Conditions: Obesity Insulin Resistance
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 26
Healthy volunteers: yes

Brief summary:
The overall goal of this project is to identify factors that "protect" some obese adults from becoming insulin resistant. Identifying mechanisms that help protect some obese adults from developing insulin resistance could lead to novel, targeted therapeutic and/or preventative strategies for obese adults who are insulin resistant.

Detailed description:
SCREENING PROCEDURES Participants will undergo a battery of initial screening tests including: a detailed health history and physical examination, a physical activity questionnaire, and a body composition assessment. All women will undergo a urine pregnancy test.

GENERAL STUDY DESIGN To avoid potential confounding hormonal effects, all women will be studied in the early follicular phase of their menstrual cycle. Subjects will be advised of what they should be eating in the 3 days leading up to their hospital visit to maintain their weight. Subjects will also complete a three day food journal before their hospital visit. The day prior to the study trial admission, subjects will need to eat pre-prepared standardized meals for breakfast, lunch, dinner and an evening snack that the study team has given them.

Subjects will be admitted to the Michigan Clinical Research Unit (MCRU) in the morning after a 12-hour, overnight fast. After arrival, subjects will rest quietly in their room for about 30 minutes. The study team will then measure subjects resting metabolic rate for about 20 minutes. After this test, an intravenous (IV) catheter will be placed for infusions and a second IV will be placed in vein of the opposite arm for blood sampling.

After the IV placement, the study team will begin a tracer-labeled glucose, or "sugar," infusion. The study team will then remove a small sample of muscle from the subjects thigh. This muscle biopsy procedure involves numbing a nickel-sized portion of the skin of the thigh with a local anesthetic, making a small incision (1/4 inch), and removing a small piece of muscle (approximately the size of 2-3 grains of rice). The incision will then be closed with a piece of sterile tape. The study team will also obtain a small sample of fat tissue from the area just underneath the skin near the belly button. This procedure involves numbing a small region of skin near the belly button with a local anesthetic, using a needle a small amount of fat tissue is removed from underneath the skin. During the procedure subjects will feel some pinching and pulling, but it should not be very painful. The injury resulting from the muscle and fat tissue biopsies is rather minor. Subjects will likely have some bruising and mild soreness, but it should not prevent them from performing their general daily activities.

Approximately 2.5 hours after admission, the study team will begin the tracer-labeled fat infusion. The tracer-labeled fats the study team are infusing occur naturally in the body. By injecting a slightly greater amount of this substance than what is already in the body the study team will be able to measure the rate of fat breakdown and fat burning. After about 50 minutes of the infusion, three blood samples will be obtained in 5 min intervals.

After, the study team will begin a hyperinsulinemic-euglycemic clamp procedure (a procedure to measure the body's sensitivity to insulin). During this procedure the study team will infuse insulin and glucose through the IV and will collect blood samples every 5 minutes for the duration of the procedure to monitor the subjects blood glucose and blood insulin concentrations. This procedure is designed to last 2 hours. The study team will change the infusion rate of glucose accordingly to ensure that the subjects blood sugar is maintained at a healthy/normal level. The study team will also infuse a small amount of potassium to make sure potassium concentrations remain at healthy/normal levels. About 30 minutes after starting this procedure to measure the body's sensitivity to insulin, the study team will collect another sample of muscle from the thigh and fat tissue from the area just underneath the skin near the belly button. After completing the hyperinsulinemic-euglycemic clamp procedure, subjects will be provided a meal, and the study team will continue to monitor the subjects blood glucose concentration until stable.

When the subjects vital signs are stable, the study team will also measure percent body fat using a Dual Energy X-ray Absorptiometry (DEXA) and intra-abdominal fat by Magnetic resonance imaging (MRI). When the DEXA scan and MRI tests have been completed, subjects will be discharged from the hospital. Depending on scheduling, however, the DEXA scan and MRI tests may be performed on a separate occasion from the study trial admission.

Arms:
arm 1: None

Interventions: intervention 1: hyperinsulinemic-euglycemic clamp
Intervention descriptions: intervention 1: None

Protocol document:
Prot_SAP_000.pdf: 
Study Protocol 
 
Project title: Protection against insulin resistance in obesity (PAIR-Study) 
ClinicalTrials.gov Identifier: NCT02717832 
Principle Investigator: Jeffrey F. Horowitz, Ph.D. 
First posted on ClinicalTrials.gov: March 24, 2016 
Document date (date last approved by IRB): March 21, 2022 
 
 


 
 
 2 
BACKGROUND AND SIGNIFICANCE 
Insulin resistance is a major contributor to the development of several obesity-related diseases. Despite the 
common link between obesity and insulin resistance, as many as one-third of obese adults appear to be 
“protected” against insulin resistance. Our preliminary findings indicate that insulin resistance can vary 
considerably even in a population of obese adults who are very similar in terms of the magnitude and distribution 
of their adiposity. The overall goal of this project is to identify factors that “protect” some obese adults 
from becoming insulin resistant. Identifying mechanisms that help protect some obese adults from developing 
insulin resistance could lead to novel, targeted therapeutic and/or preventative strategies for obese adults who 
are insulin resistant.  
Excessive availability of fatty acids in the circulation (which are largely derived from subcutaneous adipose 
tissue) is one of the key factors underlying insulin resistance in obesity. Based on our preliminary findings, we 
contend that enhanced capacity for triglyceride storage in subcutaneous adipose tissue (thereby sequestering 
fatty acids - and attenuating their release) provides important protection against insulin resistance. This notion 
is not new, however, the specific metabolic events and/or cellular factors within subcutaneous adipose tissue 
that are responsible for more effective triglyceride storage are not known. We hypothesize that obese adults who 
are protected against insulin resistance exhibit modifications in key factors that regulate adipose tissue structure 
and metabolic function (e.g., lipolytic regulation, adipogenic capacity (resulting in more, smaller fat cells), 
angiogenic capacity (enhancing capillarization), and fibrosis (altering extracellular matrix architecture))  
Although we contend that alterations in adipose tissue structure and metabolic function play a key role in 
determining insulin resistance in obesity, other factors obviously contribute as well. For example, because 
skeletal muscle is the predominant site of insulin-mediated glucose uptake, abnormalities in insulin signaling 
events within skeletal muscle also underlie systemic insulin resistance. Protein tyrosine phosphorylation (pTyr) 
plays critical role regulating key biological processes, including insulin signaling.  Using state-of-the-art mass 
spectrometry-based proteomics, we can quantify hundreds of pTyr sites simultaneously, and by doing so we 
anticipate discovering novel pTyr targets that may differentiate insulin sensitive vs. insulin resistant obese adults. 
These findings will help identify new molecular mechanisms underlying insulin resistance, and will provide novel 
targets for preventing and treating type 2 diabetes and other obesity-related diseases. 
In this project, we will measure insulin-mediated glucose uptake in a homogeneous cohort of abdominally obese 
adults, and then stratify them into three distinct groups based on their degree of insulin resistance. Our main 
comparisons will be made between the most- and least-insulin resistant groups.   
SPECIFIC AIM #1: Compare factors regulating triglyceride storage and adipogenesis in subcutaneous adipose 
tissue from insulin-sensitive obese adults compared with a well-matched cohort of insulin resistant subjects.  
Hypothesis 1: Compared with adipose tissue from obese adults who are insulin resistant, adipose tissue from 
those who remain relatively insulin sensitive will have: a) lower abundance and basal activity of lipolytic enzymes 
b) higher abundance and basal activity of esterification enzymes, c) greater anti-lipolytic response to insulin 
(measured in vivo and in vitro), d) greater expression of adipogenic markers (e.g., PPARg and C/EBPa), e) 
elevated adipogenic capacity (measured in vitro), f) higher adipocyte cell density (cells/cm2), g) greater 
angiogenic capacity (measured ex vivo), and h) less adipose tissue fibrosis. 
SPECIFIC AIM #2: Identify other important and novel factors that contribute to the protection against insulin 
resistance in some obese adults. 
Hypothesis 2A: Targeted analyses in adipose tissue and skeletal muscle will reveal that specific markers of: a) 
macrophage infiltration, b) pro-inflammatory cytokine/adipokine abundance, and c) skeletal muscle lipid 
accumulation, as well as d) hepatic lipid accumulation, will be lower in obese adults who are relatively insulin 
sensitive compared with a well-matched cohort of insulin resistant subjects.  
Hypothesis 2B: Unbiased proteomic analysis in skeletal muscle and adipose tissue will reveal novel differences 
between obese adults who are insulin sensitive compared with insulin resistant obese adults 
 


 
 
 3 
RESEARCH DESIGN AND METHODS 
Overall strategy 
• We will recruit a cohort of abdominally obese adults who will be very similar in terms of body mass index (BMI), 
fat mass, fat free mass, waist circumference, and habitual physical activity behavior (sedentary). 
• We will measure insulin sensitivity (hyperinsulinemic-euglycemic clamp) and then stratify the subjects into 3 
groups based on their degree of insulin resistance.  1) “Insulin resistant” [IR], 2) “Mildly insulin resistant” 
[MIR], and 3) “Non-insulin resistant” [NIR]. The “cutoff points” used for stratifying our subjects into these 3 
groups were defined by Stern et al., (25). 
• We will also measure systemic fatty acid kinetics (stable-isotope dilution methods) and we will collect 
subcutaneous adipose tissue biopsy samples.  
• In these adipose tissue samples, we will perform extensive, yet very relevant and integrated analyses of factors 
regulating fatty acid storage and mobilization, adipogenesis, fat cell size, capillarization, angiogenesis, and 
fibrosis. 
• We will also collect skeletal muscle biopsy samples to assess factors in muscle that may also be different in 
insulin resistant [IR] and non-insulin resistant [NIR] obese adults. 
• Several other important phenotypic measures will be performed to help characterize the subjects (e.g., 
subcutaneous and visceral adiposity [MRI analysis], , hepatic lipid accumulation accumulation and degree of 
hepatic fibrosis [MRI analysis], basal metabolic rate, blood lipid profile) 
• Our primary comparisons to assess factors contributing to protection against insulin resistance will be 
performed on the most-insulin resistant [IR] and least-insulin resistant [NIR] groups, but we will also perform 
correlational analyses using the entire subject population. 
 
Subjects 
A total of 60 obese, pre-diabetic women [n=30] and men [n=30] will be recruited for this study. Eligible subjects 
will be homogeneous in terms of the magnitude and distribution of their adiposity (BMI: 30-40 kg/m2; waist 
circumference: 88-98cm for women and 100-110cm for men), age (18-45 years old), and physical activity 
behavior (sedentary). All subjects must be weight stable (±3kg for ≥ 6 months) and they will be screened with a 
detailed health history survey.  Subjects will not be taking medications known to affect lipid or glucose 
metabolism. Anyone with evidence and/or history of cardiovascular or metabolic diseases will be excluded.  All 
women will be pre-menopausal and will have regularly occurring menses. To avoid potential confounding 
hormonal effects, all women will be studied in the early follicular phase of their menstrual cycle. 
Based on past experience, we anticipate that approximately 50% of the subjects who sign an informed consent 
document before undergoing our more involved screening procedures will not participate in this study (largely 
due to being deemed ineligible after screening or because of personal reasons).  Therefore, we anticipate a total 
of 120 subjects will sign our informed consent document.   
 
Inclusion Criteria 
• Body mass index [BMI]: 30-40 kg/m2 
• Age: 18-45 years 
• All women must be pre-menopausal 
• Non-exerciser: no regularly planned exercise/physical activity 
 
Exclusion Criteria 
• Weight instability ≥ ±3kg ≥ 6 months 
• Medications known to affect lipid and/or glucose metabolism 
• Pregnancy or actively breast feeding 
 
Additional preliminary testing will be performed on subjects deemed eligible after initial screening visit 
• Physical activity questionnaire 
• Urine pregnancy test for female subjects 
• Body composition assessment (DEXA) 


 
 
 4 
 
 


 
 
 5 
Detailed clinical study procedures 
Subjects will complete a physical activity questionnaire and a three day food journal before their hospital visit.  
On the morning of the study, subjects will be admitted to the Michigan Clinical Research Unit (MCRU) at 0700h.  
Subjects will arrive at the Michigan Clinical Research Unit (MCRU) in the University of Michigan Medical Center 
in the morning after an overnight fast.  After arrival, subjects will rest quietly in their room for 30 min.  We will 
then measure their resting metabolic rate (RMR) using a canopy-equipped metabolic cart (Vmax metabolic cart; 
Viaysis, Inc.) for 20min.  After RMR measurement, intravenous catheters (IVs) will then be placed in a forearm 
or hand vein of both arms.  Approximately 1.5h after admission to the MCRU, we will begin primed, stable isotope 
infusions of [6,6 d2]glucose (to assess the rates of hepatic glucose production and systemic glucose uptake) and 
[d5]glycerol (to assess whole body lipolytic rate). At the same time we begin the tracer infusions, we will collect 
skeletal muscle (vastus lateralis) and subcutaneous abdominal adipose tissue biopsy samples. Approximately 
2.5h after admission we will begin an infusion of [13C]palmitate (to assess the rate of systemic fatty acid 
mobilization).  Three blood samples will be collected in five minute intervals starting ~50min after beginning the 
[13C]palmitate infusion. Immediately, after collection of these blood samples we will begin a hyperinsulinemic-
euglycemic clamp (insulin infusion rate = 40 mU/m2/min) to assess insulin resistance, as described previously 
by Defronzo, et al. (6). During the clamp 
procedure we will collect small blood samples 
every 5 minutes to assess blood glucose 
concentration and the glucose infusion rate will 
be 
adjusted 
as 
necessary 
to 
maintain 
euglycermia, as described previously (6).  Thirty 
minutes after starting the hyperinsulinemic-
euglycemic clamp procedure we will collect 
another skeletal muscle and adipose tissue 
biopsy samples to acquire a sample in the 
insulin stimulated condition. Finally, we will 
collect 5 blood samples during the final 20 
minutes of the clamp.  Detailed descriptions of these procedures can be found in our previous publications (8, 9, 
16, 23), and blood sampling timeline is provided in Appendix 1 of this document.  
We will also measure percent body fat using a Dual Energy X-ray Absorptiometry [DEXA: Lunar DPX, DEXA 
Scanner] and We also plan to measure hepatic fibrosis, the amount of fat stored in the liver, intra-abdominal 
adiposity (i.e., visceral adiposity), and visceral fat (or intra-abdominal fat) using magnetic resonance imager 
(MRI) with elastography – but depending on scheduling, these tests may be performed on a separate occasion. 
The blood collection during the clinical trial will be conducted in the Michigan Clinical Research Unit (MCRU), 
which is located in room 1111 of the Cardiovascular Center (CVC).  The clinical suite is a research unit, 
specifically designed for performing clinical procedures in human subjects.  The suite contains a padded patient 
treatment table for blood draws, IV placements and biopsies.  A television is mounted on the wall for participant’s 
entertainment during the testing procedures. 
The intravenous (IV) catheter placement and blood draws will be performed by Jeff Wysocki, RN, Dr. Horowitz, 
or a qualified member of Dr. Horowitz’s research team.  Horowitz research staff members, Alison Ludzki, Suzette 
Howton, Benjamin Ryan, and Michael Schleh have completed Phlebotomy training offered through MLearning 
(supervisor: Christine Conrad, MSN, RN, CNS-BC, OCN), and they are trained and qualified to perform 
venipuncture blood draws. 
Dr. Burant will provide clinical oversight/responsibility for this study.  In general, Dr. Burant will not be present 
in the MCRU during the testing, but he will be alerted in advance to when all testing will be occurring, he will be 
available for immediate consultation by phone, and available to come to the MCRU within ~15 min to attend to 
any concerns that may need his direct attention.  In the event Dr. Burant is unavailable/out of town during a 
testing day, arrangements will be made with either Drs. Kraftson, Rothberg, Oral, or Carr to provide direct 
clinical oversight during that particular experiment.  
 


 
 
 6 
SOP for Skeletal Muscle and Subcutaneous Adipose Biopsies 
Screening and pre-visit procedures 
• Anti-coagulant medication (e.g., Coumadin, Rivaroxaban) and Lidocaine allergy/sensitivity are exclusion criteria 
for the biopsy procedure.  
• Aspirin (ASA) and/or other nonsteroidal anti-inflammatory medications (NSAIDS; e.g., Ibuprofen, Naproxen) 
must be avoided for 3 days before the biopsy procedure (Tylenol can be used)  
 
Before performing the tissue biopsies, Dr. Horowitz (or his qualified co-investigator who is performing the 
biopsies) will: 
1) confirm the participant’s identification 
2) thoroughly explain the biopsy procedures to the participant, describing what they may experience 
during the procedures. 
3) confirm the location of the procedures (e.g., left or right thigh for muscle biopsy - and left or right of the 
umbilicus for the adipose tissue biopsy/aspiration 
 
Skeletal Muscle Biopsy Procedure  
• Confirm that the participant does not have any known allergies/sensitivity to local anesthetic (e.g., 
Lidocaine/Bupivacaine) – this should also have been addressed before/during informed consent process.  
• If necessary, shave ~5” x ~5” square above the vastus lateralis  
• Put on surgical gown, mask and bonnet (assistants in procedure room also must wear mask and bonnet)  
• Disinfect skin above the vastus lateralis with betadine  
• Put on sterile gloves and all the following procedures must be performed using aseptic technique  
• Create sterile field on tray top and around the betadine covered area of the participant’s thigh  
• Infiltrate skin and underlying tissue with 2% lidocaine using a 25g x 1.5” needle (~6-10 ml of lidocaine: total 
dose of lidocaine not to exceed 4.5mg/kg)  
• Make an incision above the vastus lateralis with a #10 scalpel (cut skin and try to cut fascia if possible)  
• Apply pressure to incision site with sterile gauze until bleeding stops (or is very light)  
• While pressure is being applied to the thigh, the biopsy assistant attaches a 3-way stopcock to a 60cc syringe  
• One end of sterile pressure tubing is attached to the sterilized biopsy needle (4-5g Bergstrom or UCH biopsy 
needle), and the other end of the tubing is handed to the assistant to attach to their 3-way stopcock/60cc syringe 
set-up for suction.  
• The biopsy needle is inserted into the incision with firm pressure. The biopsy needle is inserted past the fascia, 
in the location/direction of the applied anesthetic  
• Once the biopsy needle is fully inserted, the biopsy assistant pulls back firmly on the plunger of the 60cc syringe 
to apply steady suction. The sample is collected by closure of the biopsy needle – To increase sample yield, the 
biopsy needle can be quickly rotated then opened and closed 3-5 times to obtain a few “snips” of sample before 
removing the biopsy needle from the thigh.  
• The sample is placed on a sterile absorbent pad, and pressure is applied to the biopsy site with sterile gauze  
• The biopsy assistant quickly rinses/cleans the sample with saline and gently dabs the sample on the absorbent 
pad to remove excess liquid. The sample is placed in collection vial - and placed in liquid N2.  
• While assistant is cleaning and storing the sample, manual pressure is applied to the incision site  
• The biopsy procedure can be repeated in the same incision site (or separate preprepared site) if more tissue 
is needed.  
• When the entire biopsy procedure is finished, firm direct pressure is applied to the incision site until bleeding 
stops (~10 minutes)  
• Incision site is cleaned with 70% ethanol and dried with sterile gauze  
• The incision is closed with steri-strips and an overlaying Tegaderm bandage (with absorbent pad)  
• A pressure dressing is applied to the thigh until bedtime the night of the biopsy procedure  
• Participant is instructed to avoid water submersion for 2 days and to keep the bandages on for 5 days.  
• Upon discharge, provide participant with pre-printed post-procedure care instructions with emergency contact 
information. 
 
Subcutaneous Adipose Biopsy Procedure  
• Confirm that the participant does not have any known allergies/sensitivity to local anesthetic (e.g., 
Lidocaine/Bupivacaine) – this should also have been addressed before/during informed consent process.  


 
 
 7 
• Clean the anterior abdominal wall in the periumbilical area with betadine  
• Put on sterile gloves and all the following procedures must be performed using aseptic technique  
• Create sterile field on tray top and around the betadine covered area of the participant’s abdomen  
• The location of the incision will be 5-10cm lateral to the umbilicus – infiltrate the skin and underlying tissue in 
this location with ~1-2ml of 2% lidocaine using a 25g x 1.5” needle. Then, with the lidocaine needle inserted at 
the intended incision site direct the needle at ~20 degree angle with the abdomen and anesthetize a quadrant 
of the abdomen with a 1.5” radius between the intended incision site and the umbilicus (this will anesthetize the 
sampling region). Total lidocaine dose for abdomen = 5-8ml of 2% lidocaine; total dose of lidocaine must not 
exceed 4.5mg/kg – this includes any lidocaine administered for any other procedures during the test day).  
• Make an incision in this anesthetized site (5-10cm lateral to the umbilicus) with #10 or #11 scalpel  
• Apply pressure to incision site with sterile gauze until bleeding stops (or very light)  
• While pressure is being applied to the abdomen, the biopsy assistant attaches a 3- way stopcock to a 60cc 
syringe  
• One end of sterile pressure tubing is attached to the sterilized biopsy needle (4-5g Bergstrom or UCH biopsy 
needle), and the other end of the tubing is handed to the assistant to attach to their 3-way stopcock/60cc syringe 
set-up for suction.  
• For collection of the core tissue sample, insert the biopsy needle (4-5g Bergstrom or UCH biopsy needle) into 
the incision, directed at ~20 degree angle with abdomen. The biopsy needle is inserted ~2cm in the direction of 
the anesthetized area (toward the umbilicus).  
• Once the biopsy needle is inserted ~2cm, suction is applied to the needle (by an assistant) via an attached 
extension tubing and 60cc syringe. The sample is collected by closure of the biopsy needle – and extracted.  
• The sample is placed on a sterile absorbent pad. The biopsy assistant rinses and cleans the sample with saline 
and gently dabs the sample on a on absorbent pad to remove excess liquid  
• The sample is placed in collection vial (typically containing formalin).  
• The biopsy procedure can be repeated in the same incision site if a larger core sample is needed.  
• After completing the core sample collection, more tissue can extracted using aspiration.  
• For the subcutaneous adipose tissue aspiration method, attach a sterilized aspiration needle (e.g., Spirotri 
cannula) to sterile pressure tubing, attached to sterile 60cc syringe with stopcock attached.  
• Insert aspiration needle into incision site (~3cm), retract plunger, and apply constant negative pressure with 
the syringe during the aspiration process.  
• With needle inserted into this incision site, one hand lightly pinches the surface of the biopsy site, while the 
other hand gentle maneuvers the biopsy needle under the skin for 30-60 seconds to extract tissue (tissue will 
accumulate in the syringe)  
• Remove aspiration needle from incision site –  
• The extracted tissue can be removed from the syringe either by ejecting through the tip of the syringe – or by 
removing the syringe plunger and pouring the tissue out. In both cases the tissue should be placed on a sterile 
absorbent pad for the biopsy assistant to clean and process the sample.  
• Aspiration procedure can then be repeated in the same incision, but try to direct the needle in a slightly different 
“track” within the anesthetized area in order to avoid tissue collection within exactly the same area.  
• After completion of all tissue extraction, pressure should be applied to the biopsy site for 5-10 min. • Clean 
biopsy site with alcohol, place steri-strip on site to close the small incision, and tegaderm bandage is then placed 
over the steri-strips.  
• Apply ice to biopsy site for 10-20 min  
• Participant is instructed to keep the bandages on for 5 days.  
• Upon discharge, provide participant with pre-printed post-procedure care instructions with emergency contact 
information.  
 
As an alternative approach, a 1.5in x 16g needle or a “Coleman needle” may be used for tissue aspiration instead 
of the Spirotri cannula, under the same conditions as described above. However, when using these alternative 
needles, more aspiration “passes” may be required to extract the necessary amount of adipose tissue. 
 
 


 
 
 8 
 
Diets for clinical study: Subjects will be advised what they should be eating in the 3 days leading up to their 
hospital visit to maintain energy balance (total daily energy intake will be approximated to be 370 + (21.6* fat 
free mass)] x 1.5), with a macronutrient content of approximately 55% carbohydrate, 30% fat, and 15% protein, 
which represents the macronutrient content of a “typical” western diet (2). On the experimental day, subjects will 
arrive in the overnight fasted state and will not eat anything until completion of the clamp procedure.  
 
Subject stratification 
As noted above, subjects will be stratified into 3 groups based on their degree of insulin resistance, as defined 
by their glucose disposal rate (GDR) measured via hyperinsulinemic/euglycemic clamp during the experiment. 
1) “Insulin resistant” [IR] (GDR < 28µmol/kg fat free mass [FFM]/min),  
2) “Mildly insulin resistant” [MIR] (GDR > 28µmol/kg FFM/min and < 40µmol/kg FFM/min) 
3) “Non-insulin resistant” [NIR] (GDR > 40µmol/kg FFM/min).   
 
The “cutoff points” used to stratify our subjects into these 3 groups were defined by Stern et al., (25). Stern, et 
al., used a statistical approach using hyperinsulinemic-euglycemic clamp data from 2,138 ethnically diverse non-
diabetic subjects (insulin infusion rate = 40 mU/m2/min, which is the same as we propose here).  They defined 
the “optimal insulin resistance cutoff” as being < 28 µmol/kg FFM/min. We will make every effort to closely 
balance subject characteristics across the three study groups (including sex). 
 
Primary phenotype measurements for Specific Aim #1 
During the clinical study outlined above, we will perform several clinical and sub-clinical assessments to help 
characterize our subject population. 
Insulin resistance will be assessed during the hyperinsulinemic-euglycemic clamp. The primary measure of 
insulin resistance will be calculated as the steady-state rate of glucose infusion (GDR) during the 20 min period 
at the end of the clamp. We will also assess hepatic insulin resistance (i.e., magnitude of reduction in hepatic 
glucose production (HGP) in response to insulin) using stable isotope tracer methods, and peripheral tissue 
insulin resistance (i.e., difference between whole-body GDR and HGP). Detailed methods for this procedure, 
including methods used to quantify the tracer-tracee ratio (TTR) for d2-glucose using gas chromatography/mass 
spectrometry (GC/MS) can be found in our recent publication (16). 
In vivo rates of lipolysis and fatty acid mobilization in the systemic circulation We will measure TTR for d5-glycerol 
and 13C-palmitate using the same GC/MS analysis methods we have used previously in many of our studies (4, 
17, 21, 22, 26).  
Resting energy expenditure and fat oxidation rate will be measured using indirect calorimetry (7). 
Plasma substrates and hormone concentrations. Plasma concentrations of triglyceride, fatty acid, glucose and 
insulin will be assessed using commercially available assays kits (Sigma, Wako, and ThermoDMA, Millipore, 
respectively). Plasma epinephrine and norepinephrine will be measured by HPLC with electrochemical detection 
by the pathology lab at the University of Michigan Medical Center.  
Blood lipid profile.  Plasma concentrations of total- and high-density lipoprotein cholesterol (Total-C and HDL-C) 
will be measured using commercially available colorimetric assay kits; Cholesterol E and HDL-Cholesterol E; 
WAKO Life Sciences, Inc.) and plasma low-density lipoprotein (LDL) cholesterol will be calculated as: [LDL-C] = 
[Total-C] – [HDL-C] – [Triglyceride])/5. 
Body composition and visceral adiposity.  Percent body fat will be assessed using a Dual Energy X-ray 
Absorptiometry [DEXA: Lunar DPX, DEXA Scanner].  We also plan to measure hepatic fibrosis, the amount of 
fat stored in the liver and visceral fat (or intra-abdominal fat) using magnetic resonance imager (MRI) with 
elastography as described previously (1).   
 
Liver function. Circulating markers of liver function will be assessed in serum. 
 
Quantifying factors regulating triglyceride storage and adipogenic markers. 


 
 
 9 
Isolation of adipocytes and stromal vascular fraction (SVF) will be performed by collagenase digestion and 
centrifugation methods on fresh adipose tissue biopsy samples as previously described (3, 24). Immunoblot 
analysis to quantify protein abundance of enzymes and other co-factors regulating lipolysis and fatty acid 
esterification will be performed on the purified adipocyte fraction (13, 17). We will quantify the abundance of key 
adipogenic markers in the SVF. Table 1 provides a list of some of the proteins we plan to measure.  We also 
plan to measure mRNA expression of many of these factors using the StepOnePlus real time PCR system (Life 
Technologies) with fluorescent probe-based assays (IDT). 
 
Lipolytic activity in adipose tissue explants and cultured cells (basal, stimulated and insulin inhibited). Lipolytic 
experiments will be performed on fresh adipose tissue samples ex vivo (~50mg; cultured at 5% CO2 in Krebs 
buffer + 2.5% BSA (pH 7.4)), as well as in primary cells differentiated from adipose stromal vascular cells (aSVC) 
derived from these adipose tissue biopsy samples. Lipolysis will be assessed in optimally differentiated aSVC 
(12). The differentiated cells will be washed, serum-starved for 4 hours, and incubated in basal [1 U/mL 
adenosine deaminase + 20 nM N6-(L-2-phenylisopropyl)-adenosine], stimulated (1 mM 8-bromo-cAMP), or 
stimulated + insulin (30 pM, 120 pM, or 600 pM insulin) conditions for 2 hours. Importantly, by performing these 
experiments on fresh tissue and aSVC, we will examine the lipolytic action of existing mature adipocytes, as well 
as the innate lipolytic action of derived pre-adipocytes.  
Esterification activity in adipose tissue samples.  We will measure basal GPAT and DGAT activity in adipose 
tissue lysate samples using 14C-G3P and 14C-palmitoyl-CoA, as we have reported previously (17, 23). 
Adipogenic capacity assays (in vitro). Adipose stromal vascular cells (aSVC) will be derived from fresh adipose 
tissue samples as described above.  Subcultured aSVC from individual subjects will be plated on 24-well plates. 
When confluent, cells will be incubated for 7 days in either: 1) optimal differentiation media (+IBMX and +TZD; 
(12)), 2) suboptimal differentiation media (optimal media without IBMX and TZD), or 3) control media (containing 
15% FBS). Incubation in the suboptimal media will provide information regarding whether the aSVCs from the 
different subject groups are already primed for differentiation, while the optimal media will allow us to compare 
the maximum capacity of the aSVCs to differentiate. After 7 days of incubation in these different media, cells will 
incubate in a maintenance medium (12). Ten days after the induction of differentiation, cells will be harvested for 
qPCR analysis of PPARG and FABP4 gene expression levels.  
Measurement of adipocyte cell size and cell density, and capillary density  
Adipocyte size and number in adipose tissue biopsy samples will be quantified after staining with hematoxylin 
and eosin (H&E) using MetaMorph Image Analysis software (Molecular Devices) (18, 19, 24).  Similarly, We will 
quantify the capillary:adipocyte ratio (#capillaries/cell) and capillary density (capillaries per adipocyte cell area) 
using Rhodamine-labeled GSL to stain for capillaries with standard immunohistochemistry techniques (14).  
 
Angiogenic potential (ex vivo): Quantification of angiogenic potential will be examined ex vivo by incubating 
subcutaneous adipose tissue explants in extracellular matrix components in the presence of pro-angiogenic 
factors as described in detail by Rojas-Rodriguez et al. (20). Under these conditions, the adipose tissue explants 
sprout capillaries into the extracellular matrix environment (i.e., Matrigel).  We will examine capillary “sprouting” 
over 7 days.  Quantification of capillary growth (i.e., number of capillary sprouts, growth area, and percent of 
tissue with growth) will be performed using ImageJ software.  
Extracellular matrix remodeling in adipose tissue. We will perform immunohistochemistry techniques along with 
qPCR analysis on adipose tissue samples (24) to quantify expression of some key factors involved in 
extracellular matrix remodeling in adipose tissue. Weight gain has been reported to increase expression of 
Collagen I, III, and VI, as well as Secreted Protein, Acidic and Rich in Cysteine (SPARC) (11), and we will 
compare the expression of these factors in adipose tissue samples from our insulin resistant and insulin sensitive 
obese subjects. 
 
Unbiased proteomic analysis of the phosphoproteome in skeletal muscle. The untargeted phosphoproteomic 
Table 1. Adipose proteins to measure 
 
LIPOLYSIS 
TRIGLYCERIDE 
SYNTHESIS 
ADIPOGENIC 
MARKERS 
Proteins of interest 
ATGL, CGI-58, HSL, caveolin,  
p-HSL-s565, 563, 660, Perilipin-1 
DGAT, mGPAT, AGPAT-1, PAP 
(LIPIN-1), SCD-1, SREBP-1c 
PPARg, C/EBPa, b, g, FABP4, 
Chemerin, Adiponectin, FAS 


 
 
 10 
analyses will be performed in Dr. Zhengping Yi’s laboratory at Wayne State University (~1h drive from Dr. 
Horowitz’s lab at the University of Michigan).  Dr. Yi will be performing the proteomic analysis on skeletal muscle 
samples collected in this study.  We will bring de-identified muscle samples to Dr. Yi’s laboratory at Wayne state for this 
analysis.  Dr. Yi is an expert in proteomic analysis of skeletal muscle samples. Protein isolation, and in-solution 
trypsin digestion and phosphopeptide enrichment using titanium dioxide (TiO2) will be performed as described 
previously by Dr. Yi (10, 28). Mass spectrometry, data analysis and bioinformatics will all also be performed as 
detailed previously by Dr. Yi (10, 28). We will aliquot muscle protein lysates and use some aliquots to perform 
western blot analysis on the proteins with significant differences between our insulin resistant and insulin 
sensitive subjects. We will assess the proteomic findings in context of the subjects’ muscle fiber composition, 
which we will assess using standard immunohistochemistry methods. 
Statistical analysis 
Student’s t-test will be used to assess statistical significance of our major endpoints between NIR and IR. In 
addition, we will perform linear regression analyses using our entire cohort of subjects (NIR, MIR, and IR) to 
assess relationships between each of our major endpoints and insulin resistance.  Finally, we will also conduct 
multiple regression analysis using our entire cohort to assess the relative contribution of each of our major 
endpoints to the variance of insulin resistance. A P value of £ 0.05 will be considered statistically significant. 
 
For our untargeted analyses, the false discovery rate (FDR) for both proteins and peptides will be set to 0.01. In 
addition, only phosphosites with a localization probability greater than 0.75 will be considered as “identified”, a 
threshold used commonly in phosphoproteome studies. To be used in comparisons between groups, 
phosphorylation sites need to satisfy the following criteria:  1) identified in more than half of the samples; and 2) 
with a fold change (or difference) greater than 1.5 between comparisons. For comparisons between groups for 
both our untargeted and target endpoints, a Student’s t-test will be used to assess statistical significance of our 
major endpoints for our primary comparison between the NIR and IR groups. In addition, we will perform simple 
linear regression analyses to assess relationships between each of our major endpoints and insulin resistance.  
Finally, we will also conduct multiple regression analysis to assess the relative contribution of each of our major 
endpoint measures to the variance of insulin resistance. A P value of £ 0.05 will be considered statistically 
significant. 
Sample size calculations 
Specific Aim #1: The number of subjects necessary to find statistical differences between our insulin resistant 
(IR) and our non-insulin resistant (NIR) groups in major endpoints of this study was calculated based on mean 
and standard deviation values derived from our preliminary studies and our previous publications (18, 24).  The 
sample size calculations (Table 2) indicate that at least 17 
subjects will be required for each group in this study.  Our 
primary comparisons will be between the IR and NIR 
groups, but subjects will also be stratified into the MIR 
(“mildly insulin resistant”) group.  Based on previous work 
(27), we anticipate that our subjects will be stratified 
relatively evenly into our three study groups (i.e., NIR, MIR, and IR), but it is quite possible this may not be the 
case.  Therefore, we plan to enroll a total of 60 subjects into this study in order to end with at least 17 subjects 
in both the IR and NIR groups.   
 
Specific Aim #2: The number of subjects necessary to find statistical differences between IR and NIR groups 
in key endpoints of the studies designed to address Specific Aim #2 was calculated based on mean and standard 
deviation values derived from our preliminary studies, work published in our previous publications (15, 16) and 
from previous work (14).  For Specific Aim #2, we will only 
perform the analyses on samples from subjects in the IR and 
NIR groups. The sample size calculations (Table 3) indicate 
that we will need at least 17 subjects in both groups. For our 
proteomic analyses, we will use the samples collected from 
the number of subjects required for our targeted analyses to 
be statistically significant.  
 
 
Table 2. Sample size calculations for Specific Aim #1 
Table 3. Sample size calculations for Specific Aim #2 


 
 
 11 
APPENDIX 1 - Blood sampling timeline 
The following table provides the blood sampling timeline for the infusion studies performed in the MCRU.  The 
blood volume for the trial is 199.5 mL.   
 
Time 
Sample 
Blood Volume (mL) 
 
8:25am 
EDTA tube, heparin tube, SST tubes 
45 
 
8:30am 
EDTA tube, heparin tube 
12 
  
10:20am 
EDTA tube, heparin tube, glucometer 
9.5 
  
10:25am 
EDTA tube, heparin tube, glucometer 
9.5 
  
10:30am 
EDTA tube, heparin tube, glucometer 
9.5 
 
10:30am 
BEGIN CLAMP 
 
  
10:35am 
Glucometer 
0.5 
  
10:40am 
Glucometer 
0.5 
  
10:45am 
Glucometer 
0.5 
  
10:50am 
EDTA tube, heparin tube, glucometer 
9.5 
  
10:55am 
Glucometer 
0.5 
  
11:00am 
Glucometer 
0.5 
  
11:05am 
Glucometer 
0.5 
  
11:10am 
EDTA tube, heparin tube, glucometer 
9.5 
  
11:15am 
Glucometer 
0.5 
  
11:20am 
Glucometer 
0.5 
  
11:25am 
Glucometer 
0.5 
  
11:30am 
EDTA tube, heparin tube, glucometer 
9.5 
  
11:35am 
Glucometer 
0.5 
  
11:40am 
Glucometer 
0.5 
  
11:45am 
Glucometer 
0.5 
  
11:50am 
EDTA tube, heparin tube, glucometer 
9.5 
  
11:55am 
Glucometer 
0.5 
  
12:00pm 
Glucometer 
0.5 
  
12:05pm 
Glucometer 
0.5 
  
12:10pm 
EDTA tube, heparin tube, glucometer 
12.5 
  
12:15pm 
EDTA tube, heparin tube, glucometer 
12.5 
  
12:20pm 
EDTA tube, heparin tube, glucometer 
12.5 
  
12:25pm 
EDTA tube, heparin tube, glucometer 
12.5 
  
12:30pm 
EDTA tube, heparin tube, glucometer 
12.5 
 
* 
 
Additional glucometer measures if 
clamp takes longer 
6 
 
 
 
 
 
 
Total Volume (mL) 
199.5 
 
 
 
 
 
 
 
 
 


 
 
 12 
Appendix 2 – Infusion rates  
TRACER TYPE 
 
[6,6 d2]-Glucose 
35 umol/kg bolus priming dose 
0.41 umol/kg/min infusion for 4 hours 
[d5]-Glycerol 
1.5 umol/kg bolus priming dose 
0.10 umol/kg/min infusion for 4 hours 
13C-Palmitate 
No priming dose 
0.04 umol/kg/min infusion for 3 hours 
u = “micro”, kg = kg body weight 
 
The tracer glucose and glycerol infusions begin with a bolus “priming” dose (injected over 1-2 min) – followed 
immediately by the steady state infusion at the rates and durations indicated above.  The palmitate has no 
bolus/priming dose. 
 
 
 
INSULIN AND KCl INFUSION RATE  
Time of clamp procedure 
 
0-5 min 
160 mU/m2/min 
5-10min 
80 mU/m2/min 
10-120min 
40 mU/m2/min 
 
KCl INFUSION RATES = 20Meq at 30ml/hr for all protocols 
 
 
 
 
 


 
 
 13 
REFERENCES 
1. 
Abate N, Garg A, Coleman RA, Grundy S, Peshock R. Prediction of total subcutaneous abdominal, 
intraperitoneal, and retroperitoneal adipose tissue masses in men by a single axial magnetic resonance 
imaging slice. Am J Clin Nutr 65, 1997. 
2. 
Bialostosky K, Wright JD, Kennedy-Stephenson J, McDowell M, Johnson CL. Dietary intake of 
macronutrients, micronutrients, and other dietary constituents: United States 1988-94. Vital Health Stat 
11 : 1–158, 2002. 
3. 
Cawthorn WP, Bree AJ, Yao Y, Du B, Hemati N, Martinez-Santibanez G, MacDougald OA. Wnt6, 
Wnt10a and Wnt10b inhibit adipogenesis and stimulate osteoblastogenesis through a β-catenin-
dependent mechanism. Bone 50: 477–489, 2012. 
4. 
Cornford AS, Barkan AL, Hinko A, Horowitz JF. Suppression in growth hormone during overeating 
ameliorates the increase in insulin resistance and cardiovascular disease risk. AJP: Endocrinology and 
Metabolism 303: E1264–72, 2012. 
5. 
Cunningham JJ. Body composition as a determinant of energy expenditure: a synthetic review and a 
proposed general prediction equation. American Journal of Clinical Nutrition 54: 963–969, 1991. 
6. 
DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin 
secretion and resistance. Am J Physiol 237: E214–23, 1979. 
7. 
Frayn K. Calculation of substrate oxidation rates in vivo from gas exchange. J Appl Physiol 55: 628–
634, 1983. 
8. 
Horowitz JF, Coppack SW, Paramore DS, Cryer PE, Zhao G, Klein S. Effect of short-term fasting on 
lipid kinetics in lean and obese women. Am J Physiol 276: E278–84, 1999. 
9. 
Horowitz JF, Kaufman AE, Fox AK, Harber MP. Energy deficit without reducing dietary carbohydrate 
alters resting carbohydrate oxidation and fatty acid availability. Journal of Applied Physiology 98: 1612–
1618, 2005. 
10. 
Højlund K, Bowen BP, Hwang H, Flynn CR, Madireddy L, Geetha T, Langlais P, meyn, Mandarino 
LJ, Yi Z. In vivo phosphoproteome of human skeletal muscle revealed by phosphopeptide enrichment 
and HPLC-ESI-MS/MS. J Proteome Res 8: 4954–4965, 2009. 
11. 
Johannsen DL, Tchoukalova Y, Tam CS, Covington JD, Xie W, Schwarz J-M, Bajpeyi S, Ravussin 
E. Effect of 8 weeks of overfeeding on ectopic fat deposition and insulin sensitivity: testing the “adipose 
tissue expandability” hypothesis. Diabetes Care 37: 2789–2797, 2014. 
12. 
Lee M-J, Fried SK. Optimal protocol for the differentiation and metabolic analysis of human adipose 
stromal cells. Meth Enzymol 538: 49–65, 2014. 
13. 
Li M, Paran C, Wolins NE, Horowitz JF. High muscle lipid content in obesity is not due to enhanced 
activation of key triglyceride esterification enzymes or the suppression of lipolytic proteins. AJP: 
Endocrinology and Metabolism 300: E699–707, 2011. 
14. 
Lumeng CN, DelProposto JB, Westcott DJ, Saltiel AR. Phenotypic switching of adipose tissue 
macrophages with obesity is generated by spatiotemporal differences in macrophage subtypes. 
Diabetes 57: 3239–3246, 2008. 
15. 
Nelson RK, Horowitz JF. Acute exercise ameliorates differences in insulin resistance between 


 
 
 14 
physically active and sedentary overweight adults. 39: 811–818, 2014. 
16. 
Newsom SA, Everett AC, Hinko A, Horowitz JF. A single session of low-intensity exercise is sufficient 
to enhance insulin sensitivity into the next day in obese adults. Diabetes Care 36: 2516–2522, 2013. 
17. 
Newsom SA, Schenk S, Li M, Everett AC, Horowitz JF. High fatty acid availability after exercise alters 
the regulation of muscle lipid metabolism. Metab Clin Exp 60: 852–859, 2011. 
18. 
Parlee SD, MacDougald OA. Quantifying size and number of adipocytes in adipose tissue. In: Methods 
of Adipose Tissue Biology, edited by MacDougald OA. Elsevier; 2014: Elsevier, 2014. 
19. 
Parlee SD, Simon BR, Scheller EL, Alejandro EU, Learman BS, Krishnan V, Bernal-Mizrachi E, 
MacDougald OA. Administration of saccharin to neonatal mice influences body composition of adult 
males and reduces body weight of females. Endocrinology (January 23, 2014). doi: 10.1210/en.2013-
1995. 
20. 
Rojas-Rodriguez R, Gealekman O, Kruse ME, Rosenthal B, Rao K, Min S, Bellve KD, Lifshitz LM, 
Corvera S. Adipose tissue angiogenesis assay. Meth Enzymol 537: 75–91, 2014. 
21. 
Sakharova AA, Horowitz JF, Surya S, Goldenberg N, Harber MP, Symons K, Barkan AL. Role of 
growth hormone in regulating lipolysis, proteolysis, and hepatic glucose production during fasting. 
Journal of Clinical Endocrinology & Metabolism 93: 2755–2759, 2008. 
22. 
Schenk S, Harber MP, Shrivastava CR, Burant CF, Horowitz JF. Improved insulin sensitivity after 
weight loss and exercise training is mediated by a reduction in plasma fatty acid mobilization, not 
enhanced oxidative capacity. J Physiol 587: 4949–4961, 2009. 
23. 
Schenk S, Horowitz JF. Acute exercise increases triglyceride synthesis in skeletal muscle and 
prevents fatty acid-induced insulin resistance. J Clin Invest 117: 1690–1698, 2007. 
24. 
Simon BR, Parlee SD, Learman BS, Mori H, Scheller EL, Cawthorn WP, Ning X, Gallagher K, 
Tyrberg B, Assadi-Porter FM, Evans CR, MacDougald OA. Artificial sweeteners stimulate 
adipogenesis and suppress lipolysis independently of sweet taste receptors. J Biol Chem 288: 32475–
32489, 2013. 
25. 
Stern SE, Williams K, Ferrannini E, DeFronzo RA, Bogardus C, Stern MP. Identification of 
individuals with insulin resistance using routine clinical measurements. Diabetes 54: 333–339, 2005. 
26. 
Surya S, Horowitz JF, Goldenberg N, Sakharova AA, Harber MP, Cornford AS, Symons K, Barkan 
AL. The pattern of growth hormone delivery to peripheral tissues determines insulin-like growth factor-1 
and lipolytic responses in obese subjects. J Clin Endocrinol Metab 94: 2828–2834, 2009. 
27. 
Wildman RP, Muntner P, Reynolds K, McGinn AP, Rajpathak S, Wylie-Rosett J, Sowers MR. The 
obese without cardiometabolic risk factor clustering and the normal weight with cardiometabolic risk 
factor clustering: prevalence and correlates of 2 phenotypes among the US population (NHANES 1999-
2004). Arch Intern Med 168: 1617–1624, 2008. 
28. 
Zhang X, Qi Y, Yi Z. Quantitative Phosphoproteomics Reveals Novel Phosphorylation Events in Insulin 
Signaling Regulated by Protein Phosphatase 1 Regulatory Subunit 12A. J Proteomics (June 24, 2014). 
doi: 10.1016/j.jprot.2014.06.010.