Clinical trial NCT01804049
Conditions: Prediabetes
Keywords: metformin muscle insulin resistance sarcopenia older adults impaired fasting glucose
Sponsor: VA Office of Research and Development
Start date: 2014-04-08; Completion date: 2018-08-31
Enrollment: 120 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Sarcopenia is the loss of muscle mass, strength and function with aging and is associated with increased disability, falls and fractures. Older adults with diabetes and prediabetes are insulin resistant and have a higher risk of developing sarcopenia. This study examines the use of metformin, an antidiabetic drug, for preventing the development of sarcopenia in older adults with prediabetes.

Detailed description:
The proposed study utilizes clinical and translational research approaches to study sarcopenia. Sarcopenia is common in older adults and is associated with decreased strength, increased disability, falls and fractures. There are currently few interventions to prevent or treat sarcopenia and a poor understanding of the mechanisms for sarcopenia. Given the growing number of Veterans over the age of 65, studies to prevent sarcopenia and resulting disability are important for the health, independence and well-being of this population. The investigators' preliminary studies have shown that older adults with diabetes have an accelerated loss in muscle mass and gait speed, except when treated with metformin. Older adults with prediabetes also have a greater decline in muscle mass and higher incidence of disability. Therefore, this study further investigates these findings by addressing the following aims: (1) to determine whether metformin can prevent the loss in muscle mass and physical performance and (2) to examine changes in muscle histologic characteristics associated with metformin treatment in older adults with prediabetes.

Arms: arm 1: 60 participants will be randomized to placebo pills. arm 2: 60 enrolled participants will be randomized to metformin.
Interventions: intervention 1: metformin intervention 2: placebo
Intervention descriptions: intervention 1: Metformin will be given at a dose of 850 mg orally once daily for 1 month with titration up to 850 mg orally twice daily for the remainder of the study. intervention 2: One placebo capsule by mouth once daily for 1 month followed by one placebo capsule by mouth twice daily for the remainder of the study.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
 
 
Study Protocol: Metformin and Muscle in Insulin-Resistant Older Veterans 
NCT01804049 
PI: Dr Robert Klein 
Document Date: 08-16-2016 
 
 


IRB #8860 
08/16/2016 
 
 
2 
 
Portland VA Medical Center 
Oregon Health & Science University 
RESEARCH PROTOCOL 
 
 
Protocol Title: 
Metformin and Muscle in Insulin-Resistant Older Veterans 
 
Principal Investigator: 
Robert Klein, MD 
 
Sponsor: 
VA Clinical Sciences Research & Development (CSRD) 
 
Specific Aims: 
To determine if metformin can decrease the loss of muscle mass and physical performance in insulin-resistant 
older adults and to investigate the mechanistic pathways involved. 
 
Background and Significance:  
 
Significance 
 
The proposed clinical trial may identify potential new targets for the prevention and treatment of sarcopenia. 
Sarcopenia is present in up to 24% of adults between the ages of 65 and 70 years and over 50% of adults over the 
age of 80 in the United States.1 It is associated with the clinical consequences of lower physical performance, 
decreased strength, impaired activities of daily living, greater disability, and more falls and fractures.1-3 As a 
result, the healthcare costs of sarcopenia were estimated at $18.5 billion for the US population in 2000.4 With 
adverse health, lifestyle and economic implications associated with sarcopenia, discovery of therapies to prevent 
or treat sarcopenia are important, given the growing number of veterans over the age of 65.  
 
Our preliminary data demonstrate a greater likelihood for muscle loss for older insulin-resistant men that may be 
attenuated with the use of metformin. These results strongly suggest that metformin has beneficial effects on 
muscle, and that agents acting like metformin may provide a new approach for the prevention or treatment of 
sarcopenia. To more definitively examine these effects, we will enroll older, insulin-resistant adults into a 
randomized clinical trial to determine whether metformin can slow the decline in muscle mass and physical 
performance. Furthermore, histologic measures from muscle biopsies in this clinical study will be used to 
understand the mechanism by which metformin may prevent sarcopenia. If the clinical trial supports our 
preliminary data, it will provide an exciting opportunity to develop new approaches to prevent or treat sarcopenia. 
This research is novel for its combined use of epidemiologic, clinical and translational approaches to understand 
the loss of muscle mass and function and for its investigation of potential mechanisms for the prevention or 
therapy of sarcopenia.   
Given the poor understanding of mechanisms for sarcopenia and lack of therapies for treating or preventing 
sarcopenia, there is a great need to train clinician investigators to become sarcopenia researchers. The proposed 
training plan builds on my background in endocrinology, metabolism, diabetes and epidemiology to add 
additional experience in clinical trials and translational research skills for studying muscle. The training proposed 
in this application will give me the ability to conduct epidemiologic, clinical and translational studies to 
understand sarcopenia in older veterans, thus allowing me to fill a much needed role for the field of sarcopenia 
research.  
 
 


IRB #8860 
08/16/2016 
 
 
3 
Background 
 
Relevance of Sarcopenia to Disability in U.S. veterans 
Older U.S. veterans have a high prevalence of poor physical health and functioning.5, 6 Over half of U.S. veterans 
reported difficulty with one or more activities of daily living (ADL) and instrumental activities of daily living 
(IADL).7, 8 The Vietnam War cohort surveyed in 2001 had equal or higher rates of ADL and IADL disability 
compared to WWII veterans despite being younger.9 As the Vietnam veteran cohort enters older age, prevention 
of sarcopenia will be needed to help keep them independent and functional. 
Sarcopenia, defined as low appendicular lean mass, exists in up to 24% of adults under 70 years old and over 50% 
in those over the age of 80 and is associated with self-reported disability.1  However, objective measures of 
physical performance have also been shown to predict incident disability.10-12 While low muscle mass is 
associated with disability and functional decline, it cannot completely explain the greater loss in strength or 
function that occurs in older adults.13, 14 Moreover, therapies that improve muscle mass may not necessarily 
improve muscle strength or function, and thus have limited clinical relevance.15 Future studies on the prevention 
of disability must consider the both prevention of sarcopenia and the loss of physical performance.  
Population at Risk for Sarcopenia 
The prevalence of impaired fasting glucose, impaired glucose tolerance, and diabetes increases with aging.16 
Within the Veterans Affairs system, diabetes was reported as the third most common diagnosis and was more 
common in adults over the age of 65 years.17, 18 The presence of insulin resistance is associated with an increased 
risk of sarcopenia development.19 Older adults with type 2 diabetes have a greater loss in muscle mass and 
function than adults without diabetes.20, 21 After exclusion of diabetics, studies still show that insulin resistance is 
associated with lower muscle strength and increased risk of frailty for older adults.22, 23  
Treatments for Sarcopenia 
To date, interventional studies have examined the use of testosterone, growth hormone and exercise to treat 
sarcopenia, with only exercise training demonstrating an increase in both muscle mass and strength for older 
adults without adverse effects.15, 24 Our preliminary data described below suggest that metformin may prevent the 
loss of lean mass in insulin-resistant, older men.25 However, there have been no interventional studies to 
determine the effects of metformin on preventing the loss in muscle mass and muscle function.  
Potential Mechanism for Sarcopenia Prevention with Metformin 
The skeletal muscle of aging adults differs from young adults with lower 5’ adenosine monophosphate-activated 
protein kinase (AMPK) activity, a selective decrease in type II muscle fibers, accumulation of intramyocellular 
lipid, lower capillary-to-muscle fiber ratio, and fewer mitochondria.26-30 Both metformin and exercise can 
stimulate the phosphorylation of AMPK, an enzyme that can regulate the transcription of genes responsible for 
switching muscle fiber types from glycolytic to more oxidative, fatigue-resistant fibers, lowering muscle lipid 
accumulation, stimulating angiogenesis and increasing mitochondrial biogenesis (Fig 1).31-34  
Aerobic exercise can maintain or improve muscle 
mass and function in older adults.35 Histologic changes 
in muscle accompanying exercise training in older 
adults include decreased diacylglycerol and ceramide 
content, increased capillary density, increased 
mitochondrial content and activity and a switch from 
type IIB to more oxidative IIA fibers.36-38 Similar 
studies to evaluate the effects of metformin on muscle 
mass, function and histology have not yet been 
performed in older adults.  
However, animal studies suggest that pharmacologic 
activation of AMPK results in histologic changes in 
muscle similar to exercise. Treatment of animals with 
metformin or another AMPK agonist, 5-


IRB #8860 
08/16/2016 
 
 
4 
aminoimidazole-4-carboxamide ribofuranoside (AICAR), increased fatty acid oxidation and decreased 
diacylglycerol and ceramide content in skeletal muscle.39-43 Increased expression of vascular endothelial growth 
factor and skeletal muscle angiogenesis also occur in mice treated with AICAR.44, 45 Increased activity of 
mitochondrial enzymes and expression of mitochondrial protein in muscle with AICAR and increased 
mitochondrial density with another AMPK activator, β-guanadinopropionic acid, may be mediated through 
downstream activation of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC1-α).34, 46-49 
AICAR-treated rats appeared to have a switch from type IIB fibers to more oxidative type IIX fibers similar to 
endurance trained rats.50 While AICAR and metformin in animals can lead to the histologic changes in muscle in 
our proposed mechanistic framework (Fig. 1), the effect of metformin on human muscle composition remains to 
be confirmed. 
AMPK activation in basic studies also supports an improvement in muscle mass and function. While AMPK is 
thought to increase atrophy through inhibition of mTOR from cell-based studies,51, 52 in vivo studies instead 
demonstrate increased muscle fiber area and weight with AICAR53 and increased protein synthesis with 
metformin.54 Furthermore, the treatment of sedentary mice with AICAR improved endurance and extended 
running distance.55 In contrast, AMPKβ2 knockout mice have decreased muscle fiber size, decreased exercise 
capacity and endurance.56  
These studies suggest the potential for metformin to positively affect muscle, and provide a compelling rationale 
for the positive associations between metformin use and muscle mass and function that we describe in our 
preliminary studies below. As an AMPK activator, metformin is also expected to result in histologic changes for 
decreasing intramyocellular lipid and increasing capillary density, mitochondrial density and oxidative fibers. If 
our proposed study provides evidence to support this mechanistic framework, future studies of signaling 
intermediates for these histologic changes will be pursued with stored tissue specimens. Therefore, our proposed 
study of metformin treatment in insulin-resistant older adults will provide further insight into its mechanisms for 
preventing sarcopenia and to identify other therapeutic targets for this condition. 
 
Preliminary Studies / Progress Report: 
 
The Osteoporotic Fractures in Men (MrOS) Study is a prospective cohort study of 5994 ambulatory, community-
dwelling men aged 65 and older (mean age 73.7 years, range 64-100) who were enrolled initially March 2000-
April 2002 from six U.S. clinical sites (Portland, OR; Palo Alto, CA; San Diego, CA; Minneapolis, MN; 
Birmingham, AL; and Pittsburgh, PA) for the purpose of studying osteoporotic fracture determinants.57 
Participants returned for follow-up visits in 2005-2006 and 2007-2009 and continue to participate in ongoing 
follow-up for multiple health outcomes and assessment of vital status. The Study of Osteoporotic Fractures (SOF) 
is a prospective cohort of 9,704 Caucasian women aged 65 years and older who were enrolled between 1966-1988 
from Portland, OR; Minneapolis, MN; Baltimore, MD; and the Monongahela Valley near Pittsburgh, PA for the 
study of osteoporosis and fractures.58 They were studied at baseline and returned every 2 years for a clinic visit. 
Information collected in both of these cohort studies includes dual x-ray absorptiometry scans (DXA) for body 
composition measurements (in MrOS), biochemical measurements, neuromuscular function, and questionnaires 
for medical problems, medications, demographic information, and lifestyle factors. These are unique and rich 
resources for studying longitudinal changes in older men and women. From these studies, the following 
preliminary data have been generated. 
 
Lean mass loss is associated with increased risk 
of mortality 
To examine the mortality risks of sarcopenia in 
older men, we used data from participants in the 
MrOS study who had DXA-based measurements 
of body composition on average 4.6 (range: 3.5-
5.9) years apart. Older men with ≥5% lean mass 
loss had a 1.8-fold increased risk for all-cause 
mortality compared to men who maintained lean 


IRB #8860 
08/16/2016 
 
 
5 
mass. This risk remained significantly elevated even after accounting for baseline lifestyle factors and medical 
conditions and did not differ by baseline age, BMI or self-rated health (interaction p-value<0.10) (Fig. 2). 
However, the risk of all-cause mortality associated with  ≥5% total body lean mass loss was higher for men who 
lost weight unintentionally (HR 2.09, 95% CI 1.65, 2.65) compared to men who lost weight intentionally in whom 
there was no increase in mortality risk (HR 1.06, 95% CI 0.69, 1.62). Overall these findings indicated that 
involuntary lean mass loss doubled the risk of all-cause mortality in older men. While other studies have shown 
increased morbidities with sarcopenia, this study showed that there is also an increased risk of mortality. These 
results were presented and honored with a Presidential Poster Award at the 2010 Annual American Geriatrics 
Society Meeting and were published in the Journal of the American Geriatrics Society.59  
 
Insulin resistance is associated with lean mass loss  
Baseline insulin resistance was predictive of future loss in 
lean mass in older, non-diabetic men. Insulin resistance was 
measured by the homeostasis model assessment of insulin 
resistance (HOMA-IR) in non-diabetic men in MrOS at 
baseline. There was a significant trend towards greater % 
loss in lean mass with increasing insulin resistance (Fig. 3). 
This association was unaltered by further adjustment for 
baseline physical activity and changes in physical activity 
during this period of time. The loss of lean mass associated 
with greater insulin resistance also existed independent of 
other metabolic co-morbidities like hypertension, body 
weight and dyslipidemia. These data were presented at the 
Gerontology Society of America meeting in November 
2010 and a manuscript describing these results was 
published in the Journal of the American Geriatrics 
Society.60  
 
 
Insulin resistance is associated with increasing 6-meter walk time  
The associations between baseline insulin resistance and the 
longitudinal change in 6-meter walk time was also 
examined in 3256 MrOS participants without diabetes who 
had these measurements taken 4.7 years apart. There was a 
significant trend towards increased 6-meter walk time 
(p<0.05) with greater insulin resistance as measured by the 
homeostasis model assessment of insulin resistance 
(HOMA-IR) in models accounting for age, clinic site, self-
rated health and baseline walk time, physical activity, 
hypertension and the change in lean mass (Fig. 4). 
Compared to the most insulin-sensitive men in the lowest 
quartile of HOMA-IR, insulin-resistant men in the highest 
two quartiles of HOMA-IR had a significantly greater 
increase in walk time in fully-adjusted models (p-
values<0.05). Therefore, greater insulin resistance in older 
men without diabetes is also associated with an accelerated 
loss in gait speed (data not yet published). 
   
 Metformin use may attenuate lean mass loss for older men with diabetes 
To further assess the association between insulin resistance and changes in lean mass, the loss in appendicular 
lean mass over 3.5 years was evaluated in older men who were grouped into the following categories of glycemic 
status at baseline: normoglycemia (n=1853), impaired fasting glucose (n=1403), untreated diabetes mellitus 


IRB #8860 
08/16/2016 
 
 
6 
(n=234), diabetes mellitus treated with insulin-sensitizers (n=151) and diabetes mellitus treated without insulin-
sensitizers (n=111). Even after adjustment for baseline demographics, lifestyle factors and medical comorbidities, 
the loss in appendicular lean mass was significantly attenuated in diabetic men on insulin-sensitizers compared to 
men with normoglycemia, impaired fasting glucose, untreated diabetes mellitus or diabetic men not on insulin-
sensitizers, p<0.001 for all comparisons (Fig. 4). Among diabetic men on insulin-sensitizers, metformin use was 
associated with significantly less appendicular lean mass loss (-0.41 Kg) compared to men with normoglycemia (-
0.75 Kg), p<0.01; whereas the amount of appendicular lean mass loss for men on thiazolidinediones (-0.65 Kg) 
did not differ significantly from men with normoglycemia, p=0.67.  These data suggest that use of insulin-
sensitizers, particularly metformin, may attenuate the loss of appendicular lean mass for older men with diabetes. 
Additional sensitivity analyses were performed, and these results were unchanged even after exclusion of 42 men 
on insulin. These findings have recently been published in Diabetes Care.25 
 
 
Metformin may attenuate the loss in usual gait speed for older women with diabetes 
We hypothesized that insulin sensitizer (metformin/thiazolidinediones) use would also be associated with 
attenuated loss in physical performance in older diabetic women. In the Study of Osteoporotic Fractures, a multi-
center, longitudinal cohort study, 2826 women aged ≥70 years had physical performance measures assessed 
between 1997-1998 and 4.9±0.6 years later. Least squares mean change in 6-meter walk speed was estimated in 
multivariate linear regression models for women with no diabetes (n=2711), diabetes mellitus with no insulin-
sensitizers (n=162) and diabetes mellitus with insulin-sensitizers (n=30). After accounting for baseline age, race, 
BMI, education, self-rated health, hypertension, walk speed, and use of estrogen and statins, the decline in walk 
speed was significantly greater in women with diabetes who were not using insulin-sensitizers (-0.18 m/s, 95% CI 
-0.21, -0.15) than in women with no diabetes (-0.12 m/s, 95% CI -0.12, -0.11); whereas the loss in gait speed for 
women with diabetes using insulin-sensitizers (-0.09 m/s, 95% CI -0.17, -0.04) did not differ to the loss seen in 
women with no diabetes. These results were unchanged after excluding women using insulin. These findings also 
remained unchanged after excluding 2 women on thiazolidinediones, suggesting that metformin use in older 
diabetic women may attenuate the loss in walk speed (data not yet  published).   
 


IRB #8860 
08/16/2016 
 
 
7 
 
 
Summary 
 
The longitudinal data from MrOS and SOF have helped to establish that insulin-resistant, older adults experience 
a greater loss in lean mass and physical performance. However, older adults with diabetes who were treated with 
metformin had a significant attenuation in the loss of lean mass and walk speed. These preliminary data provide 
the rationale for determining whether metformin can be used to prevent the loss of muscle mass and function. 
Although these observational results are compelling, validation with a randomized controlled trial is essential. 
Furthermore, a carefully performed trial will provide critical data about the mechanistic effects of metformin on 
muscle. 
 
Research Design and Methods 
 
Subjects and Experimental Design: 
 
Overview 
 
Older adults with impaired fasting glucose (IFG) will be randomized to into a double-blinded, placebo-controlled 
intervention study with metformin. This clinical trial has the primary outcome of change in appendicular lean 
mass and physical performance assessed between baseline and year 3 based on our prior observational findings 
above and Power calculations below. The secondary outcome of change in muscle histology over 6 months using 
muscle biopsies is planned in a substudy of participants given previous studies that show significant biological 
effects on muscle with metformin treatment during this time interval.32, 61  
 
Study Population  
 
Inclusion criteria: 120 randomized, sedentary, weight-stable, ambulatory men and women 65 years or older with 
pre-diabetes identified with IFG (100 mg/dL≤fasting glucose<126 mg/dL) on no diabetes medications. 
Participants must demonstrate that they are able to ambulate 400 meters without assistance. Participants must be 
able to fast for 12 hours prior to blood draw. 
 
Subjects who meet the above inclusion criteria and meet the additional exclusion criteria for the Muscle Biopsy 
Substudy listed below will be considered for enrollment (32 subjects) in the muscle biopsy substudy. 
 


IRB #8860 
08/16/2016 
 
 
8 
Exclusion criteria: Participants will be excluded if they plan to move away from the Portland metropolitan area 
during the 3 year study time frame. Participants with conditions that are relative contraindications to metformin or 
affect muscle mass or performance measurements will be excluded as outlined below. Specific criteria are 
delineated below.  
 
 
Exclusion Criteria 
1. Chronic medical conditions affecting muscle mass or function 
a. Active non-skin cancer 
b. Hypogonadism 
2. Medications affecting muscle mass or function – See section “Prohibited Medications.” 
3. Lifestyle factors affecting muscle 
a. Intentional/unintentional weight loss 
b. Vigorous regular exercise 
4. Contraindications to metformin 
a. Renal dysfunction defined as creatinine ≥ 1.5 mg/dL for men or ≥ 1.4 mg/dL for women or 
eGFR< 60 mL/min 
b. Liver dysfunction defined as ALT > 48 U/L, AST > 41 U/L  
c. B12 deficiency defined as B12 level < 180 pg/mL  
d. Congestive heart failure 
e. Known hypersensitivity to metformin 
f. Excessive alcohol intake (average of ≥ 2 alcoholic beverages/day over a month) 
g. Serious medical condition in the opinion of the investigator 
 
Additional Exclusion Criteria for Muscle Biopsy Substudy 
1. Conditions that increase bleeding risk 
a. Platelet count < 150 billion/L 
b. INR > 1.2 
c. aPTT > 36 seconds 
2. Medications that increase bleeding risk – See section “Prohibited Medications.” 
3. Allergy to lidocaine 
 
Prohibited Medications: 
1. Medications affecting muscle mass or function 
a. Glucocorticoids 
b. Androgen/antiandrogens 
2. Medications that increase bleeding risk (for muscle biopsy substudy) 
a. Warfarin 
b. Clopidogrel/ticlopidine 
c. Aggrenox 
d. Dabigatran 
e. Anagrelide 
 
 
Participant recruitment 
 
We will plan to screen up to 3,000 older adults (including phone screens) from VA primary care clinics (Portland, 
Vancouver, Metro East, Metro West, and West Linn) to account for those who may not qualify due to the 
presence of exclusion criteria. We plan to enroll up to 300 participants in order to randomize 120 subjects at the 
VA Portland  Health Care System (VAPORHCS) since we anticipate many participants to screen out of the study 
based on lab values after having signed a consent form. Advertisement flyers will be posted on approved bulletin 
areas on the VAPORHCS. VA primary care physicians may refer any interested and potentially eligible subjects 
to the study team. Mailing recruitment will also occur, using primary care and endocrine patient listings of those 


IRB #8860 
08/16/2016 
 
 
9 
who meet the age criteria, medication and medical history exclusions, and are not diagnosed with diabetes. 
 
The following criteria will be applied to the VISN 20 (Veterans Integrated Services Network) data warehouse to 
identify potential participants for recruiting mailing: 1) patients age 65 years and greater seen in VA primary care 
clinics (Portland, Vancouver, Metro East, Metro West and West Linn) in the past year; 2) ICD-9 codes will be 
used to exclude patients with diabetes and other exclusion diagnoses; and 3) patients will be excluded if they have 
received one or more prescriptions for diabetes medications or contraindicated medications in the past year. We 
will adhere to the VAPORHCS policy on recruitment via letters. As we indicated in the Application for a Waiver 
of Authorization and Informed Consent Process, we will use CPRS to collect names, mailing addresses, 
birthdates, telephone numbers and social security numbers. 
 
Initial contact with potential subjects will occur in clinic, by phone or by mail. If in clinic, their provider will 
approach them first to see if they are interested in the study and ask if they agree to be contacted by study 
personnel. If the patient agrees, they can initiate contact with the study team by phone or have the study 
coordinator speak to them in clinic or call them later. If initial contact is by phone, potential subjects can contact 
the study coordinator directly using information on the advertising flyers. If initial contact is by mail, potential 
subjects will receive a recruitment letter indicating the option to opt out by returning an included letter indicating 
the wish to not be contacted again. The letter will state that if a response is not received within two weeks, 
potential subjects may be contacted by phone. Included with this mailing will be an approved informational letter 
from the Chief of Primary Care. 
 
During the screening telephone call, a study coordinator will discuss the inclusion and exclusion criteria and 
provide further details on the study. If participants are interested and are potentially eligible, a Screening visit will 
be scheduled. 
 
 
Study design 
 
The proposed study duration of 3 years has been selected based on our prior observational findings over 3.5 
years.25 Eligible participants will have an initial Baseline visit, followed by randomization to placebo or 
metformin (Fig. 7). Between visits, telephone surveying of medication compliance will be obtained and any AEs 
reviewed. Participants will be seen every 6 months, and have annual study visits for measurements of muscle 
mass and performance. A substudy of participants will also include muscle biopsies at 2 visits; within 14 days 
from baseline and at the 6 month visit. 
 
Fig. 7 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


IRB #8860 
08/16/2016 
 
 
10 
 
 
Primary outcome measures  
 
To test the primary hypothesis that metformin-treated participants will have a slower decline in muscle mass and 
physical performance than those on placebo, we will be measuring total and appendicular lean mass using dual x-
ray absorptiometry and assessing physical performance with the short physical performance battery (SPPB), grip 
strength and a 400 meter walk. Our preliminary findings suggest that there should be attenuation in the loss in 
appendicular lean mass and gait speed, and additional performance measures will be explored.  
 
Secondary outcome measures  
 
The secondary hypothesis is that the muscle composition of metformin-treated participants will have a greater 
increase in number of oxidative fibers, decrease in intramyocellular lipid, increase in capillary density and 
increase in the number of mitochondria than placebo-treated participants. These outcomes have been chosen 
based on our hypothetical framework in Figure 1 that was developed from known alterations in these measures in 
animal models treated with metformin or other AMPK agonists. Quantification of intramyocellular lipid, fiber 
types, capillary density and mitochondrial content will be obtained through histochemical analyses of muscle 
biopsy specimens as described in the Muscle Biopsy section. 
 
Study Visits 
All visits will be completed at VAPORHCS and/or at OHSU. 
 
Screening Visit 
  
IRB-approved study personnel will have a full discussion with the potential subject, including information on 
study visits, procedures, risks, and benefits.  All participants will be informed of their right to withdraw from the 
study at any time. The principal investigator will answer any questions from participants at any time. Participants 
will provide written informed consent prior to any study procedures being performed. A copy of the IRB-
approved informed consent and signed HIPAA authorization form will be given to the subject. Demographic 
information (including age, gender, and race) and lifestyle factors (including physical activity, smoking history 
and alcohol intake) will be obtained. Medical history (including self-reported physician medical diagnoses), drug 
allergies/adverse effects and medications will be obtained. Height, weight and blood pressure (BP) will be 
measured. Criteria for exclusion criteria will be formally reviewed. Participants must demonstrate that they are 
able to ambulate 400 meters without assistance. Laboratory assessment for B12 levels and liver, kidney and 
clotting function will be performed. The total amount of blood drawn will be 10 ml. Labs will be drawn for 
fasting glucose to identify participants with IFG. All labs will be ordered after consent is obtained. Participants 
will be instructed to remain fasting for 12 hours prior to the study day. This visit could take up to 2 hours. 
Participants who have IFG will be  scheduled to attend a Baseline visit if the Screening visit does not reveal 
presence of an exclusion criterion.  
 
 
Baseline Visit 
 
The Baseline visit will occur in 14 days (+/- 7 days) from Screening. Participants will be instructed to remain 
fasting for 12 hours prior to the study day. This visit could take up to 3 hours. The study measures will be 
conducted by the IRB-approved study team who are all blinded to treatment allocation. The participants will first 
get a fasting blood draw at VAPORHCS. All study subjects will be provided breakfast after their fasting lab draw 
has been done. A fasting glucose level will be measured.  Subjects will proceed with the following study 
measures: review and completion of questionnaires, medication inventory, physical performance tests, and whole 
body DXA.  For those subjects who signed the consent indicating their interest in participating in the muscle 
biopsy substudy and are eligible, they will be scheduled for a Baseline muscle biopsy visit within 14 days of their 


IRB #8860 
08/16/2016 
 
 
11 
baseline visit. 
 
Because adults with IFG have a higher risk of developing type 2 diabetes, all study participants will receive 
guidelines for diet and lifestyle modification consistent with the goals of the Diabetes Prevention Program at 
baseline and yearly visits.72 These include intake of < 25% of calories as fat and engaging in ≥150 minutes of 
physical activity weekly.  
 
Baseline Muscle Biopsy Visit (if in substudy) 
 
Participants enrolled in the muscle biopsy substudy will receive a telephone call within 3 days prior to their 
biopsy visits to remind them to fast for 12 hours prior to the visit, to remind them to abstain from vigorous 
physical activity, and to ensure they are not on anticoagulant or antiplatelet medications within 7 days from the 
biopsy visit with the exception of aspirin or other anti-inflammatory medications which cannot be taken within 3 
days of the biopsy visit. 
 
This visit will occur within 14 days from Baseline, and it could take up to 3 hours. It will be completed at OHSU. 
Eligibility criteria for the muscle biopsy will again be reviewed, and if there are no contraindications, the 
participant  will undergo muscle biopsy. Participants will be given breakfast after muscle biopsy has been 
obtained. 
 
Phone calls between visits 
At 3 and 9 month time points between annual visits, telephone surveying of medication compliance will be 
obtained. Any adverse events (AE) will be reviewed. A 24-hour phone number will be provided to participants to 
call in the event of any adverse outcome between these contact points. Additional phone calls may be made if 
subjects are found to be non-compliant. 
 
All study participants will receive a telephone call to remind them to fast for 12 hours prior to their study visit.  
 
Participants enrolled in the muscle biopsy substudy will receive a telephone call within 3 days prior to their 
biopsy visits to remind them to fast for 12 hours prior to the visit, to remind them to abstain from vigorous 
physical activity, and to ensure they are not on anticoagulant or antiplatelet medications within 7 days from the 
biopsy visit.  
 
Half-Yearly Visits (6  months; Year 1 +6 months; Year 2 +6 months)  
 
Half-yearly visits will occur at 6 Months, Year 1 + 6 Months, and Year 2 + 6 Months. All subjects will go to 
VAPORHCS for these visits. For those in the substudy, only for the 6 Month visit, they will also go to OHSU 
after labwork is drawn at VAPORHCS.  
 
This visit could take up to 2 hours for participants (3 hours for participants in the muscle biopsy substudy at the 6 
Month visit). At each half-yearly visit, all participants will have fasting labwork drawn, and have questionnaires 
reviewed and completed. Subjects >80 years old will get labs checked for creatinine and eGFR. Participants in the 
muscle biopsy substudy will undergo muscle biopsy if their morning STAT labs demonstrate platelet, INR, and 
aPTT levels in the normal range. Participants will be given breakfast after fasting lab work/muscle biopsy has 
been obtained. 
 
 
Annual Visits (Years 1, 2, and 3) 
 
These annual visits could take up to 3 hours. The participants will first get a fasting blood draw. They will then be 
given breakfast before proceeding with the following study measures: review and completion of questionnaires, 
medication inventory, physical performance tests, and whole body DXA.  


IRB #8860 
08/16/2016 
 
 
12 
 
Study Drug 
 
We have chosen to study metformin in this clinical trial due to the observation that metformin use was associated 
with an attenuated loss of muscle mass and function in our preliminary studies. While both metformin and 
thiazolidinediones can upregulate AMPK and PGC1α in muscle to increase muscle fatty acid oxidation, 
angiogenesis, mitochondrial biogenesis and oxidative muscle fibers for healthier-appearing muscle,32, 34, 68 
metformin has additional benefits on mortality, microvascular and macrovascular outcomes in patients with type 2 
diabetes;69 whereas, thiazolidinediones have associated risks of adverse cardiovascular outcomes.70, 71  
 
The VA Research Pharmacy will be used to randomize, dispense, and track blinded placebo and metformin tablets 
in 3-month supplies. A covariate-adaptive randomization scheme will be used to ensure equal age, gender and 
race distributions. The investigators and study participants will be blinded to the randomization of the study drug 
assignments. A 90-day supply of study drug will be mailed every 3 months to participants with a pre-paid 
envelope for returning unused study drugs for pill counts. An additional 4-day buffer may be included to avoid 
lapses in study drug administration due to unforeseen complications in medication delivery and/or receipt. To 
minimize self-limiting gastrointestinal side effects, participants randomized to metformin will begin with an oral 
dose of 850 mg tablets once daily (taken with meals) for 1 month with titration up to 850 mg twice daily for the 
remainder of the study. We anticipate an effect on muscle using this dosage, since a significant reduction in 
peripheral insulin resistance occurs even with a metformin daily dosage of 750 mg.61 Furthermore, this dose was 
safe and effective in lowering the risk of diabetes for subjects with prediabetes in the Diabetes Prevention 
Program.75 Plans for monitoring and preventing risks associated with metformin are further discussed under 
Human Subjects. 
 
Instances where the study drug is discontinued include the following: development of a contraindication to 
metformin as described under the section for exclusion criteria; development of lactic acidosis; diagnosis of 
diabetes; hospitalization; 48 hours prior to procedures or scans that require contrast medium; or development of 
B12 deficiency. For those who are hospitalized or receive contrast medium, efforts will be made to recheck 
kidney function within 48 hours after discharge/procedure and the study drug will be restarted if there is no 
evidence of renal dysfunction. For tests or procedures that require fasting, study drug will be stopped 24 hours 
prior to test or procedure, and study drug can be resumed after the participant is allowed to eat again. Compliance 
to study drug will be tracked and accounted for in analyses. 
 
Study Procedures 
All study procedures are research-related. 
 
Exam: Exams for annual visits will occur at OHSU after fasting labwork has been drawn at VAPORHCS. At 
Screening, baseline and yearly exams, height will be measured in meters using a wall-mounted Harpenden 
stadiometer, body weight will be measured in kilograms using a balance beam scale, waist and hip circumference 
measured with a tape measure, and measurements of pulse and blood pressure will be taken.  
 


IRB #8860 
08/16/2016 
 
 
13 
Labs:  The lab samples will be collected at VAPORHCS at the Screening (after consent is obtained), Baseline, 
Half-Yearly Visits and Annual Visits, and processed at  VAPORHCS or OHSU. Fasting plasma will be drawn at 
Screening for ascertainment of IFG (100mg/dL≤fasting glucose<126mg/dL) and exclusion criteria: creatinine ≥ 
1.5 mg/dL for men or ≥1.4 mg/dL for women, eGFR<60 mL/min, B12 <180 pg/mL, ALT>48 U/L, or AST > 41 
U/L. Whole blood will be assayed for platelet counts, and platelet-poor plasma will be assayed for INR and aPTT. 
At Baseline, fasting plasma will just be assayed for glucose. At annual follow-up study visits, fasting plasma will 
be assayed for glucose, insulin, B12 (pg/mL), creatinine (mg/dL), alanine transaminase (U/L), aspartate 
aminotransferase (U/L), and estimated glomerular filtration rate (mL/min). Exclusion criteria for follow up labs 
will be identical with the following exception:  The FDA guidelines requiring the safe use of metformin have 
recently changed and the eGFR cutoff as indicated by the FDA is now eGFR < 45 mL/min/1.73 m2 which we will 
use as our exclusion criteria for continuation of study drug.  With the exception of insulin, the blood labs will be 
analyzed at VAPORHCS. Insulin will be analyzed at OHSU.  
 
A total of 10 ml of blood will be drawn at the Screening Visit. At the Baseline visit, 30 mL of blood will be 
drawn. At the half-yearly visits, a total of 20 mL of blood will be drawn. At the Annual follow-up visits, 30 mL of 
blood will be drawn.   
 
Any clinically significant abnormal laboratory findings at the scheduled end of the study should be followed up 
until satisfactory resolution or diagnosis can be made.   
 
Biorepository 
 
A separate biorepository protocol will be submitted to the IRB for approval. Additional samples of blood and 
muscle tissue (if in substudy) will be collected and stored for future analyses at the Baseline visit, muscle biopsy 
visit, half-yearly visits, and annual visits. These are described above in the Lab section and Muscle Biopsy section 
in this protocol below. 
 
Questionnaires: Prior to all study visits from Baseline to Year 3/End of Study, questionnaires to assess lifestyle,  
diet, physical activity, physical function, fatigue and medical diagnoses will be mailed to participants in their visit 
reminder letter to be completed and returned on the study visit day. A #2 pencil will be included with this mailing 
in order for participants to fill out the scantron portion of the food questionnaire. The baseline questionnaire will 
also include demographic questions like marital status, living situation, education, employment and 
socioeconomic status. The follow-up questionnaires will include study drug compliance questions to assess 
adherence to study drug. Participants will have questionnaires reviewed at the study visit by the study coordinator 
for completeness and will finish any incomplete sections at the study visit. At 1, 3 and 9 month time points 
between study visits, adherence to study drugs will be surveyed over the telephone.67 
 
Medication Use: Participants will be instructed to bring all prescription and over-the-counter medications to their 
study visits, and research staff will take an inventory of all medication names and doses. For the convenience of 
participants, a bag will be mailed along with the visit reminder letter that they may use to bring their medications 
to the visit. Participants will be given pre-paid envelopes to mail unused study drugs to the research pharmacy 
every 3 months for pill counts to track compliance.  
 
Physical Performance Tests: Performance tests will occur at VAPORHCS and OHSU. They will be conducted 
at Screening, Baseline, and the annual visits. At Screening, subjects must demonstrate they are able to walk 400 
meters. At Baseline and the annual visits, assessments of grip strength, a 400 meter walk, timed 6 meter walk, all 
of the short physical performance battery (SPPB) tests, and Instrumented Sway (ISway) test will be conducted. 
All performance tests will be instrumented with Mobility Lab sensors on bilateral upper and lower extremities, 
waist and chest using elastic straps. The ISway test consists of 3 trials where the participant stands for 30 seconds 
without moving with their arms at their side. Each trial will have measures of amplitude, velocity, frequency and 
jerkiness of postural sway in lateral and anterior-posterior directions with 42 metrics. The SPPB comprises tests 
of balance, timed walk and chair stands and is predictive of subsequent disability in older adults.11 For tests of 


IRB #8860 
08/16/2016 
 
 
14 
balance, the ability and/or duration (s) in which a participant can maintain a tandem, semi-tandem and side-by-
side stand will be recorded. Participants will be timed in the task of rising from a seated position 5 times in rapid 
succession without use of their arms. Those who are unable to complete this task without the use of their arms 
will be considered unable. The participant’s time to complete a 6 meter walking course at his/her usual pace will 
be measured with a stopwatch. Walking speed (meters/second) will be determined from the better of two trials. In 
addition to the SPPB, tests of endurance and strength will assessed with a 400 meter walk and grip strength. The 
time to complete a 400 meter walk test will also be measured with a stopwatch. Participants will be considered 
unable to perform this task if they require more than 15 minutes for the 400 meter walk or if they require use of 
assistance. Grip strength will be assessed using a Jamar dynamometer in the participant’s dominant hand.73 The 
maximum effort from two trials will be taken as the measurement of maximum grip strength (kilograms). 
 
Imaging (DXA): DXAs will occur at OHSU. At Baseline and the annual visits, whole body scans will be taken 
with a Hologic QDR 4500W DXA scanner by a certified DXA operator to determine total body lean mass and 
appendicular lean mass (kilograms), as previously described.59, 60 Standard Hologic procedures will be used to 
define regions on the whole body DXA scans using cut lines. The arms will be delineated by a cut line drawn 
between the head of the humerus and the scapula at the glenoid fossa and the leg will be delineated by a cut line 
drawn across the midpoint of the femoral neck. Appendicular lean mass will be calculated as the sum of lean mass 
in the arms and legs. Hologic whole-body QC phantoms will be scanned daily to monitor longitudinal changes so 
that correction factors can be applied to measurements to adjust for longitudinal drift.  
 
Muscle Biopsy studies (for those in the substudy): Subjects are asked to participate in the muscle biopsy studies 
based on their platelet and INR results. There will be 32 subjects in the substudy. Subjects will not enroll in the 
muscle biopsy studies or have a second muscle biopsy if they take medications that increase bleeding risk, have 
an allergy to lidocaine, or have platelet count < 150 billion/L, INR > 1.2, or aPTT > 36 seconds.  
 
At the Baseline Muscle Biopsy visit and 6 month visit, fasting muscle biopsy samples will be obtained at OHSU by a 
physician or certified nurse practitioner from the vastus lateralis muscle, 15 cm above the patella using the modified 
Bergstrom technique, as described.36, 74-76 Using a 2% lidocaine for anesthetic, a small, ¼ incision will be made in the 
skin and fascia to place a 5 mm Bergstrom biopsy needle while suction is applied through the 140 cc syringe, 
obtaining approximately 150-200 mg of muscle. The incision will be closed with sterile adhesive strips, and a 
pressure bandage will be applied for a period of 24 hours.  This procedure takes approximately 15 minutes, is a 
relatively painless, and well-tolerated in older adults.  Subjects will be instructed not to perform physical exercise 48 
hours prior to the muscle biopsy procedure to help prevent acute effects of exercise on intramyocellular lipids 
(IMCL) (7:8). They will also be given discharge instructions after the procedure for precautions to follow to prevent 
complications.  
 
All muscle specimens will be processed at OHSU. The specimen (50 mg) for histochemistry will be mounted on a 
small piece of cork, placed in cooled isopentane for 2-3 minutes and then frozen in liquid nitrogen. Another 
portion (50 mg) will be placed in a cryovial and flash frozen in liquid nitrogen for analysis of diacylglycerol and 
ceramide content.  The remaining tissue will flash frozen in liquid nitrogen, and stored for future analyses. 
 
All specimens will be stored in a -80 freezer in the biorepository  located at the Oregon Clinical & Translational 
Research Institute (OCTRI) lab until they are analyzed. All muscle specimens will be stored after being assigned 
code numbers and the information linking these code numbers to the corresponding subjects’ identities will be kept 
in a separate, secure, locked location.  This is done to protect research subject’s confidentiality. If a research subject 
decides to withdraw the specimens may continue to be stored as outlined in the approved consent documents. 
Identifiers will be stored separately from the samples. 
 
During the course of the study, 100 mg of stored muscle biopsy specimens will be prepared for histochemical 
analyses will be sectioned on a cryostat at -20 C and placed on precleaned glass slides. A longitudinal section will 
be made and imaged at 36,000 magnification for acquisition of 15-20 micrographs to determine mitochondrial 
content using digital image analytical software (Metamorph 6.3) as previously described.77 Succinate 


IRB #8860 
08/16/2016 
 
 
15 
dehydrogenase staining and quantification using image analysis of staining intensity will be assessed as 
described.75 Triglyceride content will be determined using Oil Red O staining using established methods.78 
Capillary density (number of capillaries/muscle area) will be determined by visualization using a 
tetramethylrhodamine isothiocyante (TRITC) excitation filter after thawed and air dried sections are fixed in 
0.25% formaldehyde, incubated with lectin and rinsed.36 The percentage of Type I, IIa and IIx fibers will be 
determined from manual counting 100-300 fibers in stained sections. The sections will first be incubated 
overnight at room temperature with antibodies against antihuman myosin heavy chain-7 (type I fibers) and 
myosin heavy chain-2 (type IIa fibers) and then incubated with secondary antibodies conjugated with fluorescein 
(type IIa fibers) and rhodamine (type I fibers) . Type IIx fibers will remain unstained. Images will be acquired 
with an optical microscope.36, 74 Diacylglycerol and ceramide content will be measured using high-performance 
liquid chromatography-tandem mass spectrometry  from a homogenized (using ice-cold buffer of 250 mM 
sucrose, 25 mMKCl, 50 mMTris, and 0.5 mM EDTA, pH7.4) liquid nitrogen-frozen sample as previously 
described.36  
 
Meals 
 
After fasting labwork/biopsies have been taken, participants will be provided breakfast by the OCTRI 
Bionutrition Unit.  
 


 
 
Schedule of Study Procedures 
 
 
Screening 
Visit 
Baseline 
Visita 
Baseline 
Muscle 
Biopsy 
Visitb 
3 and 9 Months, 
Year 1 +3 and +9 
Months, Year 2 +3 
and +9 Months 
Phone Callsc 
6 Month 
Visitd  
Years 1, 
2, and 3/ 
End of 
Study 
Visitd 
Year 1 + 6 
Months, 
and Year 2 
+ 6 Months 
Visitsd 
Consent 
X 
 
 
 
 
 
 
Inclusion/Exclusion 
X 
 
 
 
 
 
 
Height 
X 
 
 
 
 
X 
 
Weight, blood pressure 
X 
X 
 
 
 
X 
 
Demographics 
X 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
Questionnaires 
 
X 
 
 
X 
X 
X 
Labs 
 
X 
 
 
X 
X 
X 
     Fasting Glucose 
X 
X 
 
 
 
X 
 
     Fasting Insulin 
 
X 
 
 
 
X 
 
     ALT 
X 
 
 
 
 
X 
 
     AST 
X 
 
 
 
 
X 
 
     Creatinine 
X 
 
 
 
Xf 
X 
Xf 
     eGFRe 
X 
 
 
 
Xf 
X 
Xf 
     B12 
X 
 
 
 
 
X 
 
     Platelets 
X 
 
 
 
Xg 
 
 
     INR, aPTT 
X 
 
 
 
Xg 
 
 
Biorepository 
 
X 
 
 
X 
X 
X 
Physical Performance 
X 
X 
 
 
 
X 
 
DXA 
 
X 
 
 
 
X 
 
Muscle Biopsy (only for 
substudy)h 
 
 
X 
 
X 
 
 
Concurrent 
medications 
X 
X 
 
 
X 
X 
X 
Medication compliance 
 
 
 
X 
X 
X 
X 
AE monitoring 
 
 
 
X 
X 
X 
X 
Meals 
 
X 
X 
 
X 
X 
X 
 
aBaseline visit will occur 14 ± 7 days from Screening visit. 
bFor substudy, Baseline Muscle Biopsy visit will occur within 14 days from Baseline visit. 
cAt 3 and 9 month time points between annual visits, telephone surveying will occur. 
dHalf-yearly visits and annual visits will occur with ± 7 day visit window. 
eeGFR is calculated from the creatinine. 
fEvery 6 months, creatinine and eGFR will be obtained for subjects over 80 years old. 
gAt 6 months, STAT platelets, INR, and aPTT will be obtained for subjects in the substudy before the biopsy. 
hMuscle biopsy samples are for this study and for biorepository. 


 
 
 
Adverse events 
 
Adverse events (AEs) will be evaluated at each visit. Principal Investigator or MD Co-Investigator will determine 
the relation of the side effect to treatment as: 1) related, 2)  possibly related, or 3) not related.   
 
A serious adverse event (SAE) is defined as an AE that results in death, a life-threatening experience, inpatient 
hospitalization or prolongation of hospitalization (for a patient already hospitalized); persistent or significant 
disability or incapacity; congenital anomaly and/or birth defects; an event that jeopardizes the subject and may 
require medical or surgical treatment to prevent one of the preceding outcomes; or death.  
 
“Unanticipated” means an event that is new or greater than previously known in terms of nature, severity, or 
frequency, given the procedures described in protocol-related documents and the characteristics of the study 
population. Unanticipated problems involving risk (UPRs) are physical, psychological, social, or economic risks 
to subjects or others, including device and drug effects, and anything reportable to the FDA. 
 
All unanticipated SAEs and UPRs must be reported to the joint IRB within 5 business days of awareness, via 
OHSU eIRB UP form. SAEs and UPRs will also be reported to the VA Data Monitoring Committee.  Copies of 
the report documents will be kept in the study regulatory binder.  
 
 
Compensation  
 
Subject compensation will be offered at the end of each study visit, as follows:  
• 
$50 at Baseline, Year 1, Year 2, and Year 3/End of Study visits 
• 
$50 at Baseline Muscle Biopsy visit and at 6 month visit (if in substudy) 
• 
$5 at 6 month; $5 at Year 1 +6 month; and $5 at Year 2 +6 month visits 
 
If subject does not complete study, payment will be prorated. 
 
The payments are reasonable and commensurate with the expected contributions of the subject. Each annual study 
visit is 2-3 hours. Baseline and annual visits include a fasting blood draw, height, weight, blood pressure, 
questionnaires, performance tests, and DXA. The procedures at every 6 month study visits (in between annual 
visits) are only fasting labs and questionnaires for all participants. For the muscle biopsy substudy, there will be a 
separate 1-2 hour visit at baseline and an additional 1 hour added to the 6 month visit.  
 
Payments are fair and appropriate and do not constitute (or appear to constitute) undue pressure or influence on, 
or coercion of, the prospective research subjects to volunteer for or continue participation in the research study. 
 
 
 


IRB #8860 
08/16/2016 
 
 
18 
Statistical considerations  
 
Sample Size and Power 
 
Based on prior observational findings, there 
was a significant difference (2.2%, p< 0.01) 
in the percentage change in appendicular 
lean mass over 3.5 years between 
metformin-treated and untreated men. The 
sample size required for 80% Power to 
detect this difference and reject the null with 
SD=4% and α=0.05 for an unpaired, 2-sided 
t-test is 106 study participants (53 in each 
group). We plan to enroll 300 participants 
and, of those, randomize 120  (60 in each 
group) to allow for potential drop outs due 
to noncompliance and other reasons (7.5-
10%). While 8-11% of participants may 
progress to diabetes yearly,72, 79, 80 all efforts will be made to retain these participants in the study and to include 
them in intent-to-treat analyses. The Power and effect size estimates for gait speed (SD=0.19) for α=0.05 for 60 
participants and 53 participants per group are illustrated in the graph (Fig. 8) and based on our preliminary data in 
older women. The sample size of 60 participants per group will have 73% Power to detect a 0.09 m/s difference in 
gait speed. Within each group, 16 participants will be randomly selected and consented to undergo muscle 
biopsies at baseline and 6 months for secondary analyses of muscle histologic changes with metformin vs. 
placebo treatment. There is an expected change in these histologic measures within 6 months given prior studies 
that detect changes in AMPK activation, ATP concentration and insulin sensitivity prior to 6 months with use of 
metformin.3, 4 Estimated effect sizes of the difference in histologic measures between the two groups after 6 
months are shown below in Table 3. While this study is adequately powered for 3 year outcomes in muscle loss, 
interim analyses may be performed by a Data Monitoring Committee (managed by the VA CSRD), given blinding 
of the primary investigator to the intervention, and the trial may be terminated early if a significant difference in 
the primary outcome between metformin- and placebo-treated groups is noted before 3 years.  
 
Table 3 Estimated detectable difference in muscle histology measures at 80 and 90% Power  
 
SD 
70% 
80% 
90% 
Capillary Density (AU) 
0.35 
0.32 
0.36 
0.42 
IMCL (AU) 
0.50 
0.46 
0.51 
0.59 
SDH (AU) 
0.50 
0.46 
0.51 
0.59 
% Type I Fibers 
24.0 
21.8 
24.6 
28.4 
   SD derived from measures in older insulin-resistant adults36  
 
Statistical Analyses 
 
To assess a difference in the primary outcomes of change in muscle mass and performance between metformin- 
and placebo-treated groups, longitudinal (mixed effects) models will be used to estimate the trajectory of change. 
These models will account for correlation of outcomes within subjects and allows for those with missing follow-
up data to be included in analyses. Four measures of muscle mass and performance (baseline, year 1, 2 and 3) are 
the outcomes in separate models. These will be modeled as a linear change over time, and other functional 
changes (squared, cubic and quadratic) will be evaluated. In the best fitting model, an interaction between time 
and group (metformin vs. placebo) will be included in the model to determine if changes in muscle 
mass/performance differ between the two groups. For the secondary outcomes of muscle histology, the discrete 
change in measures of mitochondrial content, muscle capillary density, percentage of fiber types and 


IRB #8860 
08/16/2016 
 
 
19 
intramyocellular lipid content over 6 months will be calculated. The absolute and percentage changes in these 
measures will serve as the outcome in linear regression models with treatment group as the dichotomous 
exposure. Nonlinear associations between the exposure and outcomes will be evaluated with higher order terms. 
For the analyses of primary and secondary outcomes, potential confounders including demographic information, 
physical activity, diet, lifestyle factors, medical history and medication use are expected to be evenly distributed 
between the groups in a random fashion; nevertheless, we will evaluate for potential confounding by these factors 
and include them in multivariable models if they are significantly associated with our exposure and outcome and 
modify the effect size by >10%. There is no anticipated gender difference in the study outcomes, so the study has 
not been powered to detect a significant interaction by gender. However, the primary and secondary outcomes 
will also be stratified by gender to explore if there is a gender-specific muscle response to metformin. To account 
for missing data, multiple imputation modeling will constructed from participants with complete data. Participants 
with missing data will have those values imputed at each time point multiple times from these models utilizing 
available covariate data and then analyzed according to the principles of multiple imputation.82 Results will be 
reported with and without imputed values. 
 
Data Integrity 
 
For any protected health information (PHI) used for recruitment purposes (i.e. from patient lists for mailing 
recruitment letters), the information will strictly be stored on the secure VA server.  
 
Study forms will contain the subject number, and may contain PHI. All study forms with PHI will be stored 
securely in locked file cabinets in a locked office, or on a secure electronic VA drive. All PHI will be coded prior 
to release for analysis. 
 
There is a transfer of ownership of data from VAPORHCS to OHSU. The data will be managed at OHSU behind 
OHSU firewall. The Oregon Clinical & Translational Research Institute’s (OCTRI) Biomedical Informatics 
Program has the resources and expertise – software engineering, research analysts, and project management – 
available to assist researchers and staff with the storage, retrieval, sharing, and optimal use of biomedical 
information, data, and knowledge for problem solving and decision-making.  The data collection and management 
tools utilized for this grant are REDCap (Research Electronic Data Capture).  The REDCap system provides 
secure, web-based applications that are flexible enough to be used for a variety of types of research, provide an 
intuitive interface for users to enter data and have real time validation rules (with automated data type and range 
checks) at the time of entry. This system offers easy data manipulation with audit trails and reporting for 
reporting, monitoring and querying patient records, and an automated export mechanism to common statistical 
packages (SPSS, SAS, Stata, R/S-Plus). 
 
 
Human Subjects Considerations: 
 
Potential Risks, Protection from Risks, and Risk/Benefit Discussion 
 
Potential Risks  
 
This is an FDA approved drug for an unapproved purpose. However, this drug has been well-studied in and 
sometimes used for people with impaired fasting glucose to prevent progression to diabetes and risks in this 
population are no different than those in the FDA approved population.82, 92 The proposed metformin dose of 850 
mg twice daily has been previously studied safely in a population with pre-diabetes for the prevention of 
diabetes.75 This study is not intended to be used to support a significant change in the labeling or in the 
advertising of metformin, and the same route of administration will be used. With these elements, an IND is not 
necessary. 
Risks of Metformin Use 
 


IRB #8860 
08/16/2016 
 
 
20 
There are risks of gastrointestinal distress, hepatitis, or lactic acidosis. Gastrointestinal distress includes diarrhea, 
nausea, vomiting, flatulence, indigestion, abdominal discomfort/pain, constipation, and distention of the abdomen. 
Gastrointestinal symptoms are common during initiation of metformin therapy. Metformin is known to be 
substantially excreted by the kidney, and the risk of metformin accumulation and lactic acidosis increases with the 
degree of impairment of renal function. Lactic acidosis is an extremely rare, but serious, metabolic complication 
that can occur due to metformin accumulation during treatment with metformin. Occurrence of gastrointestinal 
symptoms could be due to lactic acidosis or other serious disease. A decrease in vitamin B12 levels has been 
observed in clinical trials with metformin.  Signs of lactic acidosis include feeling very weak, tired or 
uncomfortable, unusual muscle pain, trouble breathing, unusual or unexpected stomach discomfort, feeling cold, 
feeling dizzy or lightheaded, and suddenly developing a slow or irregular heartbeat. Contrast (in radiologic) 
studies with iodinated materials can lead to acute alteration of renal function and have been associated with lactic 
acidoses. 
 
There are other rare risks from metformin use. Acute congestive heart failure, acute myocardial infarction and 
other conditions characterized by hypoxemia may occur in the setting of lactic acidosis and may also cause 
prerenal azotemia (a medical condition characterized by abnormally high levels of nitrogen-containing 
compounds in the blood). Alcohol is known to potentiate the effect of metformin on lactate metabolism. Subjects 
should be warned against excessive alcohol intake, acute or chronic. Hypoglycemia does not occur in patients 
receiving metformin alone under usual circumstances of use, but could occur when caloric intake is deficient, 
when strenuous exercise is not compensated by caloric supplementation, during concomitant use with other 
glucose-lowering agents (such as sulfonylureas and insulin) or ethanol, or with substantial alcohol use. When a 
patient stabilized on any diabetic regimen is exposed to stress such as fever, trauma, infection, or surgery, a 
temporary loss of glycemic control may occur. When starting to take metformin, a metallic taste could occur. 
 
With the exclusion criteria and screening tests, these risks (except gastrointestinal intolerance) would be 
minimized. See section “Protection Against Risks of Metformin Use.” 
 
Risk of Diagnosis of Diabetes 
 
From participating in this study, there is a risk that diabetes may be detected. Participants and their primary care 
physician will be notified so that they can seek confirmatory testing and appropriate medical care from their 
primary care physician. 
 
Risks of Study Procedures 
 
Muscle biopsy: The risks of percutaneous muscle biopsy are bleeding and infection (both rare), bruising 
(infrequent) and discomfort (likely). With the use of a local anesthetic, the discomfort of muscle biopsy is 
described as about twice that of intravenous cannulation.  Some residual stiffness in the vastus lateralis can persist 
for 1-2 days (common). An elastic wrap and ice bag are applied post-biopsy to decrease the risk of bruising and 
for an anesthetic effect. Subjects from similar protocol have not experienced any adverse effects from these 
procedures other than a small amount of residual localized soreness at the biopsy site. Additional risk includes 
any unusual reaction to the elastic bandage wrap and ice, i.e. leg numbness which would indicate the elastic 
bandage had been applied too tightly or the ice left on too long; or any skin redness, irritation, and chafing from 
the applied antibiotic ointment and/or steri-strips. 
 
DXA scans: There is risk of radiation exposure. There will be multiple scans during the study which will increase 
exposure to radiation.  
 
Blood collection: There is a risk of pain, bleeding, infection, and bruising. These risks will be minimized by using 
standard blood draw procedures.  
 
Physical Performance: Participants with neuromuscular dysfunction may have a risk of falling during the 


IRB #8860 
08/16/2016 
 
 
21 
performance exams. 
 
Risk of Breach of Confidentiality 
 
There is a potential risk of breach of confidentiality. This will be minimized by coding the data and samples, 
using a unique subject code. The link matching the identifiable information to the subject code will be kept only 
by IRB-approved study personnel designated by the PI, on a password-protected drive behind the secure VA 
firewall or in locked file cabinets in a locked VA office.   Coded samples will be processed in the VAPORHCS 
and OCTRI laboratories, and then transferred (without any identifiers) to Dr. Goodpastor’s lab at Sanford-
Burnham Translational Research Institute for Metabolism and Diabetes for muscle biopsy analysis.  The signed 
consent forms and paper study documents will be kept in a locked file cabinet in a locked office or on a password-
protected drive behind the secure VA firewall.  
 
 
Adequacy of Protection from Risk 
 
The study will be reviewed and approved by the joint VAPORHCS-OHSU IRB. The investigators will adhere to 
the 
Data Monitoring Committee plan described in this protocol. Trained and IRB-approved study personnel will 
perform all data collection and information will be coded with a subject identifier to protect subject 
confidentiality. 
 
Protection Against Risks of Metformin Use 
 
By enrolling participants without liver or kidney dysfunction or congestive heart failure, the risk of lactic acidosis 
or hepatitis on metformin will be decreased. Furthermore, we intend to prevent such risks by screening liver and 
kidney function every year in all participants and quarterly in adults aged 80 or greater and stopping metformin in 
participants who develop evidence of liver/kidney dysfunction for their safety. Per 2016 FDA guidelines, 
metformin use will be discontinued if the eGFR drops below 45 mL/min/1.73 m2.  For those who are hospitalized 
or receive contrast medium, the drug will be held and efforts will be made to recheck kidney function within 48 
hours after discharge/procedure. The study drug will be restarted only if there is no evidence of renal dysfunction. 
For tests or procedures that require fasting, study drug will be stopped 24 hours prior to test or procedure, and 
study drug can be resumed after the participant is allowed to eat again. Participants with B12 deficiency will not 
be enrolled in the study, and B12 levels will be monitored throughout the study. If B12 deficiency is found, the 
participant will discontinue study drug. Initial weight loss due to gastrointestinal symptoms is usually self-limiting 
and continued metformin use is associated with weight stability.4 We have chosen a slow titration up of 
metformin dosage from 850 mg qd to 850 mg bid to allow for minimization of self-limiting gastrointestinal 
distress and plan to carefully monitor weight and dietary intake at each study visit. If participants report 
symptoms of lactic acidosis during screening for adverse events, laboratory confirmation will be sought and study 
drug cessation if found. 
 
Protection Against Risks of Diagnosis - From labs at Screening and study visits, diabetes may be diagnosed 
(defined by fasting plasma glucose≥126 mg/dL). Participants will be notified and referred to their primary care 
physician.  
 
Protection Against Risks of Study Procedures 
 
Muscle biopsy: The exclusion of participants on warfarin, clopidogrel, Aggrenox, ticlopidine, dabigatran or 
anagrelide will minimize serious bleeding with the procedure. We will also ensure that participants are not on 
these medications within a week of their second muscle biopsy. Furthermore, participants will have screening of 
INR, aPTT and platelets and will not be enrolled in the muscle biopsy substudy if there is any abnormality in 
clotting function to avoid serious bleeding. They will also have reassessment of these indices prior to the second 


IRB #8860 
08/16/2016 
 
 
22 
biopsy and will not undergo the second biopsy unless these labs are normal.  Local anesthesia with 2% lidocaine 
will be used to reduce pain associated with the muscle biopsy. To ensure safety with the anesthetic, no 
participants with an allergy to lidocaine will be enrolled. To minimize the risk of infection from muscle biopsy, 
the procedure will be performed using sterile techniques. Appropriate pressure, hemostasis and use of an elastic 
wrap and ice bag after the biopsy will decrease the risk of bruising and aid in an anesthetic effect. Participants will 
be given instructions on care of the biopsy incision site to prevent bleeding and infection after the procedure. 
 
DXA scans: The radiation exposure from one whole body DXA scan is 3.6 (4.6) µSv of radiation which is less 
than the accumulated daily background radiation dose to the whole body.3 Therefore even the amount of radiation 
from four DXA scans is nominal and should not raise the risk for adverse health effects.  
 
Blood collection: A sterile technique will be used prior to venipuncture or intravenous access and pressure will be 
applied for appropriate hemostasis after withdrawal of the needle/catheter.  
 
Physical Performance: To prevent harm from falls during the exam, neuromuscular tests will be administered by 
two staff members if the participant has a history of falling or is so frail that a fall might occur. 
 
Protection Against Risk of Breach of Confidentiality 
 
Subject confidentiality will be maintained in accordance with HIPAA regulations. The recruitment database will 
be protected by current network security in place in the VAPORHCS and all data collected prior to consent will 
be stored at VAPORHCS. After consent is signed, data will be maintained in the OCTRI/OHSU REDCap 
database. In addition, the database will be password protected, and only project personnel approved by Dr. Klein 
will be given access to them.  
 
Potential Benefits of Research 
 
Potential benefits to participants include the detection and potentially earlier treatment of diabetes mellitus. 
Participants in both metformin and placebo groups will benefit from routine lifestyle modification counseling at 
yearly visits for prevention of diabetes. 
 
Others may benefit from a greater understanding of the effect of metformin on muscle mass and function in 
insulin-resistant older adults.  
 
VA Data Monitoring Committee (DMC) 
 
A data monitoring committee will be provided by VA CSR&D. With DMC’s experience in the conduct and 
monitoring of clinical trials, it will assist with ensuring the overall safety of study participants, assessing safety 
and efficacy of the study intervention, monitoring the overall conduct of the clinical trial including 
recommendations on recruitment and retention of participants and improvement of study drug adherence, and 
providing recommendations on stopping or continuation of the trial. SAEs and UPRs will be reported to the 
DMC. For further details, see the “Instructions for the Review of New (uninitiated) Protocols by DMC Members.”   
 
Inclusion of Women and Children 
 
Because sarcopenia is a condition of older age, children will not be enrolled in this study. Women will be 
included in a proportion reflective of their gender distribution at the VAPORHCS. 
 
Criteria for Discontinuation 
 
Subjects will be withdrawn from further study drug administration in the event of any of the following: 
• 
Diagnosis of diabetes 


IRB #8860 
08/16/2016 
 
 
23 
• 
If hospitalized for any reason and after 48 hours of discharge, there is evidence of renal dysfunction 
• 
If they require contrast medium and after 48 hours of procedure, there is evidence of renal dysfunction 
• 
Developed lactic acidosis 
• 
Developed  renal insufficiency at the level indicated by the FDA (eGFR < 45 mL/min/1.73 m2). 
• 
Development of B12 deficiency 
• 
Excessive alcohol intake 
• 
Subject withdraws consent 
• 
Significant deviation from the protocol or eligibility criteria in the opinion of the investigator 
• 
Any other reason that in the opinion of the investigator, would justify removing the subject from the study 
 
There are no pre-defined stopping rules for termination of the study.  
 
 
VA Research: 
 
The following are the components of the study which will be conducted at the VAPORHCS: 
• 
Advertising 
• 
Recruitment 
• 
Study visit activities at Screening, Baseline, half-yearly visits, and annual visits 
o Consent 
o Height, weight 
o Blood pressure 
o Demographics 
o Medical history 
o Study questionnaires 
o Blood draw for labs 
o Physical performance test 
o Concurrent medications 
o AE monitoring 
o Meals 
• 
Lab analysis for fasting glucose, ALT, AST, creatinine, eGFR, B12, platelets, INR and aPTT 
• 
Study drug - storage, dispensing, randomization, tracking, and pill counts 
 
OHSU Research: 
 
The following are the components of the study which will be conducted at OHSU: 
• 
Study visit activities at Baseline, Baseline Muscle Biopsy Visit (if in substudy), 6 Month Visit (if in 
substudy) and annual visits 
o Height, weight 
o Blood pressure 
o Study questionnaires 
o Physical performance tests 
o DXA 
o Muscle biopsy 
o Concurrent medications 
o AE monitoring 
o Meals 
• 
Lab analysis for fasting insulin 
• 
Study database (REDCap) 
• 
Statistical analyses 


IRB #8860 
08/16/2016 
 
 
24 
• 
Biorepository for blood and muscle biopsy samples 
 
 


IRB #8860 
08/16/2016 
 
 
25 
Literature Cited: 
 
1. Lee CG, Boyko EJ, Strotmeyer ES, et al. Association between insulin resistance and lean mass loss 
and fat mass gain in older men without diabetes mellitus. J Am Geriatr Soc 2011;59(7):1217-1224. 
2. Lee CG, Boyko EJ, Barrett-Connor E, et al. Insulin Sensitizers May Attenuate Lean Mass Loss in Older 
Men With Diabetes. Diabetes Care 2011;34(11):2381-2386. 
3. Musi N, Hirshman MF, Nygren J, et al. Metformin increases AMP-activated protein kinase activity in 
skeletal muscle of subjects with type 2 diabetes. Diabetes 2002;51(7):2074-2081. 
4. Tamura Y, Watada H, Sato F, et al. Effects of metformin on peripheral insulin sensitivity and 
intracellular lipid contents in muscle and liver of overweight Japanese subjects. Diabetes Obes Metab 
2008;10(9):733-738. 
5. Baumgartner RN, Koehler KM, Gallagher D, et al. Epidemiology of sarcopenia among the elderly in 
New Mexico. Am J Epidemiol 1998;147(8):755-763. 
6. Janssen I, Heymsfield SB, Ross R. Low relative skeletal muscle mass (sarcopenia) in older persons is 
associated with functional impairment and physical disability. J Am Geriatr Soc 2002;50(5):889-896. 
7. Melton LJ, 3rd, Khosla S, Crowson CS, et al. Epidemiology of sarcopenia. J Am Geriatr Soc 
2000;48(6):625-630. 
8. Rabasa-Lhoret R, Bastard JP, Jan V, et al. Modified quantitative insulin sensitivity check index is better 
correlated to hyperinsulinemic glucose clamp than other fasting-based index of insulin sensitivity in 
different insulin-resistant states. J Clin Endocrinol Metab 2003;88(10):4917-4923. 
9. Festa A, D'Agostino R, Jr., Hanley AJ, et al. Differences in insulin resistance in nondiabetic subjects 
with isolated impaired glucose tolerance or isolated impaired fasting glucose. Diabetes 
2004;53(6):1549-1555. 
10. Tripathy D, Carlsson M, Almgren P, et al. Insulin secretion and insulin sensitivity in relation to glucose 
tolerance: lessons from the Botnia Study. Diabetes 2000;49(6):975-980. 
11. Cowie CC, Rust KF, Ford ES, et al. Full accounting of diabetes and pre-diabetes in the U.S. population 
in 1988-1994 and 2005-2006. Diabetes Care 2009;32(2):287-294. 
12. Park SW, Goodpaster BH, Lee JS, et al. Excessive loss of skeletal muscle mass in older adults with 
type 2 diabetes. Diabetes Care 2009;32(11):1993-1997. 
13. Suwa M, Egashira T, Nakano H, Sasaki H, Kumagai S. Metformin increases the PGC-1alpha protein 
and oxidative enzyme activities possibly via AMPK phosphorylation in skeletal muscle in vivo. J Appl 
Physiol 2006;101(6):1685-1692. 
14. Janssen I, Shepard DS, Katzmarzyk PT, Roubenoff R. The healthcare costs of sarcopenia in the United 
States. J Am Geriatr Soc 2004;52(1):80-85. 
15. Rogers WH, Kazis LE, Miller DR, et al. Comparing the health status of VA and non-VA ambulatory 
patients: the veterans' health and medical outcomes studies. J Ambul Care Manage 2004;27(3):249- 
262. 
16. Selim AJ, Berlowitz DR, Fincke G, et al. The health status of elderly veteran enrollees in the Veterans 
Health Administration. J Am Geriatr Soc 2004;52(8):1271-1276. 
17. Villa VM, Harada ND, Washington D, Damron-Rodriguez J. The health and functional status of US 
veterans aged 65+: implications for VA health programs serving an elderly, diverse veteran population. 
Am J Med Qual 2003;18(3):108-116. 
18. Crimmins EM, Saito Y, Reynolds SL. Further evidence on recent trends in the prevalence and 
incidence of disability among older Americans from two sources: the LSOA and the NHIS. J Gerontol B 
Psychol Sci Soc Sci 1997;52(2):S59-71. 
19. Villa VM, Harada ND, Washington D, Damron-Rodriguez J. Health and functioning among four war eras 
of U.S. veterans: examining the impact of war cohort membership, socioeconomic status, mental 
health, and disease prevalence. Mil Med 2002;167(9):783-789. 
20. Onder G, Penninx BW, Ferrucci L, et al. Measures of physical performance and risk for progressive and 
catastrophic disability: results from the Women's Health and Aging Study. J Gerontol A Biol Sci Med Sci 
2005;60(1):74-79. 
21. Guralnik JM, Ferrucci L, Simonsick EM, Salive ME, Wallace RB. Lower-extremity function in persons 


IRB #8860 
08/16/2016 
 
 
26 
over the age of 70 years as a predictor of subsequent disability. N Engl J Med 1995;332(9):556-561. 
22. Guralnik JM, Ferrucci L, Pieper CF, et al. Lower extremity function and subsequent disability: 
consistency across studies, predictive models, and value of gait speed alone compared with the short 
physical performance battery. J Gerontol A Biol Sci Med Sci 2000;55(4):M221-231. 
23. Goodpaster BH, Park SW, Harris TB, et al. The loss of skeletal muscle strength, mass, and quality in 
older adults: the health, aging and body composition study. J Gerontol A Biol Sci Med Sci 
2006;61(10):1059-1064. 
24. Visser M, Goodpaster BH, Kritchevsky SB, et al. Muscle mass, muscle strength, and muscle fat 
infiltration as predictors of incident mobility limitations in well-functioning older persons. J Gerontol A 
Biol Sci Med Sci 2005;60(3):324-333. 
25. Borst SE. Interventions for sarcopenia and muscle weakness in older people. Age Ageing 
2004;33(6):548-555. 
26. Hayward R. VA QUERI Diabetes Mellitus Report. Washington, D.C.2002. 
27. Miller DR, Safford MM, Pogach LM. Who has diabetes? Best estimates of diabetes prevalence in the 
Department of Veterans Affairs based on computerized patient data. Diabetes Care 2004;27 Suppl 
2:B10-21. 
28. Fink RI, Kolterman OG, Griffin J, Olefsky JM. Mechanisms of insulin resistance in aging. J Clin Invest 
1983;71(6):1523-1535. 
29. Park SW, Goodpaster BH, Strotmeyer ES, et al. Accelerated loss of skeletal muscle strength in older 
adults with type 2 diabetes: the health, aging, and body composition study. Diabetes Care 
2007;30(6):1507-1512. 
30. Barzilay JI, Blaum C, Moore T, et al. Insulin resistance and inflammation as precursors of frailty: the 
Cardiovascular Health Study. Arch Intern Med 2007;167(7):635-641. 
31. Barzilay JI, Cotsonis GA, Walston J, et al. Insulin resistance is associated with decreased quadriceps 
muscle strength in nondiabetic adults aged >or=70 years. Diabetes Care 2009;32(4):736-738. 
32. Lang T, Streeper T, Cawthon P, et al. Sarcopenia: etiology, clinical consequences, intervention, and 
assessment. Osteoporos Int 2010;21(4):543-559. 
33. Lexell J. Human aging, muscle mass, and fiber type composition. J Gerontol A Biol Sci Med Sci 
1995;50 Spec No:11-16. 
34. Goodpaster BH, Carlson CL, Visser M, et al. Attenuation of skeletal muscle and strength in the elderly: 
The Health ABC Study. J Appl Physiol 2001;90(6):2157-2165. 
35. Ryan NA, Zwetsloot KA, Westerkamp LM, et al. Lower skeletal muscle capillarization and VEGF 
expression in aged vs. young men. J Appl Physiol 2006;100(1):178-185. 
36. Crane JD, Devries MC, Safdar A, Hamadeh MJ, Tarnopolsky MA. The effect of aging on human 
skeletal muscle mitochondrial and intramyocellular lipid ultrastructure. J Gerontol A Biol Sci Med Sci 
2010;65(2):119-128. 
37. Mortensen B, Poulsen P, Wegner L, et al. Genetic and metabolic effects on skeletal muscle AMPK in 
young and older twins. Am J Physiol Endocrinol Metab 2009;297(4):E956-964. 
38. Lira VA, Benton CR, Yan Z, Bonen A. PGC-1alpha regulation by exercise training and its influences on 
muscle function and insulin sensitivity. Am J Physiol Endocrinol Metab 2010;299(2):E145-161. 
39. Stephens TJ, Chen ZP, Canny BJ, et al. Progressive increase in human skeletal muscle AMPKalpha2 
activity and ACC phosphorylation during exercise. Am J Physiol Endocrinol Metab 2002;282(3):E688- 
694. 
40. Lovell DI, Cuneo R, Gass GC. Can aerobic training improve muscle strength and power in older men? J 
Aging Phys Act 2010;18(1):14-26. 
41. Dube JJ, Amati F, Stefanovic-Racic M, et al. Exercise-induced alterations in intramyocellular lipids and 
insulin resistance: the athlete's paradox revisited. Am J Physiol Endocrinol Metab 2008;294(5):E882- 
888. 
42. Coggan AR, Spina RJ, King DS, et al. Skeletal muscle adaptations to endurance training in 60- to 70- 
yr-old men and women. J Appl Physiol 1992;72(5):1780-1786. 
43. Menshikova EV, Ritov VB, Fairfull L, et al. Effects of exercise on mitochondrial content and function in 
aging human skeletal muscle. J Gerontol A Biol Sci Med Sci 2006;61(6):534-540. 


IRB #8860 
08/16/2016 
 
 
27 
44. Smith AC, Mullen KL, Junkin KA, et al. Metformin and exercise reduce muscle FAT/CD36 and lipid 
accumulation and blunt the progression of high-fat diet-induced hyperglycemia. Am J Physiol 
Endocrinol Metab 2007;293(1):E172-181. 
45. Smith AC, Bruce CR, Dyck DJ. AMP kinase activation with AICAR simultaneously increases fatty acid 
and glucose oxidation in resting rat soleus muscle. J Physiol 2005;565(Pt 2):537-546. 
46. Lee WJ, Kim M, Park HS, et al. AMPK activation increases fatty acid oxidation in skeletal muscle by 
activating PPARalpha and PGC-1. Biochem Biophys Res Commun 2006;340(1):291-295. 
47. Smith AC, Bruce CR, Dyck DJ. AMP kinase activation with AICAR further increases fatty acid oxidation 
and blunts triacylglycerol hydrolysis in contracting rat soleus muscle. J Physiol 2005;565(Pt 2):547-553. 
48. Kaushik VK, Young ME, Dean DJ, et al. Regulation of fatty acid oxidation and glucose metabolism in 
rat soleus muscle: effects of AICAR. Am J Physiol Endocrinol Metab 2001;281(2):E335-340. 
49. Ouchi N, Shibata R, Walsh K. AMP-activated protein kinase signaling stimulates VEGF expression and 
angiogenesis in skeletal muscle. Circ Res 2005;96(8):838-846. 
50. Zwetsloot KA, Westerkamp LM, Holmes BF, Gavin TP. AMPK regulates basal skeletal muscle 
capillarization and VEGF expression, but is not necessary for the angiogenic response to exercise. J 
Physiol 2008;586(Pt 24):6021-6035. 
51. Winder WW, Holmes BF, Rubink DS, et al. Activation of AMP-activated protein kinase increases 
mitochondrial enzymes in skeletal muscle. J Appl Physiol 2000;88(6):2219-2226. 
52. Jorgensen SB, Treebak JT, Viollet B, et al. Role of AMPKalpha2 in basal, training-, and AICAR-induced 
GLUT4, hexokinase II, and mitochondrial protein expression in mouse muscle. Am J Physiol Endocrinol 
Metab 2007;292(1):E331-339. 
53. Reznick RM, Shulman GI. The role of AMP-activated protein kinase in mitochondrial biogenesis. J 
Physiol 2006;574(Pt 1):33-39. 
54. Irrcher I, Ljubicic V, Kirwan AF, Hood DA. AMP-activated protein kinase-regulated activation of the 
PGC-1alpha promoter in skeletal muscle cells. PLoS One 2008;3(10):e3614. 
55. Suwa M, Nakano H, Kumagai S. Effects of chronic AICAR treatment on fiber composition, enzyme 
activity, UCP3, and PGC-1 in rat muscles. J Appl Physiol 2003;95(3):960-968. 
56. Nakashima K, Yakabe Y. AMPK activation stimulates myofibrillar protein degradation and expression of 
atrophy-related ubiquitin ligases by increasing FOXO transcription factors in C2C12 myotubes. Biosci 
Biotechnol Biochem 2007;71(7):1650-1656. 
57. Krawiec BJ, Nystrom GJ, Frost RA, Jefferson LS, Lang CH. AMP-activated protein kinase agonists 
increase mRNA content of the muscle-specific ubiquitin ligases MAFbx and MuRF1 in C2C12 cells. Am 
J Physiol Endocrinol Metab 2007;292(6):E1555-1567. 
58. Drake JC, Alway SE, Hollander JM, Williamson DL. AICAR treatment for 14 days normalizes 
obesityinduced 
dysregulation of TORC1 signaling and translational capacity in fasted skeletal muscle. Am J 
Physiol Regul Integr Comp Physiol 2010;299(6):R1546-1554. 
59. Gore DC, Wolf SE, Sanford A, Herndon DN, Wolfe RR. Influence of metformin on glucose intolerance 
and muscle catabolism following severe burn injury. Ann Surg 2005;241(2):334-342. 
60. Narkar VA, Downes M, Yu RT, et al. AMPK and PPARdelta agonists are exercise mimetics. Cell 
2008;134(3):405-415. 
61. Steinberg GR, O'Neill HM, Dzamko NL, et al. Whole body deletion of AMP-activated protein kinase 
{beta}2 reduces muscle AMPK activity and exercise capacity. J Biol Chem 2010;285(48):37198-37209. 
62. Orwoll E, Blank JB, Barrett-Connor E, et al. Design and baseline characteristics of the osteoporotic 
fractures in men (MrOS) study--a large observational study of the determinants of fracture in older men. 
Contemp Clin Trials 2005;26(5):569-585. 
63. Cummings SR, Black DM, Nevitt MC, et al. Appendicular bone density and age predict hip fracture in 
women. The Study of Osteoporotic Fractures Research Group. Jama 1990;263(5):665-668. 
64. Lee CG, Boyko EJ, Nielson CM, et al. Mortality risk in older men associated with changes in weight, 
lean mass, and fat mass. J Am Geriatr Soc 2011;59(2):233-240. 
U.S. Census Bureau: State and County QuickFacts. Data derived from Population Estimates, American 
Community Survey, Census of Population and Housing, County Business Ownders, Building Permits, 


IRB #8860 
08/16/2016 
 
 
28 
Consolidated Federal Funds Report, Census of Governments. 2010 
66. Blank JB, Cawthon PM, Carrion-Petersen ML, et al. Overview of recruitment for the osteoporotic 
fractures in men study (MrOS). Contemp Clin Trials 2005;26(5):557-568. 
67. Subar AF, Thompson FE, Kipnis V, et al. Comparative validation of the Block, Willett, and National 
Cancer Institute food frequency questionnaires : the Eating at America's Table Study. Am J Epidemiol 
2001;154(12):1089-1099. 
68. Boucher B, Cotterchio M, Kreiger N, et al. Validity and reliability of the Block98 food-frequency 
questionnaire in a sample of Canadian women. Public Health Nutr 2006;9(1):84-93. 
69. Washburn RA, Smith KW, Jette AM, Janney CA. The Physical Activity Scale for the Elderly (PASE): 
development and evaluation. J Clin Epidemiol 1993;46(2):153-162. 
70. Svarstad BL, Chewning BA, Sleath BL, Claesson C. The Brief Medication Questionnaire: a tool for 
screening patient adherence and barriers to adherence. Patient Educ Couns 1999;37(2):113-124. 
Coletta DK, Sriwijitkamol A, Wajcberg E, et al. Pioglitazone stimulates AMP-activated protein kinase 
signalling and increases the expression of genes involved in adiponectin signalling, mitochondrial 
function and fat oxidation in human skeletal muscle in vivo: a randomised trial. Diabetologia 
2009;52(4):723-732. 
72. Holman RR, Paul SK, Bethel MA, Matthews DR, Neil HA. 10-year follow-up of intensive glucose control 
in type 2 diabetes. N Engl J Med 2008;359(15):1577-1589. 
73. Juurlink DN, Gomes T, Lipscombe LL, et al. Adverse cardiovascular events during treatment with 
pioglitazone and rosiglitazone: population based cohort study. BMJ 2009;339:b2942. 
74. Lipscombe LL, Gomes T, Levesque LE, et al. Thiazolidinediones and cardiovascular outcomes in older 
patients with diabetes. Jama 2007;298(22):2634-2643. 
75. Knowler WC, Barrett-Connor E, Fowler SE, et al. Reduction in the incidence of type 2 diabetes with 
lifestyle intervention or metformin. N Engl J Med 2002;346(6):393-403. 
76. Harkonen R, Harju R, Alaranta H. Accuracy of the Jamar dynamometer. J Hand Ther 1993;6(4):259- 
262. 
77. Amati F, Dube JJ, Carnero EA, et al. Skeletal-Muscle Triglycerides, Diacylglycerols, and Ceramides in 
Insulin Resistance: Another Paradox in Endurance-Trained Athletes? Diabetes 2011 
78. Pruchnic R, Katsiaras A, He J, et al. Exercise training increases intramyocellular lipid and oxidative 
capacity in older adults. Am J Physiol Endocrinol Metab 2004;287(5):E857-862. 
79. Evans WJ, Phinney SD, Young VR. Suction applied to a muscle biopsy maximizes sample size. Med 
Sci Sports Exerc 1982;14(1):101-102. 
80. Chomentowski P, Coen PM, Radikova Z, Goodpaster BH, Toledo FG. Skeletal muscle mitochondria in 
insulin resistance: differences in intermyofibrillar versus subsarcolemmal subpopulations and 
relationship to metabolic flexibility. J Clin Endocrinol Metab 2011;96(2):494-503. 
81. He J, Goodpaster BH, Kelley DE. Effects of weight loss and physical activity on muscle lipid content 
and droplet size. Obes Res 2004;12(5):761-769. 
82. Nathan DM, Davidson MB, DeFronzo RA, et al. Impaired fasting glucose and impaired glucose 
tolerance: implications for care. Diabetes Care 2007;30(3):753-759. 
83. Ratner RE. An update on the Diabetes Prevention Program. Endocr Pract 2006;12 Suppl 1:20-24. 
84. Bajaj M, Baig R, Suraamornkul S, et al. Effects of pioglitazone on intramyocellular fat metabolism in 
patients with type 2 diabetes mellitus. J Clin Endocrinol Metab 2010;95(4):1916-1923. 
85. Groenwold RH, Donders AR, Roes KC, Harrell FE, Jr., Moons KG. Dealing with missing outcome data 
in randomized trials and observational studies. Am J Epidemiol 2012;175(3):210-217. 
86. Goodpaster BH, Kelley DE, Thaete FL, He J, Ross R. Skeletal muscle attenuation determined by 
computed tomography is associated with skeletal muscle lipid content. J Appl Physiol 2000;89(1):104- 
110. 
87. Goodpaster BH, He J, Watkins S, Kelley DE. Skeletal muscle lipid content and insulin resistance: 
evidence for a paradox in endurance-trained athletes. J Clin Endocrinol Metab 2001;86(12):5755-5761. 
88. Lee CG, Carr MC, Murdoch SJ, et al. Adipokines, inflammation, and visceral adiposity across the 
menopausal transition: a prospective study. J Clin Endocrinol Metab 2009;94(4):1104-1110. 
Lee CG, Fujimoto WY, Brunzell JD, et al. Intra-abdominal fat accumulation is greatest at younger ages 


IRB #8860 
08/16/2016 
 
 
29 
in Japanese-American adults. Diabetes Res Clin Pract 2010;89(1):58-64. 
90. National Center for Veterans Analysis and Statistics: Veterans by State, Age Group, Period, Gender 
[internet]. 2007; http://www.va.gov/vetdata/Veteran_Population.asp. 
91. Lewis MK, Blake GM, Fogelman I. Patient dose in dual x-ray absorptiometry. Osteoporos Int 
1994;4(1):11-15. 
92. Diabetes Care 2012 Jan; 35 Suppl 1: S11-63. Standards of medical care in diabetes – 2012.