The Effect of Curcumin on the Development of Prednisolone-induced Hepatic Insulin Resistance
The Effect of Curcumin on the Development of Prednisolone-induced Hepatic Insulin Resistance
The aim of this study is to investigate whether ingestion of curcumin will prevent hepatic
 insulin resistance (assessed by homeostatic model assessment of insulin resistance (HOMA-IR))
 induced by short-term oral glucocorticoid (prednisolone) administration in overweight and
 obese participants. As a secondary endpoint it will be investigated if prednisolone
 administration induce or worsen the degree of NAFLD in overweight or obese participants using
 magnetic resonance (MR) spectroscopy (MRS), and if curcumin can ameliorate this effect. Also,
 the possible anti-inflammatory effect of curcumin will be elucidated as a range of
 inflammatory markers before and after intervention will be measured. Thus, prednisolone
 treatment is used as a model of development of pre-diabetes.
AIMS OF STUDY The aim of this study is to investigate whether ingestion of curcumin will
 prevent hepatic insulin resistance (assessed by homeostatic model assessment of insulin
 resistance (HOMA-IR)) induced by short-term oral glucocorticoid (prednisolone) administration
 in overweight and obese participants.

 BACKGROUND The increasing incidence and prevalence of obesity and its comorbidities calls for
 research in preventing these diseases. The progression to insulin resistance and increased
 blood sugar levels (i.e. prediabetes) and ultimately type 2 diabetes (T2D) in obesity is
 believed to be caused by various factors of which low-grade inflammation in adipose tissue
 and skeletal muscle tissue seems to inflict insulin resistance initiating metabolic
 deterioration towards the type 2 diabetic phenotype. Curcumin, a polyphenolic compound that
 can be extracted from the turmeric root, have been shown to exert anti-inflammatory
 properties in both in vitro and animal studies. Furthermore, animal studies indicate that
 curcumin is a modulator of gut microbiota composition in a metabolic beneficial direction.
 Studies have shown positive results in terms of lowering hepatic insulin resistance with
 curcumin in rodents and humans (measured by HOMA-IR), and another clinical trial has shown
 that curcumin can prevent worsening of HOMA-IR and development of T2D in a prediabetic
 population.

 Glucocorticoids are a group of frequently used compounds, prescribed for a wide range of
 diseases and have severe metabolic side effects as insulin resistance, T2D and non-alcoholic
 fatty liver disease. Interestingly, curcumin, a phenolic compound extracted from the turmeric
 root, and curcumin analogues have been shown to ameliorate glucocorticoid-induced
 'dysmetabolism' in rodent studies, but no human data exist. Also, results from both in vitro
 and in vivo studies with curcumin have shown promising effects in preventing NAFLD
 development, but solid human data are missing. Because of the temporary effects on glucose
 metabolism, short-term treatment with glucocorticoids can be used as an investigative model
 of these metabolic changes. Previous studies from the research unit performing the current
 study, used this setup to investigate changes in glucose homeostasis, incretin hormones and
 insulin resistance. Two different mouse studies indicate that curcumin could be beneficial in
 terms of preventing the metabolic changes associated with systemic glucocorticoid treatment,
 but no human studies have investigated if curcumin can prevent glucocorticoid-induced
 increase in HOMA-IR.

 The aim of the study is to investigate the effects of curcumin of the metabolic changes
 caused by short-term systemic prednisolone in a double-blinded, placebo-controlled trial in
 overweight and obese individuals.

 STUDY DESIGN

 Participants 27 overweight and obese individuals without diabetes

 METHODS

 In this randomised, double-blinded, placebo-controlled trial, eligible participants will
 undergo baseline assessments before being randomised to one of the following interventions:
 1) Two tablets of curcumin (each tablet containing 100 mg curcumin) twice daily in 11 days
 and 50 mg of prednisolone once daily in 10 days (capsule); 2) two tablets of placebo-curcumin
 twice daily in 11 days and 50 mg of prednisolone once daily in 10 days (capsule); 3) two
 tablets of placebo-curcumin twice daily in 11 days and one capsule of placebo-prednisolone
 once daily in 10 days. In the end of the 11-day intervention period, end-of-intervention
 assessments (similar to baseline assessments) will be performed. If necessary, a follow-up
 visit will be made.

 Information meeting and screening Prior to any protocol-related procedures, an information
 meeting will be arranged, and participants will be informed of the possibility of bringing a
 person of own choice to the visit. The information meeting is performed in undisturbed and
 confidential surroundings. The room is reserved for this purpose, and nobody will interfere
 during the meeting. It is not possible for other employees in the department to look into the
 room. During this meeting, a member of the research group will explain the purpose,
 procedures and possible risks of the study. The member of the research group will have proper
 qualifications in terms of medical knowledge, insights in the specific project and
 communications skills. It is firstly pointed out that the project is a biomedical trial.
 After this, an oral information about the project is given based on the written information.
 Questions will be answered as thoroughly as possible. The participant will be informed that
 it is possible to have a minimum of 48 hours to evaluate participation from oral information
 is given. If he does not want time to evaluate the participation, the informed consent can be
 obtained. If he wants time to evaluate participation, a new meeting will be arranged with a
 minimum of 48 hours from the information meeting. When the informed consent has been
 obtained, screening can be done, either the same day or another day, but with a maximum of
 four weeks after obtaining the informed consent. The participant will be informed that it is
 possible to withdraw the written consent at any time prior to or during the study. In the
 case that significant information about the participants health is found during the trial,
 the participant will be able to decline having this information. Information about this
 choice is confirmed in the informed consent form.

 If the participant wants to know about the results of the trial and the possible consequences
 for the participant, he will have the rights to get this information after the end of the
 study At screening, height and body weight will be measured, medication and medical history
 will be recorded, blood will be sampled for assessment of plasma/serum concentrations of
 thyroid-stimulating hormone, creatinine, creatine kinase, electrolytes, aspartate
 aminotransferase (ASAT), alanine aminotransferase (ALAT), lactate dehydrogenase, alkaline
 phosphatase, albumin, bilirubin, gamma-glutamyltransferase, viral hepatitis markers, platelet
 count, ferritin and haemoglobin, and albumin-creatinine ratio in the urine will be measured.
 A physical examination will be made including evaluation of blood pressure and pulse rate.
 Alcohol habits will be evaluated based on The Alcohol Use Disorder Identification Test
 (AUDIT) questionnaire and quantification of the weekly amount of alcohol. An MR safety
 checklist will be completed, and an MR information sheet will be given. If all inclusion
 criteria and none of the exclusion criteria are met, dates for baseline assessments and
 randomisation visit will be planned within 6 weeks from the screening visit. The participant
 will be instructed not to consume curcumin-containing food on a daily basis from screening
 until end of trial.

 Baseline assessments and randomisation visit

 If the participant is included during COVID19 pandemic, a phone call is done the day before
 the visit to ask about recent symptoms of infection, contacts to people with COVID19 symptoms
 or people with positive test for COVID19.

 Included individuals will undergo an MRS of the liver (scheduled as close as possible to the
 randomisation visit). Before intervention start, both randomisation visit and MRS will be
 performed. For MRS, participants are placed in a horizontal position. During the MRS, a
 spectroscopy of the liver will be assessed, and an estimation of visceral adipose tissue and
 subcutaneous adipose tissue will be done.

 During the last week before randomisation, the subject will collect a stool sample for the
 determination of gut bacteria count and subtype. Stool samples will be collected by the
 participant at home. Prior to defaecation a disposable collector will be placed in the toilet
 from where stool will be transferred to a tube that immediately is transferred to a transport
 container and placed at -20°C in the patient's freezer. Prior to transport to Gentofte
 Hospital, the transport container is transferred to the cooler bag and at arrival at Gentofte
 Hospital the transport container (with the sample tube) is immediately stored in at -80°C for
 later analysis. This entails DNA extraction, library preperation, MiSeq 16S rRNA sequencing
 and microbiota analysis (relative abundance, alpha/beta diversity, link to phenotype of
 interest).

 On the randomisation visit, the participant will meet in the laboratory around 8 a.m. after
 10 hours of fast (including any medication) and refrainment from tobacco or nicotine
 supplements use. The participant is allowed to drink water until 8 hours before meeting in
 department. Furthermore, the participant will be requested to avoid heavy alcohol intake
 (more than 5 units of alcohol per day) and strenuous exercise two days prior to the visit and
 instructed to use non-strenuous methods of transportation to the research facility. The
 following procedures will be conducted:

 If the test day is during COVID19 pandemic, the participant is tested for Sars-Cov2 with a
 swab procedure in the oropharynx, for immidiate PCR analysis.

 1. A fasting urine sample will be collected, and the participant is asked to empty the
 urinary bladder completely

 2. Fasting blood samples will be collected (bone markers, serum markers of muscle
 breakdown, biomarkers of NASH and fibrosis, fibroblast growths factors, triglycerides,
 cholesterols, liver parameters, HbA1c, inflammatory markers, adipokines, creatinine,
 haemoglobin) amounting 25 ml.

 3. FibroScan: The participant will be lying in a supine position for approximately 10
 minutes during which a probe using shear-waves and ultrasound is applied in the right
 side of the body below the ribs.

 4. Measurement of blood pressure, heart rate, body weight and hip and waist circumference.

 5. A questionnaire is handed out regarding calorie intake and physical activity the day
 prior to randomisation

 6. IOP is measured with icare ic100 tonometer.

 7. One cannula will be inserted in the cubital vein for collection of blood samples. The
 forearm from which blood samples are drawn will be wrapped in a heating blanket (42°C)
 throughout the experiment (for arterialisation of venous blood).

 8. Blood samples (for analysis of total amino acids, gut hormones, glucose, glucagon,
 insulin, C-peptide, urea, FFA) will be measured throughout the OGTT (see Table 1).

 9. At time 0 min, the participant will ingest 75 g of water-free glucose dissolved in 300
 ml water over a period of 5 minutes.

 10. At time 0, 30, 60, 90, 120, 150, 180, 210, 240 and after the ad libitum meal, hunger,
 satiety, fullness and prospective food consumption will be measured by VASs

 11. During the last hour (time 180 min - 240 min), REE will be measured in a supine position
 by indirect calorimetry to determine oxygen consumption (V02) and carbon dioxide
 production (VC02). A flow-through canopy gas analyser will be used, as previously
 described.

 12. At time 240 min, the subject will be asked to provide a new urine sample for measurement
 of urea concentration. The participant will be asked to empty the urinary bladder
 completely. Urine volume will be measured.

 13. At time ~250 min, the participant will ingest a standardised ad libitum meal consisting
 of minced meat, pasta, corn, carrots, peppers, cream, salt and pepper (50 energy (E)%
 carbohydrate, 37 E% fat, 13 E% protein). The participant will be instructed to eat as
 much as possible until feeling comfortable saturate. The meal should be consumed within
 a maximum of 30 minutes and by the end of the meal, time spent eating, weight of the
 meal and the total amount of energy consumed will be noted.

 14. A continuous glucose monitor will be applied on the upper arm of the participant. The
 sensor is blinded which means that the participant cannot see his blood glucose
 excursions during the 14 days period of measurements.

 At the end of the randomisation visit, the participant will be randomly assigned to one of
 three interventions (1:1:1):

 1. Two tablets of curcumin (corresponding to 200 mg) twice daily from day 1 to day 11 and
 prednisolone capsule 50 mg (oral) once daily from day 2 to day 11

 2. Two tablets of placebo-curcumin twice daily from day 1 to day 11 and prednisolone
 capsule 50 mg once daily from day 2 to day 11, or

 3. Two tablets of placebo-curcumin twice daily from day 1 to day 11 and
 placebo-prednisolone capsule once daily from day 2 to day 11 The tablets with curcumin
 and placebo-curcumin will appear identical, and the capsules with prednisolone or
 placebo-prednisolone will appear identical.

 The supplier of curcumin will also produce placebo-curcumin tablets (Indena Aps, Milan,
 Italy). The pharmacy of Region H will be responsible of production and delivery of
 prednisolone and placebo-prednisolon. Unblinded site-staff will be responsible for labelling
 and blinding of the curcumin and placebo-curcumin tablets and prednisolone and
 placebo-prednisolone capsules before the beginning of the intervention period. A person not
 otherwise involved in the study will generate a randomisation code using a random list
 generator. A randomisation list with randomisation numbers and corresponding treatment will
 be made by the unblinded site-staff. A list with randomisation number and corresponding
 packages with numbers will be made by unblinded site-staff, so that blinded site staff is
 only allocating a randomisation number at the day of randomisation. The list with the
 information about randomisation number and corresponding treatment will be kept at Center for
 Clinical Metabolic Research at Gentofte Hospital. If a participant develops adverse events
 (AEs) that demand knowledge of the content of the intervention, the list will show if the
 patient is having any active ingredients. If a participant is excluded of the trial before
 end-of-intervention visit, a new subject will be included in the same treatment-arm by taking
 over the randomisation number.

 The interventional period If the participant was tested for Sars-Cov2-infection at
 randomization visit, this test has to be negative before start of intervention. If the
 participant is included during COVID19 pandemic, a telephone call is made the day before
 intervention start, to ensure the following guidelines are followed: If the participant has
 experiences symptoms of COVID19 during the last seven days prior to intervention start or had
 close contact with COVID19-patients, intervention will not be started.

 Intervention will start soon after baseline MRS and randomisation visit. During the
 intervention period, each participant will fill out a dairy to keep track of any new
 concomitant medication, compliance and possible AEs. The participant will be asked to keep
 the alcohol and smoking habits they might have had before screening and, thus, not to consume
 more than 21 units of alcohol per week. Furthermore, they will be asked to consume no more
 than 5 units in one night. The participant will be requested to maintain the level of
 physical activity from before intervention during the interventional period. The participant
 will be informed that if he feels more hungry than usual, he is allowed to eat extra. At day
 5, 6 or 7 in the intervention period, the participant will receive a telephone call from the
 investigator or other delegated site staff, checking on AEs, concomitant medication and
 compliance, and to answer any questions from the participant. Furthermore, participants are
 instructed to contact investigator or other delegated site staff in case of any questions or
 intolerable side effects.

 The tablets and capsules are preferably ingested with a meal and a small glass of water.
 Should the participant forget to take a dose of curcumin/placebo curcumin, it can be ingested
 until 6 hours before the next planned dose. If a dosage is missed, and the next one is
 planned within 6 hours, the dosage should be passed, and the incident must be noted in the
 diary. Should the participant forget to take a dose of prednisolone/placebo-prednisolone, it
 can be ingested until 12 hours prior to the next dose. If a dosage is missed, and the next
 one is planned within the next 12 hours, the dosage should med passed, and the incident must
 be noted in the diary.

 If the participant is included during COVID19 pandemic: The participant is instructed to
 adhere to social distancing and to be aware of COVID19-symptoms, and call site staff in any
 case of symptoms during intervention period or if he has had close contact to a person who
 had a positive Sars-Cov2-test, and to immediately stop intake of intervention and
 self-isolate in this case.

 End-of-intervention assessments and visit The end-of-intervention visit will be planned on
 day 12 after start of intervention. If the participant is included during COVID19 pandemic, a
 telephone call is done the day before the visit to ask about recent symptoms of COVID19,
 recent contacts to people with COVID19 symptoms or people with positive test for COVID19.

 Stool sample collection and MRS will be performed in the period around end-of-intervention
 visit, and will be performed as described above under "baseline assessments and randomisation
 visit". Procedures during the end-of-intervention visit are similar to the ones described
 above under "Baseline assessments and randomisation visit". Also, during this day, to check
 for any effect of curcumin intervention, physical examination will be performed by a
 physician from the research team and additional blood samples (identical with the samples at
 the screening visit, except viral hepatitis markers) will be collected (which adds another 5
 ml blood samples compared to randomisation visit) and albumin-creatinine ratio in the urine
 will be measured. The continuous glucose monitor sensor is removed from the upper arm of the
 participant, and the data from the sensor is transferred to a suitable software program.
 Again, the questionnaire is handed out regarding calorie intake and physical activity the day
 prior to the End-of-intervention day. Compliance will be evaluated (if compliance is
 insufficient (missed more than ¼ of planned doses), the participant will not go through
 end-of-intervention assessments). If the participant is included during COVID19 pandemic, he
 is instructed to continue social distancing for three days after end-of-intervention visit.

 Follow-up phone call and visit Between two and four days after last day on treatment, the
 participant will receive a telephone call, where the participant is asked about withdrawal
 symptoms and symptoms of COVID19. Due to COVID19, an extra telephone call is made at day 5-8
 after end-of-intervention visit.

 If the participant feels any kind of withdrawal symptoms (tiredness, weakness, abdominal
 pain, anorexia, nausea or vomiting), or has had any symptoms of an ongoing infection after
 End-of-intervention, a plan of action is made. Judged from the physical examination, blood
 tests, results from continuous glucose monitoring, MRS and FibroScan at End-of-intervention
 visit, a follow-up visit will be arranged, if it - judged by the investigator - is necessary.
 At this follow-up visit (3-4 weeks after End-of-intervention) the investigator can do any or
 all of the following tests; physical examination, FibroScan, measurement of IOC, standard
 OGTT and blood tests, whichever is judged to be necessary to look into any late effects of
 the intervention, and if necessary, another follow-up visit after another 1-3 months. If the
 participant had steatosis on both MRS's, the participant will be offered a referral to the
 Department of Gastroenterology for further evaluation and treatment.

 BIOBANK A research biobank will be established at at Center for Clinical Metabolic Research
 at Gentofte Hospital, DK-2900 Hellerup, Denmark, during the experiments to store the blood
 tests until analyses. All participants are assigned with a trial number and will on data
 sheets and tubes only appear with the trial number. The full name, social security number and
 trial number will be stored separately. After the project is completed the biobank will be
 terminated. Excess plasma will be stored in another biobank for up to 10 years after the
 completion of the study, in case of need of re-analyses or need for further analyses. In the
 latter case, a new approval by the Regional Health Research Ethics Committee is required. The
 biobanks will be registered with Datatilsynet.

 CALCULATIONS AND STATISTICS Data will be processed and presented with the use of standard
 descriptive statistics. Areas under the curves (AUCs) are calculated by use of the trapezoid
 rule. Comparison of normally distributed data is carried out by means of repeated
 measurements analysis of variance using linear mixed modeling with "individual" as random
 factor. Freidmans test will be used for analysis of non-normally distributed data. P values
 ≤0.05 are accepted as statistically significant (significance level, α = 5%). Power of the
 study (1-β) is set to 80%, where β (20%) is the risk of accepting a hypothesis that is false.

 Power calculation: Hansen et al. studied a prednisolone-treated group of healthy individuals,
 and found an increase in HOMA-IR of 1.6 (1.3±0.1 vs. 2.9±0.3). The difference is 1.6±SD, but
 the value of this SD we unfortunately do not have. Best guess is that it is (maximum) 0.5. It
 is hypothesized that the intervention group receiving two different kinds of placebo is
 comparable to the baseline values in KB Hansens study, both before and after intervention, as
 placebo expectedly will not change HOMA-IR. And it is hypothesized that the prednisolone
 treated group is comparable with KB Hansens cohort after treatment with prednisolone. The
 wish is to achieve a reduction in the increase in HOMA-IR of 50% when ingesting curcumin,
 which gives a minimal relevant difference of 0.8.

 P values ≤ 0.05 are accepted as statistically significant (α = 5%) and the power is set to
 80%. This results in a sample size of 7 in each of the three treatment groups. . Based on
 this, we will include 9 in each of the three treatment groups, as we might have slightly more
 variance in HOMA-IR in our population compared to KH Hansen's participants. In case of
 drop-outs, we will include new participants until we have 12 completers in each group.

 The continuous glucose monitoring will provide data for up to 14 days with glucose measures
 with frequent intervals 24 hours per day. The CGM will be recording for at least 24 hours
 before start of intervention with prednisolon. CGM recordings are regarded successful if at
 least 80% of the sensor data are available for analysis. Before analysis, all data will be
 reviewed for pressure induced sensor attenuations. From these data, the following
 calculations will be made: 1) The risk of hypoglycemia and hyperglycemia will be calculated
 as the low blood glucose index (LBGI) and the high blood glucose index (HBGI) using the
 EasyGV workbook (www.phc.ox.ac.uk/research/technology-outputs/easygv) 2) coefficient of
 variability (SD/mean interstitial glucose level), 3) SD, 4) percentage of time in
 normoglycemic range (3,9-7,8 mmol/l), 5) percentage of time in three hyperglycemic ranges
 (7.9-10 mmol/l, 10.1-13.9 mmol/l, >13.9mmol/l) and 6) percentage of time in hypoglycemia
 (<3.9 mmol/l).

 SIDE EFFECTS, RISKS AND DISADVANTAGES FOR PARTICIPANTS Curcumin is a polyphenolic compound
 extracted from the turmeric root with a very low bioavailability. High doses of Meriva® have
 been well tolerated in human trials. However, most studies have been performed with
 non-formulated traditional curcumin with lower bioavailability. Nevertheless, high doses of
 non-formulated curcumin have not been associated with severe side effects, and are generally
 very well tolerated in clinical trials. Panahi et al did a study with NAFLD patients, with
 ~50 patients receiving Meriva® 1000 mg daily for 8 weeks (corresponding to 200 mg of
 curcumin). There were no reports of side effects, but 7 withdraw because of the perception of
 lack of effects. Antiga et. al. did a study with Meriva® 2000 mg daily for 12 weeks, with
 only one participant having one adverse event (diarrhoea), but presumably not caused by
 Meriva® because of other signs of viral genesis67. Mazzolani did a study with 12 patients
 receiving 1200 mg Meriva® per day for 1 year with no reports of AEs. Di Pierro et al. did a
 study on overweight participants, with a high dose of phosphatidylserine (a product very
 similar to Meriva®) with reports of no side effects.

 Prednisolone side effects are very well described, and most commonly are weight gain, skin
 abnormalities, increased serum glucose levels, insulin resistance, oedema, electrolyte
 derangement, myopathy, osteoporosis, infections, reduced symptoms of infections,
 haematological abnormalities, psychiatric diseases, mood changes, sleep disorders,
 hyperlipidaemia, hypertension and heart failure. But if the participants will experience side
 effects, they are unlikely to get severe, when treating only with 50 mg daily for 10 days, as
 they are dose and time-dependent, and with the relevant inclusion and exclusion criteria
 applied. Furthermore, they are all regarded as fully reversible after end of intervention.

 A side effect from treatment with oral corticosteroids (e.g. Prednisolone) is elevated IOP
 which in rare cases can cause the sight-threatening condition, glaucoma. In most cases
 elevated IOP is asymptomatic, which is why, it is important to measure in the patient. IOP
 can simply and easily be measured with a icare ic100 tonometer. The device is based on a
 rebound measuring principle that requires no drops, air or specialized skills for its use,
 and therefore not associated with any risk or complications. IOP is measured 3 times in each
 eye and the mean is calculated. If this is elevated the patient will be seen by an
 ophthalmologist.

 During and right after prednisolone treatment, the participants are more prone to infections
 and are likely to have fewer symptoms of infections. Participants will be asked about their
 wellbeing and symptoms of infection several times during the trial.

 If the participant is included during the COVID19 pandemic, several extra restrictions and
 assessments are applied in the study, due to the possible increased risk of a severe course
 of COVID19 in case of prednisolone treatment. This is applied only to take extraordinary
 precautions. Information on these restrictions and assessments is found in the relevant parts
 of this protocol. Participants are thoroughly instructed in the relevant changes in the
 participant information documents before screening.

 Withdrawal symptoms (after prednisolone treatment) are most commonly tiredness, weakness,
 abdominal pain, anorexia, nausea or vomiting. Due to the short treatment period and
 relatively low dose, it is very unlikely these symptoms will occur, but participants will be
 asked about these symptoms at the telephone call two to four days after last day on
 treatment.

 MRS is safe and not coupled with discomfort or pain. The scanner will be making loud noise
 while scanning, but the participant will wear proper ear protection. Some participants may
 experience claustrophobia during the scan. If the participant has steatosis on both MRS, he
 will be offered a referral to the Department of Gastroenterology.

 Penetrations of skin (blood puncture and intravenous catheter insertion and insertion of
 sensor for continuous glucose monitoring) are associated with mild discomfort and risk of
 hematomas. A theoretical complication to skin penetration is superficial phlebitis. The
 condition is not dangerous and will be treated with antibiotics should any sign of infection
 be present. The risk of superficial phlebitis is small and minimized by following clinical
 standards for the insertion of intravascular catheters including double wiping of the
 involved skin with disinfectant alcohol and other sterile procedures.

 The total blood loss will amount to approximately 365 ml distributed over a minimum of 2
 weeks, which is not in any way harmful for the participant. Only participants with
 haemoglobin >7.5 mmol/l can participate, and all participants are offered iron treatment
 after the trial.

 PARTICIPANTS PHYSICAL AND MENTAL INTEGRITY AND PRIVACY Information regarding participants of
 the project will be protected in accordance with the applicable laws of Denmark including the
 law concerning the processing of personal data. We will seek approval of the protocol by the
 Danish "Datatilsynet" via the The Capital Region of Denmark. The trial will follow the rules
 in European Union's General Data Protection Regulation.

 The protocol complies with the Helsinki Declaration (Seventh revision 2013).

 OPERATING COSTS AND ECONOMY The project is currently not supported by any medical companies
 and the responsible physician of the project has no economical affiliations with private
 companies, foundations or the like with interests in the research project. When funding for
 the project is in place the Research Ethics Committees will be notified.

 AVAILABILITY OF DATA FOR PARTICIPANTS All participants are guaranteed access to more
 information about the project. Contact: Dr. Pernille Høgh Hellmann, MD, Center for Clinical
 Metabolic Research, Gentofte Hospital, DK-2900 Hellerup; e-mail:
 pernille.hoegh.hellmann@regionh.dk; Phone: +45 20911792.

 ADJOURNMENT OF TRIAL Any participant can be withdrawn from the trial at any time, in case
 investigator finds it unsafe for the participant to continue. Also, the participant can be
 withdrawn if the participant meets any of the exclusion criteria, if compliance is
 insufficient (if participant has missed more than ¼ of planned doses, controlled at telephone
 call at day 5, 6 or 7 in the intervention period and at the end-of-intervention visit)
 evaluated by investigator, or in case exceptional circumstances make it impossible to
 complete the study. Likewise, any extraordinary event that makes it impossible to finish the
 project will result in termination of ongoing experiments. In all cases, the participant will
 be informed about the decision and the reason why. The participant may, at any time during
 the trial, voluntarily discontinue participation. In case of drop-outs, we will include new
 participants until we have 12 completers in each group.

 THE RESEARCH GROUP Pernille Høgh Hellmann MD, Jonatan Ising Bagger PhD MD, Katrine Bagge
 Hansen PhD MD, Professor Tina Vilsbøll DMSc MD, Professor Filip Krag Knop PhD, MD

 RECRUITMENT OF PARTICIPANTS Participants are recruited through the following methods: 1)
 Advertisement in newspapers or local newspapers or at www.forsøgsperson.dk. 2) Advertisement
 through flyers and posters. 3) Participants who participated in studies in the department in
 the past and gave consent that they would like to be contacted again in regard to
 participation in new studies in the department.

 Participants who responded on advertisements will receive written information on e-mail or
 regular mail, whatever is preferred. All participants will also receive the leaflet
 "Forsøgspersonens rettigheder i et sundhedsvidenskabeligt forskningsprojekt" from the Ethical
 Committee in Denmark, august 2014.

 After at least two days, the participant will be contacted by telephone and asked if he wants
 to participate in the study. If the participant has any questions, these will be answered as
 thoroughly as possible. If the participant is interested in participation, a time and place
 for the information visit will be scheduled. During the phone call, the participant is
 informed about the possibility to be accompanied by a person of own choice to all the visits
 during the trial.
Inclusion Criteria:

 -  Body mass index (BMI) >24.9 kg/m2, If participant is included during COVID19-pandemic,
 maximum BMI is 32 kg/m2

 -  Haemoglobin ≥7.5 mmol/l

 -  Written informed consent

 Exclusion Criteria:

 If the participant is screened during COVID19, extra exclusion criteria is age>59 years.

 If the participant is screened during COVID19-pandemic, following exclusion criteria is
 also applicable: 1) Diagnosed with asthma requiring medication or having uncontrolled
 asthma. 2) Beeing a current smoker. 3) diagnosed with hypertension. 4) having a job or
 engaging in daily activities, where it is not possible to adhere to the required distance
 of one meter to other people, judged by the investigator. 5) bA1c above 42 mmol/mol at
 screening.

 -  Use of glucose-lowering drugs, lipid-lowering drugs, warfarin, clopidogrel or
 non-vitamin K anti-coagulants

 -  Frequent use of anti-inflammatory drugs the last two months prior to inclusion, judged
 by the investigator

 -  Treatment with drugs with potential steatogenic side-effects within two months prior
 to inclusion

 -  Use of medication known to interact with prednisolone

 -  Use of curcumin-containing food supplements or other natural products that could cause
 confounding as evaluated by investigator

 -  Any regular drug treatment that cannot be discontinued for minimum 18 hours

 -  Previous diagnosis of T2D

 -  Diabetes (HbA1c ≥ 48 mmol/mol) found at screening

 -  Known viral, inherited or alcoholic liver disease, or any other condition known to
 affect liver

 -  Intake of more than 21 units of alcohol per week

 -  Nephropathy (eGFR < 60 ml/min/1.73 m² and/or urine albumin > 20 mg/l)

 -  In a weight management program, or planning to change life style, alcohol habits or
 eating habits during the course of the study

 -  Implanted metal objects contraindicative of MRS Claustrophobia

 -  Any condition that the investigator think would interfere with trial participation or
 with the safety of the participant.