Muscle-derived Extracellular Vesicles and Their Interactions With Adipocytes in Human Metabolic Dysfunction
Phase: NA
Conditions: Metabolism
Sponsor: Yuan Wen

Document Coversheet 
 
Study Title: Exercise-induced Skeletal Muscle Exosomes Promote Adipocyte Lipolysis 
InsƟtuƟon/Site: 
University of Kentucky 
Document (Approval/Update) Date: 
8/1/2018 
NCT Number: 
NCT04500769 
IRB Number: 
43910 
Coversheet Created: 
12/18/2023 
 

SIGNIFICANCE 
A sedentary lifestyle contributes to or exacerbates numerous diseases that represent urgent public health 
problems, including heart failure, coronary heart disease, type 2 diabetes, breast and colon cancer 1,2. Ding 
and colleagues have conservatively estimated the worldwide cost of inactivity in 2013 to be $53.8 billion 3. It is 
known that exercise confers health benefits by enhancing the function of non-skeletal muscle tissues, but the 
molecular mechanisms underlying these beneficial effects are not fully understood 4. The majority of research 
investigating the systemic benefits of exercise has focused on aerobic exercise, leading to a relative lack of 
information regarding the health benefits of resistance exercise (RE) beyond improvements in muscle function. 
The few studies that have examined the effects of RE on non-muscle tissues suggest that aerobic and 
resistance exercise may confer comparable metabolic benefits 5-9. A better understanding of the relevant 
molecular mechanisms underlying the systemic response to RE will help to generate new therapeutic options 
for patients who have limited ability to exercise, while improving exercise responses in patients who are able.  
In the past two decades, skeletal muscle has emerged as a secretory organ, producing myokines (e.g., irisin, 
GDF11, IL-6, BDNF, others) that may mediate the beneficial effects of exercise and ameliorate the negative 
effects of a sedentary lifestyle 10-12. More recently, exosomes have emerged as potential mediators of inter-
tissue signaling. Exosomes are small (~30–150 nm) membrane-bound vesicles that are secreted following the 
fusion of  multivesicular late endosomes with the plasma membrane 13. Elegant work by Valadi and colleagues 
demonstrated that exosomes contain nucleic acids (including miRNAs) that are delivered to recipient cells 
where they modulate mRNA stability 14. Notably, exosomes are easily internalized by recipient cells, making 
them an attractive vehicle for drug delivery and gene therapy 15,16. Subsequently, numerous groups have 
reported alterations in circulating miRNAs with various pathological conditions 17-19. We and others have shown 
that exosomes are released from cultured rodent and human muscle cells 20-24. Additionally, the muscle cell 
line C2C12-derived exosomes, rich in miR-133a, are capable of down-regulating target genes in NIH-3T3 cells 
25. In humans, exosomes containing miR-133b and miR-181a are released acutely following aerobic exercise 
26. Furthermore, miR-1, a muscle-specific miRNA, was increased in serum following downhill walking in 
humans 27. A fascinating new study by Whitham and coworkers showed that acute aerobic exercise promoted 
the release of exosomes that were preferentially taken up by liver 28. In contrast, our preliminary data show that 
RE induces a rapid systemic release of miR-1 containing exosomes that are preferentially delivered to adipose 
tissue (see Fig. 1-4).  
The primary role of adipose tissue is to store excess energy as lipids, and it is the principal source of serum 
non-esterified fatty acids (NEFA) during times of energy demand (i.e., fasting or exercise). As such, the 
metabolic function of adipose is critical for determining whole body metabolic outcomes. Adipocyte lipolysis is 
driven by catecholamines (norepinephrine and epinephrine), which stimulate β-adrenergic receptors (β-AR; β-
1, β-2, and β-3).  β-AR agonism enhances insulin sensitivity, lean mass, energy expenditure, and 
thermogenesis 29-33. A growing body of work indicates that adipocytes develop catecholamine resistance in the 
context of obesity and insulin resistance 34-41, potentially due to down-regulation of β-AR 35. We and others 
have shown that adipose miRNAs are altered with obesity and insulin resistance 42-44. Moreover, miRNAs in 
adipose have been shown to modulate β-AR signaling in vivo 45 and in vitro 46. These data, along with our 
preliminary data, indicate that miRNA-mediated mRNA processing can improve adipose β-AR sensitivity, 
leading to the exciting hypothesis that exosomal delivery of miRNAs to adipose could be used to ameliorate 
metabolic disease. 
Numerous studies in humans and animals have shown that aerobic exercise is beneficial to adipose tissue 
function and whole-body metabolism 10,11,47-49. Both acute and chronic aerobic exercise enhance adipocyte 
catecholamine sensitivity in humans and animals 50-56.  Although relatively few studies have investigated 
whether adipose adrenergic signaling is affected by RE, it is known that a single bout of RE can increase 
circulating NEFA and resting energy expenditure and decrease respiratory quotient for up to 24 hours 57-61, 
indicative of increased adipocyte lipolysis and muscle fatty acid oxidation. Furthermore, the lipolytic response 
to RE is impaired in obese men 62,63. Using synergist ablation, a model of RE in mice, we show that adipose 
transcriptional responses are exosome-dependent, and that serum exosomes enhance adipocyte 
catecholamine sensitivity and lipolysis for at least 24 hours (Fig. 6). To our knowledge, this is the first 
demonstration of a potential mechanism whereby RE imparts metabolic adaptations in adipose. Since adipose 
metabolic function is crucial for determining whole-body metabolic outcomes, the ability of RE-induced 
exosomes to improve adipose metabolism has significant clinical implications. 
 

Scientific Premise. The premise that RE has a beneficial effect on adipose metabolism is strongly supported 
by the literature and by our preliminary data. We will test the highly novel hypothesis that RE promotes 
adipocyte catecholamine sensitivity via muscle-derived exosomal delivery of miR-1 to adipocytes, thereby 
enhancing β-AR signaling. We will begin by determining if the effect of RE-induced exosomes on adipocyte β-
AR signaling are miR-1 dependent, and the specific mechanism of action of miR-1 in adipocytes. Next we will 
determine whether muscle fiber-derived exosomes specifically promote adipocyte lipolysis through enhanced 
β-AR signaling. Lastly, in order to obtain translational proof-of-principle, we will determine if in vitro 
mechanisms may be operating in humans by measuring serum exosome, muscle and adipose miR-1 levels 
following a single bout of RE, and testing the effects of the isolated exosomes on adipocyte catecholamine 
sensitivity. To our knowledge, we are the first group to mechanistically investigate the capacity of RE to 
enhance adipocyte catecholamine sensitivity. Furthermore, our preliminary data indicate a central role for 
exosomes and miRNAs, representing newly emerging and highly novel mechanisms of intercellular 
communication.  
 
INNOVATION 
The current proposal is highly innovative for the following reasons: 
• 
The proposed mechanism in which RE regulates adipose metabolism through exosomal delivery of the 
muscle-specific miR-1 is completely novel and, to the best of our knowledge, never been described 
before.  
• 
While aerobic exercise has been shown to influence the metabolic flexibility of adipose tissue, the 
finding that RE may also modulate the catecholamine sensitivity of adipose tissue provides a new mode 
of exercise that can be employed to potentially treat obesity; importantly, high-intensity exercise has 
been shown to have improved adherence compared to moderate-intensity training 64,65.  
• 
Establishing the use of skeletal muscle-derived exosomes as a delivery platform to regulate adipose 
tissue metabolism represents a highly unique therapeutic strategy to treat obesity.  
• 
A robust research design employing mouse and human studies to rigorously test our central hypothesis 
both mechanistically and if the proposed muscle-adipose crosstalk translates to humans.   
• 
We have assembled a distinguished team of investigators with complementary expertise in the cellular 
and molecular mechanism regulating skeletal muscle plasticity, exercise physiology, adipose tissue 
biology and metabolism.  
Collectively, these factors come together to produce a highly innovative research proposal that is expected to 
demonstrate a novel mechanism through which RE regulates adipose tissue metabolism, providing clinically 
significant insights into the potential pharmacological use of exosomes and/or miR-1 to augment or mimic the 
beneficial effects of RE on metabolic health and to treat obesity.  
 
PRELIMINARY DATA 
We recently 
reported that 
exosomes 
released from 
activated 
skeletal  muscle 
stem cells 
deliver muscle-
specific miR-206 
to fibroblasts in 
response to 
synergist 
ablation, a well-
accepted model 
of resistance 
exercise (RE) in rodents, that induces robust hypertrophy of the plantaris muscle 24, 66. This discovery inspired 
us to further explore exosome release in response to RE and their impact on putative target tissues. In 
agreement with our previous findings, muscle-specific miR-1 levels were significantly lower by 40% in the 

plantaris muscle after a single day of RE and continued to decrease for 
the next two days (Fig. 1A) 67,68. Given the relatively long half-life (5-9 
days) of miRNAs, this rapid decrease in muscle miR-1 suggested the 
intriguing possibility that miR-1 was being exported out of the myofiber 
by exosomes 69. As shown in Fig. 1B, exosomes isolated from serum of 
mice subjected to 1-3 days of RE had an increase in the abundance of 
miR-1 after 1 and 2 days, returning to baseline by day 3. We took 
advantage of the muscle specificity of miR-1 to determine if exercise-
induced exosomes were being taken up by the liver, brain or adipose 
tissue. Whereas we did not detect a change in miR-1 abundance in the 
brain or liver (data not shown), we did observe ~2.5-fold higher miR-1 
abundance in epididymal adipose after a single day of RE (Fig. 1C). 
Importantly, expression of primary miRNA-1 (both A and B) transcripts 
in adipose did not change in response to RE indicating the increase in 
miR-1 was from an exogenous source and not driven by transcription of 
the miR-1 gene within adipose cells (Fig. 1D).  
To determine if RE-induced exosomes were preferentially taken up by 
adipose tissue, we isolated exosomes from serum of mice that had 
either undergone sham surgery (Control) or synergist ablation for a 
single day (RE). Isolated exosomes were 
fluorescently labeled, reintroduced separately 
into naive mice by tail vein injection with tissue 
collected one hour later. Tissue fluorescence 
was first normalized to amount of tissue (mg) 
and then normalized to Control exosome 
fluorescence. Although most tissues showed a 
relatively modest increase in fluorescence, 
epididymal adipose showed the largest (2.3-
fold) increase in fluorescence following 
injection of RE-induced exosomes (Fig. 2). 
This finding suggests the intriguing possibility 
that RE-induced exosomes may affect many 
tissues throughout the body with adipose being 
a primary target. 
To confirm we had successfully isolated 
exosomes we performed 
Western blot analysis using 
antibodies against established 
markers of exosomes. As 
shown in Fig. 3A, we detected 
TSG101, CD81 and CD63 
proteins only in the exosome 
sample, not in myotubes (MT); 
importantly, we did not detect 
the cellular proteins α-tubulin 
or GAPDH in the exosome 
sample demonstrating the 
specificity of exosome 
isolation protocol. 
Nanoparticle tracking analysis 
showed the isolated extracellular vesicles were within the range (73-139 nm) of exosomes (~30-150 nm) and 
that exosome serum concentration did not change in response to RE (Fig. 3B-C) 70.  
We next determined if the increase in adipose miR-1 abundance after a single day of RE was the result of 
exosome delivery. This was critical because of an alternative scenario in which miR-1 is released into 
circulation by damaged muscle (as the result of RE) and travels to adipose tissue bound to lipoproteins or 

Ago2 71. Mice were treated with vehicle or GW4869 for seven days prior to either sham or synergist ablation 
surgery. GW4869 is an inhibitor of neutral sphingomyelinases which has been shown to effectively block the 
release of exosomes 72. Nanoparticle tracking analysis confirmed GW4869 treatment effectively reduced 
serum exosome concentration by over 90% (Fig. 4A). GW4869 treatment prevented the decrease in skeletal 
muscle miR-1 expression in response to a single day of RE (Fig. 4B) indicating exosome release was 
necessary for the down-regulation 
of skeletal muscle miR-1 
expression. Consistent with this 
finding, GW4869 treatment also 
prevented the increase in exosomal 
miR-1 abundance observed with 
RE and the subsequent increase in  
miR-1 abundance in adipose tissue 
(Fig. 4C-D). Together, these 
findings provide compelling 
evidence that miR-1 is exported 
from skeletal muscle by exosomes 
following RE and exosomal delivery 
of miR-1 is responsible for the 
increase in adipose miR-1 
abundance. 
In an effort to identify how miR-1 might be affecting gene expression 
in adipose tissue, we performed a microarray analysis on adipose 
collected from vehicle or GW4869 treated mice after one day of 
either sham surgery or synergist ablation. Our initial search strategy 
was to identify genes whose expression was changed in response to 
RE in the vehicle group but were unchanged in the GW4869 group 
(relative to their respective sham). Using this strategy, we identified 
the up-regulation of genes involved in adrenergic signaling (β3-AR 
and C/EBPα) and lipolysis (HSL, ATGL, TGL, PLIN1 and FABP2) in 
adipose of vehicle-treated mice following RE; however, following 
GW4869 treatment, the expression of these same genes was either 
unchanged or down-regulated (Fig. 5A). To insure the up-regulation 
of lipolytic genes in adipose was not caused by a decrease in food 
intake, we measured food intake and body weight throughout the 
experimental period. As shown in Fig. 5B-C, food intake and body 
weight remained unchanged and, in particular, for the Vehicle-RE 
group. These microarray results suggest that lipolysis was activated 
following RE through β-adrenergic signaling and prevented when 
exosome release was blocked by GW4869 treatment. 
To determine if RE in fact 
enhances catecholamine 
sensitivity of adipose tissue, we 
measured the ability of 
adipocytes isolated from mice 
subjected to either sham surgery 
(Control) or synergist ablation 
(RE) to release non-esterified 
fatty acids (NEFA) in response to 
100 uM isoproterenol. As shown 
in Fig. 6, RE adipocytes released 
significantly more NEFA into the 
media than did Control 
adipocytes in response to 

isoproterenol. These findings are consistent with the increase in the expression of β3-AR and lipolytic genes 
presented in Fig. 5.  
We next hypothesized that RE-induced exosomal miR-1 was promoting adipose β-adrenergic and lipolytic 
gene expression by targeting a repressor of C/EBPα. Specifically, we developed a working model in which the 
down-regulation of a C/EBPα repressor by miR-1 would lead to an increase in C/EBPα expression. The up-
regulation of C/EBPα would in turn drive β3-AR expression thereby enhancing catecholamine sensitivity of 
adipose and subsequently promoting lipolysis. Microarray analysis of adipose tissue (as described in Fig. 5) for 
five known inhibitors of C/EBPα expression showed ~50% lower expression of AP-2α and CKIP-1 in response 
to RE that was completely blocked by GW4869 treatment (Fig. 7A). qPCR analysis of adipose AP-2α 
expression found a significant 90% decrease in AP-2α expression following RE in the vehicle group that was 
prevented with GW4869 treatment (Fig. 7B). The RNAhybrid program identified a non-conventional miR-1 
binding site within the 3'-UTR of the AP-2α transcript with mis-matches within the seed sequence but extensive 
homology at the 3' region (Fig 7C) 73.  The possibility that this site is functional is not without precedent as 
studies have provided evidence that miRNAs do not necessarily follow the “classical” rules dictated by 
canonical seed matches 74-76. Based on the preliminary data presented in Fig. 6 and 7 we developed a working 
model that describes a novel mechanism whereby the exosomal release of miR-1 in response to RE is 
preferentially taken up by adipose tissue which in turn leads to enhanced catecholamine sensitivity through 
C/EBPα activation of β3-AR by miR-1 repression of AP-2α. 
To more specifically test the ability of 
RE-induced exosomes to alter 
adipose gene expression, we applied 
exosomes isolated from the serum of 
mice subjected to either sham 
surgery (Control) or synergist ablation 
(RE) to 3T3-L1 cells differentiated into 
adipocytes. Although we did not 
observe any significant change in 
gene expression following 24 hr 
incubation, we did see a trend for 
increased miR-1 and C/EBPα 
expression as well as decreased AP-
2α expression (Fig. 8A-B). These 
trends in gene expression are 
consistent with our working model 
and suggest with a larger “n” we will 
likely observe significant changes in 
gene expression. These preliminary data further demonstrate that we have the technical expertise to perform 
the proposed in vitro experiments using isolated exosomes and adipocytes. 
Given that 
there are 
no 
established 
skeletal 
muscle-
specific 
exosomal 
proteins 
that would 
allow for 
the 
isolation of 
muscle-
specific 
exosomes, 
we have 

used miR-1 as a readout of muscle-derived exosomes. To more 
rigorously test our working hypothesis that skeletal muscle-derived 
exosomes alter adipose metabolism, we have developed an in vitro 
model of RE using human myotubes. As shown in Fig. 9A-B, 
electrical stimulation of human myotubes appeared to increase 
myotube size compared to unstimulated myotubes and, 
importantly, resulted in a 5-fold increase in the abundance of miR-1 
in exosomes isolated from the culture media (Fig. 9C). Moreover, 
there was a small decrease (9%) in myotube miR-1 expression with 
electrical stimulation (Fig. 9D). Although we were unable to run 
statistics because of insufficient “n” (n=2), the overall trend in 
myotube size and both exosomal and myotube miR-1 abundance 
follows the same general pattern shown in Fig.1 for RE.     
As an initial effort to test whether or not our working model 
translates into humans, we carried out a pilot study. Young, 
sedentary, adults (n=10, 5 females, 5 males) performed a bout of 
RE (knee extension) to failure. Blood was collected pre- and post-
exercise (0, 30, 60 and 90 minutes). Exosomes were isolated from 
serum and miR-1 abundance quantified by qPCR. Thirty minutes 
post-exercise there was a trend for an increase in exosomal miR-1 abundance. This result suggests that both 
mice and humans respond to RE in a similar manner by releasing miR-1 into the circulation through exosomes. 
Further, this pilot demonstrates we have the necessary expertise and infrastructure to successfully conduct the 
human RE study proposed in Aim 3.  
Collectively, we have provided strong preliminary data to support a novel mechanism in which skeletal muscle, 
in response to RE, regulates adipose metabolism through the exosomal delivery of muscle-specific miR-1. 
Also, our preliminary data clearly demonstrate that we have both intellectual and technical expertise to 
successfully perform the proposed experiments.  
 
APPROACH 
An overall strength of the proposed experimental design is a shared set of techniques which will facilitate the 
comparison of results from the mouse (Aim 1), cell culture (Aim 2) and human subjects (Aim 3). The shared set 
of techniques includes exosome isolation, exosome miRNA loss- and gain-of-function studies, gene expression 
analysis and catecholamine sensitivity assay. Each of these techniques will be briefly described below and 
subsequently referred to as the SOP section. Aim-specific methods will be provided in each aim. 
Standard Operating Procedures (SOP)  
Exosome isolation. Exosomes will be isolated using Exoquick-LP (mouse serum) or ExoQuick-TC (human 
plasma and culture media) according to the manufacturer’s instructions. Briefly, following centrifugation (3000 
g ,15 min) exosome precipitation solution will be added to filtered (0.22 um) supernatant, incubated overnight 
at 4⁰C with ExoQuick solution and then centrifuged at 1,500 g for 5 minutes to remove all traces of fluid. The 
exosome pellet will be resuspended into 25ul of PBS. While the exosome field has yet to reach a consensus 
on the best method for isolating exosomes, ExoQuick has recently been shown to be as good (50-fold > yield), 
if not better, than traditional ultracentrifugation methods 77,78. We will confirm exosome isolation by Western 
blot, nanoparticle tracking analysis (ZetaView) and transmission electron microscopy 78. 
Exosome miRNA loss- and gain-of-function. Exosomes derived from Control or RE serum (~1 × 106 particles) 
will be combined with 20 pmol miR-1 mimic or inhibitor and 10 μl Exo-fect solution and then incubated at 37°C 
for 10 min. Transfected exosomes will be then mixed with 30 μl ExoQuick-TC and incubated at 4°C for 30 min. 
After centrifugation of samples (14,000 rpm for 3 min), the transfected exosomes will be suspended in PBS. 
Gene expression analysis. Total RNA will be isolated from either skeletal muscle or adipose using Direct-zol 
with 0.5 µg of DNase-treated RNA used to make cDNA using the SuperScript™ VILO™ cDNA Synthesis Kit. 
Taqman gene expression assays for the following genes: β3-AR, C/EBP2α, HSL, ATGL, TGL, PLIN1, FABP2, 
FABP4 and AP-2α. miRNAs expression/abundance of muscle, adipose and exosomes will be determined 
using the miRCury RNA isolation kit and LNA™ Universal RT microRNA PCR system.    

Catecholamine sensitivity assay. Lipolysis and anti-lipolysis will be measured in 3T3-L1 and ADHAS cells. 
Cells will be pre-treated for 5 min with adenosine deaminase and then treated with either 10 μM, 100 μM, or 1 
mM isoproterenol, 0.5 nM insulin, both insulin and 100 μM isoproterenol, or DMEM only. The cells will be 
incubated at 37°C for 1 hr with gentle rocking, and the media collected and used for NEFA measurement as 
previously described by us 79.  To correct for cell surface area, cell diameters will be measured via microscopy 
80,81. 
3T3-L1 adipocyte culture. 3T3-L1 (CL-173, ATCC) adipogenic cells will be maintained in DMEM, 10% FBS and 
differentiated into adipocytes as previously described by us 80. Briefly, 3T3-L1 cells will be grown to confluency, 
then placed in differentiation media (DM), composed of DMEM, 10% FBS, 1 μM dexamethasone, 0.5 mM 
isobutylmethylxanthine (IBMX), and 10 μg/ml insulin for four days. Cells will then be maintained in DM without 
dexamethasone and IBMX for an additional 3 days 82. Differentiation to adipocytes will be assessed by Oil Red 
O staining and expression of the adipocyte-specific mRNA FABP4. 
Adult-derived human adipocyte stem cells (ADHASC) culture. Pre-adipocytes were obtained from lipo-aspirate 
from healthy women undergoing liposuction. Lipo-aspirate was digested using collagenase type I (1 g/l KRB 
with 1% BSA) for 1 hr at 37°C with gentle shaking. The stromal-vascular fraction was separated from 
adipocytes by centrifugation, and plated in growth media (DMEM/Hams-F12, 10% fetal bovine serum, and 15 
mmol/l HEPES). Following two passages, cells were cryopreserved. Differentiation, as previously described by 
us 79,82,83; will be induced with DM containing 100 nmol/l insulin, 1.0 μmol/l dexamethasone, 0.25 mmol/l IBMX, 
0.033 mmol/l biotin, 0.017 mmol/l pantothenate, and 1.0 μmol/l rosiglitazone for three days, followed by three 
additional days using DM without IBMX and rosiglitazone. Differentiation to mature adipocytes will be assessed 
by Oil Red O staining and FABP4 gene expression.  
 
Specific Aim 1: Determine if exercise-induced exosomal miR-1 enhances adipocyte adrenergic 
signaling through activation of β3-AR expression.  
Rationale. The rationale for this aim is based on our preliminary data showing that RE-induced exosomes 
containing muscle-specific miR-1 enhance the catecholamine sensitivity of adipocyte. Further, microarray 
analysis identified a potential underlying mechanism for this enhanced catecholamine sensitivity that involves 
C/EBPα activation of β3-AR expression via miR-1 down-regulation of AP-2α, a repressor of C/EBPα. The 
purpose of this aim is to determine if this proposed mechanism is functionally operative by investigating 1) how 
RE-induced exosomes alter adipose metabolism both in vivo and in vitro, 2) the necessity of exosomal miR-1 
in regulating adipose metabolism using miR-1 loss- and gain-of-function studies and 3) validating miR-1 
predicted target gene AP-2α. Given that our preliminary data were generated using male mice, for the 
proposed studies we plan to use both male and female mice to determine if sex has an influence on exosomal 
regulation of adipose catecholamine sensitivity.     
Design. Based on preliminary data presented in Fig. 8A, we performed a power calculation to determine the 
number of mice required per group to detect significant (p < 0.05) difference between sham and synergist 
ablation groups. Male and female C57BL6/J mice (n=12/sex; 4 months of age) will undergo either sham 
(Control) or synergist ablation (RE) surgery which we have used extensively and previously described in detail 
24,66,84-86. The synergist ablation model has been successfully used for over half a century as a resistance 
exercise mimetic in rodents to uncover the molecular and cellular mechanisms regulating skeletal muscle 
hypertrophy 87. One day post-surgery, skeletal muscle (plantaris), epididymal adipose and blood will be 
collected. Skeletal muscle and a portion (100 mg) of adipose tissue will be used to isolate total RNA to quantify 
miR-1 and gene expression by qPCR. Serum will be isolated from blood for exosome isolation as described in 
the SOP section.  
Experiment 1.1. Control and RE exosomes will be incubated with 3T3-L1 adipocytes for 24 hr and then gene 
expression and catecholamine sensitivity measured as describe in the SOP.  
Experiment 1.2. To determine if miR-1 is necessary and sufficient to enhance adipocyte catecholamine 
sensitivity, Control exosomes will be transfected with miR-1 mimic (Control-plus) and RE exosomes with anti-
miR-1 oligonucleotide (RE-minus). These modified exosomes will be incubated with 3T3-L1 adipocytes for 24 
hr and then gene expression and catecholamine sensitivity measured as described in the SOP section.   
Experiment 1.3. Based on our preliminary data, we hypothesize that RE-induced exosomal miR-1 is repressing 
expression of AP-2α, a negative regulator of C/EBPα. To determine if AP-2α is a target gene of miR-1 we will 
generate a luciferase reporter gene harboring the 3'-UTR of AP-2α. We will assess the ability of miR-1 to 

repress reporter gene expression; if so, then we will repeat the experiment with a mutant AP-2α 3'-UTR 
reporter gene in which the predicted miR-1 binding site is mutated as previously described by us 24.  
Experiment 1.4. Microarray analysis of epididymal adipose showed increased expression of genes involved in 
β-adrenergic signaling and lipolysis in response to RE that was blocked by GW4869 treatment (see Fig. 5). 
The purpose of this experiment is to use qPCR to confirm the microarray results and, importantly, determine if 
the observed changes in gene expression and effects of GW4869 treatment are the same in female mice 
(n=12/sex). 
Statistical Analysis. A two-factor ANOVA (treatment X sex) will be used to determine if a significant interaction 
exists between factors for each dependent variable under consideration. If a significant interaction is detected, 
Scheffé post-hoc comparisons will be performed to identify the source of significance with p ≤ .05. 
Expected Outcomes & Alternative Approaches. The preliminary data presented demonstrate that we have the 
necessary expertise to carry out the proposed experiments so we do not anticipate any technical issues. We 
expect to confirm the change in β-adrenergic signaling and lipolytic gene expression and the enhanced 
catecholamine sensitivity of adipocytes exposed to RE-induced exosomes. Moreover, we expect to confirm the 
basis for these exosome-mediated effects is the exosomal delivery of miR-1 to adipocytes. In particular, we 
expect to find that Control exosomes containing the miR-1 mimic will promote the same up-regulation in gene 
expression and enhanced catecholamine sensitivity as do RE exosomes.  By contrast, we anticipate the 
introduction of an anti-miR-1 into RE exosomes will prevent the changes in gene expression and 
catecholamine sensitivity. What is more difficult to predict is whether or not AP-2α is a bona fide target gene of 
miR-1. The miR-1 binding site within the 3'-UTR of the AP-2α transcript does not conform to a “classical” 
miRNA binding site but the predicted thermodynamics of miR-1 binding of ~∆G= -20 is relatively favorable so 
there is a reasonable chance we will see miR-1 regulation of AP-2α expression.   
If we fail to demonstrate miR-1 regulation of AP-2α, yet show that miR-1 is necessary for mediating the effects 
of RE exosomes on adipocytes, then we will explore the possibility that miR-1 targets a different repressor of 
C/EBPα such as Sp1, Shox2, CKIP-1 or G9a. We did not initially pursue these other repressors because the 
change in their respective mRNA expression was not consistent with regulation by miR-1 (see Fig. 7); 
however, there is the possibility that miR-1 is regulating one of these repressors at the level of translation and 
not mRNA stability. Finally, if we do not confirm our working model proposing enhanced catecholamine 
sensitivity, we will begin to explore the possible influence of RE-induced exosomes on the browning of adipose 
tissue. While our microarray analysis showed a striking increase in β-adrenergic signaling and lipolytic gene 
expression, we did detect increased expression of genes known to be involved in browning (Prdm16, PGC-1α, 
UCP-1 and PPARγ), although not as robust. This finding is not surprising given that C/EBPα is known to be 
involved in regulating the conversion of white adipose tissue into brown adipose tissue , as well as many other 
signaling pathways 88. Thus, we will pursue other mechanisms potentially regulated by C/EBPα if the results of 
proposed experiments do not support our working model.     
 
Specific Aim 2: Determine if skeletal muscle fiber-derived exosomes promote adipocyte lipolysis 
through enhanced β-adrenergic signaling.  
Rationale. Based on our miR-1 preliminary data we hypothesize that skeletal muscle-derived exosomes are 
responsible for promoting lipolysis in fat in response to RE; however, the lack of an accepted marker of skeletal 
muscle-specific exosomes prevents identification of the tissue source of serum exosomes. To address this 
limitation, and test our hypothesis, we have developed an in vitro model in which electrical stimulation of 
human primary myotubes promotes an increase in the abundance of exosomal miR-1 to a similar magnitude 
as that observed in vivo with synergist ablation (see Fig. 9). Using this model system, we will be able to 
specifically determine if skeletal muscle-derived exosomes are able to promote adipocyte lipolysis through 
miR-1 activation of β3-AR expression by targeting AP-2α expression. It will also enable detailed profiling of 
miRs in muscle-derived exosomes. An additional strength of this model system is that it will allow us to 
determine if what we have observed in mouse cells translates to human cells. 
Design. Human primary myoblasts will be differentiated into myotubes as previously described by us 89,90. 
Through the Center for Muscle Biology, directed by Dr. Peterson, primary myoblasts have been isolated, 
expanded and characterized from vastus lateralis biopsies obtained from more than 100 healthy volunteers 
(https://www.uky.edu/chs/muscle/bank). We will use 10 distinct primary myoblast lines derived from both male 

(n=5) and female (n=5) donors. After 1 week of differentiation, myotubes will be electrically stimulated for 8 hr 
(12V, 1Hz, 2ms) using an Ionoptix C-Pace Electrical Pulse Stimulator. A total of four 6-well plates can be 
stimulated at a time. Cells will be harvested after an additional 16 hr with no stimulation for total RNA isolation 
to compare with cells cultured for that time period (24 hr) with no stimulation. One well of stimulated and non-
stimulated myotubes will be fixed, visualized with myosin heavy chain immunocytochemistry and imaged for 
measurement of myotube diameter using automated image analysis to quantify the hypertrophic response (see 
Fig. 9). At the time of cell harvest, media will also be collected from both stimulated and non-stimulated 
myotubes for the isolation of exosomes as described in the SOP section.  
Experiment 2.1. The purpose of this experiment is to confirm our preliminary results showing the down-
regulation of miR-1 in myotubes in response to electrical stimulation and if there is a corresponding increase in 
muscle-specific exosomal miR-1 abundance. If this relationship is confirmed, we will next treat myotubes with 
GW4869 to block exosome release to determine if the down-regulation of myotube miR-1 expression with 
electrical stimulation is mediated by the exosomal export of miR-1 as shown in vivo (see Fig. 4). The 
expression/abundance of miR-1 will be determined by qPCR as described in the SOP section.  
Experiment 2.2. Exosomes from non-stimulated and stimulated myotubes will be incubated with primary 
human adipocytes for 24 hr and then gene expression and catecholamine sensitivity measured as described in 
the SOP section. Adult-derived human adipocyte stem cells (ADHASC) were isolated previously from lipo-
aspirates 91. Dr. Kern’s lab has expanded and frozen ADHASC from 6 different individuals available for these 
experiments. We will begin with one line which has been already shown to differentiate very reproducibly and 
determine the effect of exosomes derived from stimulated and non-stimulated myotubes on gene expression 
and catecholamine sensitivity as described in the SOP section.       
Experiment 2.3. The purpose of this experiment will be to determine if muscle-specific exosomal miR-1 is 
necessary and sufficient to enhance adipocyte catecholamine sensitivity. Non-stimulated exosomes will be 
transfected with miR-1 mimic (Control-plus) while stimulated exosomes will be transfected with anti-miR-1 
oligonucleotide (Stim-minus). These modified exosomes will then be incubated with primary human adipocytes 
for 24 hr and gene expression and catecholamine sensitivity measured as described in the SOP section.  
Statistical Analysis. A two-factor ANOVA (treatment X sex) will be used to determine if a significant interaction 
exists between factors for each dependent variable under consideration. If a significant interaction is detected, 
Scheffé post-hoc comparisons will be performed to identify the source of significance with p ≤ .05. 
Expected Outcomes & Alternative Approaches. We do not anticipate any technical issues with the planned 
experiments as we have experience with all of the described techniques. We also do not expect there to be 
any sex difference in the response of myotubes to electrical stimulation in terms of miR-1 expression in both 
myotubes and exosomes as Nielsen and colleagues reported no difference in miR-1 expression between men 
and women 92. Further, we think that blocking exosome release with GW4869 treatment will prevent the down-
regulation of miR-1 as well as the increase in exosomal miR-1 abundance. Finally there is the expectation that 
muscle-derived exosomal miR-1 will effectively enhance the catecholamine sensitivity and lipolysis of 
adipocytes through the up-regulation of β3-AR signaling. Though somewhat minor, one concern we have is 
that myotube-derived exosomes will behave differently than exosomes isolated from serum; specifically, 
myotube-derived exosomes will not effectively deliver their cargo (i.e., miR-1) to adipocytes. To determine if 
this is the case, we will fluorescently label myotube-derived and serum-derived exosomes (see Fig. 2) to 
visualize their fusion kinetics with adipocytes. If we do find myotube-derived exosomes have limited fusion 
capability, we will switch to using primary myoblast cells derived from both male and female mice. We are 
confident these primary cell lines will produce functional exosomes as we have successfully used them to 
model how exosomes released from activated muscle stem cells are necessary for the proper remodeling of 
the extracellular matrix during muscle hypertrophy 24.  
This aim also provides the opportunity to profile the miR content of muscle-specific exosomes. It is reasonable 
to expect that other muscle-specific miRs (myomiRs) are packaged into exosomes and delivered to other 
tissues. For example, muscle-specific miR-133a has been reported to regulate the expression of Prdm16 and 
brown adipogenesis 93.   
 
 
 

Specific Aim 3: Determine if an acute bout of resistance exercise in humans promotes miR-1-mediated 
adipocyte lipolysis.  
Rationale. Our preliminary data in mice suggest that RE enhances adipocyte catecholamine sensitivity through 
a novel mechanism involving the delivery of muscle-specific miR-1 to adipose via exosomes. The in vitro 
analyses in Aim 1 will explore this process mechanistically, with Aim 2 extending the analyses to primary 
human myotubes and adipocytes. The purpose of this aim is to determine if our working model translates to 
humans in vivo. Analyses of serum exosomes, muscle and adipose tissue isolated before and after a bout of 
RE will determine if miR-1 potentially mediates muscle-adipose crosstalk during exercise in humans. The 
ability of isolated exosomes to induce adipocyte lipolysis in vitro by enhancing catecholamine sensitivity 
through CEBPα activation of βAR-3 expression will be assessed.   
Design. Relatively sedentary, generally healthy lean (BMI <25) and obese (BMI >30) men and women between 
18-30 years of age without study contraindications will be recruited (see inclusion/exclusion criteria in Human 
Subjects section). Potential eligibility will be assessed by self-reported height and weight, and by reporting no 
participation in regular (>1 day per week) exercise for at least the past three months. All potential participants 
will arrive fasted to the Center for Clinical and Translational Sciences (CCTS). After giving written, informed 
consent, a detailed medical history form will be filled out to screen for eligibility. Measures of physical activity 
level will be collected before baseline blood, adipose, and muscle samples are taken. We intend to recruit an 
equal number of lean and obese participants (n=40 in each group, 20 men and 20 women). Power analysis 
based on the change we observed in miR-1 abundance in exosomes following a bout of knee extensions in a 
pilot study of 10 individuals, indicated that n=36 is required in each group. We are requesting n=40 in both the 
lean and obese groups based on an expected difference in means between 1 and 1.95 and an expected 
standard deviation of 0.45, with the power set at 0.8 and alpha at 0.05. This will accommodate 10% drop out 
prior to completing all biopsies.     
Following a brief rest period, subjects will perform two lower body RE tasks to failure (knee extension and leg 
press) with blood samples drawn pre- and immediately, 30, 60 and 90 minutes post-exercise. Biopsies of the 
vastus lateralis and abdominal subcutaneous adipose tissue will be obtained pre- and post-exercise (at 
approximately 60 and 90 min post, respectively; see graphic abstract in the Human Subjects section).  
Experiment 3.1. Exosomal, muscle, and adipose miR-1 abundance will be quantified, as well as miR-1A/B 
primary transcripts in muscle and adipose by qPCR as described in the SOP section.   
Experiment 3.2.  Isolated exosomes from the 30 and 60 minute time points will be applied to differentiated 
ADHASC for 24 hr, and lipolysis in vitro and the βAR-3 pathway gene expression assessed as described in the 
SOP section.  Pre-RE exosomes will be transfected with miR-1 mimic (Pre-plus) and 30 minute post-RE 
exosomes with anti-miR-1 oligonucleotide (Post-minus) and compared to mock transfected exosomes from 
those time points. These modified exosomes will be incubated with differentiated adipocytes to parallel Aim 1 
mouse Experiments 1.1 and 1.2. 
Methods. 
Resistance exercise (RE). Following determination of each subject’s one repetition max (1-RM, see Human 
Subjects section for detailed description), subjects will perform two exercises, knee extension and leg press at 
80% of 1-RM, designed to work the quadriceps. Subjects will perform three sets of eight repetitions, with a 90-
120 second rest between sets, with a fourth set performed to failure. RE will be performed on pneumatic 
resistance devices (Keiser Sports Health Equipment, Fresno, CA).   
Muscle biopsy. A muscle biopsy will be obtained from the vastus lateralis at baseline and approximately 1 hr 
post-exercise under local anesthetic (1% Xylocaine HCl, 3cc).  A ¼ inch wide incision will be made and a 
sterile 5mm Berkstrom biopsy needle (Pelomi Industries, Denmark) inserted with suction to obtain a 100-200 
mg muscle sample. Dr. Kern, study physician and co-investigator on this project, has extensive experience 
with this procedure.  It is well-tolerated and volunteers routinely provide multiple samples 83,89,94-96. Muscle is 
snap frozen in liquid nitrogen and stored at -80oC until RNA isolation.    
Adipose Biopsy. An abdominal subcutaneous fat biopsy sample will be obtained from the lower abdominal wall 
prior to and approximately 90 min post exercise. The adipose biopsy procedure is similar to the thigh muscle 
biopsy procedure, except that 1-2 g of adipose tissue is routinely obtained 42,79,94,97,98.  
Human serum analyses. Glucose and insulin will be measured in the University of Kentucky CCTS 
Biochemical Analysis Laboratory 99,100 from baseline serum collected from fasting lean and obese participants 

to calculate HOMA-IR (Glucose*Insulin/405) as a measure of muscle insulin sensitivity. NEFA and free glycerol 
will be measured 81 both at baseline and in serum collected at multiple time points following the resistance 
exercise bout, and used to calculate a simplified “anti-lipolytic index” (NEFA*Insulin), which indicates adipocyte 
insulin sensitivity based on insulin’s ability to suppress lipolysis 101. In humans, fasting serum norepinephrine is 
positively associated with isoproterenol ED50 in isolated adipocytes. Thus, elevated fasting catecholamines 
may be  indicative of decreased adipose tissue catecholamine sensitivity 102. We will also measure serum 
epinephrine and norepinephrine by HPLC (Vanderbilt University Hormone & Analytical Services Core) 103.  
Statistical Analysis. The College of Health Sciences supports the effort of a biostatistician through the Office of 
Research to provide dedicated support to funded investigators. A one-way repeated measures ANCOVA 
adjusting by sex will be used to determine if a significant difference exists between the   groups in response to 
RE for miR-1 and miR-1A/B primary transcripts. If a significant difference is detected, LSD post-hoc 
comparisons will be performed to identify the source of significance with p ≤ .05. The Pearson correlation 
coefficient will be used to determine whether muscle, serum, and adipose miR-1 and miR-1A/B primary 
transcripts are correlated to tissue insulin sensitivity and fasting catecholamines at baseline. All statistical 
analyses will be performed using SAS© 9.4 (SAS institute, Cary NC).  
Expected outcomes and alternative approaches.  We expect that the bout of lower extremity RE will result in 
decreased miR-1 abundance in muscle at 60 minutes, a rapid and transient increase in exosomal miR-1, and a 
subsequent increase in adipose miR-1 abundance at 90 minutes. We do not anticipate changes in miR-1 
primary transcript abundance in adipose tissue which would support our hypothesis that miR-1 is delivered via 
exosomes.  miR-1 is highly expressed in muscle and we acknowledge that a single bout of RE may not 
significantly decrease muscle miR-1 expression, even if it is packaged and released in exosomes. Moreover, 
60 minutes post-exercise may not be the appropriate time point to detect this change. Our pilot time course 
blood sampling study following RE suggested that miR-1 in serum exosomes is highest at 30 minutes and is 
beginning to idecrease by 1 hr post exercise. Thus, we expect delivery to adipose should be detectable at 90 
minutes. We acknowledge that this time point may not enable us to capture any changes in downstream 
targets of miR-1, but will provide proof-of-principle for a large scale clinical exercise trial that would include 
additional post-exercise time points following an acute bout of RE, as well as a training component. 
We expect that lean individuals will be more insulin sensitive than obese individuals, and that obese 
participants will display higher fasting NEFA in the context of higher fasting insulin (due to decreased adipose 
tissue insulin sensitivity). Including both lean and obese individuals will determine if adiposity affects the miR-1 
acute response to RE.  It is possible that release of miR-1 in exosomes from muscle will be lost or blunted in 
the obese cohort. However, we hypothesize that insulin sensitivity (assessed by HOMA-IR and the anti-lipolytic 
index) will be positively correlated to the serum miR-1 excursion following the RE bout in both populations. 
NEFA and glycerol will be measured both pre- and post-RE to determine whether changes in NEFA and 
glycerol from baseline are correlated with change in miR-1 in serum and/or adipose from baseline. We will also 
determine if the induction of adipocyte lipolysis in vitro is different in exosomes isolated from lean compared to 
obese participants following RE. Further, the in vitro studies will provide the opportunity to explore additional 
aspects of adipocyte metabolism that may be regulated by miRs, which will be informed by results of Aims 1 
and 2. Overall, we expect results from this study to inform the design of a resistance exercise training trial in 
obese individuals. Moreover, the proposed studies are expected to provide the fundamental knowledge 
necessary to determine if exosomal miR-1 delivery to adipose tissue has the potential to form the basis for the 
development of a novel therapeutic strategy to treat obesity, particularly in individuals physically unable to 
exercise.    

Table 1 provides our projected timeline for the proposed experiments. As discussed above, the preliminary 
data presented demonstrate that we have the necessary expertise to perform the proposed experiments so we 
do not anticipate any major delays; however, the experiments have been laid out to accommodate unforeseen 
delays such that the proposed experiments and analyses will be completed by the end of Year 5.