Clinical trial NCT03334175
Conditions: PreDiabetes Overweight and Obesity
Sponsor: University of California, San Francisco
Start date: 2018-05-21; Completion date: 2019-08-05
Enrollment: 18 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Prediabetes is a precursor of type 2 diabetes and an independent risk factor for cardiovascular disease, and currently affects one-quarter of the population of the United States. Individuals of overweight or obese BMI are at particular high risk for incident diabetes. A major modifiable risk factor for type 2 diabetes is poor dietary quality, and improvement of dietary quality can effectively delay and even prevent type 2 diabetes. Interventions to improve dietary quality thus far, however, rely on short-term intensive clinically designed meals replacing the entire diet which have poor sustainability. Persistent improvements to daily dietary patterns are often difficult without directed guidance, and overall dietary quality in the United States remains poor. The identification of a practical, daily dietary intervention to improve dietary quality and prevent diabetes in those at high risk remains unknown. The investigators propose to enroll 40 individuals with diagnosed prediabetes into a randomized controlled pilot study and provide a daily walnut supplementation intervention to determine feasibility and acceptability of the supplement. The investigators will then determine preliminary efficacy on metabolic markers and will investigate associations between dietary quality and circulating levels of branched-chain amino acids. The goal is to implement a whole-food supplement to improve dietary quality in patients with prediabetes as a tool for future type 2 diabetes prevention.

Detailed description:

Arms: arm 1: Will receive and be instructed to consume 1-oz individually wrapped daily walnut supplement packages for 12 weeks during intervention. arm 2: Will receive diet and exercise guidance at beginning of study. At end of study, will receive 12 weeks of a walnut supply to be consumed at their discretion after the study is complete.
Interventions: intervention 1: Walnuts
Intervention descriptions: intervention 1: 1 ounce of individually wrapped, raw walnuts to be consumed daily replacing a high-refined carbohydrate food.

Protocol documents:
Prot_SAP_001.pdf: 
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 Walnuts to Achieve Lasting NUTrition to prevent Diabetes  
(WALNUT-Diabetes) 
 
 
 
 
 
 
Principal Investigator: 
Meghana Gadgil, MD 
 
Coordinating Center: 
University of California, San Francisco 
 
 
Protocol Version Date: 
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NCT:   
 
 
NCT03334175 
 
 
 
 
 
 
 


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TABLE OF CONTENTS 
PROTOCOL SECTION 
PAGE 
 
 
1.  PROJECT SUMMARY 
4 
 
 
2.  SPECIFIC AIMS AND HYPOTHESES 
5 
 
 
3. BACKGROUND AND SIGNIFICANCE 
6 
3.1.    Significance 
6 
3.2.    Rationale for our choice of Walnuts for the intervention 
7 
3.3     Innovation 
8 
  
 
4.  EXPERIENCE OF THE INVESTIGATOR 
8 
 
 
5.  RESEARCH DESIGN AND METHODS 
8 
5.1     Participants 
8 
5.2.    Study Overview 
8 
5.3.    Study Intervention 
9 
5.4     Randomization and Blinding 
9 
5.5.    Supply and Dispensing of Intervention Food 
10 
5.6.    Clinical visits and procedures 
11 
5.7.    Measures collected at each clinical visit 
13 
5.8.    Statistical analysis 
14 
5.9.    Potential problems and alternative approaches 
15 
5.10.    Study limitations 
15 
 
 
 6.  PROTECTION OF HUMAN SUBJECTS 
16 
6.1.   Risks to the Subjects 
16 
6.1.a.     Human Subject Involvement and Characteristics 
16 
 
6.1.b.     Sources of Materials 
16 
 
 
6.1.c.     Potential Risks 
16 
 6.2     Adequacy of Protection Against Risks 
17 
 
6.2.a.     Recruitment and Informed Consent 
17 
 
6.2.b.     Protection Against Risk 
17 


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 6.3     Potential Benefits of the Proposed Research to the Subjects and 
Others 
18 
 6.4     Importance of the Knowledge to be Gained 
18 
 6.5     Data and Safety Monitoring Plan 
18 
 
6.5.a.     Confidentiality 
18 
 
6.5.b.     Adverse Event Information 
18 
 
6.5.c.     Data Quality and Safety Review Plan and Monitoring 
19 
 
6.5.d.     Safety Review Plan 
420 
 
 
7.  BIBLIOGRAPHY 
43 
 
 
 


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ABBREVIATIONS 
 
AAA 
 
aromatic amino acids 
AE 
 
adverse events 
BCAA  
branched chain amino acids 
DM 
 
diabetes mellitus 
DPP 
 
diabetes prevention program 
G 
 
grams 
PI 
 
principal investigator 
SAE 
 
serious adverse experience 
 
 


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1 
PROJECT SUMMARY 
 
Prediabetes is a precursor of type 2 diabetes and an independent risk factor for 
cardiovascular disease, and currently affects one-quarter of the population of the United 
States. Individuals of overweight or obese BMI are at particular high risk for incident 
diabetes. A major modifiable risk factor for type 2 diabetes is poor dietary quality, and 
improvement of dietary quality can effectively delay and even prevent type 2 diabetes. 
Interventions to improve dietary quality thus far, however, rely on short-term intensive 
clinically designed meals replacing the entire diet which have poor sustainability. 
Persistent improvements to daily dietary patterns are often difficult without directed 
guidance, and overall dietary quality in the United States remains poor. The identification 
of a practical, daily dietary intervention to improve dietary quality and prevent diabetes in 
those at high risk remains unknown. We propose to enroll 40 individuals with diagnosed 
prediabetes into a randomized controlled pilot study and provide a daily walnut 
supplementation intervention to determine feasibility and acceptability of the supplement. 
We will then determine preliminary efficacy on metabolic markers and will investigate 
associations between dietary quality and circulating levels of branched-chain amino 
acids. Our goal is to implement a whole-food supplement to improve dietary quality in 
patients with prediabetes as a tool for future type 2 diabetes prevention. 
 


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2 
SPECIFIC AIMS AND HYPOTHESES 
Aim 1: Determine a) feasibility for recruitment and retention into a randomized controlled 
trial of daily walnut supplementation, and b) acceptability and adherence to the 
intervention containing 28g of walnuts daily, compared with usual care in individuals with 
prediabetes. 
 
Aim 2: Determine preliminary efficacy of the walnut intervention vs. usual care on change 
in a) fasting glucose, HbA1c and lipid levels, b) dietary quality score and c) BCAA and 
AAA levels. These estimates of efficacy will inform the sample size for a larger future 
randomized controlled trial. 
 
 


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3 
SIGNIFICANCE 
3.1 BACKGROUND 
Over one-quarter of the adult population in the United States has prediabetes. 
Prediabetes is a state of impaired fasting glucose and/or impaired glucose tolerance that 
is an independent risk factor for type 2 diabetes and cardiovascular disease.1,2 Up to 30% 
of people with prediabetes will progress to diabetes in 5 years at current standards.2 The 
Diabetes Prevention Program (DPP) is an intensive diet and exercise program that has 
been shown to decrease incident diabetes in those with impaired glucose tolerance,3 
however this intervention is cost and labor-intensive and is not available to all individuals 
with prediabetes. In addition, changes to diet and lifestyle must be lasting to significantly 
delay or prevent diabetes and its complications. The current standard of care to prevent 
diabetes in those with prediabetes consists only of minimal counseling during primary 
care visits without significant between-visit follow-up. 
Dietary quality is a strong, modifiable risk factor for diabetes and cardiovascular 
disease. NHANES surveys reveal stably poor dietary quality in the US population over 
many years, despite improvements in public health messaging.4 In observational studies, 
improved dietary quality is associated prospectively with lower incidence of type 2 
diabetes and cardiovascular disease.5-7 Modeling from a longitudinal cohort study 
suggests that animal protein consumption may promote progression to diabetes at a 
faster rate than plant-based protein intake.8,9 There are currently no targeted public 
health messages to assist those with prediabetes in refining their dietary quality with the 
goal of preventing diabetes. 
Walnut intake has been shown to improve insulin sensitivity, decrease 
cardiovascular risk and improve endothelial function.  Walnuts contain fiber, 
polyunsaturated fatty acids and plant-based protein that improve satiety.10 In conjunction 
with a Mediterranean diet, supplementation with walnuts has been associated with 
decreased risk of metabolic syndrome,11 cardiovascular disease12 and mortality12,13 in 
high-risk populations. 
Plant-based protein 
consumption,8,14 and nut 
intake in particular, over 
the long-term have 
shown inverse 
associations with incident 
type 2 diabetes in large 
cohort studies.15 
Individuals who regularly 
consume nuts have a 
lower BMI in 
epidemiologic studies,16 
and feeding trials have 
demonstrated an 
improvement in lipid 
subfractions with acute 
and long-term intake of 
nuts.17-19 In short-term 
trials, consumption of nuts improved endothelial function20 and insulin signaling17 without 
Figure 1: Conceptual Model 


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caloric restriction. These findings suggest that nut intake and the quality of diet has a 
major effect on the pathways involved in both the incidence and complications of 
diabetes. (Figure 1) Increasing nut consumption may also improve satiety, ideally 
displacing those foods, such as refined carbohydrates, that have been shown to 
contribute to the incidence of diabetes, cardiovascular disease and obesity.21 
Practical, daily interventions can result in improved overall dietary quality. An 
approach to add walnuts to usual dietary intake, exchanging one healthful food for other 
unhealthful foods, has the potential to improve overall dietary quality, and, in turn, 
improve surrogate markers of type 2 diabetes and lipids levels. With such cumulative 
changes, long-term improvement in dietary quality can help to decrease progression to 
type 2 diabetes in people at high risk. The PREDIMED trial added either nuts or olive oil 
to a Mediterranean-style diet, compared to a low-fat control diet and found decreased 
fasting glucose and LDL levels, as well as lower clinical outcomes of incident diabetes 
and cardiovascular disease.22,23 Little prior work, however, has assessed the effects on 
surrogate markers for diabetes, including metabolomics, of the addition of nuts to a usual 
diet in the United States and in individuals with known prediabetes who are at high risk 
for both diabetes and cardiovascular disease. 
I have examined dietary patterns and quality and their associations with risk factors for 
diabetes in a population of South Asian Americans, and found that a dietary pattern 
characterized by refined carbohydrates and high-saturated fat sweets was associated 
with higher insulin resistance (HOMA-IR) and lower HDL.24 I have also evaluated dietary 
quality, as measured by the HEI-2010 score, and its association with glycemic control in 
women with gestational diabetes mellitus, and found that higher dietary quality is 
associated with improved post-prandial glycemic control (Gadgil MD, American Journal of 
Obstetrics and Gynecology, submitted). I have an investigation currently underway to 
examine dietary quality, BCAA intake and incident gestational diabetes mellitus in women 
who are part of the Coronary Artery and Risk Development in Young Adults (CARDIA) 
longitudinal cohort study, and our preliminary results show that lower dietary quality and 
higher BCAA intake is associated with an increase in gestational diabetes incidence. As 
the preponderance of prior work has shown improved outcomes and decreased 
incidence of diabetes with improvements in dietary quality, we believe that the next step 
is to create a practical, daily intervention to aid in improving overall dietary quality. 
3.2 STUDY RATIONALE 
This study will add to the literature by investigating a practical, daily intervention for 
individuals with prediabetes to improve dietary quality and, in turn, to prevent diabetes. 
We will characterize novel metabolomics pathways from dietary patterns to DM incidence 
that may help explain the mechanism of the effect between diet and glucose tolerance in 
a high-risk population. In doing so, we will identify previously unknown targets to measure 
the preclinical effects of dietary influence on risk for diabetes. 
 
2.3 INNOVATION   
This proposed investigation will join the burgeoning movement of dietary intervention 
studies that are performed in a real-world environment, without highly controlled 


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conditions, which is more representative of dietary patterns in people at risk for metabolic 
disease. The approach we use differs from the current paradigm. We aim to use a whole-
food supplement, easily available outside of the research or clinical environment. We also 
aim to investigate changes in metabolomic measures associated with walnut 
supplementation as an intermediate outcome. Metabolomics can reflect current 
conditions for pre-existing prediabetes, and we will examine how short-term interventions 
can affect levels of these circulating markers. These data will also help us to determine if 
levels of circulating amino acid levels may be useful as markers of adherence for dietary 
interventions beyond self-report.  
4 
EXPERIENCE OF THE INVESTIGATORS 
Meghana Gadgil, MD MPH is an Assistant Professor in the Division of General Internal 
Medicine at the University of California, San Francisco. Over the past 5 years, she has 
focused her research in the areas of dietary patterns, cardiometabolic disease, and 
racial/ethnic disparities. Her prior work demonstrates associations of dietary quality and 
patterns with risk factors for metabolic disease, in both normal weight and 
overweight/obese individuals, and her objective for this study is to evaluate a walnut 
supplementation intervention in high-risk individuals with prediabetes. Her long-term goal 
is to identify and implement practical dietary interventions to improve dietary quality and 
ultimately prevent diabetes. 
5 
RESEARCH DESIGN AND METHODS 
5.1 Study Participants 
Study Population, Study Design and Eligibility criteria: We will enroll 40 individuals with 
prediabetes identified through an electronic medical record query from patients in the 
UCSF DGIM primary care clinics and through outside recruitment in the surrounding 
community. We will exclude patients with tree nut or peanut allergies, who are unwilling 
to consume a daily nut supplement, are on dietician-managed, restricted diets for any 
medical condition, have overt diabetes or are on glucose-lowering medications, or who 
are currently enrolled in a Diabetes Prevention Program activity. See full inclusion and 
exclusion criteria in next section. 
5.2 Study Overview 
This is a single-center, unblinded, randomized-controlled trial. 40 study subjects are 
planned. Each subject will consume 28g of individually wrapped raw, unsalted walnuts 
daily for a total of 12 weeks. Participants will be assigned to the treatments in random 
order. Evaluations will be taken at baseline and at end-of-study. 
Screening data will be reviewed to determine subject eligibility. Subjects who meet all 
inclusion criteria and none of the exclusion criteria will be entered into the study. 
5.3 Study Intervention 
Participants in the Usual Care group and Walnuts groups will receive usual counseling 
and care, including general dietary and lifestyle counseling provided to all patients with 
diagnosed prediabetes, recommending a diet high in vegetables, fruits, whole grains, 
lean protein and dairy. Those in the Walnuts intervention group will receive initial dietary 
counseling to describe the high refined carbohydrate foods to be replaced with the 


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intervention walnut supplement.  The intervention group will receive one, individually-
wrapped, 28g packet of raw, unsalted walnuts to be consumed daily for a total of 12 
weeks to replace specific high refined carbohydrate snacks. Individually packaged 
walnuts will be donated by the California Walnut Commission, who will not have a role in 
the design, procedures or interpretation of this study. Participants will be required to log 
their food exchanges on a daily basis. This study is designed as a non-blinded trial, 
therefore to protect against greater than usual consumption of walnuts by the Usual Care 
group during the intervention period, we will offer walnut supplementation at the end of 
the trial to these participants. Participants in the Usual Care group will be instructed to 
carry out usual diet and lifestyle changes for the 12-week duration, and will receive 
walnut supplementation, identical in dose and duration to the intervention group, after the 
completion of the study. 
The following treatment regimens will be used: 
• 
Walnuts “Now”: 28g of individually-wrapped raw, unsalted walnuts consumed daily for 
12 weeks during the study period 
• 
Walnuts “Later”: Usual diet during the study period. The participants will be provided 
with an equivalent amount of individually wrapped walnut packets for their daily use 
after the study period has concluded. 
5.4 Randomization and Blinding 
Randomization: Participants will be randomly assigned to two groups of 20 participants 
each: “Walnuts Now” Or “Walnuts Later”. We will block our randomization by two 
important potential confounders: sex (female or male) and age categories (<50 or ≥50 
years). We will generate stratum-specific sequential ID numbers to randomly allocate 
individuals to intervention groups in blocks of four. Study staff will be blinded to the 
linkage between ID numbers and group assignment. Upon enrollment, each participant's 
stratum will be identified and the next sequential ID number from that stratum was 
assigned. At the end of the baseline visit, the interviewer will open a sealed, opaque 
envelope pre-printed with the sequential ID number revealing the intervention group 
assignment to the participant.  
Blinding 
Due to the objectives of the study, the identity of test and control treatments will be 
known to investigators, research staff, or patients.   
This study is designed as a non-blinded trial, therefore to protect against greater than 
usual consumption of walnuts by the Usual Care group during the intervention period, we 
will offer walnut supplementation at the end of the trial to these participants. Participants 
in the Usual Care group will be instructed to carry out usual diet and lifestyle changes for 
the 12-week duration, and will receive walnut supplementation, identical in dose and 
duration to the intervention group, after the completion of the study.  
Because of the behavioral nature of the intervention, study staff who administer the 
intervention and study participants will not be blinded to their intervention group 
assignment, however we will blind the outcomes assessments – clinical labs and amino 
acid analyses will be done by labs without knowledge of intervention group. 
 


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5.5 Supply and Dispensing of Intervention Food at the Study Site 
A 12-week supply of individually-wrapped walnut packets will be given to intervention-
group participants at enrollment in the study. Those randomized to the control group will 
receive a 12-week supply of walnuts at the final study visit. 
The California Walnut Commission will ship the walnuts to the study site in boxes of 200 
individually-wrapped samples in 2 installments – in June 2017 and August 2017. Walnuts 
will be shipped and kept in cold storage according to the attached protocol. If a 
participant has concerns about the quality of the walnuts, they will contact the study 
coordinator who will initiate the following protocol from the California Walnut Commission: 
1) Have participant return package and remaining product 
2) Check walnut batch (lot number, date, size of package) where package obtained, if 
possible 
3) Contact Carol Berg Sloan RD, directly and immediately.  
Carol Berg Sloan RDN, FAND  
cbsrd@verizon.net  
562-221-9869  
Dosage/Dosage Regimen 
We will test a dose of 28g of walnuts daily for the experimental group participants. The 
walnuts should ideally be consumed without added salt or roasting. This whole-food 
supplement will be given in conjunction with dietary advice to replace consumption of 
added sugars and refined carbohydrates 
Dispensing 
The study coordinator will provide a 12 week supply of walnuts to the intervention 
participants at the enrollment visit. The study coordinator will provide an equivalent 
supply of walnuts to the control participants at the study conclusion visit. 
Administration Instructions  
The participants will consume one 28-gram packet of walnuts daily. This may be 
consumed at one sitting or throughout the day. Walnuts should replace added sugars or 
products with refined carbohydrates. 
Supply of Food Supplement at the Site 
The individually wrapped walnut packages will be delivered from the California Walnut 
Commission in 2 installments. The first in early June and the second in early August. 
Storage 
The individually wrapped walnuts will be stored in cold storage until they are given to the 
participants. 


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Measures of Treatment Compliance 
Subjects will be asked to keep a patient diary noting the day and date they consume their 
allocated walnut packet and any adverse events. They will be asked to bring their patient 
diary to each study visit. 
We will also test measures of walnuts consumption: whole blood RBC fatty acids 
composition; serum lipids which include LDL HDL, TC ApoA- 1, Apo B, triglycerides. 
Allowed Medications and Treatments 
Standard therapy for all medical conditions is allowed except for treatments noted in the 
exclusion criteria described above. 
5.6 CLINICAL VISITS AND PROCEDURES  
Demographics  
Demographic information (date of birth, gender, race/ethnicity) will be recorded at 
Baseline visit. 
Medical History  
Relevant medical history, including history of current disease and information regarding 
underlying diseases will be recorded at Baseline visit. 
Vital Signs 
Weight, height, blood pressure and waist circumference will be collected and body mass 
index calculated at initial baseline visit and study completion visit.  
Clinical Laboratory Measurements 
Blood will be obtained by venipuncture at the UCSF Clinical Research Services site at 
Parnassus at baseline and study completion visits. We will obtain 4 tubes of blood (serum 
and plasma and separated into aliquots. One aliquot of serum will be sent to Quest 
Diagnostics for determination of fasting glucose, lipid Apo A1- and ApoB, s and A1c 
measurements. An aliquot of plasma will be sent to the West Coast Metabolomics Center 
at the University of California, Davis, for determination of targeted amino acid 
metabolomics analysis. The remaining aliquots will be frozen at -80C for future research. 
See Table 1 for Study procedures at each visit. 
Telephone Screening 
Individuals who respond to recruitment notices will be provided with a general overview of 
the program and will complete a brief telephone survey to determine initial eligibility (age, 
prediabetes diagnosis) before being invited to the screening visit. 
Baseline clinic visit  
At the baseline visit, the study will be explained in detail, and informed consent will be 
obtained. Candidates will be told that this is a trial of walnut supplementation to improve 
dietary quality in overweight or obese individuals with prediabetes.  
 


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The research coordinator will perform measurement of height using a stadiometer and  
weight using a digital scale. Those with a BMI > 25 kg/m2 (> 23 kg/m2 if of Asian or South 
Asian ethnicity) will be eligible. The research coordinator will them perform a capillary 
blood glucose measurement using an Accu-check Aviva Connect blood glucose meter 
and Accu-Check test strips. A capillary blood glucose value of 100mg/dL and higher will 
be considered eligible for participation in the study. 
 
Eligible participants will be randomized into “Walnuts Now” and “Walnuts Later” groups. 
They will complete questionnaires about demographic and medical history, health-related 
behaviors including satiety, and medications. A research coordinator will measure height, 
weight, waist circumference and blood pressure. The participants will undergo 
venipuncture and fasting blood draw. One tube of serum will be sent to Quest for 
measurement of fasting glucose, hemoglobin A1c and serum lipids as well as 
apolipoprotein A2 and B. One tube will be aliquotted into 1mL cryogenic tubes. One 
aliquot of plasma will be shipped to the West Coast Metabolomics Center at the 
University of California, Davis for amino acid and acylcarnitine analyses. The remaining 
serum and plasma aliquots will be de-identified, frozen at -80C and stored for future 
analyses. 
 
All participants will receive handouts regarding a healthy diet to prevent diabetes.  
The research coordinator will dispense 12-week supply of individually-wrapped, raw 
walnuts to “Walnuts Now” group. 
6-week Telephone Follow-up  
The research coordinator will reach out to study participants by phone at 6 weeks to 
remind intervention participants of daily walnut intervention, give a reminder of healthy 
eating habits to all participants, and schedule Study Conclusion visit. 
Study Completion Visit 
At study completion, all participants will be invited back for a study completion visit. At 
this visit, a research assistant will record height, weight, blood pressure and waist 
circumference. The participants will undergo venipuncture and fasting blood draw. One 
tube of serum will be sent to Quest for measurement of fasting glucose, hemoglobin A1c 
and serum lipids as well as apolipoprotein A2 and B. One tube will be aliquotted into 1mL 
cryogenic tubes. One aliquot of plasma will be shipped to the West Coast Metabolomics 
Center at the University of California, Davis for amino acid and acylcarnitine analyses. 
The remaining serum and plasma aliquots will be de-identified, frozen at -80C and stored 
for future analyses. 
All participants will receive handouts regarding a healthy diet to prevent diabetes.  
The research coordinator will dispense 12-week supply of individually-wrapped, raw 
walnuts to “Walnuts Later” group. 


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5.7 Measures Collected at Each Visit 
Table 1: Measurements at each Study Visit 
Visit 
Telephone 
Screening (Week 0) 
Baseline visit  
(Week 1) 
Telephone Mid-Point (6 weeks) 
Study completion  
(12 weeks) 
Informed 
Consent 
Verbal informed 
Consent 
Written informed consent 
 
 
Questionnaires 
Initial eligibility 
screen 
Demographic questionnaire 
Block food frequency  
Self-efficacy  
 
 
Acceptability  
Palatability 
Adherence 
Block food frequency  
Self-efficacy  
Clinical 
measurements 
 
Height 
Weight 
Waist circumference 
Blood pressure 
Capillary blood glucose 
 
Height 
Weight 
Waist circumference 
Blood pressure 
Laboratory 
analyses 
 
Fasting glucose 
HbA1c 
Lipids 
BCAA, AAA, acylcarnitines 
 
 
Fasting glucose 
HbA1c 
Lipids 
BCAA, AAA, acylcarnitines 
 
Counseling 
 
Dietary and exercise 
counseling for both intervention 
and control arms 
Check-in for diet and exercise 
changes 
Adherence to walnut 
supplement for “Walnuts Now” 
group. 
 
Dispensing of 
Study 
Intervention 
 
“Walnuts Now” Intervention 
Group receives a 12-week 
supply of walnuts 
 
“Walnuts Later” Control Group 
receives a 12-week supply of 
walnuts 


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5.7 
Statistical analyses  
This trial is designed as a feasibility study and an investigation of the acceptability of the 
walnut intervention. The second aim is to establish preliminary efficacy for changes in 
fasting glucose, HbA1c and amino acid levels, for determination of sample size in a future 
clinical trial. Therefore, we have chosen to enroll a goal of 40 participants, including 20 in 
the intervention and 20 in the control groups. 
Specific Aim 1:  Aim 1: Determine a) feasibility for recruitment and retention into a 
randomized controlled trial of daily walnut supplementation, and b) acceptability and 
adherence to the intervention containing 28g of walnuts daily, compared with usual care in 
individuals with prediabetes. 
Feasibility and acceptability. We will assess feasibility based on number screened and 
enrolled in the proposed randomized clinical trial for our goal of 40 participants. Retention 
over the 12-week intervention period will be assessed by number of participants who 
complete the final study visit at the conclusion of the intervention period. Palatability will be 
assessed by a 100mm visual analog scale. We will also assess acceptability of the walnut 
intervention, and its dose and frequency through a targeted questionnaire at the end of the 
intervention period. Adherence will be measured through self-report (food diaries) as well 
as the food frequency questionnaire administered at study completion. All questionnaires 
will be administered by our research assistant. 
Aim 2: Determine preliminary efficacy measures for change in a) fasting glucose, HbA1c 
and lipid levels, b) dietary quality score and c) BCAA levels in the intervention group to 
inform a larger future randomized controlled trial. 
Fasting glucose, HbA1c and lipids: We will obtain fasting blood samples at enrollment and 
at the conclusion of the study. One aliquot of serum tube of blood will be sent for fasting 
glucose, lipids, ApoA1 and B, and HbA1c measurement, and one aliquot of plasma will be 
sent for metabolomics analyses. Additional aliquots of plasma and serum will be frozen for 
future research purposes. We will establish preliminary efficacy for each of these 
measurements. 
Dietary Quality: A Block food frequency questionnaire will be administered to participants 
at enrollment and completion of the study period. This questionnaire will use patient recall 
to gather frequency and serving size data for all participants. We will use the responses in 
this questionnaire to determine consumption of foods grouped based on likeness and 
culinary usage. These categories will then be transformed into 2 dietary scores: the 
Healthy Eating Index-2010 score, based on the Dietary Guidelines for Americans, which is 
a continuous score ranging from 0-100, with a score of 100 representing the optimal diet. 
We will also calculate the Mediterranean Diet Score, with possible values from 0-55, with 
55 representing the diet most congruent with the Mediterranean diet. 
BCAA, AAA and acylcarnitines: Targeted amino acid and acylcarnitine levels will be 
measured at baseline and at 12 weeks, post-intervention using triple quadrupole (MS/MS) 
and gas chromatography instruments. We will collaborate with the West Coast 
Metabolomic Center on the metabolomics analyses and interpretation. 
Statistical Analysis: This is designed as a parallel group controlled trial, randomized on 
dichotomized age (<50 or > years) and sex (female or male). We will use Fisher’s exact 
test and Student’s t-test to compare mean baseline demographic and clinical 


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characteristics between the intervention and control groups. Using information from the 
Block food frequency questionnaire, we will construct a continuous dietary quality score 
using the Healthy Eating Index 2010 and Mediterranean Diet Score criteria. We will use 
analysis of covariance to compute the adjusted mean for our outcome variables of fasting 
glucose, HbA1c, HDL, LDL, total cholesterol and amino acid levels. In analysis of 
covariance, the baseline value of outcome variables will be used as a covariate for the 
computation of adjusted means between the two groups after intervention. 
  
Expected Results: We believe that walnut supplementation will be acceptable at a dose of 
28g daily. We expect to be able to recruit 40 participants within 3 months, and retain >75% 
for the full 12 weeks of the intervention. We expect to see a trend towards significant 
change in the BCAA and AAA measurements and some improvements of fasting glucose 
and HbA1c in the “Walnut” group compared to the “Usual care” group.  
5.8 
Potential problems and alternative approaches  
The dose of raw, unsalted walnuts at 28g, or 1 oz, daily may not be an acceptable dose for 
study participants with diabetes. Based on our literature review, there have been several 
studies that have found a dose of 28-56g of walnuts acceptable for a similar duration. The 
Usual Care study arm may alter their diets to consume more walnuts during the study 
period given the inability to blind participants for this intervention. We plan to address this 
by offering walnuts after the study period to the control group, as an incentive to retain the 
usual care environment during the period in question. We will also be able to determine if 
the BCAA and AAA levels in the usual care group improve through the 12-week period as 
a marker of possible co-intervention.  
We have considered using an active comparator to walnuts, however with the possibility of 
confounding biological activity for other whole food supplements, we elected not to include 
one for this pilot study given our goals of feasibility, acceptability, adherence and 
preliminary efficacy of a walnut intervention. Instead, we feel that providing walnuts for all 
of the trial participants, using the approach that the intervention group will receive walnuts 
during the main study period, and the control group will continue with usual care during the 
intervention period, but then receive walnuts later as a “walnuts now” vs. “walnuts later” 
approach will help to control for confounding. If we are successful in our primary 
outcomes, we will consider an active comparator for our larger randomized controlled trial. 
5.9 
Study limitations  
Generalizability: We aim to recruit subjects who self-identify as overweight or obese, with 
prediabetes, and will assess their laboratory criteria for prediabetes at the first visit. Our 
study design is a convenience sample of volunteers who are motivated to participate in a 
clinical study, all factors which limit generalizability. Our study population should be 
representative for our target population of overweight or obese adults with prediabetes 
between the ages of 18 and 65. We will be unable to accommodate participants who do 
not speak English due to difficulties in finding qualified study personnel who speak other 
languages.  
 
 
 


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6 
PROTECTION OF HUMAN SUBJECTS 
6.1 
Risks to the Subjects 
6.1.a Human Subject Involvement and Characteristics:  We will be enrolling 
approximately 40 volunteers who are overweight or obese and who fulfill criteria for 
prediabetes. 
Eligibility criteria are listed below: 
Inclusion Criteria 
1. 
Male or female between 18-65 years of age at baseline living in the San Francisco 
Bay area. 
2. 
BMI>25 m/kg2 (or >23 m/kg2 for individuals of Asian or South Asian ethnicity) 
3. 
Documentation of prediabetes diagnosis as evidenced by the following criteria: 
a. A fasting glucose 100-125 mg/dL, or a HbA1c measurement of 5.7-6.4%, 
OR a diagnosis of “prediabetes” or “impaired fasting glucose” in the past 6 
months, identified through an electronic medical record query from patients 
at UCSF and through outside recruitment in the surrounding community 
b. We will confirm eligibility of potential participants by repeating fasting 
capillary blood glucose measurements at the baseline visit to ensure that 
they have prediabetes 
4. 
Written informed consent and ability for subject to comply with the requirements of the 
study. 
Exclusion Criteria 
1. 
Pregnant or breastfeeding women at enrollment. 
2. 
Presence of a condition or abnormality that in the opinion of the Investigator would 
compromise the safety of the patient or the quality of the data, such as diverticulosis 
or diverticulitis. 
3. 
Tree or peanut allergies 
4. 
Unwilling to consume a daily walnut supplement. 
5. 
Diagnosis of diabetes 
6. 
On glucose lowering medications 
7. 
Dietician-managed dietary intake, or personal or medical dietary restrictions that do 
not allow consumption of walnuts 
8. 
Malabsorptive conditions including intestinal bypass surgery, pancreatitis, 
inflammatory bowel disease 
All subjects should be maintained on the same medications throughout the entire study 
period, as medically feasible, with no introduction of new glucose-lowering therapies. 
 
6.1.b Sources of Materials: All personal, medical, and biochemical, data collected 
during the studies are for research purposes only, although test results that come from 


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certified laboratories can be provided to patients or their physicians upon request with the 
subject’s permission.  
 
6.1.c Potential Risks:  
 
Capillary blood glucose measurement: The baseline visit will require fingerstick collection 
of one drop of blood for determination of eligibility. This may result in pain or discomfort. 
We do not anticipate other problems from this procedure in healthy adults. 
 
Phlebotomy and venipuncture:  The baseline visit and study conclusion visit 3-month visit 
will require approximately 35-45 ml of venous blood collection. We do not anticipate any 
problems with anemia with a total of 45 ml of blood collection in our study of healthy 
volunteers. Venipuncture may result in a hematoma, which can be painful but carries no 
significant risks. Infections rarely occur from such brief interventions. 
 
 
 
Walnut supplementation: There is minimal risk associated with participation in the walnut 
intervention. There is the possibility of an allergic reaction to the walnut supplement. We 
will actively exclude any participants with a known tree nut or peanut allergy from 
enrollment in the study. There is the small possibility that an as-yet, unknown, allergy to 
walnuts will occur during the course of the study. In the event of signs or symptoms of 
allergy, the walnut intervention will be discontinued. Participants will be educated on signs 
and symptoms of a walnut allergy at the baseline visit. In order to protect the integrity and 
freshness of the walnuts, they will be kept in cold storage at the study site, and 
participants will be instructed to keep them in cold storage in their private homes before 
use. 
 
6.2 
Adequacy of Protection Against Risks 
 
6.2.a Recruitment and Informed Consent: Recruitment will be accomplished primarily 
through a medical records query, and through flyers at Parnassus and Mount Zion UCSF 
campuses. At the beginning of the baseline visit, a member of the research staff will 
review the consent form in detail with the patient and answer all questions before inviting 
the patient to sign the consent form.  A photocopy of the signed consent form with the 
Experimental Subjects' Bill of Rights is given to the patient.  The protocols are reviewed by 
the Institutional Committee on Human Research, University of California, San Francisco 
and University of California, San Diego.  All subjects will be reimbursed for the time and 
possible inconvenience associated with participation in the studies. 
 
6.2.b Protection Against Risk: The specific measures to minimize each risk are 
described in the relevant sections above, as well as the Data Safety and Monitoring Plan 
that is outlined in a subsequent section.  In addition, there will be continuous safety 
surveillance with emphasis on the potential side effects of each procedure, as detailed 
above.  Participation in the study will be discontinued if the subject fails to adhere to the 
study requirements in a way that may cause harm to him or herself or seriously interfere 
with the validity of the study results; or the investigator determines that further participation 
would be detrimental to the subject’s health or well being.    
Subjects who are injured as a result of being in this study will have treatment available. 


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The costs of such treatments may be covered by the University of California depending on 
a number of factors.   
Participation in research may involve a loss of privacy.  Research records will be kept 
confidential to the extent permitted by law.  Subjects will be identified by a code, and 
personal information from records will not be released without written permission.  
Subjects will not be personally identified in any publication about this study. 
 
6.3 
Potential Benefits of the Proposed Research to the Subjects and Others 
 
There is no guaranteed benefit to the subjects who participate in this study.  To the extent 
allowable, results of laboratory tests are made available to subjects and/or their primary 
care providers that might prove useful in their clinical management or prevention of future 
disease.  
 
 
6.4 
Importance of the Knowledge to be Gained 
 
Benefits to society may include a better understanding of the potential effectiveness of 
including walnuts in a daily diet to improve dietary quality, and to understand the 
mechanisms of improvement in blood markers after a walnut intervention. 
 
5.1.1.1 6.5 
Data and Safety Monitoring Plan 
 
6.5.a. Confidentiality  
 
1.  Protection of Subject Privacy – During this study, a limited physical examination will 
be performed and questionnaires will be administered. Fasting blood tests will be 
done periodically. Data will be kept in strict confidence. No information will be given 
to anyone without permission from the subject. Confidentiality is assured by use of 
identification codes. All data, whether generated in the laboratory or at the bedside, 
will be identified with a randomly generated identification code unique to the 
subject. Health Information Portability and Accountability Act (HIPAA) guidelines of 
the two clinical sites will be followed. (See VI. Informed Consent and HIPAA) 
 
2. Database Protection – The database is secured with password protection. The 
informatics manager receives only coded information, which is entered into the 
database under those identification codes.  Electronic communication with outside 
collaborators involves only unidentifiable information. 
 
3. Confidentiality during AE Reporting –AE reports and annual summaries will not 
include subject-identifiable material. Each will include the identification code only. 
 
6.5.b. Adverse Event Information 
 
1.  Definition - An adverse event (AE) is any untoward medical occurrence in a subject 
temporally associated with participation in the clinical study.  An adverse finding can 
include a sign, symptom, abnormal assessment (laboratory test value, vital signs, 
electrocardiogram finding, etc.) or any combination of these. 


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2. Classification of AE Severity –AEs will be labeled according to severity which is 
based on their impact on the patient, per this Event Grading Scale: 
 
Grade  1 Mild 
Transient of mild discomfort; no limitation in activity; no 
medical intervention/therapy required. 
Grade  2 Moderate 
Mild to moderate limitation in activity – some 
assistance may be needed; no medical 
intervention/therapy required. 
Grade  3 Severe 
Marked limitation in activity, some assistance usually 
required; medical intervention/therapy required, 
hospitalizations possible. 
Grade  4 Life- 
Extreme limitation in activity, significant assistance required 
 
 
Threatening  
significant medical intervention/therapy required, hospital- 
 
 
 
ization or hospice care probable. 
 
3. AE Attribution Scale –AEs will be categorized according to the likelihood that they 
are related to the study intervention.  Specifically, they will be labeled either definitely, 
probably, possibly or unrelated to the study intervention. 
 
4. Expected Risks – There is minimal risk with participating in the walnut intervention. 
Expected risks to the subject are mild gastrointestinal symptoms due to inclusion of 
walnuts in a normal daily diet. These risks are considered to be minimal and are 
addressed in the protocol and consent form. Participants will have a contact number to 
report any potential adverse events that occur in between scheduled study visits. They 
will also have an opportunity to privately discuss any other physical complaints due to 
inclusion of walnuts.  
 
 
5. SAE Reporting - SAEs that are unanticipated, serious (grades 3 and 4), and/or 
possibly related to the study intervention will be reported to the Independent Monitor, 
IRB, CTSA at UCSF, and NIH in accordance with requirements. Anticipated SAEs or 
those unrelated to the study intervention will be reported to the same 
individuals/entities in accordance with requirements. 
 
If abnormal lab values are discovered during the study, the PI will be notified and will 
address this issue as early as possible. If fasting glucose is found to be abnormally low 
(greater than or equal to 40 mg/dL and below 70 mg/dL), the participant will receive a 
call from the study coordinator asking if they are experiencing adverse effects such as 
nausea or lightheadness This mild hypoglycemia will be treated with sugar intake. A 
severely low blood sugar (<40 mg/dL) will be flagged as a critically low value by the 
study coordinator who will notify the principal investigator immediately or within half an 
hour of receiving the notification. The participant will be called to proceed to the 
emergency room and will be withdrawn from the study. Abnormal lab values will be 
reported as adverse events only when the Principal investigator deems them to be 
clinically significant.   
 
6.5.c. Data Quality and Safety Review Plan and Monitoring 


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1. Data Quality and Management 
a. Description of Plan for Data Quality and Management –  The PI will review all 
data collection forms on an ongoing basis for data completeness and accuracy as 
well as protocol compliance. A statement reflecting the results of the review will be 
sent to the NIH in the annual report.   
 
Data quality will be assessed using measures such as time from study visit to 
data entry, time to resolution of data queries, number of missing forms, and 
proportion of all study variables queried. Guidelines for concern regarding 
these measures are outlined below: 
 
 
Measure 
 
Goal Value 
 
Acceptable Value 
time from visit to data entry  
< 1 week 
 
< 2 weeks 
time to resolution of queries 
<2 week 
 
< 2 weeks 
number of missing forms 
  0 
 
< 5% 
proportion of variables queried 
  5% 
 
<10% 
 
b. Frequency of Review –The frequency of data review depends according to the 
type of data and is summarized in the following table. 
 
2.  Subject Accrual and Compliance 
a. Measurement and reporting of subject accrual, adherence to inclusion/exclusion 
criteria –Review of the rate of subject accrual, adherence to inclusion/exclusion 
criteria will occur monthly during the 3 month recruitment phase. 
 
6.5.d Safety Review Plan  
 
Study progress and safety will be reviewed monthly by the PI.  A study completion report 
will be compiled and will include a list and summarization of adverse events. In addition, 
the annual report will address (1) whether adverse event rates are consistent with pre-
study assumptions; (2) reason for dropouts from the study; (3) whether all participants met 
entry criteria. The report will be signed by the Independent Monitor and will be forwarded 
to the appropriate IRB and NIH, the UCSF CTSA on an annual basis. 
 
 
Data type 
Frequency of review 
Reviewer 
Subject accrual (adherence to 
eligibility criteria, randomization) 
Monthly 
Principal Investigator 
Adverse event rates (injuries) 
Monthly 
 
Principal Investigator 
Out of range laboratory data  
Weekly (PI) 
Principal Investigator  


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