuliiniherkkyys arvioitiin Matsuda-indeksillä, joka laskettiin kaavalla 10,000 / √[(paastoverensokeri (mg/dL) × paastoinsuliini (µU/mL)) × (keskimääräinen verensokeri suun kautta tehdyn glukoositoleranssitestin aikana (mg/dL) × keskimääräinen insuliini suun kautta tehdyn glukoositoleranssitestin aikana (µU/mL))].

Arvioitu insuliinintuntuvuuden muutos: Quicki

Insuliiniherkkyys arvioitiin kvantitatiivisen insuliiniherkkyystarkistusindeksin (QUICKI) avulla, joka laskettiin kaavalla 1 / [log(paastöinsuliini (µU/mL)) + log(paastöglukoosi (mg/dL))].

Arvioitu painoindeksin muutokset

Painoindeksi (BMI) kilogrammoina neliömetriä kohden (kg/m²) laskettiin jakamalla paino (kg) pituuden neliöllä (m²).

Arvioidut muutokset antropometriassa: Kehämmitat

Vyötärön ympärysmitta (cm) mitattiin kymmenennen kylkiluun alimman sivureunan ja lannen harjanteen välistä keskipisteestä, kohtisuorassa rungon pituusakseliin nähden. Vatsan ympärysmitta (cm) mitattiin napan korkeudelta, kohtisuorassa rungon pituusakseliin nähden. Lantion ympärysmitta (cm) mitattiin pakaroiden suurimman takapään kohdalta, kohtisuorassa rungon pituusakseliin nähden. Reiden ympärysmitta (cm) mitattiin suuren trokanterin ja sääriluun sivuisän kohdan välisestä keskipisteestä. Kaikki mittaukset suoritettiin osallistujan seisoessa pystyssä ja rentoutuneena. Jokainen mittaus suoritettiin kahdesti, ja kolmas mittaus suoritettiin, jos ero ylitti 1 %. Kahden samankaltaisimman tuloksen keskiarvo rekisteröitiin.

Arvioidut muutokset antropometriassa: Vyötärön ja lantion suhde

Vyötärön ja lantion suhde laskettiin vyötärönympärys (cm) jaettuna lantionympäryksellä (cm).

Arvioitu pituus

Pituus (metrit, m) mitattiin seinään kiinnitettyä stadiometria (TANITA-LEICESTER HR-001, Tanita Corp., Tokio, Japani) käyttäen, ja osallistujat seisoivat valmistajan ohjeiden mukaisesti.

Assessed changes in Glucose concentration

Glucose concentration (mg/dL) was measured from blood samples collected from the antecubital vein after at least 8 hours of fasting and at 30, 60, 90, 120, 150, and 180 minutes after the ingestion of 75 grams of glucose (Nuter-tec, 75 g/200 mL, orange flavor). Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine consumption for 24 hours, and avoid vigorous physical activity for at least 48 hours. Blood samples were centrifuged and plasma was analyzed using the colorimetric method performed on the Alinity CI system (Abbott Laboratories, Abbott Park, IL, USA).

Assessed changes in Insulin concentration

Insulin concentration (µU/mL) was measured from blood samples collected from the antecubital vein into serum separator tubes after at least 8 hours of fasting and at 30, 60, 90, 120, 150, and 180 minutes after the ingestion of 75 grams of glucose (Nuter-tec, 75 g/200 mL, orange flavor). Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine consumption for 24 hours, and avoid vigorous physical activity for at least 48 hours. Blood samples were centrifuged at 4000 rpm for 7 minutes, and serum was analyzed. Insulin concentrations were determined by chemiluminescent immunoassays performed on the Alinity CI system (Abbott Laboratories, Abbott Park, IL, USA).

Assessed changes in Tissue Oxygenation Index

A NIRO-200NX device was used to measure skeletal muscle tissue oxygenation index (%) through near-infrared spectroscopy (NIRS), the modified Beer-Lambert law (MBLL), and spatially resolved spectroscopy. The NIRS sensor (emitter-sensor distance of 3cm) was placed at two-thirds of the distance from the anterior superior iliac spine to the superolateral patellar border, over the vastus lateralis muscle belly. The NIRS data were continuously acquired throughout a resting energy expenditure test for 30 minutes, a maximal fat oxidation (MFO) test on a cycloergometer, and a maximal oxygen consumption (VO2max) test on a cycloergometer. Briefly, the MFO test consisted of an incremental task with 3-minute phases starting from 15W and increasing by 15W each phase until a respiratory exchange ratio of 1. After a 5-minute rest, the VO2max test was initiated at last MFO load with 15W increases each minute until VO2max determination.

Assessed changes in haemoglobin saturation delta

A NIRO-200NX device was used to measure skeletal muscle changes on oxygenated, desoxygenated, and total haemoglobin (μmol/L) through near-infrared spectroscopy (NIRS) and the modified Beer-Lambert law. The NIRS sensor (emitter-sensor distance of 3cm) was placed at two-thirds of the distance from the anterior superior iliac spine to the superolateral patellar border, over the vastus lateralis muscle belly. The NIRS data were continuously acquired throughout a resting energy expenditure test for 30 minutes, a maximal fat oxidation (MFO) test on a cycloergometer, and a maximal oxygen consumption (VO2max) test on a cycloergometer. Briefly, the MFO test consisted of an incremental task with 3-minute phases starting from 15W and increasing by 15W each phase until a respiratory exchange ratio of 1. After a 5-minute rest, the VO2max test was initiated at last MFO load with 15W increases each minute until VO2max determination.

Assessed changes in Body Weight

Body weight (kilograms, kg) was measured using a multifrequency bioelectrical impedance device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Fat Mass (kilograms of Fat Mass)

Fat mass (kilograms, kg) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Fat Mass (percentage of Fat Mass)

Fat mass (percentage of total body weight, %) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Fat-free Mass (kilograms of Fat-free Mass)

Fat-free mass (kilograms, kg) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Fat-free Mass (percentage of Fat-free Mass)

Fat-free mass (percentage of total body weight, %) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Anthropometry: Skinfold thickness

Thigh skinfold thickness was measured at two-thirds of the distance from the iliospinale point (the lowest part of the anterior superior iliac spine) to the superolateral border of the patella, over the belly of the vastus lateralis muscle. Measurements were taken on the dominant leg. Two measurements were obtained, and a third was taken if the difference between the first two exceeded 1mm. The mean of the two most similar records was recorded.

Assessed changes in interleukin-6

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum interleukin-6 (pg/mL) was measured by electrochemiluminescence immunoassay using a Cobas e 411 (Roche Diagnostics, Mannheim, Germany).

Assessed changes in C-reactive protein

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum C-reactive protein (mg/L) was determined by immunoturbidimetric assay on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in alanine aminotransferase

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum alanine aminotransferase (U/L) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in aspartate aminotransferase

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum aspartate aminotransferase (U/L) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in gamma-glutamyl transferase

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum gamma-glutamyl transferase (U/L) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in tumor necrosis factor-alpha

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® EDTA K2 separator tubes (Greiner Bio-One, Kremsmünster, Austria) to obtain plasma. Plasma was aliquoted into 500 µL tubes with 5 µL protease inhibitors, then stored at -80 °C. Plasma tumor necrosis factor-alpha (pg/mL) was measured using multiplex magnetic bead-based immunoassays (MILLIPLEX® Metabolic Hormone Panel V3, HMH3-34K-06, Merck Millipore, Darmstadt, Germany) and analyzed with a Luminex® xMap IntelliFlex System (Luminex Corp., Austin, TX, USA).

Assessed changes in triglycerides

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum triglycerides (mg/dL) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in total cholesterol

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum total cholesterol (mg/dL) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in high-density lipoprotein

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum high-density lipoprotein (mg/dL) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).

Assessed changes in low-density lipoprotein

Blood drawing was performed with participants in a fasting state for 8-10 hours and scheduled in the morning. Before blood drawing, participants were instructed to maintain their usual lifestyle, avoid alcohol and caffeine for 24 hours, and refrain from vigorous physical activity for at least 48 hours. Blood was collected by peripheral venepuncture using VACUETTE® blood serum separator tubes (Greiner Bio-One, Kremsmünster, Austria). Serum low-density lipoprotein (mg/dL) was determined by enzymatic spectrophotometric assays on the Alinity C analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).