Effects of Niacin on Intramyocellular Fatty Acid Trafficking in Upper Body Obesity and Type 2 Diabetes Mellitus
Phase: EARLY_PHASE1
Conditions: Type 2 Diabetes Mellitus, Obesity
Sponsor: Mayo Clinic

 
 
Effects of Niacin on Intramyocellular Fatty Acid 
Trafficking in Upper Body Obesity and Type 2 
Diabetes Mellitus 
 
NCT03867500 
 
3/30/2023 

Effects of Niacin on FFA in UBO and T2DM in insulin resistant states 
 
 
 
Version 2.2 
 
 
Revised 03/30/2023 
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Michael D. Jensen, MD 
 
 
CONFIDENTIAL 
 
 
Effects of Niacin on Intramyocellular Fatty Acid Trafficking in Upper Body Obesity and 
Type 2 Diabetes Mellitus 
 
 
 
 
Regulatory Sponsor: 
Michael D. Jensen, MD 
Division of Endocrinology 
 
 
 
 
Rochester, MN 55902 
 
 
 
Funding Sponsor: 
(Optional) 
National Institutes of Health/National Institute of Diabetes, 
Digestive and Kidney Disease 
 
 
 
Study Product: 
Niacin (Nicotinic acid) 
Protocol Number: (IRBe) 
 
17-009977 
 
IND Number: 
138804 
 
 
 
 
 
Initial version: [01/17/2018] Version (1.0) 
Revised version: [3/2/2021] Version (2.0)  
Revised Version: [12/22/2021]  
Revised Version: [03/30/2023] Version 2.2 
 
 

Effects of Niacin on FFA in UBO and T2DM in insulin resistant states 
 
 
 
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Table of Contents 
 
STUDY SUMMARY ...................................................................................................................................... 5 
1 
INTRODUCTION ................................................................................................................................. 7 
1.1 
BACKGROUND .................................................................................................................................... 7 
1.2 
PRECLINICAL AND CLINICAL DATA ........................................................................................................ 8 
1.3 
INVESTIGATIONAL AGENT .................................................................................................................. 10 
1.4 
DOSE RATIONALE.............................................................................................................................. 10 
1.5 
RISKS AND BENEFITS ......................................................................................................................... 11 
2 
STUDY OBJECTIVES ........................................................................................................................ 11 
3 
STUDY DESIGN ................................................................................................................................. 12 
3.1 
GENERAL DESCRIPTION ..................................................................................................................... 12 
3.2 
NUMBER OF SUBJECTS ....................................................................................................................... 14 
3.3 
DURATION OF PARTICIPATION ............................................................................................................ 14 
3.4 
PRIMARY STUDY ENDPOINTS .............................................................................................................. 16 
3.5 
SECONDARY STUDY ENDPOINTS ......................................................................................................... 16 
3.6 
PRIMARY SAFETY ENDPOINTS ............................................................................................................ 16 
3.7 
IDENTIFICATION OF SOURCE DATA ...................................................................................................... 16 
4 
SUBJECT SELECTION ENROLLMENT AND WITHDRAWAL ...................................................... 17 
4.1 
INCLUSION CRITERIA ......................................................................................................................... 17 
4.2 
EXCLUSION CRITERIA ........................................................................................................................ 18 
4.3 
SUBJECT RECRUITMENT, ENROLLMENT AND SCREENING ....................................................................... 19 
4.3.1 
Early Withdrawal of Subjects .................................................................................................... 20 
4.3.2 
When and How to Withdraw Subjects ........................................................................................ 20 
5 
STUDY DRUG .................................................................................................................................... 21 
5.1 
DESCRIPTION .................................................................................................................................... 21 
5.2 
TREATMENT REGIMEN ....................................................................................................................... 23 
SUBJECTS WILL RECEIVE A TITRATED DOSE STARTING FROM 0.6 MG/MIN TO A MAXIMUM OF 2.8 MG/MIN (LIKELY 
NEEDED DOSE = 1.4 MG/MIN) OR 0.9% NACL FOR 12 HOURS. .......................................................................... 23 
5.3 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS ................................................................ 23 
5.4 
PREPARATION AND ADMINISTRATION OF STUDY DRUG ......................................................................... 23 
5.5 
SUBJECT COMPLIANCE MONITORING ................................................................................................... 23 
5.6 
PRIOR AND CONCOMITANT THERAPY .................................................................................................. 23 
5.7 
PACKAGING ...................................................................................................................................... 24 
5.8 
MASKING/BLINDING OF STUDY........................................................................................................... 24 
5.9 
RECEIVING, STORAGE, DISPENSING AND RETURN ................................................................................. 24 
5.9.1 
Receipt of Drug Supplies .......................................................................................................... 24 
5.9.2 
Storage ................................................................................................................................... 24 
5.9.3 
Dispensing of Study Drug ......................................................................................................... 25 
5.9.4 
Return or Destruction of Study Drug ......................................................................................... 25 
6 
STUDY PROCEDURES ...................................................................................................................... 25 
6.1 
SCREENING VISIT .............................................................................................................................. 27 
6.2 
RANDOMIZATION (DAY 0) .................................................................. ERROR! BOOKMARK NOT DEFINED. 
6.3 
VISIT 1 (IN-PATIENT VISIT, 1 NIGHT STAY) ............................................................................................ 27 
6.4 
VISIT 2 (IN-PATIENT VISIT, 1 NIGHT STAY) ............................................................................................ 30 
7 
STATISTICAL PLAN ......................................................................................................................... 31 

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7.1 
SAMPLE SIZE DETERMINATION ........................................................................................................... 31 
7.2 
STATISTICAL METHODS ..................................................................................................................... 31 
7.3 
SUBJECT POPULATION(S) FOR ANALYSIS ............................................................................................. 34 
8 
SAFETY AND ADVERSE EVENTS ................................................................................................... 34 
8.1 
DEFINITIONS ..................................................................................................................................... 34 
8.2 
RECORDING OF ADVERSE EVENTS ....................................................................................................... 38 
8.3 
REPORTING OF SERIOUS ADVERSE EVENTS AND UNANTICIPATED PROBLEMS .......................................... 38 
8.3.1 
Sponsor-Investigator reporting: notifying the Mayo IRB ............................................................. 39 
8.3.2 
Sponsor-Investigator reporting: Notifying the FDA ..................................................................... 41 
8.4 
UNMASKING/UNBLINDING PROCEDURES ............................................................................................. 42 
8.5 
STOPPING RULES ............................................................................................................................... 42 
8.6 
MEDICAL MONITORING ..................................................................................................................... 43 
8.6.1 
Internal Data and Safety Monitoring Board ............................................................................... 44 
9 
DATA HANDLING AND RECORD KEEPING .................................................................................. 45 
9.1 
CONFIDENTIALITY ............................................................................................................................. 45 
9.2 
SOURCE DOCUMENTS ........................................................................................................................ 45 
9.3 
CASE REPORT FORMS ........................................................................................................................ 45 
9.4 
RECORDS RETENTION ........................................................................................................................ 48 
10 
STUDY MONITORING, AUDITING, AND INSPECTING ................................................................ 49 
10.1 
STUDY MONITORING PLAN ............................................................................................................ 49 
10.2 
AUDITING AND INSPECTING ........................................................................................................... 58 
11 
ETHICAL CONSIDERATIONS ......................................................................................................... 58 
12 
STUDY FINANCES ............................................................................................................................ 58 
12.1 
FUNDING SOURCE ......................................................................................................................... 58 
12.2 
CONFLICT OF INTEREST ................................................................................................................. 59 
12.3 
SUBJECT STIPENDS OR PAYMENTS .................................................................................................. 59 
13 
PUBLICATION PLAN ....................................................................................................................... 59 
14 
REFERENCES ................................................................................................................................... 59 
 
 
 
 
 
 
 
 

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List of Abbreviations 
 
 
LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
DSMB 
Data and Safety Monitoring Board 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
 
 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
PHI 
Protected Health Information 
PI 
Principal Investigator 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SOP 
Standard Operating Procedure 
 
 
 
 
 
 

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Study Summary 
Title 
Effects of Niacin on Intramyocellular Fatty Acid Trafficking in 
Subjects with Upper Body Obesity and Type 2 Diabetes Mellitus 
Running Title 
Effects of niacin on FFA in UBO and T2DM in insulin resistant 
states 
Protocol Number 
17-009977 
Phase 
Pilot Study 
Methodology 
Randomized, placebo-controlled; cross-over design 
Overall Study Duration 
2 years 
Subject Participation 
Duration 
6-8 weeks 
Single or Multi-Site  
Single site 
Objectives 
Evaluate the effect of niacin or placebo on intramyocellular fatty 
acid trafficking in subjects with upper body obesity and type 2 
diabetes mellitus 
Number of Subjects 
10 upper body obese and 10 Type 2 diabetic participants will be 
recruited for this study for a total of 20 participants between the 
ages of 18-65 years. 
Diagnosis and Main 
Inclusion Criteria 
Individuals with upper body obesity and Type 2 diabetics will be 
included in the study. 
Study Product, Dose, 
Route, Regimen 
Niacin (nicotinic acid).   
Dose: 0.6 mg/minute - 2.8 mg/min for 12 hours. 
Dose route: intravenous 
Dose regime: ~ 1.4 mg/min for 12 hours 
Duration of 
Administration 
17 hours on one occasion 

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Reference therapy 
The volunteers will undergo a separate study where they will 
receive intravenous saline without niacin 
Statistical Methodology 
The incorporation of plasma free fatty acids into intramyocellular 
lipids will be compared between the saline control day and the 
niacin infusion day (paired t-test). We will assess whether the 
incorporation of plasma fatty acids into intramyocellular lipids is 
related to insulin sensitivity measured by a euglycemic, 
hyperinsulinemic clamp using linear regression approaches. We 
will also assess whether the incorporation of plasma fatty acids 
into intramyocellular lipids is correlated with the activation of the 
insulin signaling cascade (Western blotting) in muscle using 
linear regression approaches. 
 
 
 
 

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1 
Introduction 
This document is a protocol for a human research study. This study will be carried out in 
accordance with the applicable United States government regulations and Mayo Clinic research 
policies and procedures.  
1.1 
Background 
Under normal circumstances adipose tissue is extremely insulin sensitive. Insulin inhibits 
adipose tissue lipolysis and thereby causes a rapid decrease in plasma free fatty acid 
concentrations. This insulin-mediated inhibition of lipolysis is impaired in upper body obese 
compared to lower body obese and normal weight adults (1-3). Humans with type 2 diabetes 
mellitus (T2DM) have an even more pronounced insulin resistance with regards to lipolysis (4, 
5). This insulin resistance results in over-exposure of muscle to free fatty acids (FFA), which can 
cause insulin resistance in muscle. Thus, it is unsurprising that adults with upper body/visceral 
obesity (UBO) and Type 2 Diabetes are typically more insulin resistant with respect to glucose 
metabolism than those with lower body obesity (LBO).  
 
These excess FFA can cause insulin resistance by interfering with insulin signaling. Increased 
FFA concentration and flux is thought to be a major contributor to insulin resistance in muscle 
(6) and to stimulate hepatic gluconeogenesis (7). Although insulin resistance is conventionally 
defined in terms of insulin’s effects on glucose metabolism, it is clear that insulin resistance is 
also present at the level of the adipocyte, with marked resistance to the antilipolytic effects of 
insulin in obesity (2) and T2DM (8, 9)  
 
Despite a large number of studies of how FFA contribute to health and disease (10-26) ,we and 
others (27) contend that the mechanisms by which fatty acids affect muscle insulin resistance 
remains to be fully delineated. The extent to which observed differences in muscle insulin action 
are the result of altered delivery (elevated adipose tissue lipolysis/FFA concentrations)  or altered 
intramyocellular FFA trafficking into lipotoxic intermediates has not been studied in an 
integrated fashion. We propose to address these questions by comprehensively assessing muscle 
fatty acid metabolism in humans with varying degrees of pre-existing and experimentally altered 
insulin sensitivity. By determining the mechanisms by which elevated plasma FFA 

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concentrations and differences in intramyocellular trafficking, we will gain a better 
understanding of insulin resistance and therefore potential therapies for insulin resistance and 
T2DM.  
 
We therefore propose to address the following questions: 1) Do UBO and T2DM differ with 
respect to muscle FFA uptake and muscle fatty acid trafficking (i.e. are lipotoxic intermediates 
from FFA greater in T2DM than UBO relative to muscle FFA exposure) 2) Will overnight, 
sustained suppression of FFA in UBO and T2DM improve insulin action with respect to glucose 
metabolism; 3) If so, will this be statistically related to the contribution of FFA to 
intramyocellular diacylglycerols (DG) and/or ceramides?  
 
Hypothesis: Compared to a saline control experiment, overnight suppression of FFA with 
intravenous niacin will decrease trafficking of plasma FFA into intramyocellular ceramides and/or 
DG and will improve insulin signaling and insulin-stimulated glucose disposal.  
 
Rationale: Niacin at high doses suppresses adipose tissue lipolysis via the PUMA-G receptor, 
independent of insulin. Suppression of FFA with Acipimox (a lipid-lowering niacin derivative) 
improved muscle insulin sensitivity with regards to glucose metabolism and Akt phosphorylation 
(66). Surprisingly, this was not accompanied by reductions in whole muscle ceramide or DG 
concentrations. While it is possible that neither DG nor ceramides were responsible for the 
abnormal insulin signaling (66), it is also possible that specific DG or ceramide species or the 
subcellular localization of those species, which were not measured by Liang et al (66), accounted 
for the improved insulin signaling/action.  
 
1.2 
Preclinical and clinical data 
Niacin, as a lipid-lowering treatment, is given chronically (for years) and safely at doses of 3g/day. 
We have given niacin (as Niaspan®) 1 g orally each hour for two hours {Ali, 2015 #107} with no 
adverse events. Intravenous niacin has been a component of the standard vitamin cocktail for 
parenteral nutrition for several decades. The daily therapeutic parenteral requirement in adults is 
40 mg per day {Bistrian, 2015 #106}. Nelson et al {Nelson, 2012 #18} administered niacin as an 

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intravenous infusion for two hours starting at 0.6mg/min and increasing to 2.8 mg/min over 30 
min; the only observed effect was asymptomatic flushing. In our proposed study we will use a one-
time dose that is less than the typical daily dose used for chronic treatment of hyperlipidemia. 
Given the safety of the route (intravenous) and the moderate dose (~1 gm over 12 hours), we 
anticipate this is a safe means to temporarily lower plasma FFA concentrations for the purposes of 
this NIH funded study. 
 
The proposed studies will measure the relative contribution of plasma FFA to 
intramyocellular signaling molecules (DG and ceramides), as well as the incorporation into imTG 
and subsequent appearance in long-chain acylcarnitines in adults with a wide range of insulin 
sensitivity with respect to glucose metabolism. By integrating information regarding the 
concentrations of lipid signaling molecules, the contribution of plasma FFA to these molecules, 
and the insulin signaling pathway we will be able to determine the extent to which varying degrees 
of insulin resistance are due to altered intramyocellular fatty acid trafficking. 
 
 
  
 
Suppression of FFA – an insulin sensitizing approach. Acipimox, a long-acting niacin 
analog, has been used to suppress FFA through a non-insulin mechanism (72) and thereby study 
the adverse effects of elevated FFA (66, 73-76). Seven days of Acipimox treatment improved 
insulin mediated stimulation of glucose disposal in T2DM, but not undifferentiated obese subjects 
(66). This treatment also increased insulin stimulated Akt phosphorylation in T2DM (66), but did 
not reduce the elevated whole muscle ceramide content in obese or T2DM and did not change 
muscle DG concentrations, which were not different between lean, obese and T2DM at baseline 
(66). Unfortunately, these authors could not document whether FFA were suppressed in a sustained 
manner, did not have measures of the DG and ceramides species, and measured only whole muscle 
content of these molecules. Furthermore, they did not characterize their non-diabetic obese 
volunteers with regards to fat distribution, which may explain why insulin-stimulated glucose 
disposal per kg fat free mass was equal to or greater in obese than lean controls. Our colleague, 
Dr. John Miles, has developed a protocol for intravenous niacin administration that suppresses 
FFA in a sustained manner (77); he has shared with us his protocol to facilitate the application for 
our own IND. By administering overnight intravenous niacin infusion we propose to test the 
hypothesis that lowering FFA in truly insulin resistant UBO and T2DM improves: 1) whole body 

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insulin sensitivity; 2) muscle insulin signaling; 3) subcellular (SS vs. IMF) content of DG and 
ceramides; 4) the contribution of plasma FFA to these molecules. 
 
In this study we propose to assess the subcellular location of DG and ceramides to better 
understand their relationship to altered insulin signaling. We will also test the hypothesis that 
sustained suppression of FFA alone can alter the trafficking of FFA into less “toxic” metabolites 
and thereby improve insulin action.   
 
1.3 
Investigational Agent 
 
Name of product: Nicotinic acid 
Brief description: Niacin will be received by Research Pharmacy in powder form. The drug will 
be administered intravenously by a Baxter infusion pump model Sigma Spectrum, after 
reconstitution by pharmacy.  
   
     
1.4 
Dose Rationale  
 
Subjects will first receive a titrated dose starting from 0.6 mg/min to a maximum of 2.8 mg/min 
(likely needed dose = 1.4 mg/min) or 0.9% NaCl. We will collect blood samples overnight 
during the infusion to determine the lowest possible dose of niacin needed to suppress plasma 
FFA concentrations to those observed in overnight, postabsorptive, lean, healthy adults. The 
infusion rate will be titrated to a maximum of 2.8 mg/min only if lower doses do not result in the 
needed reductions in plasma FFA. The dose of 2.8 mg/min has been shown to result in sustained 
suppression of plasma FFA concentrations (a reflection of adipose tissue lipolysis) to much 
lower levels than are needed for the purposes of this study {Nelson, 2012 #18}. Therefore, we 
anticipate considerably lower niacin doses will be needed. The gradual dose escalation and a pre-
study dose of 325 mg of aspirin will prevent or minimize the side effects of flushing due to 
niacin.  
 

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1.5 
Risks and Benefits 
Radiation: Radiation exposure from the studies supported by this grant include the radiation from 
the dual energy x-ray absorptiometry, and computed tomography scans of the abdomen.  
 
Blood sampling: Blood samples are collected from an indwelling intravenous catheter for this 
study. Bruising can occur with venipuncture, as can fainting. 
 
Infusion of substrates: We will infuse minute quantities of glucose and palmitate that are labeled 
with stable isotopes as part of these studies. The quality assurance plans for these compounds are 
reviewed and approved by the Mayo Clinic Radioactive Drug Research Committee as the 
designated representative of the FDA. We will also infuse insulin intravenously and give local 
anesthetics for the tissue biopsies. The insulin is infused using clinical safeguards as well as 
research pharmacy safeguards under an IND exemption from the Mayo IRB. 
 
Adipose and muscle biopsies: The risks of these procedures include pain, hematomas, bruising, 
infection, and scarring of the biopsy site.  
 
Insulin infusion: The risk of insulin infusion is hypoglycemia. 
 
Niacin: the risk of niacin infusion is flushing, pruritus, nausea or vomiting.  
 
2 
Study Objectives 
Primary Objective 
To assess the effect of overnight suppression of FFA with intravenous niacin on trafficking of 
FFA into intramyocellular ceramides and/or DG 
 
Secondary Objective 
To assess the effect of overnight suppression of FFA with intravenous niacin on insulin signaling 
and insulin-stimulated muscle glucose uptake 
 

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3 
Study Design 
This study is a non-randomized, niacin vs. saline control trial of the effects of niacin on 
intracellular fatty acid trafficking in insulin resistant states. Subjects will be screened at outpatient 
clinic visit appointments and interested qualified subjects will be consented and offered 
participation in this trial. Once consent has been obtained baseline values will be established and 
subjects will begin treatment and follow-up for up to the next 8 weeks. 
A final evaluation and collection of lab samples will be conducted at the end of the study.  
 
3.1 
General Description 
 
All volunteers will be weight stable for two months prior to the study and will consume 
an isoenergetic diet (weight stable) eating all meals (45% carbohydrate, 20% protein and 35% 
fat) from the Mayo Clinical Research Unit (CRU) for 3 days prior to the study. This will ensure 
consistency of energy intake and nutrient composition prior to the studies. Body composition 
(body fat, fat free mass (FFM), leg fat, upper body subcutaneous and visceral fat) will be 
measured using DEXA. A single slice CT abdomen will be performed to measure visceral fat. A 
treadmill exercise test will be conducted to assess fitness (peak VO2) > 1 week prior to any tracer 
studies in order to allow us to determine whether variation in muscle oxidative capacity (a 
surrogate for mitochondrial function) contributes to differences in lipotoxicity (78). We will be 
performing muscle and fat biopsies on two visits separated by a period of at least 4 weeks. 
Following admission to the CRU the evening prior to the studies a forearm intravenous catheter 
will be placed for infusion of niacin (or saline), insulin, glucose and isotopic tracers. T2DM 
volunteers taking insulin will be asked to withhold their long-acting insulin for 3 days prior to 
the study, manage their blood sugar with short acting insulin, and receive an overnight, 
monitored infusion of regular insulin to maintain blood glucose values between 4.5-5.5 mmol/L. 
At 2100 h the infusion of intravenous niacin (or saline) will begin. The initial niacin dose will be 
0.6 mg/min and, depending upon symptoms, the rate increased by 0.4 mg/min increments to a 
goal of 1.4 mg/min. We will obtain blood samples from the intravenous catheters at 2200, 1200, 
0200 and 0400 h to measure plasma FFA concentrations. The niacin infusion will be continued 
until1420 h. The niacin infusion will be gradually weaned off in a pattern designed to avoid a 

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rebound increase in FFA concentrations. Depending upon the degree of overnight suppression of 
FFA concentrations, the dose may be adjusted downwards or upwards (maximum rate of 2.8 
mg/min) in subsequent studies. At 0400 h an infusion of [U-13C]palmitate (330 nmol/min) will 
be initiated to trace FFA contribution to intramyocellular lipids in the euinsulinemic state 
(13,15).  At 0600 h an infusion of [6-2H2]glucose will be started to trace glucose metabolism and 
continued till the end of the study.  Resting energy expenditure will be measured by indirect 
calorimetry to permit interpretation of FFA flux data. A series of blood samples will be collected 
between 0830 and 0900 h to measure steady state plasma palmitate and glucose enrichment and 
concentrations. The volunteers will then undergo a vastus lateralis muscle biopsy as well as 
abdominal subcutaneous adipose biopsy together for measurement fatty acid metabolites and to 
provide material for the “pre-clamp” measures of insulin signaling; the niacin infusion will then 
be discontinued.  
 
Beginning at 0915 h the volunteers will receive a primed, constant infusion of insulin (1 
mU/kg/min) together with an infusion of 50% dextrose labeled with [6-2H2]glucose in order to 
maintain euglycemia. At the start of the insulin clamp the [U-13C]palmitate will be stopped and 
[2H9]palmitate (400 nmol/min) will be started to measure the contribution of FFA to 
intramyocellular lipids during hyperinsulinemia (35). At the end of the 5 hour insulin clamp a 
second muscle biopsy will be collected from the contralateral leg from the first biopsy to avoid 
the effects of local trauma. This biopsy is taken at 5 h to allow us to measure the accumulation of 
[2H9]palmitate in the intramyocellular lipid metabolites listed above, as well as to detect the loss 
of [U-13C]palmitate from the metabolites as a function of their turnover. A series of blood 
samples for glucose and palmitate enrichment will be collected just prior to each of these 
biopsies. 
 
The change in the ratio of phosphorylated to total insulin signaling effector proteins between the 
first and second muscle biopsy will be used to assess the insulin-mediated activation of insulin 
signaling pathways. We will assess insulin signaling in biopsies taken under basal and insulin 
stimulated conditions at the level of Akt phosphorylation using capillary Western blot 
approaches to quantify phosphorylation (80). Glucose disappearance rates under euglycemic, 
hyperinsulinemic conditions are an excellent reflection of muscle glucose uptake, and this will be 

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used as our index of muscle glucose disposal. Indirect calorimetry will be done prior to the first 
and second muscle biopsies to provide context for substrate utilization and energy expenditure.  
We will  perform abdominal subcutaneous adipose tissue biopsies in order to measure fat cell 
size and the inflammatory characteristics of adipose tissue because these factors have been 
related to insulin resistance in muscle. The biopsies will be done using sterile technique under 
local anesthesia and will be done on different sides of the abdomen.  
 
3.2 
Number of Subjects 
 
Ten upper body obese and 10 Type 2 diabetics (5 male and 5 female in each group) will be recruited 
to participate in this study to assure we have truly insulin resistant participants in whom 
improvements in insulin action can be expected.  
 
3.3 
Duration of Participation 
 
The duration of participation for each subject would take anywhere between 6 weeks to 3 
months, including the 4-8 week interval period, all the visits and overnight stay at the CRTU. 
The overall recruitment and completion of the study will take about 4 years due to disruptions in 
scheduling from the COVID pandemic.  
 
 

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Flowchart of Study Events 
 
 
 
 
Screening 
Niacin study 
Placebo (saline) 
Participants taking insulin will be 
asked to withhold their long-
acting insulin 3 days prior and 
use a short-acting insulin and an 
overnight 
regular 
insulin 
infusion.  
Washout 
 4-8 weeks 

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3.4 
Primary Study Endpoints 
 
To determine the contribution of plasma palmitate ([U-13C] and [2H9]palmitate) to muscle 
triglycerides, DG and ceramides in saline control and niacin (FFA suppressed) conditions. 
 
 
3.5 
Secondary Study Endpoints 
 
We will also measure the insulin signaling assays and glucose disposal rates, specifically to test 
the effects of the lower FFA concentrations on insulin signaling and glucose disposal  
 
We will assess whether the incorporation of plasma palmitate into muscle DG is related to 
changes in the proximal activation by phosphorylation of the insulin signaling pathway during 
the insulin clamp.  
3.6 
Primary Safety Endpoints 
 
The only know adverse effects of short term exposure to niacin are skin flushing and pruritus. 
We will document the duration and severity of any symptoms during the niacin infusion. (ICH 
E6, section 6.8) 
 
3.7 
Identification of Source Data 
 
The following source data will be directly recorded on the Case Report Form (CRF):  
• Patient Reported Outcomes – flushing 
• Smoking Status 
 

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The following source data will not be directly collected in the Case Report Form (CRF), but will 
be captured in supportive documentation (study source documents, EMR, study staff maintained 
excel spreadsheets): 
• Laboratory results and clinical interpretation of the values.  
• Blood pressure, temperature, pulse rate. Will be recorded in the EMR by CRTU nursing 
staff.  
• Height and weight are directly recorded in excel files by study staff. 
• Patient Questionnaire (VAS) flushing are recorded in the EMR by CRTU nursing staff. 
• CT scan analysis 
 
4 
Subject Selection Enrollment and Withdrawal 
4.1 
Inclusion Criteria 
 
Subjects who meet the following criteria may be included in the study: 
 
1. Males and females between 18 and 65 years of age who are able to comprehend 
instructions, follow study procedures, willing to sign an informed consent form, and 
consume an isoenergetic diet eating all meals from Mayo GCRC for at least 3 days prior 
to study.  
2. Overweight/Obese volunteers will have a BMI 29.0 – 40.0 kg/m2 
o Upper body/visceral obesity (UBO) in women will be those with a waist-hip 
ratio (WHR) > 0.85 and/or increased visceral fat by single slice CT scan and/or 
biochemical evidence of metabolic syndrome as determined by the investigators.  
Upper body obesity in men will be defined as a waist-hip ratio of >0.95 and/or 
increased visceral fat by single slice CT scan and/or biochemical evidence of 
metabolic syndrome. 
3. Volunteers with Type 2 Diabetes will have HbA1C between 6.5 and 11.5%. 
Hold metformin and sulfonylurea for 1 week prior study 
Avoid pioglitazone  due to long half life 

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4. Resting vital Signs are within the following range:  95 mmHg < systolic blood pressure < 
160 mmHg 
45 mmHg < diastolic blood pressure < 100 mmHg 
40 bpm < heart rate <100 bpm 
5. Female subjects are eligible if they meet the following criteria: 
o Are not pregnant or nursing 
o All women of childbearing potential will have a negative serum pregnancy test at 
screening and a negative urine pregnancy test within 48 hours before administering 
study drug. 
o All women of childbearing potential will use an appropriate contraceptive method 
including barrier method, oral contraceptive medication, contraceptive device or 
abstinence while participating in the study. 
6. Recent or Current research participation? 
If Yes look at consent form and f/u visits: 
• Medications that alter fat metabolism possibly given: if yes, exclude 
• Weight possible changing in the study 
• Amount of blood drawn during the study (if our study plus this one draw ≥ 450 ml 
these should be separated by 8 weeks 
7. Previous labs: 
• HbA1C < 6.5% for non-diabetic UBO 
• Fasting glucose < 126 mg/dl for non-diabetic UBO 
• Hb ≥ 11.0 for women and ≥ 12 for men 
• platelets > 100 000 
 
4.2 
Exclusion Criteria 
The following will exclude potential subjects from the study: 
1. Individuals with a history of a disease process such as: 
• Ischemic heart disease 
• Atherosclerotic valvular disease 
• Blood pressure greater than 160/95 despite antihypertensive medication 

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2. Smokers > 20 cigarettes per week 
3. Concomitant use of medications that can alter serum lipid profile: 
• High dose fish oil (>3g per day),  
• STATINS (if yes hold for 6 weeks and receive PCP’s approval),  
• Niacin  
• Fibrates 
• thiazolidinediones 
• Beta-blockers  
• Atypical antipsychotics      
      
 
 
 
4. Allergy to Lidocaine, niacin and/or Niaspan 
5. Subjects with any of the following parameters greater than 1.5 times the upper limit of 
normal: 
a. Serum creatinine 
b. Alkaline phosphatase 
c. Aspartate aminotransferase (AST) unless participant has fatty liver disease, in 
which case AST can be < 2 times upper limit normal 
d. Alanine aminotransferase (ALT) unless participant has fatty liver disease, in which 
case AST can be < 2 times upper limit normal 
e. Total bilirubin (unless the patient has documented Gilbert’s syndrome) 
 
4.3 
Subject Recruitment, Enrollment and Screening 
Subjects will be contacted by letters, email and telephone calls  (maximum number of contact 
attempts with subject will be 3). Copies of the letters, email scripts and telephone scripts will be 
submitted to IRB.  
 
At the time of enrollment the investigator or personnel involved in recruitment will address the 
following:  

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Amount of time needed for reading or reviewing the consent document Amount of time needed 
for the prospective subject to make a decision 
The location or methods of communicating and the related privacy needs for the initial and on-
going discussions. 
The need for including a medical interpreter, Legally Authorized Representative, family 
member(s), witness, or advocate who may need to be present and observe the discussions within 
the informed consent process. 
 
The screening procedure will involve the following: 
• Evaluation and documentation of inclusion/exclusion criteria 
• Blood draws to measure labs (see table 1) 
• Serum pregnancy test unless postmenopausal 
• DEXA scan, Data from DEXA scans conducted in the CRTU for other protocols within a 
3 month time period may be used in place of repeating the scan in order to reduce 
radiation risk.   
• CT scan.* 
• Meeting with the registered dietician to discuss food preferences, food allergies, etc. for 
the three-day meal plans.  
• Maximum physical exertion level will be examined. This will include periodic 
monitoring of heart rate and blood pressure. An EKG will also be a part of this test. 
* Single slice abdominal L2-L3 CT scans conducted in a previous 3 month window, may be used 
in place of repeating the CT scan.  
4.3.1 Early Withdrawal of Subjects 
4.3.2 When and How to Withdraw Subjects 
 
 
Subjects must be withdrawn from the study for any of the following reasons: 
•  
Withdrawal of informed consent 
•  
Pregnancy 
•  
If the investigator concludes that it would be in the subject’s best interest for any reason 
Subjects may voluntarily withdraw from the study for any reason at any time. 

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If withdrawal occurs for any reason, the investigator will document the reason for withdrawal in 
the source notes.   
 
 
4.3.3 Data Collection and Follow-up for Withdrawn Subjects 
 
 
Subjects who withdraw after enrollment will be replaced to complete the study. 
 
5 
Study Drug  
5.1 
Description 
 
The investigational product to be studied will be an infusion of nicotinic acid (niacin) or saline. 
The chemical formula is C6H5NO2. It is a white, crystalline, powder that is reconstituted for 
intravenous infusion. It is soluble in water.  
 
A. Mechanism of Action 
As B complex vitamins, niacin and niacinamide have identical physiological effects. However, 
these vitamins differ in some of their actions. Niacin produces peripheral vasodilation and 
flushing, an effect that generally does not occur with niacinamide. Niacinamide is formed in vivo 
from metabolism of niacin. Niacinamide is an essential precursor of nicotinamide adenine 
dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which are the 
physiologically active forms of niacin. Serving as coenzymes for several dehydrogenases, NAD 
and NADP are functional groups of electron-transfer agents active in cellular respiration, 
glycolysis, and lipid synthesis. When taken in amounts considerably greater than needed to meet 
vitamin requirements, niacin causes a partial suppression of the release of free fatty acids from 
adipocytes. 
Niacin can lower serum triglyceride and LDL-cholesterol concentrations. Rate of hepatic TG 
synthesis are decreased and thereby decreased VLDL. Subsequent clinically lowered apo B and 
LDL concentrations are found (81). 
 
 

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B. Pharmacokinetics 
Absorption 
Nicotinic acid undergoes rapid first-pass hepatic metabolism after oral ingestion, which 
we will avoid by infusing it intravenously. Niacin concentrations in the systemic 
circulation are dose-dependent.  
A) Bioavailability – intravenous – 100% 
B) Effects of Food – none known with intravenous niacin 
 
Distribution 
Nicotinic acid is widely distributed and in concentrated in liver, spleen, and adipose 
tissue.  
 
Metabolism 
When administered orally the pharmacokinetic profile of niacin is complicated due to 
extensive first-pass metabolism. The profile is dose-rate specific and, at the doses used to 
treat dyslipidemia, saturable. In humans, one pathway of metabolizing niacin is through a 
simple conjugation step with glycine to form nicotinuric acid (NUA). NUA is excreted in 
the urine, although there may be a small amount of reversible metabolism back to niacin. 
The other metabolic pathway results in the formation of nicotinamide adenine 
dinucleotide (NAD). It is unclear whether nicotinamide is formed as a precursor to, or 
following the synthesis of, NAD. Nicotinamide is further metabolized to at least N-
methylnicotinamide (MNA) and nicotinamide-N-oxide (NNO). MNA is further 
metabolized to two other compounds, N-methyl2-pyridone-5-carboxamide (2PY) and N-
methyl-4-pyridone-5-carboxamide (4PY). The formation of 2PY appears to predominate 
over 4PY in humans. The activity of these metabolites is unknown. 
 
Excretion 
Following single and multiple doses, approximately 60 to 76% of the niacin dose 
administered orally is recovered in urine as niacin and metabolites; up to 12% was 
recovered as unchanged niacin after multiple dosing. The ratio of metabolites recovered 
in the urine was dependent on the dose administered. Niacin is excreted as nicotinuric 

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acid in urine after conjugation with glycine. Whether the same distribution of metabolites 
in the urine occurs with intravenous as with oral administration is unknown. 
5.2 
Treatment Regimen 
Subjects will receive a titrated dose starting from 0.6 mg/min to a maximum of 2.8 mg/min 
(likely needed dose = 1.4 mg/min) or 0.9% NaCl for 12 hours.  
5.3 
Method for Assigning Subjects to Treatment Groups 
 
Subjects will first receive niacin and for the second study they will receive intravenous saline as 
the placebo.  
 
Preparation and Administration of Study Drug 
Nicotinic acid will be received in powder form and compounded at Research Pharmacy 
following Compounding Procedures and an approved compounding formula sheet, for 
intravenous administration which runs in a Baxter infusion pump model Sigma Spectrum. The 
drug will be aliquoted in single dose amber vials of 10 mL each and refrigerated at a temperature 
of 2-8°C. 
5.4 
Subject Compliance Monitoring 
We are not expecting non-compliance as the drug administration is a constant monitored 
intravenous infusion. In cases where a subject requests to stop study drug, it will be recorded and 
reported as a note to file or a protocol deviation.  
 
5.5 
Prior and Concomitant Therapy 
The following medications will be withheld 3 days prior to beginning of the study: 
• Participants with Type 2 Diabetes on insulin will have their long-acting insulin replaced 
with a short-acting insulin.   

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5.6 
Packaging 
Contents of kits/boxes associated with labelling: The study drug or saline/placebo is labelled 
with a patient specific label by the RESEARCH pharmacy. This label shall bear the statement 
"Caution: New Drug--Limited by Federal (or United States) law to investigational use." 
 
5.7 
Masking/Blinding of Study  
This study is not a blinded study. 
 
5.8 
Receiving, Storage, Dispensing and Return 
5.8.1 Receipt of Drug Supplies 
Nicotinic acid will be obtained in powder form and be compounded in the Research Pharmacy 
for intravenous administration, following Compounding Procedures and an approved 
compounding formula worksheet specific to each patient.  
Upon receipt of the drug, an inventory will be performed and logged by research pharmacy 
technicians accepting the shipment. 
 
5.8.2 Storage 
Niacin storage: The investigational drug will be packaged in 10 mL amber vials and be refrigerated 
away from light in a temperature setting of 2-8°C. After compounding, a sterility date applied is 
90 days with refrigerated storage. Niacin preparation is sterilized by a sterile filtration procedure 
using a sterile 0.2 micron filter. 
 
Placebo storage: 0.9% NaCl will be stored at USP controlled temperature.   
 
Storage location: Study drug and placebo will be stored at SMH Research Pharmacy that is 
secure and can be only accessed by research pharmacy personnel.  
 
Light conditions: Amber glass vials provide light protection. 
 

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Container closure components: 10 mL amber Type 1 glass vials Wheaten item # 223761 are used 
for the container.  The vials are depyrogenated through a washing cycle and are sterilized through 
a steam sterilization cycle. 
The stoppers are West Pharmaceutical Flurotec 20 mm stoppers item #19700021.  We purchase 
them as ‘ready to sterilize’ and subsequently sterilize them through repackaging and a steam 
sterilization cycle. 
 
Sterility and bacterial endotoxins test methods: Sterility testing is accomplished through membrane 
filtration, using Millipore Steritest canisters.  In a sterile environment, the contents of an 
appropriate number of vials (based on USP 71) are filtered through the canister set and are flushed 
with 0.9% sodium chloride for injection, USP.  The canisters are incubated at appropriate 
temperatures for the media for 14 days. 
Endotoxin testing is completed by an outside laboratory (ARL Bio Pharma).  Limits are calculating 
according to USP 85 or if present, the USP monograph for the injection. 
 
5.8.3 Dispensing of Study Drug 
 
Regular study drug reconciliation will be performed to document drug assigned, drug dispensed, 
drug returns, and drug remaining.  This reconciliation will be logged on the drug reconciliation 
form and reported by Research Pharmacy. 
5.8.4 Return or Destruction of Study Drug 
All medications would be given on site. Any patient specific unused medication will be 
destroyed on site.  
 
6 
Study Procedures 
 
Table 1 below lists all of the assessments and indicates with an “x” the visits when they are 
performed. Subjects should be seen for all visits on the designated day and time. 
 
Subjects who are discontinued before completing the study, and those who withdraw prematurely 
from the study for any reason, should be scheduled for a visit as soon as possible, at which all of 
the assessments listed for the “end of Study” visit will be performed. Study visits will occur at 
Screening, Visit 1 and Visit 2/End of Study. 

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The maximum duration of the clinical investigation is up to 8 weeks from enrollment (signing of 
consent, which allows for a 14 day screening period). 
Schedule of Events 
 
 
S
d
A
i i
Screening 
Inpatient Visit 1 
Inpatient Visit 2 
                                   Study Day 
-14 
0 
28-56 
Written informed consent 
X 
 
 
History and demographics 
X 
 
 
Concurrent med reconciliation 
X 
 
 
Height 
X 
 
 
Weight 
X 
X 
X 
Vitals 
X 
X 
X 
Laboratory tests a 
X* 
 
 
Single-slice abdominal CT 
X 
 
 
DEXA scan 
X* 
 
 
Adverse event evaluation 
X 
X 
X 
Pregnancy test 
Xb 
Xc 
 
Study Agent (niacin administration) 
 
X 
 
Insulin and glucose administration 
 
X 
X 
Isotope administration 
 
X 
X 
Resting energy expenditure 
 
X 
X 
 Muscle Biopsies 
 
X 
X 
Adipose biopsies 
 
X 
X 

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a: Complete metabolic panel, CBC w/differential, Lipid Panel, HbgA1C, liver function tests if not part 
of complete metabolic panel 
b:Serum pregnancy test (women of childbearing potential) at screening 
c: Urine pregnancy test (women of childbearing potential) ≤ 48 hours prior to study drug 
administration. 
*Research DEXA scan, labs, and CT scan completed in the previous 3 months may be used in place 
of repeating these.  
 
6.1 
Screening Visit  
 
The data that will be collected on each subject at the screening visit are as follows: 
• Confirmation of informed consent 
• Vital signs (Blood pressure, pulse rate, respiratory rate, and body temperature) 
• Body weight and height 
• Confirmation of meeting all the Inclusion criteria and none of the exclusion criteria 
• Demographics and medical history  
• Pregnancy test – serum pregnancy test (for females of childbearing potential) 
• Laboratory examinations: Complete Blood Count, Fasting lipid profile, Alkaline 
Phosphatase, Electrolyte Panel, AST, ALT, HbA1c (for diabetics), Fasting Glucose, Serum 
Creatinine.  Overnight fasting is required and will be documented in the EMR by CRTU 
nursing staff. 
• Body composition as measured by Dual-energy x-ray absorptiometry* (see section 4.3) 
and abdominal CT scan * (see section 4.3). 
 
  
 
6.2 
Visit 1 (In-patient visit, 1 night stay) 
At visit 1 the subjects will undergo the following: 
 

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• Admission to in-patient facility the evening prior to scheduled biopsies 
• Negative urine pregnancy test (for females of childbearing potential only) 48 hours prior 
to dosing. 
• Vital signs (blood pressure, pulse rate, respiratory rate, and body temperature) 
• Body weight 
• Concomitant and non-drug therapies 
• Niacin or saline infusion started the evening prior 
 
Volunteers will be asked to stop taking any aspirin or ibuprofen from three days before to three 
days after their overnight stays.  
 
All participants will consume an isoenergetic diet (weight stable, 45% carbohydrate, 20% protein 
and 35% fat) from the Mayo Clinical Research and Trials Unit (CRTU) for 1-3 days prior to the 
niacin infusion study and 3 days before the saline control study. This will assure consistency of 
energy intake and nutrient composition prior to the study saline control study that is helpful in 
reducing variability in plasma FFA concentrations.  
Following admission to the CRU the evening prior to the studies a forearm intravenous catheter 
will be placed for infusion of niacin, insulin, glucose and isotopic tracers. T2DM volunteers 
taking insulin will be asked to withhold their long-acting insulin for 3 days prior to the study, 
manage their blood sugar with short acting insulin, and receive an overnight, monitored infusion 
of regular insulin to maintain blood glucose values between 4.5-5.5 mmol/L. At 2100  h the 
infusion of intravenous niacin (or saline) will begin. The initial niacin dose will be 0.6 mg/min 
and, depending upon symptoms, the rate increased by 0.4 mg/min increments to a goal of 1.4 
mg/min. A retrograde intravenous catheter will be placed to allow for blood sampling at 2200 h, 
2400 h, 0200 and 0400 h. The niacin/saline infusion will be continued until 0900 h the following 
morning. The niacin infusion will be weaned off gradually in order to prevent a rebound increase 
in plasma FFA.  Depending upon the degree of overnight suppression of FFA concentrations, the 
dose may be adjusted downwards or upwards in subsequent studies to a maximum of 2.8 mg/min 
in subsequent studies. At 0400 h an infusion of [U-13C]palmitate (330 nmol/min) will be initiated 
to trace FFA contribution to intramyocellular lipids in the euinsulinemic state (13,15).  At 0600 h 
an infusion of [6-2H2]glucose will be started to trace glucose metabolism and continued till the 

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end of the study.  Resting energy expenditure will be measured by indirect calorimetry to permit 
interpretation of FFA flux data. A series of blood samples will be collected between 0830 and 
0900 h to measure steady state plasma palmitate and glucose enrichment and concentrations.  
 
The volunteers will then undergo a vastus lateralis muscle biopsy for measurement fatty acid 
metabolites and to provide material for the “pre-clamp” measures of insulin signaling and the 
niacin infusion will be tapered to 25% of their overnight dose.  
 
Insulin regulation of muscle glucose uptake and fatty acid metabolism will be assessed using a 
primed, constant infusion of insulin at a dose of 1.0 mU/kg/min. Arterialized venous blood 
samples will be collected from the same retrograde IV catheter using the hot box with a 
temperature of 131 degrees Fahrenheit, to allow measurement of hormone, tracers and substrate 
concentrations over the 30 min prior to the insulin clamp and the last 30 min of the insulin 
clamp. Blood glucose will be measured every 10 min throughout to allow the glucose infusion 
rate to be adjusted to maintain euglycemia. Resting energy expenditure will be measured by 
indirect calorimetry to permit interpretation of FFA flux data. (79).  
 
Adipose tissue biopsies will be collected from the abdominal region under local anesthesia using 
sterile technique (82) at the same time as the first and last muscle biopsies. The adipose tissue 
samples (2-3 g from each site) will be immediately rinsed of blood and transported to our 
laboratory (~ 3 min walk from the CRTU).  
 
There we will divide the specimen into smaller aliquots for collagenase digestion to be used for 
cell sizing (83), and immunohistochemistry.  
 
The volunteers will be able to order a general meal after the biopsies. After this, the participants 
will be discharged from the CRTU. 
 

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6.3 
Visit 2 (In-patient visit, 1 night stay) 
After ~4 weeks subjects will return to the CRTU and the same procedure and guidelines as Visit 
1 will be repeated with the saline/placebo.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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7 
Statistical Plan 
7.1 
Sample Size Determination 
 
Power calculation: 
Acipimox increased insulin-stimulated glucose disposal in undifferentiated obese subjects by 
~10% (P = 0.07) and in T2DM by ~ 50%, whereas insulin-stimulated Akt phosphorylation 
increased by ~80% in both groups (66); IRS-1 tyrosine phosphorylation increased only in obese 
by 300%. In our hands, the reproducibility of insulin-stimulated glucose disposal is within ~5% 
with no intervention. If we obtain similar responses as Liang et al (66), we would have 90% 
power to detect a 10% improvement in insulin sensitivity in the obese group at a P of 0.05 with 5 
subjects per group. However, because women may respond differently than men to changes in 
FFA (85), we propose to include more participants in order to allow sex-specific analysis. By 
selecting UBO volunteers we will likely have a more insulin resistant group than Liang (66), 
which should improve our statistical power to detect changes in lipid metabolites and insulin 
signaling. We can also expect some biological heterogeneity in the responses, which should be 
helpful in that we intend to understand the extent to which changes in insulin sensitivity relate to 
changes in intramyocellular DG and ceramides (86) and insulin signaling, for example by 
grouping participants post hoc to groups that have the greatest and least response to FFA 
suppression. 
 
7.2 
Statistical Methods 
 
Descriptive Statistics 
Baseline values for demographic, clinical, and outcome variables (primary and secondary) will 
be tabulated for the treatment groups.  These analyses will help identify potential confounding 
variables to be used as covariates in sensitivity analyses.  Distributions across subgroups used in 
randomization will be compared to assess whether the randomization was successful in 
equalizing distributions of these prognostic variables across treatment groups.   

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Our hypothesis will be tested by measuring the concentration of plasma palmitate (and total 
FFA) as well as the enrichment ([U-13C] and [2H9]palmitate), and therefor flux of palmitate. We 
will measure the incorporation of [U-13C] and [2H9]palmitate in DG and ceramides relative to 
plasma palmitate, as well as the insulin signaling assays and glucose disposal rates, specifically 
to test the effects of the lower FFA concentrations and lesser contribution of plasma FFA 
combined with improvements in insulin signaling and glucose disposal (ANCOVA). If glucose 
disposal and insulin signaling improvements in each group are accompanied by selective 
reductions in either ceramides or DG (especially the saturated species), this will suggest which of 
these is most likely responsible for the baseline impairments relative to the lean control state. It 
is possible that the UBO and T2DM will have different responses in ceramides and DG. For ex-
ample, the portion of insulin resistance due to lipotoxicity in T2DM could be the combined effect 
of both DG and ceramides, whereas in UBO may be just one of these compounds. If the insulin 
signaling measures coincide with the changes in ceramides and DG in such a way as to confirm 
the pathways that have been described using cell/animal models are operative in humans, in vivo, 
this will provide an important confirmation that we can translate basic findings to human 
physiology. To the extent reduced lipolysis reduces FFA during the insulin clamp, we anticipate 
a lesser contribution of the [9-2H]palmitate to DG and ceramides (repeated measures ANOVA). 
 
A subtext to the DG and ceramide enrichment measures relative to plasma and palmitoyl-
CoA is that, to the extent these molecules are synthesized from sources other than FFA, this will 
provide clues to alternate pathways that may be active in generating lipid signaling molecules. 
We will be able to understand whether excess ceramides are due to de novo synthesis as opposed 
to sphingomyelin hydrolysis and whether elevated DG is due to de novo synthesis or other 
sources, potentially including phospholipid hydrolysis. We believe that our ability to go beyond 
measures of concentrations by making more mechanistic assessments of the source of 
lipotoxicity is a strength of the proposed studies.   
 
Handling of Missing Data: 
The frequency and pattern of missing data will be evaluated to assess whether  the data is 
missing completely at random, or whether a pattern exists that may suggest a  systematic bias 

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with respect to the presence or absence of data. If the missing data appear to  be missing 
completely at random, and if the amount of missing data is relatively small,  
the analysis will utilize all available data. Depending on the pattern and amount of missing data, 
multiple imputation methods may be considered, however given the proposed study has 
relatively modest sample size, the amount of missing data is anticipated to be small. 
 
Primary Hypothesis:  
In order to test Hypothesis 1 we will use the following pieces of data: a) subsarcolemmal (SS - 
largely equivalent to membrane associated DG (68)) DG concentrations, focusing primarily, but 
not solely, on 16:0/18:0, 16:0/16:0 and 18:0/18:0, as well as the enrichment in 16:0/18:0, 
16:0/16:0 and 16:0/18:1 relative to plasma palmitate. This will provide us with as an index of de 
novo synthesis of DG from FFA; b) the baseline and insulin-stimulated changes in 
phosphorylation of Akt (66, 80); c) the increase in glucose disposal as a function of the increase 
in plasma insulin concentrations.  
We will assess whether the SS DG concentrations and the incorporation of plasma FFA into DG 
(ratio of DG palmitate:plasma palmitate) differs between UBO and T2DM (ANOVA).  
 
We will also test whether the concentrations or incorporation of plasma palmitate into the SS vs. 
IMF DG is related to changes in the proximal activation by phosphorylation of the insulin 
signaling pathway during the insulin clamp.  
 
Secondary Hypotheses:  
 
To address hypothesis 2 we will use a similar statistical approach, except using intramyocellular 
ceramide concentrations and enrichments relative to plasma palmitate in conjunction with Akt 
phosphorylation. Robust Akt phosphorylation in the face of high ceramide 
concentrations/synthesis from FFA would argue against the ceramide-PP2A mechanism of 
insulin resistance. 
 
Interim Analysis 
No interim analysis is proposed. 

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7.3 
Subject Population(s) for Analysis 
 
• All-treated population:  Any subject entered into the study started the drug infusion and/or 
biopsy procedure. 
• Protocol-compliant population:  Any subject who was entered and received the protocol 
required study drug exposure and required protocol processing 
• All-completed population:  Only subjects who completed ALL study related procedures 
and follow-up will be included.  
8 
Safety and Adverse Events 
Safety Review Plan –Study progress and safety will be reviewed quarterly with the research 
fellow and study coordinator and, because these are relatively small studies, the data summarized 
at 6 month intervals to allow for a large enough sample to assess outcomes.  Progress reports, 
including patient recruitment and AE's will be provided to the Endocrine Research Committee 
following each of the 6 month reviews in the event that no participants are accrued in the study a 
report will not be provided.  An annual report will be compiled and will include a list and 
summarization of adverse events at the time of the non-competing renewal.  In addition, the 
annual report will address (1) whether adverse event rates are consistent with pre-study 
assumptions; (2) reason for dropouts from the study; (3) whether all participants met entry 
criteria; (4) whether continuation of the study is justified on the basis that additional data are 
needed to accomplish the stated aims of the study.  The annual report will be reviewed by the 
Endocrine Research Committee and the results included in the minutes. These will be forwarded 
to the IRB and NIDDK and, if applicable, the Mayo CTSA CRU and FDA as indicated.  
8.1 
Definitions 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
Any unanticipated problem or adverse event that meets the following three criteria:  
• Serious: Serious problems or events that results in significant harm, (which may be 
physical, psychological, financial, social, economic, or legal) or increased risk for the 

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subject or others (including individuals who are not research subjects). These include: (1) 
death; (2) life threatening adverse experience; (3) hospitalization - inpatient, new, or 
prolonged; (4) disability/incapacity - persistent or significant; (5) birth defect/anomaly; (6) 
breach of confidentiality and (7) other problems, events, or new information (i.e. 
publications, DSMB reports, interim findings, product labeling change) that in the opinion 
of the local investigator may adversely affect the rights, safety, or welfare of the subjects 
or others, or substantially compromise the research data, AND 
• Unanticipated: (i.e. unexpected) problems or events are those that are not already 
described as potential risks in the protocol, consent document, not listed in the 
Investigator’s Brochure, or not part of an underlying disease. A problem or event is 
"unanticipated" when it was unforeseeable at the time of its occurrence. A problem or 
event is "unanticipated" when it occurs at an increased frequency or at an increased 
severity than expected, AND 
• Related: A problem or event is "related" if it is possibly related to the research procedures. 
 
 
Adverse Event 
An untoward or undesirable experience associated with the use of a medical product (i.e. drug, 
device, biologic) in a patient or research subject. 
Serious Adverse Event 
Adverse events are classified as serious or non-serious.  Serious problems/events can be well 
defined and include; 
• death 
• life threatening adverse experience 
• hospitalization 
• inpatient, new, or prolonged; disability/incapacity 
• persistent or significant disability or incapacity 
• birth defect/anomaly 
 

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and/or per protocol may be problems/events that in the opinion of the sponsor-investigator 
may have adversely affected the rights, safety, or welfare of the subjects or others, or 
substantially compromised the research data. 
 
Other important medical events are those that may not be immediately life threatening, but are 
clearly of major clinical significance.  They may jeopardize the subject, and may require 
intervention to prevent one of the other serious outcomes noted above. 
 
All adverse events that do not meet any of the criteria for serious, should be regarded as non-
serious adverse events.  
 
Adverse Event Reporting Period 
For this study, the follow-up period is up to 6 weeks from the 1st biopsy visit. Any adverse event 
occurring during the course of the study period will be reported.  
 
Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  A preexisting condition 
should be recorded as an adverse event if the frequency, intensity, or the character of the 
condition worsens during the study period. 
 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting 
condition.  At the end of the study, any new clinically significant findings/abnormalities that 
meet the definition of an adverse event must also be recorded and documented as an adverse 
event.  
 
Post-study Adverse Event 
All unresolved adverse events should be followed by the sponsor-investigator until the events are 
resolved, the subject is lost to follow-up, or the adverse event is otherwise explained.  At the last 
scheduled visit, the sponsor-investigator should instruct each subject to report, to the sponsor-

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investigator, any subsequent event(s) that the subject, or the subject’s personal physician, 
believes might reasonably be related to participation in this study.  
 
Abnormal Laboratory Values 
Research lab results taken between study drug administration and study completion are expected 
to be abnormal as a result of adipose tissue and muscle biopsies (catecholamines) done during 
the study. Screening lab values are reviewed by the physician principal investigator in the EMR 
and documented as reviewed in the EMR. Clinical lab results obtained by a primary care 
provider or other physician that is not involved in the study in the time that a participant receives 
the study drug will not be assessed for abnormality.   
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization should be 
documented and reported as a serious adverse event unless specifically instructed otherwise in 
this protocol.  Any condition responsible for surgery should be documented as an adverse event 
if the condition meets the criteria for an adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an 
adverse event in the following circumstances:  
• Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures 
for a preexisting condition.  Surgery should not be reported as an outcome of an adverse 
event if the purpose of the surgery was elective or diagnostic and the outcome was 
uneventful. 
• Hospitalization or prolonged hospitalization required to allow efficacy measurement for 
the study. 
• Hospitalization or prolonged hospitalization for therapy of the target disease of the study, 
unless it is a worsening or increase in frequency of hospital admissions as judged by the 
clinical investigator. 
 
  

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8.2 
Recording of Adverse Events 
At each contact with the subject, the study team will seek information on adverse events by 
specific questioning and, as appropriate, by examination.  Information on all adverse events will 
be recorded immediately in the source document, and also in the appropriate adverse event 
section of the case report form (CRF) or in a separate adverse event worksheet.  All clearly 
related signs, symptoms, and abnormal diagnostic, laboratory or procedure results will be 
recorded in the source document. 
 
Adverse events are considered events that are not already described as routine research risks in 
the consent form. We will not report pain at time of the biopsy as an AE, nor will we report 
bruising from biopsy or line placement procedures as these are nearly guaranteed to occur with 
all participants. All adverse events occurring during the study period will be recorded.  The 
clinical course of each event would be followed until resolution, stabilization, or until it has been 
ultimately determined that the study treatment or participation is not the probable cause.  Serious 
adverse events that are still ongoing at the end of the study period must be followed up, to 
determine the final outcome.  Any serious adverse event that occurs during the Adverse Event 
Reporting Period and is considered to be at least possibly related to the study treatment or study 
participation will be recorded and reported immediately. 
 
We will use the following standardized system for classification and grading of Adverse Events 
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_8.5x11.pdf 
 
8.3 
Reporting of Serious Adverse Events and Unanticipated Problems 
 
When an adverse event has been identified, the study team will take appropriate action necessary 
to protect the study participant and then complete the Study Adverse Event Worksheet and log.  
The sponsor-investigator will evaluate the event and determine the necessary follow-up and 
reporting required. 
 

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8.3.1 Sponsor-Investigator reporting: notifying the Mayo IRB 
 
The IRB requirements reflect the guidance documents released by the Office of Human Research 
Protections (OHRP), and the Food and Drug Administration (FDA) in early 2007 and are 
respectively entitled “Guidance on Reviewing and Reporting Unanticipated Problems Involving 
Risks to Subjects or Others and Adverse Events” and “Guidance for Clinical Investigators, 
Sponsors, and IRBs: Adverse Event Reporting – Improving Human Subject Protection.” 
 
The sponsor-investigator will report to the Mayo IRB any UPIRTSOs and Non-UPIRTSOs 
according to the Mayo IRB Policy and Procedures. 
 
According to Mayo IRB Policy any serious adverse event (SAE) which the Principal Investigator 
has determined to be a UPIRTSO would be reported to the Mayo IRB as soon as possible but no 
later than 5 working days after the investigator first learns of the problem/event:  
 
The following information to collect when developing any forms for documentation of adverse 
events. Adverse events are considered events that are not already described as routine research 
risks in the consent form. We will not report pain at time of the biopsy as an AE, nor will we 
report bruising from biopsy or line placement procedures as these are nearly guaranteed to occur 
with all participants.  
Information collected on the adverse event worksheet (and entered in the research database):  
• Subject’s name:  
• Medical record number:  
• Disease/histology (if applicable):  
• The date the adverse event occurred:  
• Description of the adverse event:  
• Relationship of the adverse event to the research (drug, procedure, or intervention):  
• If the adverse event was expected:  
• The severity of the adverse event: (use a table to define severity scale 1-5**) 
• If any intervention was necessary:  
• Resolution: (was the incident resolved spontaneously, or after discontinuing treatment) 

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• Date of Resolution:  
 
The sponsor-investigator will review all adverse event reports to determine if specific reports 
need to be made to the IRB and FDA.  The sponsor-investigator will sign and date the adverse 
event report when it is reviewed.  For this protocol, only directly related SAEs/UPIRTSOs will 
be reported to the IRB. 
 
The use of a table of tables to define Relationship of an Event to the Research and to define the 
severity of each event. 
 
Relationship Index  
The relationship of an AE to the  Investigational Drug is a clinical decision by the sponsor-
investigator (PI) based on all available information at the time of the completion of the CRF and 
is graded as follows: 
 
1. Not related: a reaction for which sufficient information exists to indicate that the etiology is 
unrelated to the study drug; the subject did not receive the study medication or the temporal 
sequence of the AE onset relative to administration of the study medication is not reasonable or 
the event is c1early related to other factors such as the subject' s clinical state, therapeutic 
intervention or concomitant therapy. 
 
2. Unlikely: a clinical event, including laboratory test abnormality, with a temporal relationship 
to drug administration which makes a causal relationship improbable and in which other drugs, 
chemicals, or underlying disease provide plausible explanations. 
 
3. Possible: a clinical event, including laboratory test abnormality, with a reasonable time 
sequence to administration of the drug but which could also be explained by concurrent disease 
or other drugs or chemicals; information on drug withdrawals may be lacking are unclear. 
 
4. Probable: a clinical event including laboratory test abnormality, with a reasonable time 
sequence to administration of the drug, unlikely to be attributed to concurrent disease or other 

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drugs or chemicals and which follows a clinically reasonable response on withdrawal (de-
challenge): re-challenge information is not required to fulfi1 this definition. 
 
5. Definite: a reaction that follows a reasonable temporal sequence from administration of the 
drug, or in which the drug level has been established in body fluids or tissues, that follows a 
known or expected response pattern to the suspected drug, and that is confirmed by improvement 
on stopping or reducing the dosage of the drug, and reappearance of the reaction on repeated 
exposure (re-challenge). 
 
 
Severity Index 
The maximum intensity of an AE during a day should be graded according to the definitions 
below and recorded in details as indicated on the CRF. If the intensity of an AE changes over a 
number of days, then separate entries should be made having distinct onset dates.  
 
1. Mild: AEs are usually transient, requiring no special treatment, and do not interfere with 
patient’s daily activities. 
 
2. Moderate: AEs typically introduce a low level of inconvenience or concern to the patient and 
may interfere with daily activities but are usually ameliorated by simple therapeutic measures. 
 
3. Severe: AEs interrupt a patient's usual daily activity and traditionally require systemic drug 
therapy or other treatment. 
 
8.3.2 Sponsor-Investigator reporting: Notifying the FDA 
 
 
The sponsor-investigator will report to the FDA all unexpected, serious suspected adverse 
reactions according to the required IND Safety Reporting timelines, formats and requirements. 
 

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Unexpected fatal or life threatening suspected adverse reactions where there is evidence to 
suggest a causal relationship between the study drug/placebo and the adverse event, will be 
reported as a serious suspected adverse reaction.  This will be reported to the FDA on FDA Form 
3500A, no later than 7 calendar days after the sponsor-investigator’s initial receipt of the 
information about the event. 
 
Other unexpected serious suspected adverse reactions where there is evidence to suggest a causal 
relationship between the study drug/placebo and the adverse event, will be reported as a serious 
suspected adverse reaction.  This will be reported to the FDA on FDA Form 3500A, no later than 
15 calendar days after the sponsor-investigator’s initial receipt of the information about the 
event. 
 
Any clinically important increase in the rate of serious suspected adverse reactions over those 
listed in the protocol or product insert will be reported as a serious suspected adverse reaction.  
This will be reported to the FDA on FDA Form 3500A no later than 15 calendar days after the 
sponsor-investigator’s initial receipt of the information about the event. 
 
Findings from other studies in human or animals that suggest a significant risk in humans 
exposed to the drug will be reported.  This will be reported to the FDA on FDA Form 3500A, no 
later than 15 calendar days after the sponsor-investigators initial receipt of the information about 
the event. 
 
8.4 
Unmasking/Unblinding Procedures 
 
Not applicable to our study design.  
8.5 
Stopping Rules  
 
This is unlikely as our study does not involve a treatment or interventional strategy and subjects 
are dismissed on the day of biopsies. However the entire study may be discontinued at the 
discretion of the PI based on the occurrence of the following: 

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• Adverse effects that seriously impact the risk-benefit ratio have been observed 
• Medical or ethical reasons affecting the continued performance of the study 
• Difficulties in recruitment of subjects 
• Any new information becomes available during the trial that necessitates stopping the 
trial 
• Other situations that may warrant stopping the trial 
 
Subjects will be informed that they are free to withdraw from the study at any time for any 
reason. The Principal Investigator (PI) may remove a subject from the study if, in the PI’s 
opinion, it is not in the best interest of the subject to continue the study.  Subjects may be 
discontinued due to a change in compliance with an inclusion/exclusion criterion that is 
clinically relevant and affects subject safety, occurrence of AEs, occurrence of pregnancy, or 
administration of non-permitted concomitant medication that might affect subject safety or study 
assessments/objectives.  Notification of discontinuation will be made immediately to the 
Sponsor’s Medical Monitor.  In case of premature discontinuation of study participation, efforts 
will be made to perform end of study/follow up assessments.  The date the subject is withdrawn 
from the study and the reason for discontinuation will be recorded on the subject’s Case Report 
Form (CRF).  All withdrawn subjects will be followed until resolution of any AEs or until any 
unresolved AEs are judged by the PI to have stabilized. 
 
8.6 
Medical Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of the study at his/her 
site.  This safety monitoring will include careful assessment and appropriate reporting of adverse 
events as noted above, as well as the construction and implementation of a site data and safety-
monitoring plan (see section 10  “Study Monitoring, Auditing, and Inspecting”).  Medical 
monitoring will include a regular assessment of the number and type of serious adverse events. 
 
The investigator will allocate adequate time for such monitoring activities.  The Investigator will 
also ensure that the monitor or other compliance or quality assurance reviewer is given access to 

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all the study-related documents and study related facilities (e.g. pharmacy, diagnostic laboratory, 
etc.), and has adequate space to conduct the monitoring visit. 
 
 
 
Safety Review Plan –Study progress and safety will be reviewed monthly with the research 
fellow and study coordinator and, because these are relatively small studies, the data summarized 
at 6 month intervals to allow for a large enough sample to assess outcomes.  Progress reports, 
including patient recruitment and AE's, if any occur, will be provided to the Endocrine Research 
Committee following each of the 6 month reviews if any studies have been done.  An annual report 
will be compiled and will include a list and summarization of adverse events at the time of the 
non-competing renewal.  In addition, the annual report will address (1) whether adverse event rates 
are consistent with pre-study assumptions; (2) reason for dropouts from the study; (3) whether all 
participants met entry criteria; (4) whether continuation of the study is justified on the basis that 
additional data are needed to accomplish the stated aims of the study.  The annual report will be 
reviewed by the Endocrine Research Committee if any studies have been done and the results 
included in the minutes. These will be forwarded to the IRB and NIDDK if applicable, the Mayo 
CTSA CRU and FDA as indicated.   
8.6.1 Internal Data and Safety Monitoring Board 
Designation of an Independent Monitor – The Endocrine Research Committee, composed of 
experienced investigators with appointments in the Division of Endocrinology, will serve as the 
independent monitor for the parts of the study. The PI will present relevant information to  
the committee at the indicated intervals and answer questions. The findings and 
recommendations of the committee will be included in the meeting minutes. If the PI presenting 
the data is the Chair of the Endocrine Research Committee another member of the committee 
will serve as chair for that meeting. The Endocrine Research Committee meets monthly. The  
nursing staff in the CRTU will also be involved with the monitoring of the respective protocols. 
 

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9 
Data Handling and Record Keeping 
9.1 
Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  
Those regulations require a signed subject authorization informing the subject of the following:  
• What protected health information (PHI) will be collected from subjects in this study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research subject to revoke their authorization for use of their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of subject 
authorization.  For subjects that have revoked authorization to collect or use PHI, attempts 
should be made to obtain permission to collect at least vital status (long term survival status that 
the subject is alive) at the end of their scheduled study period. 
9.2 
Source Documents 
Source data is all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial.  Source 
data are contained in source documents.  Examples of these original documents, and data records 
include: hospital records, clinical and office charts, laboratory notes, memoranda, subjects’ 
diaries or evaluation checklists, pharmacy dispensing records, recorded data from automated 
instruments, copies or transcriptions certified after verification as being accurate and complete, 
microfiches, photographic negatives, microfilm or magnetic media, x-rays, subject files, and 
records kept at the pharmacy, at the laboratories, and at medico-technical departments involved 
in the clinical trial. 
9.3 
Case Report Forms 
The study case report form (CRF) is the primary data collection instrument for the study. All 
missing data will be explained. All data requested on the CRF will be recorded in the study excel 
files. Clinical plasma data is maintained in the EMR. The Inclusion/exclusion sheet will serve as 

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the CRF for smoking status, and waist hip measurements and medications taken at time of 
screen. If the space on the CRF is left blank because the procedure was not done, or question was 
not answered it will be documented as “N/D”. If the item is not applicable to the individual case, 
it will be documented as “N/A” Errors will be identified by a strike-through and corrected data. 
 
Data Management 
 
Study data will be managed in a study specific Excel database. 
 
Data Security and Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA). 
Those regulations require a signed subject authorization informing the subject of the following:  
• What protected health information (PHI) will be collected from subjects in this study  
• Who will have access to that information and why  
• Who will use or disclose that information  
• The rights of a research subject to revoke their authorization for use of their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of subject 
authorization. For subjects that have revoked authorization to collect or use PHI, attempts should 
be made to obtain permission to collect at least vital status (long term survival status that the 
subject is alive) at the end of their scheduled study period.  
As part of the screening for these studies a medical history and physical examination will be 
performed, blood tests and body composition measurements will be made. None of the research 
data will be entered into the Mayo Electronic Medical Record. Any paper records that are 
generated are kept in Dr. Jensen's laboratory, which is locked at all times to prevent hallway 
entry. Because it often takes several months from the initial volunteer contact until the studies 
are complete, and because we need to communicate with multiple units (Radiology, Nuclear 
Medicine, Mayo CTSA CRTU, etc.) over the course scheduling, it is not possible to de-identify 
paperwork immediately. The paperwork with patient identifiers is kept in a locked room when 

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not in use. Whenever possible the paper records generated will be such that once the study is 
complete the identifiers can be removed. A paper matching the subject name and other 
identifying information with the subject code should be kept in a separate place from the study 
information. No information will be given to anyone without permission from the subject. If 
samples are sent outside Mayo for analysis they are de-identified per Mayo Biospecimens 
Committee rules. 
 
Data Quality Assurance 
 
1) Description of Plan for Data Quality and Management: The PI will review all data collection 
forms on an ongoing basis for data completeness and accuracy as well as protocol compliance. A 
statement reflecting the results of the review will be sent to the NIDDK in the annual report 
(non-competing continuation). Although there are additional reports to be produced by the study 
coordinator as a result of this DSMP, there are no substantive changes to the study protocol that 
might require review by the NIDDK.  
 
2) Frequency of Review. The personnel and frequency of data review in these studies varies 
depending upon the source and type of data. The frequency of data review for this study is 
summarized in the following table.  
Data type  
Frequency of review  
Reviewer  
Subject accrual (adherence to 
protocol regarding 
demographics, 
inclusion/exclusion)  
Monthly  
Study coordinator, research 
fellow and Principal 
Investigator  
Adverse event rates (injuries)  
Quarterly  
Study coordinator, research 
fellow, Principal Investigator 
and Endocrine Research 
Committee as independent 
monitor  
Out of range research 
laboratory data  
As results become available  
Research fellow and Principal 
Investigator*  
Overview of data collection as 
regards statistical power 
implications of drop outs and 
missing data  
Yearly  
Research Fellow and Principal 
Investigator 
* the research data generated by these studies is unique, entirely novel and requires profound 
expertise/experience to identify analytical, experimental or other research laboratory problems that 
may have arisen.  

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B. Subject Accrual and Compliance  
 
1) Measurement and reporting of subject accrual, adherence to inclusion/exclusion criteria 
Review of the rate of subject accrual, adherence to inclusion/exclusion criteria will occur during 
the weekly meetings Dr. Jensen has with the research fellow and/or study coordinator working 
on each protocol. In addition to these reviews Dr. Jensen will conduct an informal quarterly 
progress report with the fellow in charge of each protocol where more comprehensive overall 
progress is discussed and we determine whether changes need to be made to our recruitment 
strategy. Given that small numbers of volunteers are recruited for each protocol and some are 
linked to clinical patient care populations we will track whether overall participation in the 2 
different projects combined include participants that meet our ethnic diversity goals.  
 
2) Measurement and reporting of participant compliance to treatment protocol- the only 
compliance issues relate to volunteers returning for the second study. 
 
 
Data Clarification Process 
A report will be run in Excel to identify missing elements in the data capture. A designated study 
team member will make corrections in the data and the principle investigator will verify. 
 
9.4 
Records Retention 
The sponsor-investigator will retain the specified records and reports for; 
1. Up to 2 years after the marketing application is approved for the drug; or, if a marketing 
application is not submitted or approved for the drug, until 2 years after shipment and 
delivery of the drug for investigational use is discontinued and the FDA has been so 
notified. OR 
2. As outlined in the Mayo Clinic Research Policy Manual –“Retention of and Access to 
Research Data Policy” 
   

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Whichever is longer 
 
10 Study Monitoring, Auditing, and Inspecting 
10.1 Study Monitoring Plan 
 
The investigator will allocate adequate time for such monitoring activities.  The Investigator will 
also ensure that the monitor or other compliance or quality assurance reviewer is given access to 
all the study-related documents and study related facilities (e.g. pharmacy, diagnostic laboratory, 
etc.), and has adequate space to conduct the monitoring visit. 
 
I. Study Identification Information 
 
A. NIH Study Number – “RO1 DK45343” 
B. Study Title – “Regional Fatty Acid Metabolism in Humans” 
C. Name of Principal Investigator (PI) – Michael D. Jensen, M.D.” 
 
II. Study Overview 
 
A. Brief Description of the Purpose of the Study – This proposal includes the study “Muscle 
lipid metabolism and insulin resistance following suppression of FFA in individuals with 
obesity and type 2 diabetes mellitus”. This study includes the administration of 
isotopically labeled fatty acids, muscle biopsies, blood and breath sampling. The Data 
Safety Monitoring Plan (DSMP) for these studies involves monitoring for potential 
adverse events by the PI together with independent observers - the Mayo Clinical 
Research and Trials Unit nursing personnel; adverse events will be reported to the NIH 
and to the Mayo Clinic IRB. 
 
B. Adherence Statement –The Data Safety Monitoring Plan (DSMP) outlined below for 
RO1 DK45343 will adhere to the protocols approved by the Mayo Clinic IRB.   
 

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III. Confidentiality  
 
A.  Protection of Subject Privacy – As part of the screening for these studies a medical 
history and physical examination will be performed, blood tests and body composition 
measurements will be made. None of the research data will be entered into the Mayo 
Electronic Medical Record. Any paper records that are generated are kept in Dr. Jensen's 
laboratory, which is locked at all times to prevent hallway entry. Because it often takes 
several months from the initial volunteer contact until the studies are complete, and 
because we need to communicate with multiple units (Radiology, Nuclear Medicine, 
Mayo CTSA CRTU, etc.) over the course scheduling, it is not possible to de-identify 
paperwork immediately. The paperwork with patient identifiers is kept in a locked room 
when not in use. Whenever possible the paper records generated will be such that once 
the study is complete the identifiers can be removed. A paper matching the subject name 
and other identifying information with the subject code should be kept in a separate place 
from the study information. No information will be given to anyone without permission 
from the subject. If samples are sent outside Mayo for analysis they are de-identified per 
Mayo Biospecimens Committee rules.  
 
B. Database Protection – Electronic research data will be kept on password protected 
computer servers that can be accessed only by those with human studies/privacy training 
and only with Dr. Jensen's direct approval.  Electronic communication with outside 
collaborators involves only unidentifiable information. 
 
C. Confidentiality during AE Reporting –AE reports and annual summaries will not include 
participant-identifiable material. Each will include the identification code only. 
 
IV. Adverse Event Information 
 
A.  Definition - An adverse event (AE) is any untoward medical occurrence in a subject 
temporally associated with participation in the clinical study.  An adverse finding can 

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include a sign, symptom, abnormal assessment (laboratory test value, vital signs, 
electrocardiogram finding, etc.) or any combination of these. 
 
A Serious Adverse Event (SAE) is any adverse event that results in one or more of the 
following outcomes: 
• Death 
• A life-threatening event 
• Inpatient hospitalization or prolongation of existing hospitalization  
• A persistent or significant disability/incapacity 
• A congenital anomaly or birth defect 
• Important medical event based upon appropriate medical judgment 
 
 
B. Classification of AE Severity – AE's will be labeled according to severity which is based 
on their impact on the volunteer.  An AE will be termed ‘mild’ if it does not have a major 
impact on the participant, ‘moderate’ if it causes considerable inconvenience and ‘severe’ 
if it causes a substantial disruption to the volunteer's well-being.   
 
C. AE Attribution Scale – AE's will be categorized according to the likelihood that they are 
related to the study intervention.  Specifically, they will be labeled either definitely, 
probably, possibly or unrelated to the study intervention. 
 
D. Expected Risks – 
Vascular access.  Catheter insertion, intravenous infusion, and blood withdrawal are 
associated with small risks of phlebitis.  This will be minimized by careful attention to 
sterile technique.  As superficial phlebitis occurs, it will be treated conservatively with heat 
and, when appropriate, antibiotics.  Superficial burns to the hand when using the hot hand 
technique to obtain arterialized venous samples are possible.  This has not been a problem 
with the brief use of the heated box.  
 

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Muscle biopsies.  The risks of these procedures include pain, hematomas, bruising, 
infection, and scarring of the biopsy site.  In <1% of biopsies, a small area of numbness 
can develop due to cutting of a superficial sensory nerve. We advise volunteers that this 
can occur and make them aware that if it does, the re-growth of the nerve will take 6-12 
months. 
 
Risk Monitoring/Risk Reduction: 
The risk of pain from muscle biopsies is virtually 100%, and the volunteers are given this 
information. We reduce the risk of infection by performing all biopsies under sterile 
conditions. We have yet to have an infection related to a muscle biopsy in the last 20 years.  
Nonetheless, each volunteer is told of the signs symptoms of infection and is told to call 
the study coordinator immediately should any of they occur. A record is kept of all adverse 
reactions to biopsies. If an increase in the number of adverse reactions relative to historical 
controls is observed during the course of a particular study a careful assessment of 
technique and approaches is made. The protocol might then be modified or additional 
instruction or supervision of the individual performing the biopsies could be undertaken. 
The subsequent results are then audited to determine how effective the intervention has 
been. 
 
Radiation exposure.  Dual energy x-ray absorptiometry and the CT scan result in small 
amounts of radiation exposure. The lowest dose that can be reliably results in good image 
quality for will be employed.  In all instances the amount of radiation that a volunteer will 
receive is well below the level that results in a significant risk of harmful effects.  
 
Infusion of substances (intravenously or subcutaneously) for studies.  A number of 
substances are administered as part of the studies supported by this grant.  These include 
the administration hormones, niacin, isotopic tracers and local anesthetics. We do expect 
to see flushing with niacin infusion. This would be managed by reassuring the subject that 

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it is self-resolving and causes no potential harm in the short or long-term and subjects will 
be advised to keep themselves hydrated to ease the symptoms.  
 
Risk Monitoring/Risk Reduction: 
1.  
In order to assure the safety of tracers administered to volunteers, a series of safety 
checkpoints are in place.  This begins with the ordering of the tracer from the company. 
When compound is received the logbook is checked, recorded and initialed to assure that 
the substance ordered is the substance that is received.   
2. 
 If the compound is to be prepared for intravenous infusion, the substance is 
prepared in a Class 2 sterile hood by individuals who have been certified by the pharmacy 
at Mayo to be competent with respect to the proper procedures. Periodic competency tests 
are performed to assure continued sterile technique.  If substances are to be made for single 
unit dosage administration and stored in the investigator’s laboratory, the Class 3 
pharmaceutical room at Rochester Methodist Hospital is used by a pharmacy technician 
and pharmacist trained and experienced in the procedures. All lots of tracers are tested for 
pyrogens and sent for cultures. No tracers are administered to volunteers until the results 
of the pyrogen testing and culture are available. The tracers are prepared for the final 
intravenous infusion in the Class 2 sterile hood also by individuals trained to pharmacy 
specifications in proper technique. 
3. 
 All tracers are administered in the Clinical Research and Trials Unit where 
comprehensive hospital facilities are available should a study participant have an allergic 
and/or untoward reaction to either tracers, hormones, anesthetics, or other substances. 
4. 
 For administration of anesthetics, care is taken to administer the minimum amount 
of lidocaine necessary in order to achieve adequate anesthesia.  We are careful to limit the 
amount administered to much less than that which could cause cardiac arrhythmias.  
The most prominent risks to each individual volunteer are pain and bruising related to 
vascular access and adipose biopsies. These are addressed in the protocol and consent form.  
 
 

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E. SAE Reporting 
• 
SAEs that are unanticipated, serious, and/or possibly related to the study intervention 
will be reported to the independent monitors for each separate protocol as indicated 
above, the IRB, the Mayo CTSA Clinical Study and Trials Unit, NIDDK and other 
oversight organizations as appropriate.  
• 
Anticipated or unrelated SAEs are reported to the IRB, the Mayo CTSA Clinical 
Study and Trials Unit and NIDDK in accordance with their requirements. As the 
incidence of pain and bruising with biopsies is 100% none of the oversight 
organizations or nursing staff has requested this information.  
 
Because these studies do not involve interventions/treatments or follow up laboratory 
testing, we do not anticipate that abnormal lab values will need to be addressed as they 
would only be discovered at screening and would make the volunteer ineligible for the 
study. 
 
V. Data Quality and Safety Review Plan and Monitoring 
 
 
A. Data Quality and Management 
 
 
1) Description of Plan for Data Quality and Management: The PI will review all data 
collection forms on an ongoing basis for data completeness and accuracy as well 
as protocol compliance.  A statement reflecting the results of the review will be 
sent to the NIDDK in the annual report (non-competing continuation).  Although 
there are additional reports to be produced by the study coordinator as a result of 
this DSMP, there are no substantive changes to the study protocol that might 
require review by the NIDDK.  
 
2) Frequency of Review. The personnel and frequency of data review in these 
studies varies depending upon the source and type of data. The frequency of data 
review for this study is summarized in the following table. 
 

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* the research data generated by these studies is unique, entirely novel and requires profound 
expertise/experience to identify analytical, experimental or other research laboratory 
problems that may have arisen. 
B. Subject Accrual and Compliance 
 
1) Measurement and reporting of subject accrual, adherence to inclusion/exclusion 
criteria Review of the rate of subject accrual, adherence to inclusion/exclusion 
criteria will occur during the weekly meetings Dr. Jensen has with the research 
fellow and/or study coordinator working on each protocol. In addition to these 
reviews Dr. Jensen will conduct an informal quarterly progress report with the 
fellow in charge of each protocol where more comprehensive overall progress is 
discussed and we determine whether changes need to be made to our recruitment 
strategy.  Given that small numbers of volunteers are recruited for each protocol 
and some are linked to clinical patient care populations we will track whether 
overall participation in the 2 different projects combined include participants that 
meet our ethnic diversity goals.   
Data type 
Frequency of review 
Reviewer 
Subject accrual (adherence to 
protocol regarding demographics, 
inclusion/exclusion) 
Quarterly 
 
Study coordinator, research 
fellow and Principal Investigator 
Adverse event rates (injuries) 
Quarterly 
Study coordinator, research 
fellow, Principal Investigator 
and Endocrine Research 
Committee as independent 
monitor 
Out of range research laboratory 
data  
As results become available  
Research fellow and Principal 
Investigator* 
Overview of data collection as 
regards 
statistical 
power 
implications of drop outs and 
missing data 
Yearly 
Research Fellow and Principal 
Investigator 

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2) Measurement and reporting of participant compliance to treatment protocol – The 
weight loss intervention provided as part of these studies is purely voluntary and 
no compliance issues are anticipated.   
 
C. Justification of Sample Size – We used published data regarding the improvement in an 
insulin sensitivity from a study that used Acipimox to determine that 10 subjects/group will 
be sufficient to detect a clinically meaningful change in insulin action with 90% power at a P 
of <0.05.  
 
D. Stopping Rules - Because this is not a treatment studies per se, but instead one 
physiological measurements that take advantage of a clinical treatment protocol with its 
own monitoring, there is no stopping rule based on interim data analysis clearly 
demonstrates an effect. This study will be stopped prior to its completion if:  (1) adverse 
effects that significantly impact the risk-benefit ratio have been observed; (2) study 
recruitment or retention becomes futile; (3) any new information becomes available 
during the trial that necessitates stopping the trial; or (4) other situations occur that might 
warrant stopping the trial.  
 
E. Designation of an Independent Monitor – The Endocrine Research Committee, composed 
of experienced investigators with appointments in the Division of Endocrinology, will 
serve as the independent monitor for the parts of the study indicated in the table. The PI 
will present relevant information to the committee at the indicated intervals and answer 
questions. The findings and recommendations of the committee will be included in the 
meeting minutes. If the PI presenting the data is the Chair of the Endocrine Research 
Committee another member of the committee will serve as chair for that meeting. The 
Endocrine Research Committee meets monthly. The nursing staff in the Departments of 
Surgery, Nuclear Medicine and the CRTU will also be involved with the monitoring of 
the respective protocols.   
 

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F. Safety Review Plan –Study progress and safety will be reviewed monthly with the 
research fellow and study coordinator and, because these are relatively small studies, the 
data summarized at 6 month intervals to allow for a large enough sample to assess 
outcomes.  Progress reports, including patient recruitment and AE's will be provided to 
the Endocrine Research Committee following each of the 6 month reviews.  An annual 
report will be compiled and will include a list and summarization of adverse events at the 
time of the non-competing renewal.  In addition, the annual report will address (1) 
whether adverse event rates are consistent with pre-study assumptions; (2) reason for 
dropouts from the study; (3) whether all participants met entry criteria; (4) whether 
continuation of the study is justified on the basis that additional data are needed to 
accomplish the stated aims of the study.  The annual report will be reviewed by the 
Endocrine Research Committee and the results included in the minutes. These will be 
forwarded to the IRB and NIDDK and, if applicable, the Mayo CTSA CRTU and FDA as 
indicated.   
 
VI. Informed Consent 
 
Written informed consent will be obtained from each subject at entry into the study. 
Informed consent is obtained by the following process: 
 
 
•  The subject will be asked to review the study consent form; 
 
•  The PI or an IRB approved individual authorized to obtain consent will meet with the 
subject to review the form, to confirm the subject’s understanding of the study, and to 
answer any questions that the subject might have; and 
•  Once the subject demonstrates understanding of the study and agrees to participate in the 
study, the consent will be signed in the presence of the PI or an IRB approved individual 
authorized to obtain consent. The consent is entered into the Mayo electronic medical 
record. 

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10.2 Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the IRB, the 
sponsor, and government regulatory agencies, of all study related documents (e.g. source 
documents, regulatory documents, data collection instruments, study data etc.).  The investigator 
will ensure the capability for inspections of applicable study-related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection by 
government regulatory authorities and applicable compliance offices. 
11 Ethical Considerations 
This study is to be conducted according to United States government regulations and 
Institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted local Institutional 
Review Board (IRB), in agreement with local legal prescriptions, for formal approval of the 
study.  The decision of the IRB concerning the conduct of the study will be made in writing to 
the sponsor-investigator before commencement of this study. 
 
All subjects for this study will be provided a consent form describing this study and providing 
sufficient information for subjects to make an informed decision about their participation in this 
study.  This consent form will be submitted with the protocol for review and approval by the IRB 
for the study.  The formal consent of a subject, using the Approved IRB consent form, must be 
obtained before that subject undergoes any study procedure.  The consent form must be signed 
by the subject or the subject’s legally authorized representative, and the individual obtaining the 
informed consent. 
 
12 Study Finances 
12.1 Funding Source 
 

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The study is financed through a grant from the US National Institute of Health.  
12.2 Conflict of Interest 
Not applicable  
12.3 Subject Stipends or Payments 
The remuneration will be $
per visit. Study compensation will be prorated if needed. 
13 Publication Plan 
The study team at Mayo Clinic holds the primary responsibility for publication of the results of 
the study. 
 
14 References 
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Rothman DL, Magnusson I, Cline GW, Gerard D, Kahn CR, Shulman RG, et al. Decreased muscle glucose 
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obesity phenotypes. Diabetes. 1999;48:1586-92. 
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Roust LR, Jensen MD. Postprandial free fatty acid kinetics are abnormal in upper body obesity. Diabetes. 
1993;42:1567-73. 
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Basu A, Basu R, Shah P, Vella A, Rizza RA, Jensen MD. Systemic and regional free fatty acid metabolism 
in type 2 diabetes. American journal of physiology. 2001;280:E1000-E6. 
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Groop L, Saloranta C, Shank M, Bonadonna RC, Ferrannini E, DeFronzo RA. The role of free fatty acid 
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Chavez JA, Knotts TA, Wang L-P, Li G, Dobrowsky RT, Florant GL, et al. A role for ceramide, but not 
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62. 
Straczkowski M, Kowalska I, Baranowski M, Nikolajuk A, Otziomek E, Zabielski P, et al. Increased 
skeletal muscle ceramide level in men at risk of developing type 2 diabetes. Diabetologia. 2007;50(11):2366-73. 
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Adams JM, 2nd, Pratipanawatr T, Berria R, Wang E, DeFronzo RA, Sullards MC, et al. Ceramide content 
is increased in skeletal muscle from obese insulin-resistant humans. Diabetes. 2004;53(1):25-31. 
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Skovbro M, Baranowski M, Skov-Jensen C, Flint A, Dela F, Gorski J, et al. Human skeletal muscle 
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65. 
Lee JS, Pinnamaneni SK, Eo SJ, Cho IH, Pyo JH, Kim CK, et al. Saturated, but not n-6 polyunsaturated, 
fatty acids induce insulin resistance: role of intramuscular accumulation of lipid metabolites. J Appl Physiol. 
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