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Trial registered on ANZCTR


Registration number
ACTRN12611000979954
Ethics application status
Approved
Date submitted
13/09/2011
Date registered
14/09/2011
Date last updated
25/01/2016
Type of registration
Retrospectively registered

Titles & IDs
Public title
The effect of meals rich in carbohydrate and fat on metabolism and inflammation
Scientific title
Postprandial effect of carbohydrate and fat mixed meals on the regulation of metabolism, inflammation and immune function in healthy subjects with or without a family history of type 2 diabetes and in type 2 diabetes patients
Secondary ID [1] 263036 0
St Vincent's Hospital Research Ethics Reference H06/147
Universal Trial Number (UTN)
Trial acronym
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Type 2 diabetes 270762 0
Family history of type 2 diabetes 270763 0
Condition category
Condition code
Metabolic and Endocrine 270944 270944 0 0
Diabetes

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
Meals:
High carbohydrate
High fat
Mixed high carbohydrate and high fat

Meal composition:
High carbohydrate:
130 g of Heinz canned spaghetti in tomato sauce and cheese, 140 g of white bread, 40 g toasted Muesly, 30 g jam, 40 ml of cordial concentrate in 250 ml water, 150 g plain fat free yoghurt, 5 g sugar (950 kcal, 12.6 g fat, 158 g carbohydrate, 51 g protein).

High fat meal:
100 g eggs fried in 15 ml canola oil, 30 g Ryvita crisp-bread, 10 g butter, 52.5 cheddar cheese, 30 g mixed nuts, 125 g strawberries, 40 g whipping cream, 4 g sugar (1066 kcal, 81 g fat, 25 g carbohydrate, 59 g protein).

High carbohydrate high fat meal:
100 g eggs fried in 15 ml canola oil, 160 g bagel, 7 g butter, 40 g jam, 21 g cheddar cheese, 20 g mixed nuts (1169 kcal, 75 g fat, 79 g carbohydrate, 45 g protein).

Research Plan and procedures:
Each subject attends the Clinical Research unit in the morning after an overnight fast (10-12 hours) for five visits:

Screening Visit (visit 1):
Detailed information, informed consent.
Medical history, clinical check, blood pressure, weight, height, oral glucose tolerance test (OGTT).
At the end of screening, if eligible to participate in the study, the clamp visit (visit 2) and meal visits (visits 3-5) will be scheduled.
The clamp visit will be booked 7-14 days after the screening visit and the first meal visit (visit 3) will be scheduled 14 days after visit 2.

Clamp visit (visit 2):
Fasting blood samples
Indirect calorimetry
Heart rate variability at resting and during the intravenous glucose tolerance test (IVGTT) and hyperinsulinaemic euglycaemic clamp (see detailed procedure below).
Dual X-ray absorptiometry (DXA) and computerized tomography (CT).

Visit 3,4 and 5 are identical and performed 7-14 days apart:
Baseline (fasting) indirect calorimetry, heart rate variability
Test meal 1/2/3 in a randomized order. Specifically, the subjects will be randomized for the first meal and will then have the 2 other meal types in a randomized order.

Blood samples are taken before and 30, 60, 120, 180 and 240 min after the meal for measuring cytokines, glucose, insulin, hormones, triglyceride.
Indirect calorimetry is repeated at 3.5-4.0 hours to evaluate postprandial fat and carbohydrate oxidation.
Heart rate variability at 30-min intervals in the postprandial state.

Subjects undergo the following procedures:
Oral Glucose Tolerance Test (OGTT)- A 75g glucose drink is given and blood glucose is measured at -10, 0, 30, 60, 90 and 120 minutes.

Intravenous Glucose Tolerance Test (IVGTT) is performed to assess B-cell function and the heart rate variability response to endogenous insulin. Blood is drawn at -10, 0, 1, 2, 3, 4, 5, 6, 8, 10, 20, 30, 40, 50, 60 min after infusion of 0.3 g/kg dextrose 25% (maximum 25 g or 100 ml).


Hyperinsulinaemic euglycaemic clamp is performed to assess whole-body insulin sensitivity and heart rate variability response to supra-physiological hyperinsulinaemia. Insulin is infused intravenously at 60 mU/m2 body surface area/min. Glucose is infused at a rate sufficient to maintain blood glucose at about 5 mmol/L (euglycaemia). The steady state glucose infusion rate (GIR) is defined as the average rate of glucose delivery during the last 30 min period of the clamp. Two venous cannulae will be needed, one at each forearm. The antecubital vein is usually used. Before the insertion of the cannulae the usual antiseptic procedures are applied. Blood is drawn every 10 minutes during the clamp.

Indirect calorimetry will be performed fasting and at clamp steady stae (from 90-120 min) or at 3.5-4 h post meal. This technique is used to evaluate resting energy expenditure and the carbohydrate and fat oxidation rate. A transparent plastic hood is connected to the measuring device and placed over the subjects head for a 30 min period. Calculations of O2 consumption and CO2 production are done by continuous measurements of inspired and expired air diluted in a constant air flow. Subjects will be asked to remain motionless and awake.

Dual X-ray absorptiometry (DXA) is performed to quantify body fat, central fat and lean body mass. Lunar Prodigy (Lunar Radiation Corporation, software version 7.51) whole body scanner at St Vincent’s Hospital is used. The scanner emits low energy X-rays. The scan takes about 5 minutes and the radiation dose is less than 1 mrem (equal to about 12 h background radiation).

CT scan - Cross-sectional CT slices (at L2/3 and L4/5) to evaluate visceral and subcutaneous abdominal fat. Liver fat content will also be evaluated (1 slice).
Intervention code [1] 269380 0
Early detection / Screening
Comparator / control treatment
Non
Control group
Uncontrolled

Outcomes
Primary outcome [1] 279612 0
The effect of hyperinsulinamia on the autonomic nervous system

The autonomic nervous system activity will be assessed using a heart rate sensor (SphygmoCor, AtCor Medical Inc., Australia). Heart rate (HR, bpm) and both time and frequency domain measures of heart rate variability, root mean square of successive differences (RMSSD) between adjacent R-R intervals, low frequency (LF) power (0.04 - 0.15 Hz), high frequency (HF) power (0.15 - 0.4 Hz), and the LF/HF ratio will be derived. Power spectral density for the LF and HF components of heart rate variability are calculated using fast Fourier transformation. RMSSD is sensitive to vagal cardiac control, both sympathetic and vagal activity are considered to be the main determinants of the LF component, whereas the HF component is thought to predominantly reflect vagal activity. The LF/HF ratio is therefore considered to be an indicator of sympatho-vagal balance.
Timepoint [1] 279612 0
After 30 min rest, HRV will be assessed in 2 lots of 8-10 min. During IVGTT, clamp and meals, HRV will be assessed every 30 min for 10 min.
Secondary outcome [1] 294033 0
The effect of meal composition on the function of immune cells and inflammatory markers


Immune cell preparation and flow cytometry analysis:
Fresh whole blood sample will be stained with fluorochrome-conjugated antibodies to various cell surface markers purchased from BD Biosciences (San Diego, CA) at baseline and every 1-hour post meal. All analyses will be performed on a BD FACSCaliburTM (BD Biosciences, San Diego, CA) with an excitation laser line Argon (488nm) and Red diode (635nm), and running CellQuest software (version 3.3 from BD Biosciences). Data Analysis software FlowJo version 7 from TreeStar Inc will be used. For comparative quantification of surface activation marker expression, the mean fluorescence intensity (MFI) of the marker will be divided by the MFI of the unstained control to give relative MFI (rMFI). For quantification of Th1/Th2 cells we use intracellular cytokine staining for the key cytokines interferon-gamma (for Th1) and IL-4 (for Th2) (BD Bioscience Pharmingen, San Diego, CA). Briefly, peripheral blood mononuclear cells (PBMCs) will be activated with PMA (160ng/ml) and Ionomycin (1000ng/ml) for 4 hours at 37C in the presence of GolgiPlug (BD Bioscience, San Diego, CA), allowing the identification of T-helper cell subsets. After surface staining for CD4+ and CD8+, cells will be permeabilized using BD Cytofix/Cytoperm? reagents (BD Bioscience Pharmingen, San Diego, CA), stained for intracellular cytokines and analysed immediately by flow cytometry.

Circulating inflammatory markers:

High sensitivity C-reactive protein (hsCRP) will be measured using a Beckman Coulter Synchron LX system Chemistry Analyser, with reagents and calibrators supplied by Beckman Coulter Inc. (Sydney, Australia).
Interleukin (IL)6, monocute chemoattractant protein (MCP)1 and soluble intercellular adhesion molecule (sICAM)-1 will be measured using commercial high sensitivity ELISA (R&D Systems, Minneapolis MN, USA).
Timepoint [1] 294033 0
Immune cell surface markers and Th1/Th2 will be evaluated at fasting and every hour post meal for 4 hours and inflammation markers at fasting and at 3-h postprandially

Eligibility
Key inclusion criteria
Healthy men and women or type 2 diabetes patients
Minimum age
50 Years
Maximum age
65 Years
Gender
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
Type 2 diabetes patients:
HbA1c>10% or insulin treatment
Cardiovascular disease, cancer.
Healthy individuals:
Any underlying disease

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)
Allocation is not concealed.
Methods used to generate the sequence in which subjects will be randomised (sequence generation)
The subjects will be randomized for the first meal and will then have the 2 other meal types in a randomized order based on a randomization list.

Research Randomizer (Version 3.0) http://www.randomizer.org/
Masking / blinding
Open (masking not used)
Who is / are masked / blinded?



Intervention assignment
Crossover
Other design features
Phase
Not Applicable
Type of endpoint(s)
Efficacy
Statistical methods / analysis

Recruitment
Recruitment status
Completed
Date of first participant enrolment
Anticipated
Actual
Date of last participant enrolment
Anticipated
Actual
Date of last data collection
Anticipated
Actual
Sample size
Target
Accrual to date
Final
Recruitment in Australia
Recruitment state(s)
NSW

Funding & Sponsors
Funding source category [1] 269837 0
Charities/Societies/Foundations
Name [1] 269837 0
Diabetes Australia Research Trust
Address [1] 269837 0
GPO BOX 3156
CANBERRA ACT 2601
Country [1] 269837 0
Australia
Funding source category [2] 269838 0
Commercial sector/Industry
Name [2] 269838 0
Neuro Science Research Pfeizer Australia
Address [2] 269838 0
P O Box 57
West Ryde NSW 2114
Country [2] 269838 0
Australia
Primary sponsor type
Other
Name
Garvan Institute of Medical Research
Address
384 Victoria Street
Darlinghurst NSW 2010
Country
Australia
Secondary sponsor category [1] 268869 0
None
Name [1] 268869 0
Address [1] 268869 0
Country [1] 268869 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 271807 0
St Vincent's Hospital Human Research Ethics Committee
Ethics committee address [1] 271807 0
Research Office
Level 6, de Lacy Building
St Vincent's Hospital
Darlinghurst NSW 2010
Ethics committee country [1] 271807 0
Australia
Date submitted for ethics approval [1] 271807 0
Approval date [1] 271807 0
08/01/2007
Ethics approval number [1] 271807 0
H06/147

Summary
Brief summary
Type 2 diabetes is an increasingly common disorder characterized by a poor response of the body’s tissues to insulin and impaired secretion of insulin from the insulin producing cells in the pancreas. The mechanisms causing these two defects are still poorly understood. Moreover, there are signs of chronic inflammation in the body, but its role in diabetes and the contribution of the immune system to this process is not known. We believe that studying the metabolic responses to glucose and insulin in subjects with or without a family history of diabetes will advance our understanding of early underlying impairments, possibly contributing to the development of diabetes.
Trial website
Trial related presentations / publications
Viardot A, Heilbronn LK, Samocha-Bonet D, Mackay F, Campbell LV, Samaras K (2012) Obesity is associated with activated and insulin resistant immune cells. Diabetes Metab Res Rev 28:447-454

Jenkins AB, Batterham M, Samocha-Bonet D, Tonks K, Greenfield JR, Campbell LV (2013) Segregation of a latent high adiposity phenotype in families with a history of type 2 diabetes mellitus implicates rare obesity-susceptibility genetic variants with large effects in diabetes-related obesity. PLoS One 8:e70435
Public notes

Contacts
Principal investigator
Name 33151 0
Dr Dorit Samocha-Bonet
Address 33151 0
384 Victoria Street, Darlinghurst, NSW 2010
Country 33151 0
Australia
Phone 33151 0
+61292958309
Fax 33151 0
Email 33151 0
d.samochabonet@garvan.org.au
Contact person for public queries
Name 16398 0
Ms Lynne Schofield
Address 16398 0
384 Victoria Street
Darlinghurst NSW 2010
Country 16398 0
Australia
Phone 16398 0
+61292958230
Fax 16398 0
Email 16398 0
l.schofield@garvan.org.au
Contact person for scientific queries
Name 7326 0
Dr Dorit Samocha-Bonet
Address 7326 0
384 Victoria Street
Darlinghurst NSW 2010
Country 7326 0
Australia
Phone 7326 0
+61292958309
Fax 7326 0
Email 7326 0
d.samochabonet@garvan.org.au

No information has been provided regarding IPD availability
Summary results
Have study results been published in a peer-reviewed journal?
Other publications
Have study results been made publicly available in another format?
Results – basic reporting
Results – plain English summary