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Trial registered on ANZCTR


Registration number
ACTRN12623000260628
Ethics application status
Approved
Date submitted
8/02/2023
Date registered
10/03/2023
Date last updated
1/11/2024
Date data sharing statement initially provided
10/03/2023
Type of registration
Prospectively registered

Titles & IDs
Public title
Effect of Nutritional INterventions on Autophagy (The NINA Study)
Scientific title
Effect of Nutritional INterventions on Autophagy in healthy adults (The NINA Study)
Secondary ID [1] 305322 0
Nil known
Universal Trial Number (UTN)
Trial acronym
The NINA Study
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Autophagic flux in healthy subjects 323642 0
Condition category
Condition code
Metabolic and Endocrine 321180 321180 0 0
Normal metabolism and endocrine development and function
Inflammatory and Immune System 326189 326189 0 0
Normal development and function of the immune system

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
This study is a single-site, single blinded, randomised crossover trial that aims to measure the effect of a four week modification of dietary macronutrient intake on autophagic flux (i.e. a cell mechanism that cleanses the cells) in fasting healthy individuals.

Participants will be randomised to one of two study diets for four weeks before crossing over to the other diet, with a four week washout period between diets. Both diets are healthy and balanced and are designed by a dietitian. Each diet has a different macronutrient profile. The reference diet has an ‘average’ macronutrient profile, whereas the intervention diet has an 'altered' macronutrient profile. Participants will be provided with individual 7-day rotating menus and will be asked to complete menu checklists daily to record any food prescribed that was not eaten and any food eaten that was not prescribed.

The study design is comprised of an online screening survey (30 minutes), a screening visit (1 hour) followed by four, 1.5 hour metabolic visits (M1, M2, M3, M4). All participants will have similar visit schedules (minimum 28 days on diet) and measures of adherence.

Eligible participants will attend the first metabolic visit (M1) where they will be randomised to one of the two study diets and will be asked to adhere to this diet for a four week (minimum 28 day) intervention period. After the first diet intervention, participants will attend the second metabolic visit (M2), following this visit they will commence the four week washout period where participants will be allowed to consume their regular diet. At the end of the washout period, participants will attend the third metabolic visit (M3) and be allocated to the diet that they were not allocated to during the first four weeks. After a further four weeks on the second diet intervention, participants will attend the fourth metabolic visit (M4) which is the end of the study.

At each metabolic visit participants will arrive at the South Australian Health and Medical Research Institute in the morning after a 12 hour overnight fast. Body weight, height, waist circumference, hip circumference, hand grip strength, body composition, and blood pressure (following 10 minute seated rest) will be measured using standardised procedures. Following this fasting blood samples will be drawn. Participants will also complete a series of questionnaires to assess quality of life, sleep habits, eating behaviours, and physical activity. At the end of both dietary periods (visits M2 and M4), participants will be asked to complete a survey to record diet preference and satisfaction.

POST COMPLETION:
This project has ethical approval by the University of Adelaide HREC for limited disclosure to participants. Please find the information below that was approved to be disclosed once the last participant has completed their last appointment.

This research involves limited disclosure in relation to the difference in protein amounts between the two study diets. To prevent study participants from trying to guess which diet is the ‘Average Protein Diet’ and which diet is the ‘Reduced Protein Diet’ and therefore avoid bias, the participant information sheet does not outline that the purpose of the study is to investigate the effect of dietary protein intake on autophagy (Attachment 2). The wording has been kept general and states that the study aims to measure the effect of diet composition on autophagy in humans. The project involves low risk participants only, therefore we anticipate that the limited disclosure will not affect participants adversely. After their participation has ended, participants will be provided with information about the aim of the research and an explanation of why the omission was necessary and offered the opportunity to withdraw any data or sample provided by them. There is no likely reason for thinking that participants would not have consented if they had been fully aware of what the research involved (National Statement Chapter 2.3).

The identity of the two study diets used as interventions are as follows:

Intervention (2 x 4 weeks = 8 weeks)

This is a crossover study. Participants will be first be randomly assigned to one of the two diets from M1 to M2. From M3 to M4, they will be assigned to the study diet they have not been assigned to from M1 to M2.

• Average Protein Diet: a control comparator diet that is similar to the ‘typical Australian diet’, will be delivered at calculated energy balance based on equations we have previously validated. This will be a nutritious whole foods diet with a macronutrient profile of 20 % protein, 30 % fat (<10 % saturated) and 50 % carbohydrate, with 30 g of fibre. This group will receive the same information and study visits as other group.

• Reduced Protein Diet: This diet will be similar to the control diet, except macronutrients will be given at 10 % protein, 30 % fat (<10 % saturated fat), 60 % carbohydrate, ~30 g of fibre/day. This diet will provide 0.7g of protein per kg of body weight (the recommended minimum daily protein intake).
Intervention code [1] 321733 0
Treatment: Other
Intervention code [2] 321734 0
Prevention
Comparator / control treatment
The reference diet will be the balanced diet with the 'average macronutrient' profile.
Control group
Active

Outcomes
Primary outcome [1] 329042 0
Change in autophagic flux between an average macronutrient diet compared to altered macronutrient diet (randomly assigned crossover design). Assessed by measuring the amount of the autophagic cargo LC3 (with or without the lysosome inhibitor chloroquine) in peripheral blood mononuclear cells by ELISA.
Timepoint [1] 329042 0
After 4 weeks adherence to diet 1 and after 4 weeks adherence to diet 2 (12 hour overnight fasted blood samples).
Secondary outcome [1] 401252 0
Change in autophagy-related markers, namely p-mTOR, p-S6, p-AMPK, P62, LAMP1, BECLIN-1.
Timepoint [1] 401252 0
After 4 weeks adherence to diet 1 and after 4 weeks adherence to diet 2 (12 hour overnight fasted blood samples).
Secondary outcome [2] 401253 0
Change in body weight measured as a part of bioelectrical impedance analysis (BIA), assessed at each metabolic visit.
Timepoint [2] 401253 0
After 4 weeks adherence to diet 1 and after 4 weeks adherence to diet 2 (12 hour overnight fast).
Secondary outcome [3] 401254 0
Change in blood metabolic markers: glucose, HDL, LDL, triglycerides, free fatty acids, insulin, c-peptide, adiponectin, c-reactive protein, GLP-1, GIP, ghrelin, free amino acids in plasma.
Timepoint [3] 401254 0
After 4 weeks adherence to diet 1 and after 4 weeks adherence to diet 2 (12 hour overnight fasted blood samples).
Secondary outcome [4] 401255 0
Change in blood pressure using an automatic sphygmomanometer.
Timepoint [4] 401255 0
After 4 weeks adherence to diet 1 and after 4 weeks adherence to diet 2 (12 hour overnight fast).
Secondary outcome [5] 401256 0
Change in muscular strength using a digital hand grip dynamometer.
Timepoint [5] 401256 0
After 4 weeks adherence to diet 1 and after 4 weeks adherence to diet 2 (12 hour overnight fast).
Secondary outcome [6] 401257 0
Change in quality of life using the SF36 validated questionnaire.
Timepoint [6] 401257 0
After 4 weeks adherence to diet 1 and after 4 weeks adherence to diet 2 (12 hour overnight fast).
Secondary outcome [7] 401258 0
A question about which diet the participant preferred, asked at the end of the study.
Timepoint [7] 401258 0
Metabolic visit 4, at the end of the 12 week study period.
Secondary outcome [8] 418836 0
Assessment of eating behaviour (cognitive restraint, disinhibition, and hunger) using the TFEQ validated questionnaire.
Timepoint [8] 418836 0
Questionnaire used at each metabolic visit.
Secondary outcome [9] 418838 0
Assessment of physical activity using the IPAQ validated questionnaire.
Timepoint [9] 418838 0
Questionnaire used at the screening visit, and metabolic visits 2, 3, and 4.
Secondary outcome [10] 418839 0
Assessment of self-rated sleep habits using the PSQI validated questionnaire.
Timepoint [10] 418839 0
Questionnaire used at each metabolic visit.
Secondary outcome [11] 418842 0
Assessment of body composition using bioelectical impedance and other anthropometric measures.
Timepoint [11] 418842 0
Measurement at every metabolic visit.

Eligibility
Key inclusion criteria
1, Aged between 20 and 50 years of age
2. BMI between 18.5 and 29.9 kg/m2
Minimum age
20 Years
Maximum age
50 Years
Sex
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
1. Diagnosis or evidence of neurodegenerative disorder such as Alzheimer’s disease.

2. Any co-morbidities that are likely to change the activity of the lysosomal system, such as cancer, diabetes, cardiovascular disease/conditions (stroke, heart attack, high blood pressure), renal or liver disease, hemoglobin disorder or anaemia, gastrointestinal surgery or disease, major psychiatric disorders (schizophrenia, addiction, eating disorders, unstable major depressive disorder), neurological disorders, and/or any other condition deemed likely to affect the results by the study principal investigator.

3. Taking any medications that, in the opinion of the investigator, might change autophagic activity; including, but not restricted to, chronic medications or supplements that change appetite, body composition or metabolism (e.g. medications used to lower blood glucose, antidiabetic medications), anti-inflammatory medications, medications used in the treatment of cancer or cardiovascular disorders.

4. Female participants taking hormonal contraceptive will not be excluded from the study. It is preferable but not essential that women who have a menstrual cycle attend the M1 baseline study appointment between day 1 and day 10 of their menstrual cycle. Date of first day of menstrual cycle will be documented as well as cycle length in days at both Screening Visit and Metabolic Visits.

5. Anyone who is not weight stable (lost or gained > 5% body weight in the last 3 months).

6. Current alcohol abuse (>14 standard drinks/week) and/or substance use disorder.

7. Current smoker.

8. Vegan.

9. Lactose intolerance.

10. Pregnant or breastfeeding women or women planning a pregnancy.

11. Women who have been through menopause or peri-menopausal.

12. Self-reported dietary practice or restriction that may affect autophagy (including calorie restriction, intermittent fasting and ketogenic diets).

13. Self-reported high consumption of protein or supplements.

14. Anyone who has an eating disorder, food allergies or practices that are likely to interfere with compliance (eg. coeliac, gluten intolerance, and nut allergies).

15. Anyone who is unable to comprehend the study protocol (i.e. due to English language or cognitive difficulties).

16. Anyone with a pacemaker or other internal electronic medical devices.

Study design
Purpose of the study
Prevention
Allocation to intervention
Randomised controlled trial
Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)
Central randomisation via an electronically generated randomisation schedule built into REDCap.
Methods used to generate the sequence in which subjects will be randomised (sequence generation)
Simple randomisation using a randomisation table created by computer software (i.e. computerised sequence generation)
Masking / blinding
Blinded (masking used)
Who is / are masked / blinded?
The people receiving the treatment/s


Intervention assignment
Crossover
Other design features
Phase
Not Applicable
Type of endpoint/s
Efficacy
Statistical methods / analysis
Sample size calculation:
With existing data available to estimate the expected change in LC3II flux (i.e. autophagic flux) between diets, we have assumed a change within-subject of 15%. Based on a previously observed change in LC3II flux mean of 277.862 ng LC3BII/mg protein/h and a SD of 90.675 in healthy subjects, assuming a change of 15% within subject between both diets, alpha = 0.05 and a power of 80 %, this requires a sample size of 53 participants based on a paired Student’s t-test (two-tailed). Assuming a 15% dropout rate, we will recruit n = 63 participants.

Analysis:
Outcomes will be analysed using linear mixed effects models to estimate the effect of diet, expressed as a difference in means with 95% confidence interval and 2-sided p-value. Models will take into account potential period effects and baseline measurement.

Recruitment
Recruitment status
Completed
Date of first participant enrolment
Anticipated
15/03/2023
Actual
11/05/2023
Date of last participant enrolment
Anticipated
30/08/2024
Actual
8/07/2024
Date of last data collection
Anticipated
30/09/2024
Actual
3/10/2024
Sample size
Target
63
Accrual to date
Final
63
Recruitment in Australia
Recruitment state(s)
SA
Recruitment postcode(s) [1] 35354 0
5000 - Adelaide

Funding & Sponsors
Funding source category [1] 309687 0
Charities/Societies/Foundations
Name [1] 309687 0
BrightFocus Foundation
Country [1] 309687 0
United States of America
Primary sponsor type
Individual
Name
Dr Timothy Sargeant
Address
Level 6 North
South Australian Health and Medical Research Institute (SAHMRI)
North Terrace
Adelaide, South Australia
5000
Country
Australia
Secondary sponsor category [1] 310709 0
None
Name [1] 310709 0
Address [1] 310709 0
Country [1] 310709 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 309455 0
University of Adelaide Human Research Ethics Committee
Ethics committee address [1] 309455 0
Ethics committee country [1] 309455 0
Australia
Date submitted for ethics approval [1] 309455 0
28/05/2021
Approval date [1] 309455 0
30/08/2021
Ethics approval number [1] 309455 0
H-2021-154

Summary
Brief summary
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 114218 0
Dr Tim Sargeant
Address 114218 0
Hopwood Centre for Neurobiology
South Australian Health and Medical Research Institute
North Terrace, Adelaide
South Australia
5000
Country 114218 0
Australia
Phone 114218 0
+61 881284940
Fax 114218 0
Email 114218 0
tim.sargeant@sahmri.com
Contact person for public queries
Name 114219 0
Tim Sargeant
Address 114219 0
Hopwood Centre for Neurobiology
South Australian Health and Medical Research Institute
North Terrace, Adelaide
South Australia
5000
Country 114219 0
Australia
Phone 114219 0
+61 8 8128 4940
Fax 114219 0
Email 114219 0
tim.sargeant@sahmri.com
Contact person for scientific queries
Name 114220 0
Tim Sargeant
Address 114220 0
Hopwood Centre for Neurobiology
South Australian Health and Medical Research Institute
North Terrace, Adelaide
South Australia
5000
Country 114220 0
Australia
Phone 114220 0
+61 8 8128 4940
Fax 114220 0
Email 114220 0
tim.sargeant@sahmri.com

Data sharing statement
Will individual participant data (IPD) for this trial be available (including data dictionaries)?
No
No/undecided IPD sharing reason/comment
Study findings will be made available in a way so that no individual is identifiable without their consent.


What supporting documents are/will be available?

No Supporting Document Provided



Results publications and other study-related documents

Documents added manually
No documents have been uploaded by study researchers.

Documents added automatically
No additional documents have been identified.