Genomic Outcomes of Metformin
Phase: PHASE4
Conditions: Coronary Artery Disease, Mild Cognitive Impairment, Obesity, Abdominal, Hypertension
Sponsor: Wake Forest University Health Sciences

NCT02986659 
Date: 9/4/2018 
 
Study Title: Genomic Outcomes of Metformin (GO MET) 
 
Principal Investigator, Co-investigator(s): Jingzhong Ding, Yongmei Liu, Barbara Nicklas, Stephen 
Kritchevsky, William Applegate, Mike Miller, Jamie Justice, Anthony Molina, and Don McClain 
 
Sponsor or funding source: Wake Forest Claude D Pepper Center 
 
Background, Rationale and Context 
The number of older adults is projected to increase dramatically by 2050.  Aging-related diseases and 
conditions still seriously compromise the quality of life among most older adults.  Several pharmaceutical 
agents (1-4), such as metformin, have been tested to extend lifespan and delay aging-related diseases and 
dysfunctions in mice. Metformin, a biguanide antidiabetic drug, reduces the risk for developing type-2 
diabetes in persons at risk by over one-third with few adverse effects (e.g., gastrointestinal irritation) (5).  
Metformin prevents type-2 diabetes primarily through decreasing hepatic glucose synthesis (6), as well as 
enhancing insulin sensitivity and increasing peripheral glucose uptake (7-9).  The molecular mechanisms 
remain unclear, although a number of potential mechanism such as activation of AMP-activated protein 
kinase (AMPK) and inhibition of mitochondrial glycerophosphate dehydrogenase have been proposed, 
see figure 1 (10;11).  The fact that metformin treatment in persons with type-2 diabetes has been 
associated with reduced risk of other aging-related diseases and conditions, including cardiovascular 
disease (12), cancer (13) and 
cognitive decline (14) supports 
the possibility of the beneficial 
effects of metformin on healthy 
aging.  It is imperative to 
capitalize on these leads to extend 
health span among older adults.    
 
 
To translate animal findings to 
human intervention trials, 
appropriate aging biomarkers are 
needed. Methylomic and 
transcriptomic profiles in relevant 
cells may reflect molecular 
features that mediate effects of 
both genetic and environmental 
factors on aging-related 
functional decline and disease. 
The roles of monocytes have 
been implicated in development 
of many aging-related diseases 
such as cardiovascular disease, 
cancer and neurodegenerative 
disease (15).  In a cross-sectional 
association study of 1,200 monocyte samples, we identified 1,794 age-associated methylation sites (16) 
and 2,704 age-associated transcripts (17), which were over-represented in two networks (autophagy and 
oxidative phosphorylation) and suggestive of decline in those functions with age. Both autophagy (18) 
and oxidative phosphorylation (19) are considered as key contributors to the aging process, and their 
dysfunctions have been linked to aging-related diseases (20;21). Changes in these aging-related omic 
biomarkers may be early indicators of cellular damage or disruption that eventually leads to age-related 
dysfunctions.  Assessment of these aging biomarkers in response to therapeutic intervention may also 
AMPK
PI3K
Akt
ACC
AMP
p66shc
IRS-1
IRS-2
Metformin
Rapamycin
Ras
Protein Synthesis
Autophagy
Stress Defense
Inflammation
Erk1/2
Healthspan and
Longevity
Resveratrol
NAD+
NADH
Cellular Survival
Cytokine
Receptor
ATP
SIRT1
mTOR
ATG13
p70S6K
4EBP1
Bax
p53
FOXO
1
2
3
NF-kB
Adiponectin
Receptor
INSR
IGF-1R
PAI-1
TNFa
IL-6
Adiponectin
Insulin
Metformin
IGF-1
Figure 1. Metformin targets multiple pathways of aging 

 
Protocol version: 9.4.18 
2 
provide molecular insight for personalizing treatment.  We propose a pilot study to examine changes in 
aging-related omic profiles after 3 months of metformin treatment in 35 monocyte samples from older 
adults using a randomized, double-blind, placebo-controlled crossover study design. 
 
Objectives 
Our overarching goal of the pilot study is to evaluate the utility of using the aging-related omic 
biomarkers as an indicator of pharmacologic responses in the anti-aging therapeutic intervention trials 
through the following specific aims.  Although this pilot study does not have sufficient power to 
definitively test all the aims, it will provide essential preliminary data for developing a full scale research 
program. 
• 
Aim 1A: To test the effects of the metformin treatment on transcriptomic profiles and related 
functional changes in human monocytes, 
• 
Aim 1B: To explore the effects of the metformin treatment on methylomic profiles in human 
monocytes, 
• 
Aim 2: To investigate the longitudinal relationship between transcriptional and functional 
changes in human monocytes during the metformin treatment and  
• 
Aim 3: To test the effects of the metformin treatment on frailty and other aging-related physical 
and cognitive measures and investigate the longitudinal relationship between these changes and 
transcriptional changes. 
 
Methods and Measures 
 
Design 
A randomized, double-blind, placebo-controlled crossover trial in 35 participants using metformin and 
matching placebo will be used. In the absence of a treatment by sequence interaction effect, this design 
can increase study power for evaluating treatment effects by allowing each participant to be his/her own 
control. The period effect may be minimum because the primary outcomes, methylation and 
transcriptional measures, are relatively stable overtime.  
 
Setting 
All visits will occur in the Geriatric Clinical Research Unit (G-CRU) or the Geriatric Research Center 
(GRC) in the Sticht Center on Aging. 
 
Subjects selection criteria 
All prospective participants in and around Forsyth County, NC will initially be screened via telephone to 
determine general eligibility. They will be asked about their age, medical history, and current 
medications. All participants must meet the inclusion/exclusion criteria below.   
Inclusion Criteria 
Age 65 – 79 
Must meet criteria from one or more of the following groups:  
 
 
      Group 1 (Can have 1 or 2 of these, but not all 3) 
• 
History of coronary artery disease  (MI/heart attack, stroke, heart failure, or peripheral 
artery disease) 
• 
Cancer, with no active treatment in the last year  
• 
MCI (MoCA >18<26 –inclusive of 1 point if <12 years of education 
 
 
      Group 2 
• 
Decline physical function (walking speed < 1 m/s) 
                   Group 3 (Either or both) 
• 
Abdominal obesity (>88cm women, >102cm men) AND hypertension (treated or resting   
 
blood pressure >140/90 

 
Protocol version: 9.4.18 
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• 
Abdominal obesity (>88cm women, >102cm men) AND hyperlipidemia (treated or   
 
fasting total cholesterol >240 
English literacy 
Willing to provide informed consent  
Exclusion Criteria 
 eGFR <45 
Type 2 diabetes (HbA1c>6.5) or type 1 diabetes 
Any tobacco or nicotine product use in the past year  
Low vitamin B12 Levels (< 300 pg/mL) 
Self-reported severe difficulty or inability to walk 400m or climb 10 steps (from Q 2 and 19 
on PAT-D) 
Self-reported difficulty or inability to perform basic ADL functions (from Q 10, 13, 14, 16 on 
PAT-D) 
Excessive alcohol use (>14 drinks/week) 
Cancer requiring treatment in past year (except skin) 
Dementia – diagnosed and/or MoCA score <18 
Parkinson’s or other neurological disease 
Chronic liver disease or cirrhosis 
End stage renal disease or on dialysis 
Rheumatic conditions (Rheumatoid arthritis, lupus, and any other autoimmune disease the PI 
deems them to be ineligible for) 
Thyroid problems the PI deems them to be ineligible for 
Recent or recurrent exacerbation of gout  
Involved in another interventional study 
Hemoglobin <8 or diagnosed with anemia 
Recent unintentional weight change (+/- 10 lbs. in the last 12 months) 
BMI <18.5 
Likely to not follow the protocol  
 
PI deems unfit to participate 
Already taking Metformin or any other drug intended to treat diabetes 
 
Screening, consent and randomization. Individuals who pass the telephone screening will be scheduled 
for a visit to the Geriatric Research Clinic (GRC) in the Sticht Center on Aging for a screening visit (SV). 
Before any data collection, all participants will provide written informed consent and complete a HIPAA 
authorization form in accordance with the Wake Forest School of Medicine Institutional Review Board 
policies. Individuals who meet all eligibility criteria and provide informed consent will be invited back for 
the first baseline visit (BV). At the end of BV, participants will be randomized to one of the two study 
intervention sequences (metformin followed by placebo or placebo followed by metformin) using a web-
based randomization scheme (developed by Dr. Miller). The randomization sequences will be unknown to 
the research staff. Intervention assignment will be generated using a program that the study coordinator 
will access from his/her PC through the study website.   
 
Schedule and organization of assessment visits 
All assessments will be conducted during 4 visits (before and after each intervention) according to the 
chart below. All examiners are trained in the standardized conduct of all assessments before data 
collection.  Participants will be instructed to wear appropriate and comfortable clothing, and standardized 
written instructions will be provided prior to each study visit. 
 
 
 
 

 
Protocol version: 9.4.18 
4 
 
Assessment timeline                                               Visit code 
SV 
BV 
On 
trial 
Mid 
 
On 
trial 
 
FV 
Activity/assessment                                                       Week number 
-4 
0 
12-14 
24-26 
Informed consent, review inclusion/exclusion criteria, demographics, 
weight, height, BP and pulse, fasting screening blood draw (metabolic 
blood panel, lipid panel, CBC, Vit B12, Hba1c), eSPPB, cognitive 
screen (MoCA), waist circumference,  medical history, medications 
PAT-D,  
X 
 
 
 
 
 
Blood collection for methylomic and transcriptomic, bioenergetic 
capacity, CBC, and blood for storage, DSST, leg press, grip, , MATsf, 
and 6MWD  
 
X 
 
 
 
 
Randomization 
 
X 
 
 
 
 
Crossover – Dispense New Medication 
 
 
 
X 
 
 
Vitals, blood collection for methylomic and transcriptomic, bioenergetic 
capacity, and blood for storage, CBC, CMP, lipids, Hba1c,  eSPPB, 
MoCA, DSST, leg press, grip, PAT-D, MAT-sf, 6MWD 
 
 
 
X 
 
X 
Health Maintenance  
 
 
 
 
 
X 
The screening visit (SV) will be conducted in the morning following an 8-hour fast for measurement of 
lipids, blood count, Hba1c, Vitamin B12, and a metabolic panel for screening purposes. Vitals (blood 
pressure, pulse, height, weight, and waist circumference) are collected and the blood draw completed, 
participants will be given a snack and continue with the remainder of the visit. First, the expanded short 
physical performance battery (eSPPB) will be administered and scored and then undergo a cognitive 
screen, assessed using the Montreal Cognitive Assessment (MoCA), and a medical history, review of 
medications and dietary supplements We will also ask about the participants demographics and physical 
abilities using the Pepper Assessment Tool for Disability (PAT-D), 
 
The first baseline visit (BV), will be conducted in the morning following an 8-hour fast for blood 
collection for methylomic and transcriptomic measures, bioenergetic capacity, CBC, as well as blood for 
storage. After these measures are complete, participants will be given a snack and will continue with the 
rest of the visit consisting of lower extremity muscle power (Nottingham Power Rig), grip strength, 6 
minute walk distance test (6MWD), and the Mobility Assessment Tool – short form (MAT-sf).  Cognitive 
function will be assessed using the Digit Symbol Test (DSST). Once the visit is complete, the participant 
will be randomized and placed in one of the following groups discussed below. Participants will be 
provided with an information sheet that will provide additional details on how and when to take the 
medication, who to call if they have any problems, possible side effects, and what to do should they 
encounter any side effects.  
 
Interventions and Interactions 
Eligible participants will receive both interventions (3-month metformin treatment and 3-month placebo). 
Randomization will be used to determine the order in which the participants receive each intervention 
(i.e., 15 participants with metformin treatment followed by placebo, and 15 with placebo followed by 
metformin treatment). Neither the participants nor the investigators know the intervention order of a 
participant. Metformin reaches peak plasma concentration within one to eight hours, has an average 
elimination half-life of 6.2 hours in plasma, and is cleared but not metabolized from the body by tubular 
secretion.  Treatment with metformin and placebo will be initiated at a dose of 425 mg taken orally once a 
day at night for 7 days. After the week, the participant will be called to assess tolerance and will be asked 
to increase dose to one pill at night at a dose of 850 mg for one week. At the end of the second week, 
participants will be called again and if they tolerated the second dose, they will be asked to take two 
850mg pills, one in the morning and one at night, for a total dose of 1700 mg which is within the range of 
the usual effective dose of 1500 to 2000 mg/day for the remainder of the 3 months.  This dosing regimen 
was created in order to avoid or reduce possible gastrointestinal side effects. This can be modified if the 

 
Protocol version: 9.4.18 
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participant is experiencing any side effects and will be handled on a case by case basis. Each study visit 
where stored blood is obtained (BV, Mid, FV) may be rescheduled if the subject has a fever, infection, or 
has taken antibiotics within 24hrs of this scheduled visit. 
 
All baseline assessments will be completed again at 12 weeks and again at 24 weeks as listed in the 
assessment table.  
 
Outcome Measure(s) 
The primary outcomes will be the eigengene scores (and related individual transcriptional measures) for 
the two transcriptional networks, the autophagy and oxidative phosphorylation.  The eigengene score is 
defined as the first principal component of a transcriptional network. The secondary outcomes will 
include functional, methylation and other aging-related measures.  
 
Individual methylation sites and transcripts, especially the eigengenes of autophage, oxidative 
phosphorylation and protein synthesis networks Methylomic and transcriptomic profiles in monocytes 
will be quantified using the Illumina HumanMethylation450 BeadChip and the Illumina HumanHT-12 v4 
Expression BeadChips, respectively, at Dr. Liu’s laboratory (22).  Blood will be collected in Vacutainer 
CPTTM cell separation tubes containing sodium citrate (Becton Dickinson, Rutherford, NJ) to separate 
peripheral blood mononuclear cells.  Then, monocytes will be isolated with the anti-CD14 coated 
magnetic beads using AutoMACs automated magnetic separation unit (Miltenyi Biotec, Bergisch 
Gladbach, Germanry). DNA and RNA will be isolated simultaneously using the AllPrep DNA/RNA Mini 
Kit (Qiagen, Inc., Hilden, Germany).  
 
Functional measures: An autophagic profile in monocytes including macroautophagy, microautophagy 
and chaperone-mediated autophagy will be measured using fluorescent reporters at Dr. Ana Maria 
Cuervo’s lab in Albert Einstein College of Medicine. 
 
Mitochondrial Function: Respirometric profiling will be used to assess bioenergetic capacity, 
respiratory control and electron transport chain function at Dr. Molina’s lab (23). This will be performed 
by examining the oxygen consumption profile of blood monocytes. Specific outcomes of mitochondria 
function include respiratory control ratio (RCR), state 3 (maximal oxygen consumption rate), and state 4 
(oxygen consumption upon inhibition of ATP synthase). 
 
Other Data 
 
Vital Signs Prior to randomization data are collected on sitting blood pressure, heart rate, and weight. 
Body height is measured once prior to randomization. The blood pressure assessments will allow the 
determination of the incidence of hypertension and serve as basis for a temporary exclusion. The other 
measures are collected primarily for descriptive purposes. 
 
Demographics and co-morbidities: Medical information regarding co-morbidities and medications will 
be ascertained at the screening visit.  Demographic data such as socio-economic status, education level, 
age, and race also will be recorded and used as covariates if necessary. Information about preventative 
health screenings and other health maintenance information is asked at the follow up visit. 
 
Physical performance will be assessed using the expanded Short Physical Performance Battery (SPPB) 
(24). The expanded SPPB consists of 5 repeated chair stands, standing balance (semi- and full-tandem 
stands and a single leg stand for 30 seconds), a 4-m walk to assess usual gait speed, and a narrow 4-m 
walk test of balance (walking at usual pace within lines of tape spaced 20 cm apart). Scores for the 
traditional 0-12 point SPPB can also be obtained from these tests. We will also assess physical 

 
Protocol version: 9.4.18 
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performance using the six minute walk distance test (6MWD). The participant is asked to walk at a pace 
they can maintain for six minutes and the distance covered will be recorded.  
Lower extremity muscle power will be measured using the Nottingham Power Rig, a safe, convenient 
method for assessing power output from the lower limb which has been used reliably in older adults. (25) 
Participants will sit in a chair and unilaterally depress a foot lever attached to a flywheel as hard and as 
fast as they can. Power output, derived from the acceleration of the flywheel, will be recorded in Watts. 
Power will be averaged for each leg following five trials at maximal effort. Participants that have had a 
unilateral hip or knee replacement should not have that side tested.  
 
Grip strength will be measured twice in each hand to the nearest 2 kg using an isometric Hydraulic Hand 
Dynamometer (Jamar, Bolingbrook, IL) and the mean value from the stronger hand used. Participants will 
be excluded from performing the test if they report hand-pain or recent hand or wrist surgery.   
 
Cognitive function will be assessed during screening using the Montreal Cognitive Assessment (MoCA) 
(26), participants must score ≥18 to be eligible. We will also assess psychomotor speed, attention, and 
working memory using the Digit Symbol Substitution Test (DSST) (27; 28). Participants are given a 
series of numbered symbols and then asked to draw the appropriate symbols below a list of random 
numbers. The score is the number of correctly made matches in 2 minutes (120 seconds).  
 
Body composition will be measured by circumference measurements of the waist which is made with a 
tape measure.   
 
Blood lipids (total cholesterol, HDL-Cholesterol, Triglycerides and calculated LDL levels) will be 
measured at a clinical lab and all results will be entered into the data system. 
 
Activities of daily living will be assessed using the Mobility Assessment Tool – Short Form (MAT-sf). 
This is a novel, computerized tool for self-assessment of functional performance designed to reduce bias 
from factors such as age, gender and body image. The Pepper Assessment Tool for Disability (PAT-D) 
will also be used to assess the participant’s ability to complete an array of important day-to-day activities 
without difficulty and, for a subset of activities, without personal assistance. 
 
Medication adherence: Although we will attempt to enhance adherence by monthly calls to participants, 
there may be inter-individual variability in adherence to the medication. At the end of each phase, 
participants will be asked to return any unused pills for a pill count to determine participant adherence. 
Our primary analyses will be an intent-to-treat analysis of the effects of the metformin intervention, but 
secondary analyses will include adherence as a covariate. 
 
Analytical Plan  
We will use an analysis of variance model for crossover design to assess the effect of metformin on 
transcriptional profiles and other aging-related outcomes. The factors in the analysis will include the 
baseline measure, sequence, participant within sequence, period, and metformin treatment. We will 
explore for the metformin by sequence interaction and will base estimates of the metformin effect on 
whether this interaction effect is significant.  We will also perform exploratory, linear regression analysis 
with transcriptional changes as the explanatory variables and changes in other aging-related measures as 
the outcome variables. The analysis will be focused on the eigengene scores of the autophagy and 
oxidative phosphorylation transcriptional networks (and related individual transcriptional measures).  As 
this is a pilot study, focus of analyses will be on estimation of effects and constructing appropriate 95% 
confidence intervals for these effects.   
 
 
Human Subjects Protection 

 
Protocol version: 9.4.18 
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Risk to participants 
The Diabetes Prevention Program (DPP) study, a placebo-controlled study of metformin vs. lifestyle 
intervention in persons with pre-diabetes (included subjects with fasting blood glucose 90-125 mg/dL, n = 
3,234) provides insight into side effects of long-term metformin administration in the elderly and non-
diabetics.29 Of note, the most common side effect was not hypoglycemia, but gastrointestinal symptoms 
(see table below). 
 
 
  
As far as common side effects, GI side effect and diarrhea are transient and usually pass after a week of 
therapy. In the DPP study, 3-5% of subjects had continuous diarrhea; this portion of subjects would be 
excluded in the present trial during the run-in, dose-titration period. No one had metformin associated 
lactic acidosis. Hypoglycemic events were either not in evidence or reported. 
 
Weight loss is a common occurrence with metformin and will be monitored. In such a case some patients 
may complain of hypoglycemic symptoms (rapid heartbeat, feeling light-headed or fainting, and nausea), 
and in this case subjects will be asked to refrain from taking their next dose. Subjects may not need 
medical interventions, but will be given instruction for fruit juice or other carbohydrate and to call the 
coordinator immediately to come in for a blood glucose check. Levels will be checked by finger 
(Accucheck) and labs to LabCorp to confirm levels.  If blood glucose levels are < 60 mg/dL they will be 
removed from the study and replaced by an additional participant. No more side effects were noted in the 
elderly; in fact, they had a better adherence to therapy.  Young and elderly had the same rates of GI side 
effects and diarrhea. 
 
Subject Recruitment Methods 
We will recruit these individuals using community-based recruitment strategies including newspaper ads 
and mass mailings. We will also advertise in the VITAL newsletter (BG99-559) and participate in 
community outreach events. 
 
Informed Consent 
Written informed consent will be obtained from each subject. The informed consent process will follow 

 
Protocol version: 9.4.18 
8 
the procedures of the WFSM Institutional Review Board. The study interviewers will explain the purpose, 
methods and extent of the study to prospective participants. The potential participant is asked to read the 
informed consent form and ask questions. The form is written in simple easy to understand language. We 
require study staff to review all of the key aspects of the study verbally with the potential participants. 
Staff is provided with a structured checklist for this purpose. Staff is then required to question potential 
participants to ascertain whether s/he has understood the information. Potential participants who are 
illiterate or have impaired vision must have the consent read to them, followed by review of the checklist, 
opportunity for questions, and discussion. This process will take place in a quiet, private room. A copy of 
the signed and dated consent form will be given to participants, and the original document will be placed 
in subjects’ individual study files, which will be stored in a secure location. In compliance with the Health 
Insurance Portability and Accountability Act (HIPAA) and the Standards for Privacy of Individually 
Identifiable Health Information of the Department of Health and Human Services, we will access personal 
health information only after obtaining informed consent. 
 
Confidentiality and Privacy 
All data are obtained for research purposes only. Confidentiality of data is maintained by using research 
identification numbers which uniquely identify each individual. Data will be used only in aggregate and 
no identifying characteristics of individuals will be published or presented. The information collected has 
a low potential for abuse because the data do not address sensitive issues. Nevertheless, appropriate 
measures are taken to prevent unauthorized use of study information. Research records are kept in a 
locked room. Data access will be limited to study staff. Information linking IDs to individuals is kept on a 
secure, password-protected server to which only authorized study personnel will have access. Data and 
records will be kept locked and secured, with any computer data password protected. A web-based data 
entry system will be created for this study which will allow access only to study staff (user id and 
password required to access). Blinded staff will not have access to blinded study information on the web-
based database. Computer files are stored on file servers, which are backed up each night and stored for 
easy retrieval in case of emergency.  Files may not be obtained from the research unit by persons other 
than the research personnel, who are asked to sign a document agreeing to maintain the confidentiality of 
the information. No data files distributed for analysis will include personal information. No reference to 
any individual participant will appear in reports, presentations, or publications that may arise from the 
study. After the study is completed, the data will be stored with other completed research studies in a 
locked secure area. 
 
Data and Safety Monitoring 
The principal investigator along with the study physician (William Applegate) will be responsible for the 
overall monitoring of the data and safety of study participants. The study physician will be unmasked to 
treatment assignment as needed and appropriate. The principal investigator will also be assisted by other 
members of the study staff. All participants will have a protocol-driven medical monitoring plan to 
consistently assess for adverse events or medical safety concerns. In addition, participant safety in all 
Wake Forest Pepper Center OAIC-supported studies is monitored by a Data and Safety Monitoring 
Committee (DSMC). It reviews study recruitment, drop-outs, protocol changes, losses to follow-up, and 
adverse events every 6 months. The DSMC has authority to recommend changing or stopping the 
protocol. In the latter case, the PI will immediately act upon the recommendation according to 
institutional policy and in consultation with the Wake Forest Pepper Center OAIC Executive Committee. 
 
Reporting of Unanticipated Problems, Adverse Events or Deviations 
Any unanticipated problems, serious and unexpected adverse events, deviations or protocol changes will 
be promptly reported by the principal investigator or designated member of the research team to the IRB 
and sponsor. 
 
 

 
Protocol version: 9.4.18 
9 
Use of biological samples by other investigators 
Biological samples may be used by investigators other than the investigators of the current study. The use 
will be limited to non-commercial purposes. The names and other personal identifiers of the study 
participants will not be sent to any recipients of the blood samples. 
 
Storage and disposal of biological material 
Blood samples will be stored at Wake Forest University Medical Center for up to twenty years after the 
end of the trial at which time the samples will be destroyed. Biological specimens will be stored in locked 
-70oC alarmed freezers located in a locked room. The lab coordinator and the PIs have access to the keys 
of the freezers. All the specimens will have numerical study IDs with no personal identifiers of the 
participants. These are stored under the Pepper Center Tissue Repository (IRB#1219). 
 
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