Relationship Between Insulin Resistance and Statin Induced Type 2 Diabetes, and Integrative Personal Omics Profiling
Phase: PHASE4
Conditions: Hyperlipidemia, Insulin Resistance, Type 2 Diabetes
Sponsor: Stanford University

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STANFORD UNIVERSITY SCHOOL OF MEDICINE 
DIVISION OF CARDIVASCULAR MEDICINE 
 
 
Relationship Between Insulin Resistance and Statin Induced Type 2 Diabetes, and 
Integrative Personal Omics Profiling 
IRB Protocol Number: 33347 
NCT Number: NCT02437084 
December 20, 2016 
 
STUDY TITLE 
2 
ABBREVIATIONS 
2 
BRIEF DESCRIPTION: BACKGROUND, GOALS, GENERAL APPROACH 
3 
KEY STUDY PERSONNEL 
4 
FUNDING SOURCES 
5 
1. SIGNIFICANCE 
6 
2. RATIONALE, HYPOTHESIS AND OUTCOME MEASURES 
7-8 
3. STUDY DESIGN 
9 
SAMPLE SIZE 
9 
STUDY POPULATION 
9 
STUDY LOCATION 
9 
DURATION 
9 
4. STUDY PROCEDURES 
10 
RECRUITMENT 
10 
PARTICIPANT VISITS AND PROCEDURES 
10 
VISIT 1. SCREENING VISIT 
10 
VISIT 2. ORAL GLUCOSE TOLERANCE TEST 
10-11 
VISIT 3. GRADED GLUCOSE INFUSION TEST 
11 
VISIT 4. INSULIN SUPPRESSION TEST AND INTEGRATED PERSONAL 
OMICS PROFILING, iPOP 
11-12 
VISIT 5-7. BIWEEKLY FOLLOW UP 
12 
VISIT 8: REPEAT ORAL GLUCOSE TOLERANCE TEST 
12 
VISIT 9: REPEAT GRADED GLUCOSE INFUSION 
12 
VISIT 10: REPEAT INSULIN SUPPRESSION TEST AND iPOP 
12 
VISIT 11: ONE-MONTH OFF STATIN: iPOP 
12 
VISIT 12: TWO-MONTH OFF STATIN: iPOP (LAST STUDY VISIT) 
12 
STUDY MEDICATION MONITORING 
12-13 
5. STATISTICAL CONSIDERATIONS 
14 
APPENDIX 
15 
A. INCLUSION/EXCLUSION CRITERIA 
15 
B. PARTICIPANT EXPECTATIONS SCHEDULE EXAMPLE 
16 
C. OUTCOME MEASUREMENT METHODS 
17 
D. REFERENCES 
18 

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STUDY TITLE 
Relationship Between Insulin Resistance and Statin Induced Type 2 Diabetes, and 
Integrative Personal Omics Profiling 
 
 
 
ABBREVIATIONS 
The following abbreviations will be used throughout this protocol: 
Atherosclerotic Cardiovascular Disease: ASCVD 
Type 2 Diabetes Mellitus: T2D 
Metabolic syndrome: MetS 
LDL cholesterol: LDL-C 
HMG-coenzyme A reductase: HMGCR 
High-sensitivity C-reactive protein hs-CRP 
Oral Glucose Tolerance Test: OGTT 
Insulin Suppression Test: IST 
Graded Glucose Infusion Test: GGIT 
Area under the curve: AUC 
Glucose-stimulated Insulin Secretion Rate: ISR 
Integrated Personal Omics Profiling: iPOP 

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Brief Description: 
Background: 
There is general agreement that statin-treatment of patients to lower plasma cholesterol levels 
can increase the incidence of type 2 diabetes mellitus (T2D) in some individuals1-5. The 
physiologic mechanism for the increased risk for T2D from statin treatment is unknown but 
could result from effects on insulin sensitivity or insulin secretion. This study will evaluate how 
the medication atorvastatin (trade name Lipitor) works in non-diabetic individuals in regards to 
its effect on insulin sensitivity and insulin secretion to help further understand the possible cause 
of the increased occurrence of T2D in people who are at risk for T2D. This research study will 
also examine what metabolic characteristics and variables (for example insulin resistance, high 
triglycerides, or both) will identify those people at highest risk of statin-induced T2D. 
 
The goals of this study are to: 
1) determine the effect of high-intensity atorvastatin (40 mg/day) for ~ 10 weeks on 
insulin sensitivity and insulin secretion (defined with gold standard methods) (PRIMARY 
OUTCOMES) as well as other glycemic traits (SECONDARY OUTCOMES); 
2) compare a number of cardio-metabolic characteristics (e.g. weight, lipids) before, 
during, and after administration of atorvastatin; 
3) determine if significant deterioration of insulin action and/or secretion following statin 
treatment will be confined to those with baseline insulin resistance (PRE-SPECIFIED 
SUBGROUP ANALYSES); 
4) perform Personal Omics Profiling (iPOP) 6,7 before and after taking atorvastatin to 
examine treatment-associated changes in all baseline variables and to analyze not only 
previously-known drug efficacy but also untargeted drug efficacy (EXPLORATORY 
ANALYSES). 
General approach: 
This will be an open-label study to evaluate the diabetogenic effect of atorvastatin (40 mg/day 
for 10 weeks) on both insulin action and insulin secretion in nondiabetic individuals. To ensure 
we recruit individuals across a broad range of insulin sensitivity, we will target recruitment to 
enrich for those with combined increases in LDL-C and TG concentrations (see SIGNIFICANCE 
and RATIONALE). The experimental population will consist of ~75 apparently healthy, non- 
diabetic volunteers eligible for statin therapy but without pre-existing atherosclerotic 
cardiovascular disease. Following baseline assessments of co-primary outcome measures: 
insulin sensitivity (by insulin suppression test, IST) and insulin secretion (by graded glucose 
infusion test, GGIT), participants will be placed on a weight maintenance diet and treated with 
40 mg/day of atorvastatin. All baseline measurements will be repeated ~10 weeks later with 
iPOP8 measurements done at baseline, at weeks 2, 4, and 10 on atorvastatin, and at weeks 4 
and 8 off atorvastatin. 

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KEY STUDY PERSONNEL 
Principal Investigator 
Joshua W. Knowles, MD, PhD 
Assistant Professor, Cardiovascular Medicine, Stanford University 
Co-Principal Investigator 
Sun Kim, MD 
Associate Professor, Division of Endocrinology, Stanford University 
Co-Investigator 
Gerald Reaven*, MD 
Professor of Medicine, Stanford University 
* Deceased 
Collaborator 
Michael P. Snyder, PhD 
Stanford W. Ascherman, MD, FACS, Professor in Genetics 
Division of Genetics, Stanford University 
University – Scholar – Postdoctoral Scholar 
Ming-Shian Tsai, PhD 
Division of Genetics, Stanford University 
Senior Research Scientist 
Fahim Abbasi, MD 
Division of Cardiovascular Medicine, Stanford University 
Study Coordinator 
Cindy Lamendola, MSN, ANP 
Nurse Practitioner and Clinical Research Nurse Manager 
Division of Cardiovascular Medicine, Stanford University 
Assistant Clinical Research Coordinator 
Pragya Tripathi, MBBS 
Division of Cardiovascular Medicine, Stanford University 
Assistant Clinical Research Coordinator 
Vander Harris, BA 
Division of Cardiovascular Medicine, Stanford University 
Assistant Clinical Research Coordinator 
Chelsea S. Harris, BA 
Division of Cardiovascular Medicine, Stanford University 
Assistant Clinical Research Coordinator 
Fakhar Abbas, MD 
Research Assistant 
Collaborator 
Peter Reaven, MD 
Professor Division of Endocrinologist 
Carl T Hayden VA Medical Center 

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FUNDING SOURCES 
The Doris Duke Charitable Foundation, grant #2016097 provided major funding for this trial. 
Dr. Knowles is supported by the NIH through grants: P30DK116074 (to the Stanford Diabetes 
Research Center), R01 DK116750, R01 DK120565, R01 DK106236; and by the American 
Diabetes Association (grant #1-19-JDF-108). 
Dr. Kim is supported by the Bose Family Foundation and P30DK116074. 
The Washington University at St. Louis Diabetes Research Center central lab is supported by 
NIH grant P30 DK020579. 
Dr. Snyder is supported by the Metabolic Health Center through Lucille Packard Children’s 
Hospital, the Stanford PHIND Initiative and the NIH R01 AT01023203 

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1. SIGNIFICANCE 
Statins and the risk of T2D: Statin treatment is associated with an increase in incident T2D.1-4 5 
This adverse outcome seems to be a class effect, although there is some evidence for a dose 
dependent relationship. After analyses of large statin clinical trial data showed an increased risk 
of T2D associated with statin use, initial follow-up studies focused on the clinical impact of this 
risk. More recently, attention has been given to understanding why statins increase risk of T2D 
and the clinical characteristics that help identify those at increased risk. 
Mechanism of statin-induced T2D: It is unclear whether statins increase the risk of T2D by 
decreasing insulin action, secretion, or both. Several manuscripts have been published that 
substantially increase understanding of the link between statin use and incident T2D. Swerdlow, 
et al.2 based on evidence from genetic analysis and randomized trials, concluded that the 
increased risk of T2D noted with statins is at least “partially explained by HMG-coenzyme A 
reductase (HMGCR) inhibition.” They also noted an association of weight gain with HMGCR 
variants in statin-treated patients, leading to the notion that decreases in insulin sensitivity 
contribute to statin-induced diabetes. In that context, Cederberg, at al.9 have shown in a large 
prospective study (n=8749 men) that participants treated with statins (n=2142) had a 46% 
increase in incident T2D, associated with a 24% decrease in insulin sensitivity and a 12% 
decrease in insulin secretion assessed by surrogate measures. 
Identifying those with at enhanced risk of statin-induced T2D: Studies of 3 randomized 
clinical trials with atorvastatin by David Waters’ group 1,3,4 have demonstrated that “baseline 
fasting glucose, body mass index, hypertension, and fasting triglycerides were independent 
predictors of T2D.” These abnormalities form a cluster, initially referred to as Syndrome X (or 
sometimes referred to as the Metabolic Syndrome, MetS) and attributed to insulin resistance.10 
Since insulin resistance is a predictor of developing T2D, it seems likely that the more insulin 
resistant the individuals are before treatment, the greater is their risk to for statin-induced T2D. 
In that context, relatively little attention has been given to the role that metabolic heterogeneity 
in patients with elevated LDL-C concentrations might play in statin-induced T2D. Specifically, 
subjects with elevated LDL-C concentrations, whose plasma triglyceride (TG) concentrations 
are also elevated, are insulin resistant, hyperinsulinemic, and glucose intolerant as compared to 
those with isolated LDL-C levels. As such, this subset of patients with elevated LDL-C 
concentrations can be viewed as being at a “tipping point,” and any adverse effect of statins on 
insulin action and/or secretion, irrespective of how mediated, places them at enhanced risk to 
develop statin-induced diabetes. Indeed, we have shown (Kohli et al)1 that patients with both 
insulin resistance (as estimated by high TGs) and prediabetes are at particularly high risk of 
statin-induced T2D. 
While there are other possible approaches to identify individuals most at risk to develop T2D 
when treated with statins, e.g., a diagnosis of the metabolic syndrome (MetS), a comparison of 
291 apparently healthy individuals in our database, grouped together on the basis of having the 
MetS vs. a plasma TG concentration ≥ 150 mg/dL, revealed comparable cardio-metabolic risk 
profiles. Given this information, it seemed reasonable to continue evaluating the ability of a 
plasma TG concentration of ≥ 150 mg/dL as a surrogate for insulin resistance to identify those 
most at risk to develop T2D when taking statins. 
We seek to combine our understanding of the phenotypic heterogeneity of individuals with 
elevated LDL-C concentrations with quantification of insulin action and secretion with gold 
standard methods to address 2 important unanswered questions: Do statins primarily affect 
insulin resistance or insulin secretion?; Are there subsets of individuals at highest risk of statin- 
induced T2D? 

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2. RATIONALE, HYPOTHESIS AND OUTCOME MEASURES 
Rationale: 
T2D develops when insulin resistant individuals cannot maintain the degree of compensatory 
hyperinsulinemia needed to maintain normal glucose tolerance. However, significant 
fundamental questions remain. For example, what is the cellular/molecular link between statin 
treatment and changes in insulin action and secretion? 
This proposal is based on the premise that studying the effect of statins on insulin action and 
insulin secretion using “gold standard” methods will help determine if statins adversely affect the 
risk of T2D by increasing insulin resistance or decreasing insulin secretion. We use the insulin 
suppression test (with the read-out of steady-state plasma glucose, SSPG) to ascertain insulin 
sensitivity and the graded glucose infusion test (with the readout of insulin secretion rate, ISR) 
to ascertain insulin secretion both before and after statin treatment in non-diabetic individuals. 
We hypothesize that treatment with atorvastatin 40 mg/day for approximately 10 weeks will 
impair insulin sensitivity and/or insulin secretion and that this effect may be exacerbated in 
those with underlying insulin resistance. Thus, we plan to look at the effect of atorvastatin not 
only in all participants but also in subsets of individuals with baseline insulin resistance (which 
will be enriched for by recruiting volunteers with elevated plasma TG levels (≥150 mg/dL) at 
baseline. The rationale for this is that plasma TGs are a surrogate measure for insulin 
resistance with a modest correlation with the direct measure of insulin resistance (steady-state 
plasma glucose) measured by the insulin suppression test. Clinically, subjects with elevated 
TGs prior to statin treatment would have substantial clinical benefit from statins, and one of 
investigators' secondary goals is to demonstrate that a simple measurement of plasma TG 
concentration (as a surrogate for insulin resistance) can help identify those most at risk of statin 
induced derangements in glycemic control. Consequently, we propose to enroll nondiabetic 
volunteers at high-risk for T2D, free of known atherosclerotic cardiovascular disease (ASCVD), 
not receiving statins, eligible for statin therapy according to ACC/AHA (American College for 
Cardiology/American Heart Association) 2013 guidelines.11 We will also target recruitment 
efforts to enrich for subjects with plasma TG concentration ≥ 150 mg/dL to ensure that we enroll 
subjects across the range of insulin sensitivity. 
Hypothesis: We hypothesize that high intensity atorvastatin treatment for approximately 10 
weeks will impair insulin sensitivity and/or insulin secretion and that this effect may be 
exacerbated in those with underlying insulin resistance. 
Primary Outcome Measures: 
• 
Change in insulin sensitivity 
o Insulin Sensitivity: Steady-state plasma glucose concentration (mg/dL) measured 
during the insulin suppression test (IST) 
o Time Frame: Change from baseline to 9 -10 weeks in insulin sensitivity 
• 
Change in insulin secretion 
o Insulin secretion: Insulin secretion rate AUC (pmol/min x 4 h) measured during the 
graded glucose infusion test (GGIT) 
o Time Frame: Change from baseline to 9 -10 weeks in insulin secretion 
Secondary Outcome Measures: 
• 
Change in fasting plasma glucose and fasting plasma insulin 
o Fasting plasma glucose and insulin 
o Time Frame: Change from baseline to 8 - 10 weeks in fasting plasma glucose and 
insulin 

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• 
Change in OGTT glucose AUC and insulin AUC 
o OGTT glucose AUC (mg/dL x 2 h) and OGTT insulin AUC (mU/L x 2h) 
o Time Frame: Change from baseline to 8 weeks in OGTT glucose AUC and insulin 
AUC 
 
Prespecified Subgroup Analyses 
• 
Determine if significant deterioration of insulin action and/or secretion following statin 
treatment will be confined to those with baseline insulin resistance. 
 
Exploratory analyses: 
• 
Personal Omics Profiling Personalized medicine is expected to benefit from the combination 
of genomic information with the global monitoring of molecular components and 
physiological states6,7. To further extend Dr. Snyder's previous research of integrated 
Personal Omics Profiling (iPOP), which monitors genomic, transcriptomic, proteomic, 
metabolomic, and autoantibodyomic information over time, the investigators propose to 
analyze iPOP of apparently healthy volunteers with dyslipidemia longitudinally before, 
during, and after taking atorvastatin. In this pilot substudy, by performing unprecedented 
depth of omics analysis, both previously known and untargeted efficacies of atorvastatin will 
be analyzed. Overall, the endeavor will improve our understanding of how to perform a 
personal omics profile when taking statins and help us develop recommendations for better 
use of statins. 

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3. STUDY DESIGN 
Sample Size 
• 
We aim to recruit and retain 75 total participants in this study. 
Study Population (see Appendix A for detailed inclusion and exclusion criteria) 
• 
Healthy adults 30 - 70 years old. 
• 
BMI: 20 - 37 kg/m2. 
• 
Persons without diabetes as defined by having fasting plasma glucose < 126 mg/dL and 
not taking glucose lowering medications. 
• 
Individuals eligible for statin therapy for primary prevention of ASCVD based on LDL-C ≥ 
130 mg/dL, > 5% ASCVD risk over 10 years, or hs-CRP ≥ 2.0 mg/L. 
Study Location 
• 
Research Participants will be seen and have all study procedures at: Clinical and 
Translational Research Unit (CTRU) at 800 Welch Road, Palo Alto, CA 94304. 
Duration 
• 
We anticipate that the entire study will take 4 - 5 years through the end of data analysis. 
Each eligible candidate who voluntarily consents to participate in the study will be active 
in the study for a total of 5 months from screening to the end of their last visit. 
• 
Month: 1 
o Prepare IRB/Human Subjects materials (2 months total). 
• 
Month: 2-3 
o Prepare all recruitment materials. 
o Prepare all study protocols and materials. 
o Finish IRB protocol approval. 
• 
Months: 4 
o Begin study recruitment. 
o Each patient will be in study for approximately 5 months. 
o Goals is 15 - 20 participant enrollment per year. 
• 
Months: 5-12 
o Begin screening and enrolling study participants. Each participant will complete 
the ~ 5 -month protocol of the study. 
o Data clean-up on an ongoing basis. 
• 
Month: 30 
o Send out OGTT, IST and GGIT blood samples for the first 30 participants for 
measurement of glucose, insulin, and C-peptide. 
• 
Month: 48 
o Last participant completes the study. 
• 
Months: 49-50 
o Finish record update for remaining blood samples and ship out OGTT, IST, and 
GGIT samples for measurement of glucose, insulin, and C-peptide. 
o Start rechecking and clean-up of data and begin data analysis. 
• 
Month: 52 
o Receive the lab analysis results. 
o Continue with study data analysis and prepare manuscript. 

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4. STUDY PROCEDURES 
Recruitment 
Preliminary recruitment strategies will include: 
Volunteers will be recruited from the San Francisco Bay Area through advertisements in 
newspapers, posted flyers, and the social networking site NextDoor as well as from the 
Preventive Cardiology Clinics at Stanford Health Care. Our goal is to ensure recruitment across 
a broad range of insulin sensitivity. Prior work from our group and others has shown that high 
plasma TG concentrations are associated with increased insulin resistance as assessed by 
reference measures. Therefore, we will target advertisements to enrich for individuals with high 
TG levels (³ 150 mg/dL) as a surrogate for increased insulin resistance. 
Participant Visits and Procedures 
Potential participants will be screened initially when they call or email in response to recruitment 
ads, or a letter from their MD, describing the study as follows: 
Preliminary intake will occur over the phone. Potential participants will be asked their height and 
weight, if they have any major health problems, specifically T2D, cardiovascular disease or 
kidney disease, and a list of their current medications. We will also ask potential participants if 
they know their lipid profiles, if they have been on statins in the past, and if they are currently on 
a statin or other lipid lowering medication. Demographic information (name, date of birth and 
address) is required to establish screening appointment in CTRU and a medical record number. 
If they meet preliminary inclusion/exclusion criteria, an appointment will be made for a screening 
visit. The consent will be emailed prior to the visit to allow them to review before the screening 
visit. 
Name, height, weight, and phone number are requested on research line to call back 
appropriate people. That information is written down and is discarded after ~ 6 months. Any 
other information we obtain over the phone is also discarded in confidential bin. 
 
Visit 1 Screening visit. 
For the first visit, potential participants will be invited to the Stanford CTRU for screening. 
Participants must fast (excluding water) for 8 -10 hours prior to the appointment. They will be 
given a copy of the consent to first read on their own. Once they are ready, a member of the 
research group will review and explain the study to the patient and answer any questions. If they 
agree to participate, they will sign the consent form, which will be witnessed and signed by the 
researcher, and a copy of the consent form will be given the participant. 
Once consent is signed, a detailed medical history will be taken to further determine eligibility 
criteria. Participants will undergo measurement of height, weight, blood pressure, and 
calculation of BMI. Blood will be drawn and hematocrit and fasting plasma glucose will be 
analyzed in the CTRU lab and lipid concentrations, hs-CRP, liver function tests and serum 
creatinine will be determined by standard laboratory techniques in Stanford Health Care (SHC) 
laboratory. 
If there are no contraindications to participating based on further data obtained at the screening 
visit including laboratory results noted above, then they will proceed to be scheduled for an oral 
glucose tolerance test. 
 
Visit 2: 
Oral Glucose Tolerance Test (OGTT): This test will take approximately 3 hours. 
The OGTT involves a series of blood samples taken before and during 120 minutes after 
drinking Dexola/Glucola containing 75-gram glucose. This test will be done before and after 

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taking the study medication atorvastatin 40/day. Participants will have fasted for 12 hours from 
the previous evening. One intravenous (IV) catheter will be inserted in their arm for the purpose 
of drawing blood samples to determine blood glucose level during this procedure at zero, 30 
min, 60 min, 90 min, and 120 min. The total amount of blood taken for this test will be 33.5 ml. 
The purpose of this procedure is to define: 
i) 
Glycemic status: Participants will be classified as having normal glucose tolerance 
(NGT), isolated impaired fasting glucose (IFG), isolated impaired glucose tolerance 
(IGT), or combined IFG/IGT12. In addition, the total integrated plasma glucose response 
during the OGTT will be calculated by the trapezoidal method (Glucose-AUC). 
ii) 
β-cell function: Calculations of the Insulin Secretion- Sensitivity Index-2 (ISSI-2) will be 
used to quantify insulin secretory function 13, if for some reason a graded glucose 
infusion study is not done. The ISSI-2 is a validated OGTT- derived method to measure 
of β-cell function, analogous to the disposition index obtained from the intravenous 
glucose tolerance test 14. It is calculated by multiplying the insulin secretory response 
during the OGTT (Insulin-AUC/Glucose-AUC) by insulin resistance (Matsuda index). 
 
The fasting and 2-hour glucose results will be discussed with the study participant and a copy of 
the results will be given to them. 
Visit 3. 
Graded Glucose Infusion, GGIT 15,16: This test will take approximately 6 hours. 
This test is designed to assess the ability of the pancreas to produce insulin in response to a 
graded glucose infusion. During this test, subjects will have two small IV catheters placed, one 
in each arm. One IV will be used for drawing samples and the other for the infusion of glucose. 
During the GGIT, continuous intravenous infusions of glucose will be given at progressively 
increasing rates: 1, 2, 3, 4, 5, 6, and 8 kg/min in six infusion periods of 40-min duration. Blood 
samples will be collected for measurements of glucose, insulin, and C-peptide concentrations at 
fasting and at 30 min and 40 min into each infusion period. The two values during the last 10 
min of each infusion period will be averaged. The amount of blood taken for this test will 47.5 
ml. 
 
Visit 4. 
Insulin Suppression test, IST 17,18: This test will take approximately 6 hours. 
This test is designed to determine whole body insulin sensitivity. Following an overnight fast, 
subjects will have an IV placed in each arm. One for collection of blood and the other for 
infusion with octreotide (0.27 μg/m2/min), insulin (32 mU/m2/min), and glucose (267 mg/m2/min) 
for 180 minutes. During the test, endogenous insulin is suppressed and all individuals are given 
the same concentration of insulin, based on their body surface area. Blood is drawn every 30 
minutes for 150 minutes and then at 10-minute intervals from 150 to 180 minutes of the infusion 
to measure plasma glucose and insulin concentrations. The mean of the last four values is used 
as the steady-state plasma insulin (SSPI) and glucose (SSPG) concentrations for each 
individual. As SSPI concentrations are similar in all subjects during the IST, the SSPG 
concentration provides a direct measure of the ability of insulin to mediate disposal of an infused 
glucose load; the higher the SSPG concentration, the more insulin resistant the individual. Labs 
to check kidney and liver function plus a lipid panel and a urine pregnancy test (if appropriate) 
will be done at this visit. Blood drawn or IST will be 58.5 ml and 5 ml for SHC labs. 
Labs for iPOP will be drawn at this time and additional samples will be obtained for 
transcriptome, microbiome, metabolome, and proteome analysis in blood; nasal, tongue, skin 
surface swabs; urine; and stool. Total blood draw is 54 ml for omics. Participants will fill out 
questionnaires at the time of iPOP samples. The questionnaires will be on physical activity, 

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stress and diet. Laboratory measures will include complete blood count, comprehensive 
metabolic profile, hemoglobin A1c, insulin and IgM levels. 
• 
The study drug, atorvastatin 40 mg will be given to study participants, once all 
labs have been reviewed and participant qualifies. 
Visit 5-7 
Visits will be every 2 weeks for a total of 10 weeks on study medication (statin). Participants will 
be assessed for any side effects or adverse events (AE) on the statin. Adherence to study 
medication will be assessed at each visit. 
At each visit, weight and vital signs will be done as well as iPOP lab testing as described above 
except no iPOP will be done at visit 6. 
Visit 8: 
Weight, vital signs, and OGTT described above. 
Visit 9: 
Repeat GGIT as described above. 
Visit 10: 
Repeat IST and iPOP lab testing and samples as described above. 
At the end of this visit, the statin will be stopped and the study participant would be scheduled 
for 4- and 8-week follow up visits. 
Visit 11: 
One month off statin study visit – weight, vital signs, and iPOP laboratory testing and samples 
will be done as described above. 
Visit 12: 
Last study visit – weight, vital signs, and iPOP laboratory testing and samples will be done as 
described above. 
Participants will be asked to fill out questionnaire about her/his physical activity status, food and 
eating habits, and stress at the time of each iPOP. 
We will not be providing any iPOP results to study participants per protocol. 
 
Study Medication Management of Atorvastatin 40 mg 
Atorvastatin: The most commonly reported adverse reactions (incidence ≥ 2%) in patients 
treated with atorvastatin 40 mg in placebo-controlled trials regardless of causality were: 
nasopharyngitis-7%, arthralgia-10.6%, diarrhea-14.1 %, pain in extremity- 9.3%, and urinary 
tract infection-8.0%. 
WARNINGS AND PRECAUTIONS: Skeletal Muscle 
Rare cases of rhabdomyolysis with acute renal failure secondary to myoglobinuria have been 
reported with LIPITOR and with other drugs in this class. A history of renal (kidney) impairment 
may be a risk factor for the development of rhabdomyolysis. People with kidney disease will not 
qualify for this study. 
Lipitor, like other statins, occasionally causes myopathy, defined as muscle aches or muscle 
weakness in conjunction with increases in creatine phosphokinase (CPK) (enzyme measuring 
muscle breakdown) of values >10 times of upper limits of normal-ULN. The use of higher doses 
of atorvastatin with certain drugs such as cyclosporine and strong CYP3A4 inhibitors (e.g., 
clarithromycin, itraconazole, and HIV protease inhibitors) increases the risk of 
myopathy/rhabdomyolysis. We will only be using 40 mg not the highest dose. 

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Myopathy should be considered in any patient with diffuse myalgias, muscle tenderness or 
weakness, and/or marked elevation of CPK. Patients should be advised to report promptly 
unexplained muscle pain, tenderness, or weakness, particularly if accompanied by malaise or 
fever or if muscle signs and symptoms persist after discontinuing Lipitor. Lipitor therapy should 
be discontinued if markedly elevated CPK levels occur or myopathy is diagnosed or suspected. 
There have been rare reports of immune-mediated necrotizing myopathy (IMNM), an 
autoimmune myopathy, associated with statin use. IMNM is characterized by: proximal muscle 
weakness and elevated serum creatine kinase, which persist despite discontinuation of statin 
treatment; muscle biopsy showing necrotizing myopathy without significant inflammation; and 
improvement with immunosuppressive agents. 
The risk of myopathy during treatment with drugs in this class is increased with concurrent 
administration of cyclosporine, fibric acid derivatives, erythromycin, clarithromycin, certain 
medications for treating HIV or hepatitis C, protease inhibitor telaprevir, niacin, or azole 
antifungals. Creatine phosphokinase (CPK) determinations may be considered in such 
situations, but there is no assurance that such monitoring will prevent the occurrence of severe 
myopathy. Patients with HIV or Hepatitis C will not qualify for this study 
At each visit, we assessed for any sided effects or adverse effects (AE). A lipid panel with 
calculated LDL and comprehensive metabolic profile will be drawn (for iPOP) at weeks 2, 4, and 
10 while on the study drug. If there are any side effects or myalgia, a creatinine kinase will be 
checked, study medication will be stopped until symptoms are resolved and then restarted. If 
symptoms return, with or without laboratory abnormalities, the atorvastatin dose will be 
decreased to 20 mg/day. 

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5. STATISTICAL CONSIDERATIONS 
 
Based on our prior work19, we calculated that with 60 subjects, we would be able to detect an 
8% change in SSPG concentration and an 8% change in ISRAUC after atorvastatin therapy with 
80% power and two-side significance level of 5% using a paired samples t test. Thus, we 
estimated needing to enroll 75 subjects with an anticipated a dropout rate of 20%. 
Summary statistics will be reported as median (interquartile range) or number (percent) of 
participants unless otherwise specified. Shapiro-Wilk tests will be used to assess normality of 
data, and variables that are not normally distributed will be log-transformed. Percent changes in 
variables will be calculated by the formula: [(end-of study value) - (baseline value) / baseline 
value] x 100. Paired samples t tests will be used to compare baseline and end-of-study means. 
One sample t tests will be employed to evaluate whether percent changes in variables are 
significantly different from zero (no change). Pearson correlation coefficients will be calculated 
to determine the strength of association between variables of interest. Prespecified subgroup 
analyses will be carried out by stratifying for insulin resistant versus insulin sensitive subjects. 
The SSPG concentration median will be used to define subjects as being insulin resistant or 
insulin sensitive. Insulin resistant and insulin sensitive group means will be compared by 
independent sample t tests and proportions by chi-square tests or Fisher’s exact tests. 
Statistical analyses will be performed by using statistical software IBM SPSS version 26.0. 

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APPENDIX 
 
A. Inclusion/Exclusion Criteria 
 
Eligibility Criteria 
Inclusion Criteria: 
• 
Healthy adults 30 - 70 years old. 
• 
BMI: 20 - 37 kg/m2. 
• 
Persons without diabetes as defined by having fasting plasma glucose < 126 mg/dL and 
not taking glucose lowering medications. 
• 
Individuals eligible for statin therapy for primary prevention of ASCVD based on LDL-C ≥ 
130 mg/dL, > 5% ASCVD risk over 10 years, or hs-CRP ≥ 2.0 mg/L. 
 
 
Exclusion Criteria: 
• 
Younger than 30 or older than 70 years. 
• 
Persons with any significant co-morbidities, such as diabetes (fasting glucose ≥ 126 
mg/dL or use of glucose lowering medications), active coronary artery disease or heart 
failure, accelerated or malignant hypertension, kidney disease (creatinine ≥ 1.5 mg/dL), 
liver disease (alanine aminotransferase > 2 times upper limit of normal), or severe 
anemia (hematocrit < 30%). 
• 
Individuals taking any medications for weight loss or known to influence insulin 
sensitivity. 
• 
Pregnant or lactating. 
• 
Women unwilling to use an effective birth control method. 
• 
History of statin intolerance. 

Save Date: 11/1/20 
16 
 
B. Participant Expectations Schedule Example 
 
Schedule of Study Visits 
 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
Visit 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
Week 
Week 
Week 
Week 
Week 
Week 
Week 
Week 
Week 
Week 
Week 
Week 
-3 
-2 
-1 
0 
2 
4 
6 
8 
9 
10 
14 
18 
Screening 
X 
Lipids 
X 
X 
X 
X 
X 
OGTT 
X 
X 
GGIT 
X 
X 
IST 
X 
X 
Follow-up 
X 
X 
X 
X 
X 
Atorvastatin 
40 mg 
Start 
Stop 
 
 
 
Individuals on a stain at the screening visit will undergo a 4-week statin washout before the 
baseline metabolic tests. GGIT indicates graded glucose infusion test; IST, insulin suppression 
test; and OGTT, oral glucose tolerance test. 

Save Date: 11/1/20 
17 
 
C. Outcome Measurement Methods 
 
Glucose, Insulin, C-Peptide (Blood): Insulin and C-peptide will be measured by radioimmunoassay 
(Millipore, St. Charles, MO). These will be analyzed by the Core Laboratory for Clinical Studies at the 
Washington University School of Medicine in St Louis, MO. Homeostasis model assessment of insulin 
resistance (HOMA-IR) will be calculated by the following formula: (fasting insulin (mU/L) x fasting glucose 
(mmol/L))/22.5. 
 
Lipids (Blood) LDL-C will be calculated according to Friedewald (1972). 
Anthropometrics. Height will be measured to the nearest mm on a standard wall-mounted stadiometer. Body 
weight will be measured to the nearest 0.1 kg, in duplicate, on a calibrated clinical scale. Body mass index 
(BMI) will be calculated as weight in kg divided by height in m2, and waist circumference will be measured at 
the midpoint between upper iliac crest and lower end of the rib cage in mid respiration while subjects are 
standing. Blood pressure will be measured by using an automated blood pressure monitor with appropriately 
sized arm cuff. Before blood pressure measurement, participants will sit quietly in a chair for 5 minutes with 
arm supported at the heart level. 
Weight and vital signs will be measured at every visit, and waist circumference will be done at baseline and 
post study IST. 

Save Date: 11/1/20 
18 
 
D. References 
 
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2. 
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diabetes, and bodyweight: evidence from genetic analysis and randomised trials. Lancet 2015;385:351-61. 
3. 
Kohli P, Waters DD, Nemr R, et al. Risk of New-Onset Diabetes and Cardiovascular Risk Reduction 
From High-Dose Statin Therapy in Pre-Diabetics and Non-Pre-Diabetics: An Analysis From TNT and IDEAL. 
Journal of the American College of Cardiology 2015;65:402-4. 
4. 
Waters DD, Ho JE, Boekholdt SM, et al. Cardiovascular event reduction versus new-onset diabetes 
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5. 
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14. 
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Clin Endocrinol Metab 1997;82:1834-8. 
16. 
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17. 
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18. 
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19. 
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