Clinical trial NCT02833415
Conditions: Obesity, Visceral
Keywords: Visceral Obesity Adipose Diabetes
Sponsor: University of Texas Southwestern Medical Center
Start date: 2016-03-01; Completion date: 2018-11-01
Enrollment: 40 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This study is a clinical study to investigate the gluconeogenesis pathway related to visceral adipose tissue (VAT) in obese individuals without type 2 diabetes and the effects of empagliflozin (EMPA) on glucose homeostasis in viscerally-obese individuals using functional studies of glycerol metabolism in hepatic gluconeogenesis using a well-validated nuclear magnetic resonance (NMR) spectroscopy platform.

Detailed description:
Diabetes mellitus type II is the consequence of insulin resistance and pancreatic beta cell failure resulting from a variety of metabolic insults, one of which is excess body adiposity/obesity. In the diabetic individual, hepatic gluconeogenesis may go uninhibited due to failure of the body's normal feedback mechanisms to appropriately incorporate glucose into cells via insulin signaling, leading to excess gluconeogenesis and hyperglycemia. The substrate for this excess glucose derives from multiple sources in the liver including dietary glycerol, adipose-derived glycerol from lipolysis, and substrates from the citric acid cycle. In the normal state, lipolysis is maintained at a steady state in equilibrium between stored dietary triglycerides and free fatty acids. However, in situations of triglyceride excess (e.g. in the obese state), lipolysis may become overactive resulting in increased free fatty acids and adipose-derived glycerol. This excess glycerol drives hepatic gluconeogenesis and is incorporated into glucose and released into the blood, leading to hyperglycemia, and ultimately diabetes and its clinical sequelae.

A popular hypothesis linking visceral fat with excess gluconeogenesis is delivery of glycerol arising from mesenteric triglyceride turnover directly into the portal circulation and to the liver. Glycerol is a primary substrate for gluconeogenesis in the liver. Under normal conditions, hepatic gluconeogenesis begins from glycerol ingested in the diet which is converted to glycerol-3-phosphate and subsequently dihydroxyacetone phosphate (DHAP) in the liver. DHAP is converted to fructose-1,6-bisphosphate which undergoes a series of reactions to become a single 6-carbon glucose molecule. Adipocytes contribute glycerol to hepatic gluconeogenesis through lipolysis of triglyceride stores. Although glycerol-gluconeogenesis has been extensively studied in animals, the traditional reliance on radioactive tracers makes translation to humans difficult for many reasons. We aim to use new techniques to explore the mechanisms behind altered glucose metabolism related to excess visceral adiposity in obese adults by quantifying the relative contributions of varying substrates to liver-derived glucose. One such method uses 13C3 labeled glycerol to trace the incorporation of glycerol from dietary sources to hepatic gluconeogenesis. This technology utilizes nuclear magnetic resonance (NMR) spectroscopy, a technique that does not require ionizing radiation and has been extensively validated, to analyze the NMR spectra of plasma glucose and quantify the "percent enrichment" of the circulating glucose molecules with labeled glycerol. In turn, differences in enrichment reflect variability in hepatic glucose metabolism as it relates to the contribution of glycerol from visceral adipose tissue to gluconeogenesis.

The rationale of this project is to utilize existing technology to investigate the impact of excess visceral adiposity on glycerol metabolism in hepatic gluconeogenesis in obese adults without diabetes and to explore the effects of treatment with EMPA on visceral adiposity related glucose homeostasis.

Arms: arm 1: Empagliflozin 10 mg by mouth daily for 3 months. arm 2: Placebo one tablet daily for 3 months
Interventions: intervention 1: [U-13C3] glycerol intervention 2: Empagliflozin intervention 3: Placebo (for Empagliflozin)
Intervention descriptions: intervention 1: Ingestion of \[U-13C3\] glycerol based on human's body weight such as (50 mg/kg body weight). intervention 2: Active drug intervention 3: Placebo tablet manufactured to mimic EMPA 10 mg tablet.

Protocol documents:
Prot_SAP_000.pdf: 
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Page 1 of 22 
 
Title: 
Visceral Adiposity and Diabetes: Translating Form to Function 
Using Imaging 
 
 
 
 
Principal Investigator: 
Ian J. Neeland, MD 
 
 
 
 
 
Funding Sponsor: 
National Institutes of Health 
National Institute of Diabetes and Digestive and Kidney Diseases 
Bethesda, MD 20892 
Phone:  301-496-3583 
 
 
 
 
IND Number:  N/A 
 
 
 
 
Version 8: October 13, 2017 
 
 
 
 
The University of Texas Southwestern Medical Center at Dallas 
Institutional Review Board 
Protocol (Investigator Initiated Study) 


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1. Introduction and Purpose:  
One out of three adults in the US is obese and forecasts predict that >50% of the US population will be 
obese by 2030.1  Although obesity is associated with increased risk for diabetes, many obese individuals 
remain free of metabolic disease.2  One factor that contributes to the heterogeneity of risk among obese 
individuals is visceral adipose tissue (VAT) distribution.3  Excess VAT in obese adults is associated with 
insulin resistance, atherogenic dyslipidemia, and hepatic steatosis.4  We have previously shown that 
higher VAT mass is associated with a significant increase in the risk of developing diabetes and that 
this effect is independent of body mass index (BMI).5  One hypothesis linking excess VAT to the 
development of diabetes is that VAT alters glucose homeostasis by contributing excess glycerol from 
overactive lipolysis to stimulate hepatic gluconeogenesis.6,7  However, this hypothesis has yet to be 
tested in human subjects.  Furthermore, the effect of Empagliflozin (EMPA) on glucose homeostasis 
associated with visceral adiposity has not been explored previously.   
 
My long term goal is to understand how VAT contributes to diabetes risk and to identify strategies to 
reduce the metabolic consequences of excess VAT.   
My objectives here are to:  
 
Elucidate the mechanistic relationship between VAT and abnormal glucose homeostasis. 
 
Determine the effect of EMPA, a sodium glucose co-transporter 2 inhibitor shown to reduce 
surrogate markers and direct imaging-based assessments of VAT,8,9 on glucose homeostasis in 
obese adults with differing VAT.   
I will test the central hypothesis that visceral adiposity plays a key role in the pathogenesis of diabetes 
and that EMPA may positively impact glucose homeostasis in viscerally-obese individuals using 
functional studies of glycerol metabolism in hepatic gluconeogenesis using a well-validated nuclear 
magnetic resonance (NMR) spectroscopy platform.  The rationale of this project is to utilize emerging 
technology to investigate potential mechanisms underlying diabetes risk related to excess VAT and 
explore the potential beneficial effects of EMPA on these pathways.  I will achieve the scientific 
objectives of this application by pursuing the following two specific aims: 
 
1. Elucidate the relationship between VAT mass and glucose homeostasis by measuring 
enrichment of glycerol in blood glucose by NMR spectroscopy.  Here we will measure the 
differential enrichment of exogenous 13C3-labeled glycerol vs. adipose-derived glycerol in blood glucose 
among non-diabetic obese adults with high vs. low VAT using a single venous blood sample. 
Hypothesis 1: Participants with high VAT will have greater adipose contribution of glycerol and therefore 
lower 13C3 glycerol enrichment compared with age, sex, and BMI-matched participants with low VAT.  
2. Determine the effect of EMPA on glucose homeostasis in non-diabetic obese adults with 
differing VAT mass.  In this aim we will measure changes in the enrichment of 13C3 glycerol in response 
to treatment with EMPA or placebo among age, sex, and BMI-matched participants stratified by high 
vs. low VAT. 
Hypothesis 2: 13C3 glycerol enrichment will increase from baseline (signifying less contribution from 
adipose tissue) among EMPA-treated participants with high VAT, with no change in EMPA-treated low 
VAT or placebo-treated participants. 
 
2. Background: 
 
 
Diabetes mellitus type II is the consequence of insulin resistance and pancreatic beta cell failure 
resulting from a variety of metabolic insults, one of which is excess body adiposity/obesity.  In the 


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diabetic individual, hepatic gluconeogenesis may go uninhibited due to failure of the body’s normal 
feedback mechanisms to appropriately incorporate glucose into cells via insulin signaling, leading to 
excess gluconeogenesis and hyperglycemia. The substrate for this excess glucose derives from 
multiple sources in the liver including dietary glycerol, adipose-derived glycerol from lipolysis, and 
substrates from the citric acid cycle.  In the normal state, lipolysis is maintained at a steady state in 
equilibrium between stored dietary triglycerides and free fatty acids.  However, in situations of 
triglyceride excess (e.g. in the obese state), lipolysis may become overactive resulting in increased free 
fatty acids and adipose-derived glycerol.  This excess glycerol drives hepatic gluconeogenesis and is 
incorporated into glucose and released into the blood, leading to hyperglycemia,  and ultimately 
diabetes and its clinical sequelae. 
 
A popular hypothesis linking visceral fat with excess gluconeogenesis is delivery of glycerol arising from 
mesenteric triglyceride turnover directly into the portal circulation and to the liver.  Glycerol is a primary 
substrate for gluconeogenesis in the liver.  Under normal conditions, hepatic gluconeogenesis begins 
from glycerol ingested in the diet which is converted to glycerol-3-phosphate and subsequently 
dihydroxyacetone phosphate (DHAP) in the liver.  DHAP is converted to fructose-1,6-bisphosphate 
which undergoes a series of reactions to become a single 6-carbon glucose molecule.  Adipocytes 
contribute glycerol to hepatic gluconeogenesis through lipolysis of triglyceride stores (Figure 1).  
Although glycerol-gluconeogenesis has been extensively studied in animals, the traditional reliance on 
radioactive tracers makes translation to humans difficult for many reasons.  We aim to use new 
techniques to explore the mechanisms behind altered glucose metabolism related to excess visceral 
adiposity in obese adults by quantifying the relative contributions of varying substrates to liver-derived 
glucose.  One such method uses 13C3 labeled glycerol to trace the incorporation of glycerol from dietary 
sources to hepatic gluconeogenesis.  This technology utilizes nuclear magnetic resonance (NMR) 
spectroscopy, a technique that does not require ionizing radiation and has been extensively validated, 
to analyze the NMR spectra of plasma glucose and quantify the “percent enrichment” of the circulating 
glucose molecules with labeled glycerol.  In turn, differences in enrichment reflect variability in hepatic 
glucose metabolism as it relates to the contribution of glycerol from visceral adipose tissue to 
gluconeogenesis.  
 
The rationale of this project is to utilize existing technology to investigate the impact of excess visceral 
adiposity on glycerol metabolism in hepatic gluconeogenesis in obese adults without diabetes and to 
explore the effects of treatment with EMPA on visceral adiposity related glucose homeostasis.  Our 
hypothesis is that among individuals with excess visceral adipose tissue, high lipolytic turnover results 
in excess adipose-derived glycerol shunting to the liver and hyperactive gluconeogenesis.  This 
phenomenon will be reflected in a relatively low enrichment of dietary (labeled) glycerol when analyzed 
by NMR spectroscopy when compared with obese individuals with low visceral adiposity. We also 
hypothesize that 13C3 glycerol enrichment will increase from baseline (signifying less contribution from 
adipose tissue) among EMPA-treated participants with high VAT, with no change in EMPA-treated low 
VAT or placebo-treated participants. 
 
3. Concise Summary of Project: 
Study Population: 
Obese (BMI ≥ 30 kg/m2) participants who are referred, or enrolled in the DHS, without prevalent type 2 
diabetes who have previously undergone VAT imaging by MRI33 or DXA34 or will have an MRI as part 
of this study will be recruited.  Participants with high VAT will be selected and matched (age, sex, BMI) 
with an equal number of low VAT participants, defined by the highest and lowest age-and-sex stratified 
decile of VAT in the study population.  Individuals with prevalent dyslipidemia or liver disease that could 
influence 13C3 glycerol enrichment will be excluded.  Recruitment is highly likely to be successful given 
that >450 DHS participants have been recruited to participate in additional mechanistic studies to date.    
 
Exposure variables: 


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For the DHS subjects visceral adipose tissue (intraperitoneal + retroperitoneal) was measured by a 
1.5-T MRI system (Intera, Philips Medical Systems, Best, The Netherlands) between 2000 and 2002 
using a prospectively designed and validated method of fat mass prediction from a single MRI slice at 
the L2-L3 inter-vertebral level (Figure 2).  Single slice measurement of visceral adipose tissue mass at 
this inter-vertebral level has been shown to be highly concordant with total abdominal fat mass 
measured at all inter-vertebral levels (R2=85-96%).  Multiple studies have been published 
demonstrating the validity of this method of visceral fat quantification.  Body composition was 
measured using dual x-ray absorptiometry (DXA, Delphi W scanner, Hologic Inc, Bedford, MA and 
Discovery software [version 12.2]) and proprietary software will be used to calculate visceral adipose 
tissue mass from the DXA data. For referral subjects, an MRI prior to glycerol administration will 
measure abdominal visceral adipose tissue (VAT). These participants will undergo a neck-to-knee 
scan on a 3T Philips Ingenia MRI scanner lasting approximately 6 minutes. No intravenous contrast 
will be administered during the study. The scan will be performed in the AIRC by expert technologists 
trained in the conduct of clinical research. The de-identified, HIPAA-compliant images will then be 
interpreted offline by expert readers blinded to the participant’s study assignment and a report will be 
generated for use in the data analysis. 
 
Body composition will be measured using height, weight, waist circumference, hip circumference; 
body fat percentage will be calculated using both skinfold thickness at 7 different anatomical sites and 
bio-impedence analysis. 
 
Plasma blood glucose (mg/dL) measured by YSI glucose analyzer to determine glycemic status prior 
to study intervention.   
 
Blood samples will also be used to measure hemaglobin A1C, adipocytokines, markers of 
inflammation, plasma insulin, alanine, catecholamines, and nonesterified free fatty acids.   
 
Intervention: 
[U-13C3] glycerol 50 mg/kg (obtained from Cambridge Isotopes, Andover, MA) will be given to 
participants to drink.  It is non-radioactive and non-toxic.  There is no known risk to drinking glycerol.  
Ingestion of this item is similar to drinking a commericial sugar sweetened beverage.  If a participant 
cannot tolerate the drink initially, they will be given a second attempt at ingestion.  If after two attempts 
they cannot ingest the entire item, they will be removed from the study and a replacement participant 
will be recruited. 
 
Aim 2 will be an extension of Aim 1 in which all participants from Aim 1 will receive empagloflozin study 
drug or placebo for 12 weeks and then return for repeat testing.  Participants stratified by VAT (high vs. 
low) will be randomized to either treatment or placebo using a random number generator (0-100, 
http://www.random.org) followed by the creation of numbered envelopes; ≤median will be assigned to 
treatment and >median will be assigned to placebo.  The minimum approved dose of empagliflozin (10 
mg tablet daily by mouth taken in the morning with or without food) will be used in Aim 2 of this study; 
this dose was chosen to minimize the chances of adverse events (specifically hypoglycemia, 
dehydration, and urinary tract infections) while still maintaining efficacy in clinical trials and preliminary 
data.  We expect that this dose will be well tolerated, with the most common side effects being urinary 
tract infection or yeast infection (in women).  
 
Outcome variables:  


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[U-13C3] glycerol enrichment in plasma blood glucose over time will be measured by nuclear magnetic 
resonance spectroscopy.  
 
 
 
4. Study Procedures: 
Study procedures are outlined below.  Study procedures will be performed at baseline and then 
repeated after 3 months of study drug administration.  There will also be two (2) interval visits during 
which study participants will meet with the study coordinator to review study drug adherence, obtain 
the next month’s supply of study drug, and perform surveillance for any adverse effects. 
 
Study drug will be either empagliflozin (10 mg once daily by mouth) or matching placebo (once daily 
by mouth). Both active drug and placebo will be obtained from the manufacturer (Boehringer-
Ingelheim) and will be administered by study staff (blinded to treatment allocation) in oral tablet form.  
Participants will randomized to either active drug or placebo using a random number generator (0-
100, http://www.random.org); ≤median will be assigned to emagliflozin and >median will be assigned 
to placebo.     
 
Study Procedures Visits 1 and 4 
1. Arrive at AIRC 
2. Review study procedures 
3. Sign written informed consent 
4. Urine pregnancy test to verify non-pregnant status (female only) 
5. Measurement of height, weight, body composition, heart rate, blood pressure, and temperature. 
6. Possible neck-to-knee MRI 
7. IV insertion by AIRC nurse for blood draws 
8. Baseline blood draw. 
9. Ingestion of [U-13C3] glycerol (50 mg/kg body weight)  
10. Blood draws: +30, +60, +90, +120, +150, +180 min. Each blood draw will be 41 ml for a total of 
312 ml (includes 25 ml waste) 
11. IV discontinued 
12. Lunch and discharge from AIRC.  Study participation completed.  
Each study visit 1 and 4 will be approximately 5 hours.   
 
Study Procedures Visits 2 and 3 
 
1. Arrive at AIRC 
 
2. Return study drug pill bottle and review study drug adherence 
 
3. Assess for any interval adverse effects and document as necessary 
 
4. Dispense study drug to be used in the next study visit interval (1 month) 
 
5. Discharge from the AIRC. 
 


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Study participants do not need to be fasting for Visits 2 or 3 and no blood will be drawn at these 
interval visits. They are for study drug adherence/monitoring and drug dispensing only.  Each study 
visit 2 and 3 will be approximately 30-60 minutes. 
 
After the ingestion of the glycerol and during the blood draws, volunteers will be made comfortable in 
the AIRC procedure room.  Conditions that would result in the subject exiting the study prior to the 
expected completion date include withdrawl of informed consent, intolerance of the glycerol drink, 
intolerance of study procedures (IV insertion, blood draws), or meeting exclusion criteria that were not 
previously known through telephone screening. Research subjects will not be responsible for any 
costs of the study.   
 
  
 
5. Sub-Study Procedures: N/A 
 
6. Criteria for Inclusion of Subjects: 
      - Obese, defined as BMI ≥ 30 kg/m2, at both time of abdominal fat imaging and at study entry. 
      - Ages 30-65 
      - No prevalent diagnosis of type 2 diabetes mellitus, either at the time of abdominal fat imaging or     
        at study entry.   
- 
Previous abdominal fat quantification by magnetic resonance imaging in the Dallas Heart Study 
or possible neck-to-knee MRI for VAT measurement may be performed. 
   
7. Criteria for Exclusion of Subjects: 
      - Pregnant or breastfeeding 
      - Incarcerated 
      - Chronic kidney or liver disease 
      - History of frequent (>2/year) urinary tract infections 
 
      - Non-obese either at time of abdominal fat imaging or at present. 
      - Greater than 10% change in body weight (kg) between time of abdominal fat imaging and  
         present.  
      - Has donated blood within last 6 weeks 
      - Cannot give informed consent, understand the protocol, or tolerate any aspect of the protocol 
- 
If undergoing MRI, persons with metal implants contraindicated for 3Tesla MRI exams will be 
excluded. Severe claustrophobia will also be assessed prior to an MRI exam. 
 
The inclusion/exclusion criteria above were chosen because: 
- 
Subjects must be obese and non-diabetic to meet the study objectives and test the study 
hypothesis. 
- 
Pregnant or breastfeeding women, incarcerated individuals, and children/minors are excluded 
because these are special populations with higher risk and exclusion of these participants 
minimizes risk of the study. 
- 
Chronic kidney or liver disease can possibly affect the outcome measurement in the study and 
confound the results.  Additionally, for Aim 3, empagliflozin should be avoided in persons with 
chronic kidney or liver disease, so participants with these disorders will be excluded to minimize 
risks to human subjects. 


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- 
Since empagliflozin can increase risk for urinary tract infections, participants with a history of 
frequent urinary tract infections will be excluded to minimize risk to human subjects. 
 
8. Sources of Research Material: 
 
The research materials that will be used for this proposal include demographic records and data 
(age, sex, weight, height, body mass index, abdominal MRI and dual x-ray absorptiometry imaging 
data currently collected and maintained in the Dallas Heart Study database), blood samples, and 
urine samples (females capable of pregnancy only).  The blood and urine specimens will be taken 
at the time of study procedures and processed immediately.  No samples will be stored.  The 
database records that will be utilized include age, sex, race, cardiometabolic risk factor status, and 
data generated from blood tests and imaging phenotyping as part of the DHS study protocol.  
 
Prospective data to be obtained include current age, weight, height, waist circumference, hip 
circumference, calculated body fat percentage using both skinfold thickness at 7 different 
anatomical sites and bio-impedance analysis, a brief medical history, blood samples, and a urine 
pregnancy test (females only). 
 
For Aim 2, the FDA-approved drug, empagliflozin 10 mg by mouth daily, will be administered to 
those participants randomized to drug treatment.  A matching placebo will be administered to 
participants not randomized to empagliflozin treatment. Both active drug and placebo will be 
obtained from the manufacturer (Boehringer-Ingelheim). 
 
No member of the study team will have access to individually identifiable private information about 
human subjects in the DHS. The DHS study coordinators have access to this information but are 
not part of the study team.  
 
Every study participant was assigned a randomly generated subject ID that is not linked to 
identifiable information. All data generated from a study participant is labeled with only their unique 
subject ID, including blood specimens and database records. The database records are kept on 
password protected servers in the DHS research center and managed by the study coordinators 
and statistical team. Investigators must have approved and authorized proposal to gain access to 
these data and all data transfers are managed by the database and statistical core. All analyses for 
this project will be conducted on site by the DHS statistical core. Measurements made during this 
study will be linked to participants in the database by the subject ID and merged into the database 
by the DHS statistical core.  
 
9. Recruitment Methods and Consenting Process: 
Obese (BMI ≥ 30 kg/m2) participants who are referred, or enrolled in the DHS, without prevalent type 
2 diabetes who have previously undergone VAT imaging by MRI or DXA will be recruited.  
Participants with high VAT will be selected and matched (age, sex, BMI) with an equal number of low 
VAT participants, defined by the highest and lowest age-and-sex stratified decile of VAT in the study 
population.  Individuals with prevalent dyslipidemia or liver disease that could influence 13-C3 glycerol 
enrichment will be excluded.  Recruitment is highly likely to be successful given that >450 DHS 
participants have been recruited to participate in additional mechanistic studies to date.   All 
participants will be volunteers and not recruited from a clinical practice. Both genders as well as 
minorities will be actively recruited for participation in this study.  Participants will be recruited for a 
visit via telephone by the primary investigator (IN).  A confidential telephone translation service will be 
used if the participant is a non-English speaker.  All attempts to ascertain inclusion/exclusion criteria 
will be made prior to the visit.  Subjects will participate after having the purpose and benefits of the 
study explained in detail by a research nurse.  In addition, a detailed list of potential adverse reactions 
will be reviewed as part of informed consent.  These participants will be assured that the lack of 
participation in the study will in no way affect their ability to obtain medical care at UTSW.  


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Documentation of permission to participate in this study will be obtained using a UTSW IRB approved 
written informed consent form.  No waiver of informed consent will be requested. Participants will be 
paid an incentive to participate in the research. Parking tickets from the Clements Imaging Building 
valet service will be validated.   
 
A partial waiver of HIPPA authorization for screening purposes will be requested.  This is a waiver 
used only for collection of initial screening data to determine eligibility and/or recruit potential research 
subjects. Authorization by the subject will be obtained at the time of consent.  This is justified because 
it is not practical to obtain a signed HIPPA Authorization form prior to review of the records of potential 
participants because review of records is required to screen for a participant's eligibility for study 
participation. Over 3000 HIPPA Authorization forms (all participants in phase 3 of the Dallas Heart 
Study) would need to be completed prior to screening of records to determine the eligibility of study 
participants if records cannot be reviewed for eligibility purposes beforehand. This is not practical and 
the study could not be effectively conducted in this manner.  Inclusion and exclusion criteria could not 
be determined without access to the PHI.  This would also not be possible with the use of de-identified 
information. 
 
10. Potential Risks: 
 
Adverse Reaction to Empagliflozin 
Known adverse effects of empagliflozin include hypoglycemia (0.4%), dehydration (0.3%), impaired 
renal function (change in eGFR -1.0 mL/min/1.73 m2 compared placebo), and an increased risk of 
urinary tract infection (9.3%) or vaginal yeast infection (5.4%).  The most common adverse reactions 
associated with empagliflozin were urinary tract infections and vaginal yeast infections. 
 
Risks of IV Insertion and Blood Drawing  
Participants may experience a small amount of pain in the arm when the needle is inserted into the 
vein.  Risks associated with drawing blood from the arm include minimal discomfort and/or bruising.  
Infection, excess bleeding, clotting, and/or fainting also are possible, although unlikely.  Participants will 
have less than 1½ cups of blood collected because they are in this research study. 
 
13C Labeled Glycerol  
Labeled glycerol is non-radioactive and non-toxic and there is no known risk to drinking labeled glycerol. 
The glycerol will taste sweet and will be mixed with a glass of drinking water. 
 
MRI 
The subject’s experience during this procedure is identical to what they would experience during a 
clinical MRI (magnetic resonance imaging).  MRI involves the use of a magnet and radio frequency 
energy.  Therefore, patients who have implanted metal devices, such as pacemakers, certain aneurysm 
clips, or metal in the eye will be excluded.  If subjects have any potential for metal in the body, the 
technologist or investigators should be informed before entering the magnet room. Because of the 
strong magnetic field associated with the scanner, it is rare, but possible, that a metallic object could fly 
through the air toward the scanner and hit the subject.  To reduce this risk, everyone near the magnet 
will remove all metal from his or her clothing or pockets when in the scanning environment. There are 
no known risks or adverse effects resulting directly from exposure to magnetic fields and radio frequency 
energy used in this study. Some people feel claustrophobic in the MR scanner. If the subject is unable 
to tolerate being in the scanner, the scan can be stopped at any time. In addition, the MR scanner 
produces tapping sounds during operation, which may reach very loud levels. To minimize any 
discomfort from this noise, the subject will be provided with disposable earplugs and/or headphones 
that suppress external noise levels but do not eliminate voice communication with the scanner operator. 
In some cases, it is possible that the subject might experience neurostimulation effects, such as muscle 
twitches and tingling sensations, due to the rapid switching of magnetic field gradients used in these 
examinations. There are no known risks associated with these effects. 
 


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All subjects will be carefully evaluated by trained personnel prior to entering the MR scanner to exclude 
metal objects in or on their body. Any female of childbearing age will be screened and subjected to a 
pregnancy test, if indicated, prior to any MR procedure. 
 
The MR scans in this study are designed for research, not for medical purposes.  They are not useful 
for finding problems or diseases.  In the event that a researcher discovers a possible abnormality, the 
subject will be informed. The subject will then consult with their primary care physician to discuss further 
tests and/or follow-up and treatment. 
 
 
Risks to an Embryo, Fetus or Breast-fed Infant 
Females: If females are part of this study while pregnant or breast-feeding an infant, it is possible that 
they may expose the unborn child or infant to risks.  For that reason, pregnant and breast-feeding 
females cannot participate in the study.  If participants can become pregnant, a pregnancy test will be 
done from a urine sample, and it must be negative before they can be a part of this study.  If they do 
become pregnant during this study, they must tell the researchers immediately, and they will be 
removed from the study. 
 
Loss of Confidentiality 
There is a potential risk of loss of confidentiality any time information is collected.  Every effort will be 
made to keep participant information confidential; however, this cannot be guaranteed. 
 
Other Risks 
There may be other side effects that are unknown at this time. If participants are concerned about other, 
unknown side effects, they will discuss this with the researchers. 
 
11. Subject Safety and Data Monitoring: 
Type of Research Data or Events to be Monitored: 
All research activities described in the project summary will be monitored in addition to study data 
accrual, protocol deviations, protocol violations, unanticipated problems, and adverse events. 
 
Methods and Frequency of Analysis: 
All research staff will have appropriate training to ensure the safety of participants, the appropriate 
conduct of research, and the integrity of safety and/or efficacy of data.  This training includes completion 
of all University-required training in the policies and procedures to protect the rights and welfare of 
people who participate in research.   
 
Subjects will be fully informed about the study requirements throughout the conduct of the trial by the 
study personnel.  At the beginning of the study, it will be noted in the research record that the subject 
received complete information about the study requirements.  Study personnel will give participants 
information relevant to continued participation.   
 
The PI will perform monitoring activities. Monitoring will be performed weekly by the PI. The conclusions 
of the monitoring will be reported to the IRB in writing if there is any change in the risk/benefit ratio for 
subjects or if there is any new information which may affect a subject’s willingness to continue 
participation. Monitoring will include consideration for (1) progress of the trial, (2) data quality, (3) 
timeliness of data collection, (4) recruitment, (5) accrual and retention, (6) risk vs. benefit for 
participants, (7) protocol violations, and (8) other factors that could affect the outcome of the study.  
Monitoring will be documented in the research record. 
 
Person(s) Responsible for Data Monitoring: 


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The persons responsible for data monitoring in this research are Ian Neeland, MD and Bienka Milton, 
MPH BS.  Dr. Neeland will oversee study operations on a day-to-day basis. Ms. Milton is a certified 
study coordinator. Ms. Milton will be responsible for aiding in subject recruitment and retention, protocol 
execution, and general study coordination.  
 
Reporting Unanticipated Problems, Adverse Events, Protocol Deviations and Protocol Violations: 
The principal investigator and research coordinator will monitor subject involvement in the study on a 
daily basis. The PI will report all classes of unexpected adverse events and any serious adverse events 
within 48 hours of recognition to the Federal agencies and University and hospital offices as specified 
by the IRB.  The PI will submit a summary of adverse events in continuing review reports to the IRB at 
intervals specified by the IRB.  Subject identifiers will be excluded from all reports. 
 
Stopping Rules: 
Subjects will be informed of the voluntary nature of the research and their option to stop study 
procedures for any reason.  They will also be advised that the PI may stop their participation if it is 
deemed to be in the subject’s best interest, if new information becomes available during the research, 
or if a subject cannot follow study instructions or keep appointments.   
 
Procedures and Time Frames for Communicating Outcomes: 
Medical literature for scientific or therapeutic developments that may have an impact on the safety of 
the participants or the ethics of the study will be monitored by the PI.   
 
Investigational New Drug (IND) Exemption: 
Empagliflozin has been FDA approved as an adjunct to diet and exercise to improve glycemic control 
in adults with type 2 diabetes The empagliflozin intervention component of the proposed study should 
be IND exempt based on meeting IND exemption criteria.  Specifically, 
 
IND exemption criteria: 
(1) it is not intended to be reported to FDA 
in 
support of a new indication for use or to 
support any other significant change in the 
labeling for the drug; 
As a small pilot clinical study looking at an 
intermediate phenotype (glycerol enrichment in blood 
glucose), this will not support a label change or new 
indication. 
(2) it is not intended to support a significant 
change in the advertising for the product; 
See response to (1). 
(3) it does not involve a route of 
administration or dosage level, use in a 
subject population, or other factor that 
significantly increases the risks (or 
decreases the acceptability of the risks) 
associated with the use of the drug product; 
The route of administration and dosage level are 
FDA approved for use in patients with type 2 
diabetes.  The lowest approved dosage will be used 
for a short period of time to minimize risks. The 
subject population will be obese volunteers who are 
otherwise healthy; it is expected that use of the drug 
product will not significantly increase risks in this 
population.    
(4) it is conducted in compliance with the 
requirements for IRB review and informed 
consent [21 CFR parts 56 and 50, 
respectively]; 
IRB review is being obtained for the 
intervention/clinical trial aim of this study. 
(5) it is conducted in compliance with the 
requirements concerning the promotion and 
sale of drugs [21 CFR 312.7]; 
Use of empagliflozin in this capacity will be 
investigational; promotional claims of safety and 


Version 8, 10/13/17 
Page 11 of 22 
effectiveness for obesity or diabetes will not be 
made. 
(6) it does not intend to invoke 21 CFR 
50.24. 
We will obtain written informed consent. 
 
12. Procedures to Maintain Confidentiality: 
Subjects will be assigned an identification code. A subject's name and code will be contained within a 
small notebook and will be kept in a locked cabinet in the research coordinator's locked office.  The 
research chart will contain personal health information and will be kept in locked file cabinet in the 
research coordinator's locked office.  All other data on a computer will be de-identified with the subject's 
identification code.  Blood samples waiting for analysis will not be labeled with the subject's name, but 
with the assigned identification code. 
 
The study database will be password protected. No data will be sent over the internet unless it is 
encrypted. All email with subject-identifiable information will be password protected.  Only key personnel 
will have access to the information in the study database on an as-needed basis. Key personnel may 
not alter the data in the database or directly view all of it without specific cause and approval of the PI.  
Subjects will be advised that representatives of the Sponsor and the UT Southwestern Medical Center 
IRB may review their medical and research records to assure the quality of information used in this 
research. A HIPAA “Authorization for Disclosure” will be obtained from each study participant. A 
Certificate of Confidentiality is not needed for this study.  The research coordinator will keep the original 
signature copy of the informed consent document in the research records, which will be kept in a locked 
file cabinet in a locked office.  Each subject will receive a photocopy of consent documents.   
 
Neither the PI nor any other key personnel have any real or perceived conflict of interest in the research 
outcome. All have submitted a report of financial interest to the Conflict of Interest Office at UT 
Southwestern within the past twelve months. 
 
13. Potential Benefits: 
 
There is a potential benefit of the proposed research to study participants in the form of weight loss. 
However, researcher cannot guarantee any direct benefit to the study participants.  Knowledge 
gained from this project will likely lead to better understanding of diabetes in the general population 
and across ethnic minorities and women as well as potentially lead to improved targets of therapies 
aimed at reducing diabetes risk.  
 
The risks outlined in this proposal are reasonable for this societal benefit as it may lead to improved 
care to reduce diabetes risk, a leading cause of morbidity and mortality in the U.S.  
 
14. Biostatistics – Statistical Analysis Plan:  
Aim 1 Sample size calculation: Assuming 20% 13C3 enrichment in subjects with low VAT (based on prior 
work), and an expected enrichment of between 15-18% among those with high VAT (SD 2%), for Aim 
1 we expect to require 20 subjects per group to achieve 87% power to detect a 2% difference between 
groups at an alpha level of 0.05.  Because we expect some patients may be deemed ineligible or 
withdraw consent in the interim between recruitment/informed consent is signed and undergoing study 
procedures, we will recruit up to a total of 50 subjects in order to ensure that 40 total subjects complete 
the study procedures (see sample size table). The study will be stopped after 40 subjects complete all 
study procedures and no further patients will be included in the study.  
Variable 
Mean 
Difference 
 
Standard 
Deviation 
Type 1 error 
(α) 
Power 
(1-β) 
 
Total N 


Version 8, 10/13/17 
Page 12 of 22 
 
 
 
 
 
 
 
Aim 1 Statistical analysis: Data will be expressed as means ± standard deviations.  The primary 
outcome of mean [U-13C3] enrichment of blood glucose at study end will be compared between high 
VAT and low VAT groups using the Student’s t-test.  Plots of [U-13C3] enrichment over time for each 
group will be constructed to compare the trajectory of glycerol enrichment over time between groups 
and compared using a mixed linear model.  The association between VAT and change in plasma blood 
glucose with glycerol challenge will be analyzed within each subject using a paired t-test and between 
groups by mean change in plasma glucose using the Student’s t-test.  The relationship between VAT 
and glycerol enrichment will also be modeled using linear regression and adjusted for potential 
confounders of age, ethnicity, family history of diabetes, triglycerides, lean mass, liver fat, abdominal 
subcutaneous fat mass, and biomarkers. 
 
Aim 2 Sample size calculation: Assuming a mean change of 2% (SD 1.5%) [U-13C3] enrichment after 
treatment with EMPA, for Aim 2 we expect to require 8 paired measurements in each arm to achieve 
84% power to detect this difference at an alpha level of 0.05.  Assuming an estimated 20% dropout rate 
consistent across groups we will still have sufficient power to detect this mean difference within each 
treatment arm using the planned enrollment strategy.   
 
Aim 2 Statistical analysis: See Aim 1 above.  The primary outcome of change in mean [U-13C3] 
enrichment of blood glucose within each treatment assignment will be tested using the paired t-test.  
Secondary outcomes comparing post-treatment glycerol enrichment across groups will be tested with 
ANOVA.    
 
For all statistical testing, a 2-sided p-value <0.05 will be considered statistically significant.  All 
statistical analyses will be performed using SAS version 9.2 software (SAS Corporation, Cary, NC).  
 
15. References: 
1. 
Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Blaha MJ, Dai S, Ford ES, Fox CS, 
Franco S, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, Huffman MD, Judd SE, Kissela 
BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Mackey RH, Magid DJ, Marcus GM, Marelli A, 
Matchar DB, McGuire DK, Mohler ER, 3rd, Moy CS, Mussolino ME, Neumar RW, Nichol G, Pandey 
DK, Paynter NP, Reeves MJ, Sorlie PD, Stein J, Towfighi A, Turan TN, Virani SS, Wong ND, Woo D, 
Turner MB, American Heart Association Statistics C, Stroke Statistics S. Heart disease and stroke 
statistics--2014 update: A report from the american heart association. Circulation. 2014;129:e28-e292 
2. 
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. 2008;168:1617-1624 
3. 
Cornier MA, Despres JP, Davis N, Grossniklaus DA, Klein S, Lamarche B, Lopez-Jimenez F, 
Rao G, St-Onge MP, Towfighi A, Poirier P. Assessing adiposity: A scientific statement from the 
american heart association. Circulation. 2011;124:1996-2019 
4. 
Neeland IJ, Ayers CR, Rohatgi AK, Turer AT, Berry JD, Das SR, Vega GL, Khera A, McGuire 
DK, Grundy SM, de Lemos JA. Associations of visceral and abdominal subcutaneous adipose tissue 
with markers of cardiac and metabolic risk in obese adults. Obesity (Silver Spring). 2013;21:E439-447 
[U-13C3] 
glycerol 
enrichment 
2% 
2% 
0.05 
0.87 
40 
3% 
2% 
0.05 
0.89 
20 
5% 
2% 
0.05 
0.97 
12 
7% 
2% 
0.05 
0.89 
6 


Version 8, 10/13/17 
Page 13 of 22 
5. 
Neeland IJ, Turer AT, Ayers CR, Powell-Wiley TM, Vega GL, Farzaneh-Far R, Grundy SM, 
Khera A, McGuire DK, de Lemos JA. Dysfunctional adiposity and the risk of prediabetes and type 2 
diabetes in obese adults. JAMA. 2012;308:1150-1159 
6. 
Puhakainen I, Koivisto VA, Yki-Jarvinen H. Lipolysis and gluconeogenesis from glycerol are 
increased in patients with noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab. 
1992;75:789-794 
7. 
Gaidhu MP, Anthony NM, Patel P, Hawke TJ, Ceddia RB. Dysregulation of lipolysis and lipid 
metabolism in visceral and subcutaneous adipocytes by high-fat diet: Role of atgl, hsl, and ampk. 
American journal of physiology. Cell physiology. 2010;298:C961-971 
8. 
Haring HU, Merker L, Seewaldt-Becker E, Weimer M, Meinicke T, Woerle HJ, Broedl UC. 
Empagliflozin as add-on to metformin plus sulfonylurea in patients with type 2 diabetes: A 24-week, 
randomized, double-blind, placebo-controlled trial. Diabetes Care. 2013;36:3396-3404 
9. 
Kovacs CS, Seshiah V, Swallow R, Jones R, Rattunde H, Woerle HJ, Broedl UC. Empagliflozin 
improves glycaemic and weight control as add-on therapy to pioglitazone or pioglitazone plus metformin 
in patients with type 2 diabetes: A 24-week, randomized, placebo-controlled trial. Diabetes Obes Metab. 
2014;16:147-158 
10. 
Flegal KM, Carroll MD, Ogden CL, Curtin LR. Prevalence and trends in obesity among us adults, 
1999-2008. JAMA. 2010;303:235-241 
11. 
Fox CS, Pencina MJ, Meigs JB, Vasan RS, Levitzky YS, D'Agostino RB, Sr. Trends in the 
incidence of type 2 diabetes mellitus from the 1970s to the 1990s: The framingham heart study. 
Circulation. 2006;113:2914-2918 
12. 
Wijeysundera HC, Machado M, Farahati F, Wang X, Witteman W, van der Velde G, Tu JV, Lee 
DS, Goodman SG, Petrella R, O'Flaherty M, Krahn M, Capewell S. Association of temporal trends in 
risk factors and treatment uptake with coronary heart disease mortality, 1994-2005. JAMA. 
2010;303:1841-1847 
13. 
Jensen MD, Ryan DH, Apovian CM, Ard JD, Comuzzie AG, Donato KA, Hu FB, Hubbard VS, 
Jakicic JM, Kushner RF, Loria CM, Millen BE, Nonas CA, Pi-Sunyer FX, Stevens J, Stevens VJ, 
Wadden TA, Wolfe BM, Yanovski SZ, Jordan HS, Kendall KA, Lux LJ, Mentor-Marcel R, Morgan LC, 
Trisolini MG, Wnek J, Anderson JL, Halperin JL, Albert NM, Bozkurt B, Brindis RG, Curtis LH, DeMets 
D, Hochman JS, Kovacs RJ, Ohman EM, Pressler SJ, Sellke FW, Shen WK, Smith SC, Jr., Tomaselli 
GF. 2013 aha/acc/tos guideline for the management of overweight and obesity in adults: A report of the 
american college of cardiology/american heart association task force on practice guidelines and the 
obesity society. Circulation. 2014;129:S102-138 
14. 
Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin JM, Walker EA, Nathan DM. 
Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med. 
2002;346:393-403 
15. 
O'Neil PM, Smith SR, Weissman NJ, Fidler MC, Sanchez M, Zhang J, Raether B, Anderson CM, 
Shanahan WR. Randomized placebo-controlled clinical trial of lorcaserin for weight loss in type 2 
diabetes mellitus: The bloom-dm study. Obesity (Silver Spring). 2012;20:1426-1436 
16. 
Schauer PR, Kashyap SR, Wolski K, Brethauer SA, Kirwan JP, Pothier CE, Thomas S, Abood 
B, Nissen SE, Bhatt DL. Bariatric surgery versus intensive medical therapy in obese patients with 
diabetes. N Engl J Med. 2012;366:1567-1576 
17. 
Ferrannini E, Natali A, Bell P, Cavallo-Perin P, Lalic N, Mingrone G. Insulin resistance and 
hypersecretion in obesity. European group for the study of insulin resistance (egir). J Clin Invest. 
1997;100:1166-1173 
18. 
Fox CS, Massaro JM, Hoffmann U, Pou KM, Maurovich-Horvat P, Liu CY, Vasan RS, Murabito 
JM, Meigs JB, Cupples LA, D'Agostino RB, Sr., O'Donnell CJ. Abdominal visceral and subcutaneous 


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adipose tissue compartments: Association with metabolic risk factors in the framingham heart study. 
Circulation. 2007;116:39-48 
19. 
Liu J, Fox CS, Hickson DA, May WD, Hairston KG, Carr JJ, Taylor HA. Impact of abdominal 
visceral and subcutaneous adipose tissue on cardiometabolic risk factors: The jackson heart study. J 
Clin Endocrinol Metab. 2010;95:5419-5426 
20. 
Boyko EJ, Fujimoto WY, Leonetti DL, Newell-Morris L. Visceral adiposity and risk of type 2 
diabetes: A prospective study among japanese americans. Diabetes Care. 2000;23:465-471 
21. 
Despres JP, Moorjani S, Lupien PJ, Tremblay A, Nadeau A, Bouchard C. Regional distribution 
of body fat, plasma lipoproteins, and cardiovascular disease. Arteriosclerosis. 1990;10:497-511 
22. 
Pouliot MC, Despres JP, Nadeau A, Moorjani S, Prud'Homme D, Lupien PJ, Tremblay A, 
Bouchard C. Visceral obesity in men. Associations with glucose tolerance, plasma insulin, and 
lipoprotein levels. Diabetes. 1992;41:826-834 
23. 
Ross R, Aru J, Freeman J, Hudson R, Janssen I. Abdominal adiposity and insulin resistance in 
obese men. Am J Physiol Endocrinol Metab. 2002;282:E657-663 
24. 
Ross R, Freeman J, Hudson R, Janssen I. Abdominal obesity, muscle composition, and insulin 
resistance in premenopausal women. J Clin Endocrinol Metab. 2002;87:5044-5051 
25. 
Bacha F, Saad R, Gungor N, Janosky J, Arslanian SA. Obesity, regional fat distribution, and 
syndrome x in obese black versus white adolescents: Race differential in diabetogenic and atherogenic 
risk factors. J Clin Endocrinol Metab. 2003;88:2534-2540 
26. 
Bogardus C, Lillioja S, Howard BV, Reaven G, Mott D. Relationships between insulin secretion, 
insulin action, and fasting plasma glucose concentration in nondiabetic and noninsulin-dependent 
diabetic subjects. J Clin Invest. 1984;74:1238-1246 
27. 
Nurjhan N, Kennedy F, Consoli A, Martin C, Miles J, Gerich J. Quantification of the glycolytic 
origin of plasma glycerol: Implications for the use of the rate of appearance of plasma glycerol as an 
index of lipolysis in vivo. Metabolism. 1988;37:386-389 
28. 
Ciapaite J, van den Broek NM, Te Brinke H, Nicolay K, Jeneson JA, Houten SM, Prompers JJ. 
Differential effects of short- and long-term high-fat diet feeding on hepatic fatty acid metabolism in rats. 
Biochimica et biophysica acta. 2011;1811:441-451 
29. 
Podolin DA, Wei Y, Pagliassotti MJ. Effects of a high-fat diet and voluntary wheel running on 
gluconeogenesis and lipolysis in rats. J Appl Physiol (1985). 1999;86:1374-1380 
30. 
Baba H, Zhang XJ, Wolfe RR. Glycerol gluconeogenesis in fasting humans. Nutrition. 
1995;11:149-153 
31. 
Nurjhan N, Consoli A, Gerich J. Increased lipolysis and its consequences on gluconeogenesis 
in non-insulin-dependent diabetes mellitus. J Clin Invest. 1992;89:169-175 
32. 
Howe HR, 3rd, Heidal K, Choi MD, Kraus RM, Boyle K, Hickner RC. Increased adipose tissue 
lipolysis after a 2-week high-fat diet in sedentary overweight/obese men. Metabolism. 2011;60:976-981 
33. 
Abate N, Garg A, Coleman R, Grundy SM, Peshock RM. 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. 1997;65:403-408 
34. 
Vega GL, Adams-Huet B, Peshock R, Willett D, Shah B, Grundy SM. Influence of body fat 
content and distribution on variation in metabolic risk. J Clin Endocrinol Metab. 2006;91:4459-4466 
35. 
Jin ES, Beddow SA, Malloy CR, Samuel VT. Hepatic glucose production pathways after three 
days of a high-fat diet. Metabolism. 2013;62:152-162 
36. 
Jin ES, Sherry AD, Malloy CR. Metabolism of glycerol, glucose, and lactate in the citric acid 
cycle prior to incorporation into hepatic acylglycerols. J Biol Chem. 2013;288:14488-14496 


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Page 15 of 22 
37. 
Rasouli N, Raue U, Miles LM, Lu T, Di Gregorio GB, Elbein SC, Kern PA. Pioglitazone improves 
insulin sensitivity through reduction in muscle lipid and redistribution of lipid into adipose tissue. Am J 
Physiol Endocrinol Metab. 2005;288:E930-934 
38. 
Lord J, Thomas R, Fox B, Acharya U, Wilkin T. The effect of metformin on fat distribution and 
the metabolic syndrome in women with polycystic ovary syndrome--a randomised, double-blind, 
placebo-controlled trial. BJOG : an international journal of obstetrics and gynaecology. 2006;113:817-
824 
39. 
Gonzalez-Ortiz M, Martinez-Abundis E, Robles-Cervantes JA, Ramos-Zavala MG. Effect of 
exenatide on fat deposition and a metabolic profile in patients with metabolic syndrome. Metab Syndr 
Relat Disord. 2011;9:31-34 
 
Figure Legend 
 
Figure 1.  
Title: Schematic of Glycerol Metabolism in Gluconeogenesis in Liver and Adipose Tissues 
Caption: Glucose as a product of hepatic gluconeogenesis is comprised of a 6-carbon molecule 
derived from glycerol via multiple different metabolic pathways including exogenous glycerol from 
dietary sources and adipose tissue-derived glycerol produced from lipolysis of triglyceride stores.  In 
the liver, glycerol (Gly) is converted to glycerol-3-phosphate (G3P) and subsequently 
dihydroxyacetone phosphate (DHAP) which is in equilibrium with glyceraldehyde-3-phosphate 
(GA3P).  Both DHAP and GA3P are converted to fructose-1,6-bisphosphate which is serially 
enzymatically converted to fructose-6-phosphate, glucose-6-phosphate, and ultimately glucose.  In 
adipose tissue, triglycerides (TG) undergo lipolysis to form free fatty acids (FFA) and glycerol (Gly) 
which then shuttles to the liver to enter into the same gluconeogenesis pathway as that of exogenous 
glycerol.  Therefore, free plasma glucose can be comprised of two glycerol molecules from either 
exogenous or lipolytic sources in any combination. 
 
Figure 2. 
Title: Representative examples of abdominal fat by MRI in two subjects with divergent metabolic 
phenotypes in the Dallas Heart Study.  
Caption: A. Transverse abdominal MRI images of visceral (VAT) and abdominal subcutaneous (SAT) 
adipose tissue in a 21-year-old black female with BMI of 36 kg/m2 and total body fat of 4.2 kg (41%) 
demonstrates very low VAT (0.22 kg/m2) and high SAT (4.45 kg/m2).  B. In contrast, images of VAT 
and in a 59-year-old white male with a BMI of 31 kg/m2 and total body fat of 4.0 kg (34%) 
demonstrate very high VAT (1.80 kg/m2) and low SAT (1.46 kg/m2). 
 
 
 
Figure 1. 
 


Version 8, 10/13/17 
Page 16 of 22 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2. 
 
A.                                                                         B. 
 
Orally ingested 
[U-13C3]glycerol 
     C3  
Unlabeled glycerol 
from lipolysis 
         C3 
Free plasma glucose (C6) by NMR:                                                   
Schematic of Glycerol Metabolism in 
Gluconeogenesis in Liver and Adipose 
Tissues 


Version 8, 10/13/17 
Page 17 of 22 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Glycerol Study Protocol Flowsheet 
 


Version 8, 10/13/17 
Page 18 of 22 
Advanced Imaging Research Center 
eIRB File #: 012015-064   
Visceral Adiposity and Diabetes: Translating Form to Function Using Imaging 
Subject Name:   
 
 
Visit Date:   
Scheduled 
Time 
Actual 
Time 
Initials AIRC Outpatient Procedure 
0800 
 
 
Meet patient in lobby of Rogers MRI Center and verify name & 
DOB. 
0810 
 
 
Explain study procedures and obtain informed consent.  Verify 
NPO except water since midnight. 
0825 
 
 
Obtain urine sample for pregnancy test (female only) 
0830 
 
 
Obtain vital signs and anthropometric measurements.   
T______ P_______BP_____/_____ R______ Body Fat % ______ 
 
Ht_______Wt_______BMI____  Waist Circ____  Hip Circ____ 
0845 
 
 
Insert peripheral IV with 150ml bag of normal saline to KVO.  
Verify good blood return and flushes easily.   
0855 
 
 
Collect 40ml blood sample in purple top and 1ml blood sample 
for blood sugar.   
YSI Result: ________/________mg/dl 
0915  
-0930 
 
 
Prepare for subjects receiving [U-13C3]glycerol in ½ cup 
filtered water: 
________kg x 0.05 gm/kg x 1 ml/1.302 gm = _________ml 
Lot#: ______________  Exp: n/a 
0930 
 
 
 
Have subject drink Glycerol.  Follow with 1 cup filtered water to 
rinse bottle.  
1000 
+30 
 
 
Collect 1ml for blood sugar and 40ml blood sample in purple 
top tubes.    
 
YSI Result: ________/________mg/dl 
1030 
+60 
 
 
Collect 1ml for blood sugar and 40ml blood sample in purple 
top tubes.    
 
YSI Result: ________/________mg/dl 
1100 
+90 
 
 
Collect 1ml for blood sugar and 40ml blood sample in purple 
top tubes.    
 
YSI Result: ________/________mg/dl 
1130 
+120 
 
 
Collect 1ml for blood sugar and 40ml blood sample in purple 
top tubes.    
 
YSI Result: ________/________mg/dl 
1200 
+150 
 
 
Collect 1ml for blood sugar and 40ml blood sample in purple 
top tubes.    
 
YSI Result: ________/________mg/dl 


Version 8, 10/13/17 
Page 19 of 22 
Advanced Imaging Research Center 
eIRB File #: 012015-064   
Visceral Adiposity and Diabetes: Translating Form to Function Using Imaging 
Subject Name:   
 
 
Visit Date:   
Scheduled 
Time 
Actual 
Time 
Initials AIRC Outpatient Procedure 
1230 
+180 
 
 
Collect 1ml for blood sugar and 40ml blood sample in purple 
top tubes.    
 
YSI Result: ________/________mg/dl 
 
 
 
Discontinue peripheral IV line after last blood sample obtained. 
 
 
 
Obtain vital signs.  BP: _______/_______   P: ______ T: 
________°F  R: _____   
 
 
 
Provide lunch.  
 
 
 
Perform MRI scan if indicated 
 
 
 
On Study Day 1 Provide Emagliflozin/Placebo and Instructions. 
 
 
 
Discharge subject from AIRC. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Version 8, 10/13/17 
Page 20 of 22 
 
 
 
 
 
 
Glycerol Study Procedure Grid 
 
 
 
 
 
 
 
 
 
 
Advanced Imaging Research Center 
eIRB File #: 012015-064  
Visceral Adiposity and Diabetes: Translating Form to Function Using Imaging  
Study Time 
Plasma (Pre) 
(40ml) 
Glucose 
(1ml) 
NMR 
(40ml) 
Comments 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Arrive @ AIRC 
 
 
 
 
Start IV x 1 
-60 (Pre) 
X 
X 
 
 
0 
 
 
 
Glycerol consumed 
+15 
 
X 
X 
 
+30 
 
X 
X 
 
+60 
 
X 
X 
 
+90 
 
X 
X 
 
+120 
 
X 
X 
 
+150 
 
X 
X 
 
+180 
 
X 
X 
 
 
 
 
 
D/C IV & provide 
lunch 
Tubes 
10ml purple top  
tube x 1 
1ml microfuge 
tube 
10ml purple top 
 tube x 4 
 
Processing 
On ice  spin  
plasma in small 
vial  
REFRIGERATOR  
for Dr. 
Jin/Neeland 
IMMEDIATELY 
spin  run  
record result  
plasma in cryo 
vial for Dr. 
Jin/Neeland 
On ice  spin  
plasma divide 
into 3 10ml 
plastic vials  
REFRIGERATOR  
for Dr. 
Jin/Neeland 
 


Version 8, 10/13/17 
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VISIT 2 and VISIT 3 STUDY MEDICATION COMPLIANCE FORM 
 
DID SUBJECT RETURN UNUSED AND/OR EMPTY MEDICATION CONTAINERS?     
 YES      
  NO 
If no, comment below:    
__________________________________________________________________________________________
________________________________________________________________________________ 
 
 
Date of Last Dose Dispensed: _____/_____/_____ 
 
 
                                                  Day     Mon   Year 
 
Total Number of Tablets Dispensed at last visit: __________  
 
Total Number of Tablets Returned Today: ____________ 
 
Last Dose Kit #: _________________     
 
Last Dose Lot #:_________________ 
 
                                               
                                       TOTAL NUMBER OF TABLETS USED 
COMPLIANCE % =     ___________________________________________________    X   100 
 
                                        TOTAL NUMBER OF TABLETS REQUIRED PER PROTOCOL 
                                                      
                                              _________ /________ X 100=___________ 
 
 
Is Subject compliant with Medication?  
 YES      
 NO 
If no, comment below and counsel/educate subject again:    
__________________________________________________________________________________________
________________________________________________________________________________ 
 
If medication compliance percentage is <80%, please re-educate subjects.   The subject can be withdrawn from 
study after 2 visits of medication noncompliance.  The principal investigator should be notified for each 
subject’s visit of not meeting medication compliance.  It is up to the discretion of the principal investigator to 
allow the subject to remain in the study. 
 
MEDICATION RE-DISPENSED                                   
 YES      
 NO 
If no, comment below:    
__________________________________________________________________________________________
________________________________________________________________________________ 
 
 
Kit #: ____________________ Lot#: _____________________    Expiration Date: _________________      
 
 
NUMBER OF TABLETS DISPENSED?  ________ 
        
MEDICATION DOSAGE GIVEN TO AND REVIEWED WITH SUBJECT          
 YES     
 NO                                       
 
 
 
 
 


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