The Impact of Insulin Therapy on Protein Turnover in Pre-Diabetic Cystic Fibrosis Patients
Phase: PHASE2
Conditions: Cystic Fibrosis
Sponsor: University of Minnesota

PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
PROTOCOL COVER PAGE 
Protocol Title
The Impact of Insulin Therapy on Protein Turnover in Pre-
Diabe9c Cys9c Fibrosis Pa9ents
Principal 
Inves9gator/Faculty 
Advisor
Name: Antoine?e Moran
Department: Pediatric Endocrinology
Telephone Number: 612-624-4446
Email Address: moran001@umn.edu
Student Inves9gator
Name:
Current Academic Status (Student, Fellow, Resident): 
 
Department:
Telephone Number:
Ins9tu9onal Email Address:
Scien9ﬁc 
Assessment
Na9onally-based, federal funding organiza9ons
IND/IDE # (if 
applicable)
N/A
IND/IDE Holder
N/A
Inves9ga9onal Drug 
Services # (if 
applicable)
#4817
Version Number/
Date:
Version 10/28 June 2021 
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
REVISION HISTORY 
Revision #
Version Date
Summary of Changes
Consent Change?
6
11 June 2019
Physical exam, visit windows, and 
safety levels added to the protocol, 
as well as minor administra9ve 
changes 
Yes
7
4 February 
2020
Would like to include a non-English 
speaking par9cipant 
Yes, added short 
form
8
11 February 
2020
Spanish consent form and would 
like to add a remote consent 
process for the second parent if 
they are unable to make it. 
Yes, added Spanish 
version 
9
6 July 2020
Changing the inclusion criteria to 
say 24 months of having an 
abnormal OGTT. Due to COVID, the 
CF center is not doing rou9ne 
OGTTs thus their results would not 
be within the 12 months. 
No 
9.1
8 February 
2021
Adding an addi9onal 12 control 
par9cipants to be studied. 
No 
10
28 June 2021
Removing the CGM part of the 
protocol. We would like 30 controls 
in total. 
Yes
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
Table of Contents 
1.
Objectives 
 
................................................................................................................5
2.
Background 
 
..............................................................................................................5
3.
Study Endpoints/Events/Outcomes 
 
.........................................................................6
4.
Study Intervention(s)/Investigational Agent(s) 
 
.......................................................6
5.
Procedures Involved 
 
................................................................................................7
6.
Data and Specimen Banking 
 
..................................................................................10
7.
Sharing of Results with Participants 
 
......................................................................10
8.
Study Population 
 
...................................................................................................10
9.
Local Number of Participants 
 
................................................................................13
10.
Local Recruitment Methods 
 
..................................................................................13
11.
Withdrawal of Participants 
 
....................................................................................14
12.
Risks to Participants 
 
..............................................................................................14
13.
Potential Benefits to Participants 
 
...........................................................................15
14.
Statistical Considerations 
 
......................................................................................15
15.
Confidentiality 
 
.......................................................................................................17
16.
Provisions to Monitor the Data to Ensure the Safety of Participants 
 
....................18
In addition to the two study visits, the study nurse coordinator is in weekly contact with 
study participants and all adverse events are recorded on case report forms. The safety 
data including all adverse events are reviewed monthly by the investigators. The only 
significant potential risk directly associated with this study is that of severe 
hypoglycemia, if the insulin dose is too high for the subjects.  In fact, we have now 
studied 24 subjects and have not had one single episode of severe hypoglycemia, so the 
risk is low. The only adverse events that have occurred are respiratory infections as is 
expected in cystic fibrosis. The investigators are all pediatric endocrinologists, well 
experienced with insulin administration and management of hypoglycemia.  Efficacy data 
will not be reviewed until study end. No conditions have been identified that would 
trigger an immediate suspension of the research. 
 
..............................................................18
17.
Provisions to Protect the Privacy Interests of Participants 
 
....................................18
18.
Compensation for Research-Related Injury 
 
..........................................................19
19.
Consent Process 
 
.....................................................................................................19
20.
Setting 
 
....................................................................................................................21
21.
Multi-Site Research 
 
...............................................................................................21
22.
Resources Available 
 
...............................................................................................21
23.
References .............................................................................................................22
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
ABBREVIATIONS/DEFINITIONS 
•
CF – Cys9c Fibrosis 
•
CFRD – Cys9c Fibrosis Related Diabetes 
•
CGM – Con9nuous Glucose Monitor 
•
BMI – Body Mass Index 
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
1.
Objec9ves 
1.1 Purpose: The current protocol describes a double-blind, placebo-controlled 
trial to determine whether insulin therapy improves protein catabolism in youth with CF 
and abnormal glucose tolerance, and to explore diﬀerences in eﬃcacy between mul9ple 
daily pre-meal insulin dosing (as is currently standard for early CFRD) versus a more 
convenient once daily basal insulin dose (as has been used in small uncontrolled pilot 
studies). The ﬁndings of this study will provide a mechanis9c ra9onale for ins9tu9ng 
insulin in youth with CF and pre-diabetes, and will inform both research studies and 
clinical prac9ce as to the best regimen for insulin delivery in this popula9on. 
Speciﬁc Aim 1 
To conduct a double-blind, placebo-controlled study of the impact of daily insulin 
therapy on meal s9mulated protein turnover in 60 youth age 10-25 years with CF and 
abnormal glucose tolerance. Assessment will occur before and afer 1 month of insulin 
therapy. 
Hypothesis 1: In this insulin insuﬃcient CF popula7on, daily insulin replacement 
therapy will reduce meal s7mulated protein catabolism from pre-insulin baseline, 
and will produce greater reduc7ons from baseline than in placebo-treated 
pa7ents. 
 
 
Speciﬁc Aim 2 
To ascertain diﬀerences in the impact of 1X daily basal insulin versus 3X daily rapid-
ac9ng insulin on meal s9mulated protein turnover in 60 youth age 10-25 years with CF 
and abnormal glucose tolerance. 
Hypothesis 2: A single daily dose of basal insulin will safely provide the same 
therapeu7c beneﬁt as the current standard method of insulin delivery of pre-meal 
3x daily rapid ac7ng insulin. 
Speciﬁc Aim 3 
In a novel exploratory aim, we will assess the turnover of 3 individual proteins previously 
implicated in CF pathophysiology--- albumin, transferrin, and clusterin--- to determine 
the extent to which their synthesis and accumula9on diﬀers at baseline from normal 
controls and is subsequently aﬀected by insulin therapy. Twenty matched healthy 
controls will be assessed at baseline only (no insulin therapy).  
2.
Background 
2.1. Signiﬁcance of Research Ques9on/Purpose: Given the universal prevalence 
of insulin insuﬃciency in CF, the high life9me risk of developing diabetes, 
the clinical impact of insulin insuﬃciency on protein catabolism and survival 
in CF, and the cri9cal importance of maintaining body weight and LBM in 
this popula9on, there is an urgent need to determine whether insulin 
replacement therapy should be ins9tuted for anabolic purposes prior to the 
actual onset of diabetes and, if so, to ascertain the op9mal regimen. 
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
2.2. Preliminary Data: In a placebo-controlled clinical trial, insulin therapy 
improved body mass index (BMI) and LBM in pa9ents with very early CFRD 
(CFRD without fas9ng hyperglycemia), and this is now standard care for 
these pa9ents. 
2.3. Exis9ng Literature: N/A  
3.
Study Endpoints/Events/Outcomes 
3.1.
Primary Endpoint/Event/Outcome:  Synthesis rates and rela9ve 
accumula9on of Individual proteins (albumin, transferrin, clusterin): the rate of 
plasma protein synthesis and breakdown can be measured using the amino acid 
isotopes infused during the isotopic meal study. Frac9onal synthesis rates of 
these proteins will be measured from the rate of incorpora9on of [13C6]Phe. We 
will purify these proteins by immunoaﬃnity chromatography and the isotopic 
enrichment in the respec9ve proteins and plasma Phe will be measured as 
previously describe. The calcula9on of frac9onal synthesis rates of these proteins 
is as described previously using plasma [13C6]Phe as precursor. We will measure 
the isotopic enrichment in these proteins for 2 weeks afer the isotope infusion is 
discon9nued in order to monitor whether the isotope label is retained at a 
greater or lower rate as a measure of the accumula9on of these proteins. If the 
isotope label is retained longer we will consider it an indica9on of lower 
turnover. 
3.2. Secondary Endpoint(s)/Event(s)/Outcome(s):  
Oxida9ve damage---In studies in type 1 diabetes, transient insulin 
depriva9on caused accelerated plasma protein oxida9ve damage. We will 
assess the extent of oxida9ve damage by pos?ransla9onal modiﬁca9ons 
and deamida9on in new and old isoforms of albumin, transferrin, clusterin, 
and other proteins. We will determine if insulin treatment prevents or 
reduces oxida9ve damage to speciﬁc proteins. 
Protein Metabolomics—We will use ultra-performance liquid 
chromatography tandem mass spectrometry to measure 45 amino acids 
and their metabolites at weeks 0 and 6. It has been shown that amino acids 
(especially branched chain and aroma9c amino acids) are sensi9ve 
predictors of developing diabetes in the future, and that branched chain 
amino acids, aroma9c amino acids and several metabolites of amino acids 
such as citrulline and alpha-amino-adipoic acid are highly sensi9ve to 
insulin ac9on. These amino acids and metabolites will be a secondary 
outcome to determine insulin eﬀect. 
4.
Study Interven9on(s)/Inves9ga9onal Agent(s) 
4.1. Descrip9on:  
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
1.  Subjects are admi?ed to the Clinical Research Unit here on the UMN 
campus in the early morning and 2 IVs are placed. 
2.  At ~6am subjects are given a priming dose of [15N]-Phe (0.7 mg/kg), 
followed by con9nuous infusion over the next 8 hours of 15[N]-Phe (0.7 
mg/kg). 
3.  2H5-Phe infusion begins at 9me 0 and con9nues for 5hr at a variable 
rate to mimic meal 13C6-Phe Ra. 
4.  During the second phase of the study, CF pa9ents will have been 
receiving insulin (basal or bolus) or placebo for 1 month. They will receive 
their usual insulin/placebo dose during the protein turnover study. 
5.  To calculate protein dynamics, free amino acids are extracted from 
plasma using ace9c acid and analyzed as their t-butyldimethylsilyl ester 
deriva9ves. The molar percent excess (MPE) of phenylalanine isotopes will 
then be calculated above background 
4.2. Drug/Device Handling:  Study drug, insulin or placebo (insulin diluent) will 
be obtained from the manufacturer in standard insulin vials. The 
inves9ga9onal pharmacy will cover the label so that the study medica9on 
will be blinded. 
• IDS #4817 
5.
Procedures Involved 
5.1. Study Design: This double-blind, placebo-controlled trial of insulin therapy 
in CF youth with abnormal glucose tolerance will assess the impact of 2 
diﬀerent insulin regimens on protein turnover. In Phase 1, stable isotope 
measurement of meal-s9mulated protein ﬂux will be done in CF pa9ents 
and normal controls, followed by bi-weekly fas9ng blood draws x2 wks to 
assess individual proteins. In Phase 2, CF pa9ents will receive once daily 
basal insulin, 3X daily rapid-ac9ng insulin, or injectable placebo for 6 weeks. 
Afer the 1st month, the meal-s9mulated protein ﬂux study will be 
repeated, followed by bi-weekly fas9ng blood draws x2 wks to assess 
individual proteins. Power analysis suggests 15 pa9ents are needed per 
group; 20 are planned to account for drop-outs due to acute illness and 
non-adherence (although we expect good adherence since the treatment 
period is short). 
5.2. Study Procedures:  
Phase 1: Baseline Assessment, No insulin therapy, All CF pa9ents and 
controls will complete this phase 
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
Pre-Visit: CF pa9ents will complete a dummy injec9on experience at the 
9me of consent to see if they are okay with insulin injec9ons. A collec9on 
of their latest physical examina9on will be grabbed from data records to 
make sure they are healthy. A physical exam, including tanner staging, can 
be completed at the pre-visit phase for CF pa9ents.    
Visit 0 (Baseline): 
Meal-s9mulated protein ﬂux study (Jello Part): Control par9cipants will be 
consented and a physical examina9on will be conducted by the PI at the 
beginning of this visit, CF pa9ents have this done previously at a pre-visit. 
The study will start early in the morning (5am).   They must have fasted 
since 8PM the night beforehand and encouraged to drink lots of water to 
help with IV placement. Two IVs will be placed, one for the isotope 
infusions and one for blood draws. Afer a 3 hour intravenous priming 
infusion, they will consume a jello test meal at 9me 0 and blood will be 
periodically sampled for the next 5 hours. The blood draws during the 
infusions are for cytokines, glucose, c-pep9des, insulin, and isotopes. 
Par9cipants that have CF and their parent/guardian will be asked to 
complete the Cys9c Fibrosis Ques9onnaire-Revised, a standardized 
ques9onnaire created by the cys9c ﬁbrosis founda9on, during the infusion. 
Also during the infusion part, a cer9ﬁed die99an will come in and do a 
consult in regards to coun9ng carbohydrates. The consult is necessary 
because it is important to know card coun9ng when taking insulin. Once 
the infusion is completed we will give them a meal and take them over to 
get a DXA scan for determina9on of lean body mass. Visit 1 (week 1, +/- 1 
day): outpa9ent blood draw to assess individual proteins  
Visit 2 (week 2, +/- 1 day): For control pa9ents they will complete an 
outpa9ent blood draw to assess individual proteins. End of study 
par9cipa9on.  
For CF pa9ents: they will complete the same outpa9ent blood draw and 
start Phase 2.  
Phase 2: Insulin therapy; All CF pa9ents into 3 groups, No controls 
Randomized into group (once daily basal insulin, 3x daily rapid ac9ng 
insulin, or placebo) Insulin (basal or rapid) or placebo, will be supplied by 
the manufacturer in iden9cal standard insulin vials. Par9cipants will be 
instructed in subcutaneous injec9on technique. Study medica9on 
adjustments will be made under the supervision of the research teams, 
who are quite familiar with insulin management in pa9ents with CF, and 
who will be available at all 9mes for support.  
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
Visit 3 (Week 6, +/- 7 days): Complete the meal-s9mulated protein ﬂux 
study part again from Phase 1 along with the DXA scan, CFQ-R, and CGM 
placement. There will not be a die99an consult at this visit.  
Visit 4 (Week 7, +/- 1 day): outpa9ent blood draw to assess individual 
proteins and removal of con9nuous glucose monitor 
Visit 5 (Week 8, +/- 1 day): outpa9ent blood draw to assess individual 
proteins and end study.  
Between Visits: 
Study personnel will contact (e.g. telephone, email) pa9ents every three 
days while insulin is being adjusted and weekly thereafer in addi9on to 
their study visits. During these contacts staﬀ will review and adjust (if 
needed) the medica9on dose, review hypoglycemia symptoms and 
management, monitor acute illness or adverse events, provide educa9on, 
and encourage adherence. 
Par9cipants will also be asked record their blood glucose levels at random 
9mes during the en9re study. These need to be recorded to determine 
clinically if insulin adjustment is necessary.  
Pa9ents will receive all usual CF care; study par9cipa9on will not impact 
standard-of-care treatment. 
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
5.3. Study Dura9on: Each study par9cipant will be in the study for an 8 week 
dura9on.
•
An9cipated dura9on to recruit all study par9cipants is 4.5 years. 
•
The an9cipated dura9on to complete all study procedures, including 
any long-term follow-up, and data analysis is 5 years. 
5.4. Individually Iden9ﬁable Health Informa9on: Research par9cipants in this 
study will sign an Authoriza9on to Use or Disclose Protected Health 
Informa9on for Research Purposes. 
5.5. Use of radia9on: See a?ached UHS applica9on 
6.
Data and Specimen Banking 
N/A 
7.
Sharing of Results with Par9cipants 
7.1. Afer all subjects have completed the study and the results are analyzed 
(about 5 years total), the PI will let the subjects know the overall study 
results and whether they received insulin or placebo. 
8.
Study Popula9on 
8.1. Inclusion Criteria:  
1
2
3
4
0
*Blood
Baseline 
Assessment, 
No Insulin (CF 
n=60, Controls 
CGM
8
7
6
5
 
CGM
*Blood
*Blood
*Blood
PHASE 1
PHASE 2
Week
Jell-O Protein Study 
DXA 
HbA1c 
Cytokines
Jell-O Protein Study 
DXA 
HbA1c 
Cytokines
*Blood=outpa9ent fas9ng blood draw for labeled 
protein
CF Pa9ents Only (No Healthy Controls) 
Once daily basal insulin levemir (n=20) 
3x daily rapid-ac9ng insulin novolog (n=20) 
Injected Placebo (N=20, 1/2 once daily, 1/2 3x daily)
Start insulin
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
1.  Diagnosis of cys9c ﬁbrosis, age 10-25 years  
2.  A standard rou9ne annual OGTT performed within 24 months of   
randomiza9on  
3.  Abnormal glucose tolerance, with a fas9ng glucose level <126 mg/dl and 
     a.  The 1-hr OGTT glucose is ≥200 mg/dl but the 2-hr glucose is <140 
(INDET), OR 
     b.  The 2-hour OGTT glucose is 140-199 mg/dl (impaired glucose 
tolerance, IGT). 
8.2. Exclusion Criteria:  
1.  Diagnosis of CFRD, Consensus Conference deﬁni9on  
2.  Previous organ transplant, or transplant imminent during study period 
3.  BMI percen9le >95  
4.  Treatment with systemic glucocor9coids (nasal or inhaled 
glucocor9coids are acceptable) 
5.  Therapy with growth hormone or Megace  
6.  Nighxme con9nuous drip gastrostomy/jejunostomy feedings 
7.  Pregnancy or breast-feeding or plans to become pregnant during study 
period 
8.  Any change in medica9ons during the 3 months prior to the study 
     •  Excep9on 1: Any an9bio9cs given for an acute illness greater than 6  
week prior to study start. An9bio9c therapy is a rou9ne treatment plan for 
acute illnesses in this popula9on and is not a relevant medica9on therapy 
change for this study.  
     •  Excep9on 2: the new corrector/poten9ator combina9on drug 
lumacafor/ivacafor is expected to get FDA approval in early 2015, and 
most CF pa9ents with severe genotypes, including many eligible for this 
proposal, will receive this drug. This is not a contraindica9on to 
par9cipa9on in the current proposal (and par9cipa9on in other studies is 
not contraindicated in the PROSPECT post-marke9ng drug study). Though 
the primary eﬀects of the combina9on therapy appear to be apparent afer 
1 month, we will wait 6 months afer ini9a9on of lumacafor/ivacafor 
before enrollment in this study to make sure subjects are in a steady state. 
9.  Any an9cipated change in medica9on during the 3 month study period 
10.  Acute illness in the 6 weeks prior to enrollment 
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
8.3. Screening: Screening begins with iden9ﬁca9on of eligible subjects using the 
standard OGTT recommended annually for all CF pa9ents. Currently at 
UMN we have 121 CF pa9ents age 10-25, 58 of whom have IGT or INDET 
and meet study entrance criteria. Nine new pa9ents per year in this age 
range are diagnosed with IGT or INDET at UM and thus become eligible for 
the study. An addi9onal 46 non-diabe9c children and youth age 10-25 years 
are followed at our Children’s Hospitals and Clinics of Minnesota CF 
aﬃliate, 20 of whom currently have abnormal glucose tolerance. Three to 
four new pa9ents per year become eligible. Our experience from previous 
CF studies in this age group suggests ~60% will agree to par9cipate. Over 
the study period we an9cipate that 45-50 CF pa9ents will par9cipate from 
UM and 10-15 from Children’s. Normal control subjects will be recruited 
from ﬂiers placed in the clinics and the University campus. In addi9on, 
siblings of CF par9cipants will be invited to par9cipate as controls. CF 
subjects will be randomized in a 1:1:1 ra9o to basal insulin, pre-meal 
insulin, or injectable placebo (half 3x/daily pre-meal, half 1x/daily), 
stra9ﬁed by 2 age intervals (10-17, 18-25y) and gender. Randomiza9on will 
be done with computer-generated lists for each strata, in randomly 
alterna9ng blocks of 3 and 6. The research subject, inves9gator and clinic 
personnel will be blinded to assignment. Healthy controls will be matched 
for gender, age and BMI percen9le. The sta9s9cian will review control 
recruitment afer every 5 subjects to ensure they are representa9ve of the 
pa9ent groups and determine whether speciﬁc control ages or genders 
should be recruited. 
8.4. Vulnerable Popula9ons:  
☒ Children 
☐ Pregnant women/Fetuses/Neonates 
☐ Prisoners 
☐ Adults lacking capacity to consent and/or adults with diminished 
capacity to consent, including, but not limited to, those with acute 
medical condi9ons, psychiatric disorders, neurologic disorders, 
developmental disorders, and behavioral disorders 
☐ Approached for par9cipa9on in research during a stressful situa9on such 
as emergency room sexng, childbirth (labor), etc. 
☐ Disadvantaged in the distribu9on of social goods and services such as 
income, housing, or healthcare 
☐ Serious health condi9on for which there are no sa9sfactory standard 
treatments 
☐ Fear of nega9ve consequences for not par9cipa9ng in the research (e.g. 
ins9tu9onaliza9on, deporta9on, disclosure of s9gma9zing behavior) 
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
☐ Any other circumstance/dynamic that could increase vulnerability to 
coercion or exploita9on that might inﬂuence consent to research or 
decision to con9nue in research 
☐ Undervalued or disenfranchised social group 
☐ Members of the military 
☒ Non-English speakers 
☐ Those unable to read (illiterate) 
☐ Employees of the researcher 
☐ Students of the researcher 
☐ None of the above 
8.5. Addi9onal Safeguards:  
Given the universal prevalence of insulin insuﬃciency in CF, the high 
life9me risk of developing diabetes, the clinical impact of insulin 
insuﬃciency on protein catabolism and survival in CF, and the cri9cal 
importance of maintaining body weight and LBM in this popula9on, there is 
an urgent need to determine whether insulin replacement therapy should 
be ins9tuted for anabolic purposes prior to the actual onset of diabetes 
and, if so, to ascertain the op9mal regimen. The current protocol describes 
a double-blind, placebo-controlled trial to determine whether insulin 
therapy improves protein catabolism in youth with CF and abnormal 
glucose tolerance, and to explore diﬀerences in eﬃcacy between mul9ple 
daily pre-meal insulin dosing (as is currently standard for early CFRD) versus 
a more convenient once daily basal insulin dose (as has been used in small 
uncontrolled pilot studies). The ﬁndings of this study will provide a 
mechanis9c ra9onale for ins9tu9ng insulin in youth with CF and pre-
diabetes, and will inform both research studies and clinical prac9ce as to 
the best regimen for insulin delivery in this popula9on. 
          Both parents must give their permission unless one parent is deceased, 
          unknown, incompetent, or not reasonably available, or when only one 
          parent has legal responsibility for the care and custody of the child. 
 
          
                    The minor will be able to give assent and if he/she chooses not to 
                      par9cipate, the choice will be honored. 
  
9.
Local Number of Par9cipants 
9.1. Local Number of Par9cipants to be Consented: We have consented 40 CF 
pa9ents, and need 30 control pa9ents in total.  
10.
Local Recruitment Methods 
10.1. Recruitment Process: Normal control subjects will be recruited by ﬂyers 
located around the University campus and word of mouth.  CF pa9ents who 
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
have had abnormal glucose tolerance documented in the last 24 months 
will be contacted by Dr. Moran, who will describe the research and the fact 
that it is voluntary, and determine if pa9ents are interested and eligible.  Dr. 
Moran maintains the CF OGTT Database and follows these pa9ents 
clinically. 
10.2. Iden9ﬁca9on of Poten9al Par9cipants: Dr. Moran or members of her 
research team will make ini9al contact with CF pa9ents. Normal controls 
will contact research staﬀ- contact informa9on will be provided on 
adver9sements around campus.  Dr. Moran will conﬁrm with the CF oﬃce 
that the pa9ent has signed an agreement to release their PHI contained in 
their medical records and to be contacted for research purposes. Only a 
handful of the more than 500 CF pa9ents at the UM CF Center have not 
provided such consent. 
10.3. Recruitment Materials: A recruitment ﬂyer will be used for Healthy 
Controls.  A phone call, an email, or face-to-face contact may be used by 
the PI for poten9al CF subjects. 
10.4. Payment:  CF pa9ents: max $400;  
                          $140 per jello meal study x2 
                          $15 per blood draw x4 
                          $60 for bonus comple9on  
                          Healthy controls: max $200 
                         $140 per the clamp 
                         $15 per blood draw x2 
                         $30 bonus for comple9on 
Study subjects will be receiving the compensa9on that will be mailed within 6 
weeks of  each of their visits.  
 
           
11.
Withdrawal of Par9cipants 
11.1. Withdrawal Circumstances: If the subject becomes ill or hospitalized during 
the study period, the PI will inform them that they will be withdrawn. 
11.2. Withdrawal Procedures: All data collected up to the point of withdrawal will 
be kept by the inves9gator.  The inves9gator will let the subject know that 
they might be contacted in the future to possibly restart the study. 
12.
Risks to Par9cipants 
12.1. Foreseeable Risks:  
                          1. The CF pa9ents who will receive insulin and they are already insulin 
insuﬀcientand have abnormal glucose tolerance. Hypoglecemia is always a risk with 
 
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insulintherapy. They could also have hypoglemic reac9ons during the jello part of the 
study. This will be closely monitored and they will receive juice if symptoms arise.  
                         2. There is poten9al for discomfort as the iv is being inserted and at the iv 
site.  
                         3. The stable isotopes to be infused are naturally occurring isotope of 
amino acids and thus the amino acids are not an9cipated to pose any risk. During 
prepara9on of any iv solu9on there is the poten9al for contamina9on which is why the 
isotopes will be prepared by the inves9ga9onal pharmacy under their usual sterile 
protocol. 
                         4. Conﬁden9ality may be breached.  
                         5. Nausea from ea9ng the Jello.  
                         6. DXA is an x-ray that has very li?le radia9on exposure.  
7. The con9nuous glucose monitor may cause some discomfort, inﬂamma9on or 
irrita9on at the site.   
13.
Poten9al Beneﬁts to Par9cipants 
13.1. Poten9al Beneﬁts: It is possible that there is no beneﬁt for par9cipants 
because they may not receive ac9ve drug. There is a possibility that insulin 
replacement therapy may be eﬀec9ve in breaking the cycle of underweight 
and overall clinical deteriora9on because of its potent anabolic eﬀects on 
protein turnover. 
14.
Sta9s9cal Considera9ons 
This study will compare the three parallel treatment groups (placebo, basal insulin, and 
pre-meal insulin) on the basis of within-subject changes from week 0 (baseline) to week 
8 (afer 1 month of assigned treatment). The primary endpoints are within-subject 
diﬀerences from week 0 to week 8 in protein turnover parameters, change in Phe ﬂux 
(ΔQP) and in conversion of Phe to Tyr (ΔQPT). Treatment groups will be compared by 
pairwise contrasts in analysis of variance, adjusted for baseline protein turnover. The 
primary-endpoint analysis will be per-protocol, performed on adherent subjects who 
received at least 70% of study medica9on in order to assess insulin eﬀects. In addi9on, 
as part of planning for a larger trial, an intent-to-treat analysis will be performed, 
including all randomized par9cipants to assess insulin treatment eﬀects in prac9ce. 
Exploratory analyses will examine associa9ons between diﬀerences in protein turnover 
parameters and mechanis9c endpoints: baseline pulmonary func9on, BMI percen9le, 
dietary intake (from diet records), observed plasma glucose mean and MAGE (from 
CGM), cytokines, and treatment adherence.    
Analysis of secondary protein turnover endpoints will be exploratory extensions of 
previous work (37, 38, 57, 58). For the 3 proteins of interest (albumin, transferrin, 
clusterin), both the frac9onal synthesis rate and rela9ve isotopic enrichment (ra9o of 
isotopic enrichment to the baseline enrichment) will be calculated from each of the 4 
weekly blood samples afer both the week 0 and week 8 labeled-infusion studies. At 
these same points we will also assess the accumula9on of damaged proteins, since 
 
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PROTOCOL TITLE: The Impact of Insulin Therapy on Protein Turnover in Pre-Diabe9c 
Cys9c Fibrosis Pa9ents  
VERSION DATE: 28 June 2021
oxida9ve stress is likely to be reduced during insulin therapy. The baseline (wks 0-4) 
comparison will be between CF par9cipants and healthy controls, while the comparison 
of the treatments will use measurements from both weeks 0-4 and 8-12. The proﬁles of 
these rate changes over 9me will be compared visually, by summary measures such as 
slope if applicable, and by mixed eﬀects linear model for longitudinal trends, with 
par9cipants treated as a random eﬀect to model the correla9on between repeated 
observa9ons from the same subject.   
Protein metabolomics will be assessed during the in-pa9ent studies at week 0 and week 
8. The baseline comparison will be between CF par9cipants and healthy controls, while 
the comparison of the treatments will use changes from week 0 to week 8. The 
comparison of protein metabolomics will use principal components analysis of the 
baseline protein and metabolite measurements to ﬁnd separated clusters of CF 
par9cipants versus healthy controls, and principal components analysis of the week 8 
measurements to compare clusters between the three treatment groups. 
Secondary mechanis9c endpoints are measured weekly during both baseline and 
interven9on phases: 3-point glucose proﬁles, mean and MAGE plasma glucose will be 
measured at weeks 1-12 using CGM.  We will use a mixed-eﬀects linear model to 
compare these longitudinal proﬁles of plasma glucose between treatments, adjus9ng for 
the appropriate baseline value and trea9ng par9cipants as a random eﬀect to model the 
correla9on between repeated observa9ons from the same par9cipant.  
The safety analysis will es9mate and compare rates of hypoglycemia and subclinical 
hypoglycemia (iden9ﬁed form CGM) between treatments using Poisson regression.  In 
addi9on, rates of non-adherence will be compared between treatments. 
Power Analysis  
We an9cipate that 45 par9cipants, 15 from each of 3 arms, will provide suﬃcient power 
to detect clinically signiﬁcant improvements in protein turnover (ΔQP and ΔQPT) at 
baseline, based on comparisons between healthy controls and insulin-insuﬃcient CF 
pa9ents in our previous study (18). Details are given below. We have inﬂated the sample 
size to 20 per arm to allow replacement of drop-outs and non-adherent par9cipants, 
par9cipants who develop diabetes, or suﬀer an acute exacerba9on. The most common 
reason for drop out is likely to be acute illness, although the brief (3 month) 9me span 
will help reduce this.  Also, given the short 9me span of this study we an9cipate 
adherence will be good. This is in line with other studies in CF where about 80% of 
subjects are adherent at least 80% of the 9me (24, 65). Because many of our subjects 
will be adolescents, one might expect somewhat worse adherence. However, we believe 
this will be oﬀset by the short dura9on of the study and by mechanisms to encourage 
adherence such as school nurse supervision on school days and frequent contact with 
study personnel. Study drop-out may occur due to development of fas9ng 
hyperglycemia or acute illness. These are discussed in greater detail in the human 
 
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subjects sec9on. We expect less than 2% of subjects to develop CFRD during the 3 
month study period. Pa9ents who experience acute illness during the study may repeat 
it when they are well. Thus, we have inﬂated our group size to account for 20% non-
adherence, 1-2% development of diabetes, and 11-12% acute illness.  
Because studies of the eﬀect of chronic insulin therapy have not been conducted 
previously in CF pa9ents, precise es9mates of treatment eﬀect cannot be made. We 
previously demonstrated signiﬁcant insulin treatment eﬀects protein on de novo 
synthesis in 14 non-CF study par9cipants, 7 with type 1 diabetes and 7 without 
(66). Previous work (24) gave es9mates for CF pa9ents of the standard devia9on (SD) of 
changes in protein turnover parameters ΔQP and ΔQPT, diﬀerences between condi9ons 
of insulin infusion and baseline fas9ng. The primary endpoint is the within-subject 
change in ΔQP and ΔQPT from week 0 (baseline) to week 8 (afer 1 month of assigned 
treatment), and we es9mated the SD of these changes as (SD of ΔQX)
 , 
assuming ρ=.6 for within-subject correla9on, for ΔQP = 4.0 and ΔQPT = 1.26. The table 
below reports these standard devia9ons and shows corresponding minimum diﬀerences 
that the study would have 80% power to detect with sample sizes of 15 or 20 in each 
group.  For context, these minimum detectable diﬀerences are also given as percent of 
the baseline value in the CF pa9ents.  Observed mean diﬀerences in ΔQP and ΔQPT 
between CF pa9ents and healthy controls were larger than the minimum detectable 
diﬀerences for n = 15/group (24).  To the degree that either insulin treatment is able to 
normalize protein turnover in the CF pa9ents, these observed diﬀerences suggest 
possible treatment eﬀects.  
The unblinded study sta9s9cian will perform new sample size calcula9ons afer the ﬁrst 
15 CF subjects (5 in each group), and, if necessary, addi9onal subjects will be studied.  
15.
Conﬁden9ality 
2(1 −ρ)
N per group
Minimum 
detectable 
d ﬀ
Minimum detectable 
diﬀerence as % of 
b
l
Observed mean 
diﬀerence (CF pa9ents – 
h
l h
l )
ΔQP (SD = 3.6)
 baseline mean = 50.8
6.0
15
3.8
7%
20
3.3
6%
ΔQPT (SD = 
)
baseline mean = 6.43
1.5
15
1.2
19%
20
1.0
16%
 
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15.1. Data Security: The PI, Dr. Moran, will store and maintain data indeﬁnitely 
on her password protected UM desktop computer in her locked oﬃce 
which is maintained by the AHC. 
16.
Provisions to Monitor the Data to Ensure the Safety of Par9cipants 
16.1. Data Integrity Monitoring.  
The protein turnover por9on of the study will only be done at the University of 
Minnesota where the staﬀ have the necessary exper9se to perform these 
complicated medical studies. Dr. Moran is in regular (at least monthly) 
communica9on with Dr. Larson Ode at the University of Iowa to ensure all other 
study procedures are handled in a consistent fashion. In order to ensure the 
validity of subject characteris9cs that may impact the rigor of the study, we will 
con9nue to work to make sure we have approximately equal gender distribu9on 
and that we have a similar number of pediatric (age 10-18) and young adult (age 
19-15) subjects. In order to ensure robust and unbiased results, we are working 
with collaborators in Italy who are considered the world experts in metabolic 
mathema9cal modeling.  We send them the raw mass spectroscopy data and 
they perform the mathema9cal analysis. They are blinded to treatment arm, 
which will help ensure the validity of the data. 
16.2. Data Safety Monitoring.  
In addi9on to the two study visits, the study nurse coordinator is in weekly 
contact with study par9cipants and all adverse events are recorded on case 
report forms. The safety data including all adverse events are reviewed monthly 
by the inves9gators. The only signiﬁcant poten9al risk directly associated with 
this study is that of severe hypoglycemia, if the insulin dose is too high for the 
subjects.  In fact, we have now studied 24 subjects and have not had one single 
episode of severe hypoglycemia, so the risk is low. The only adverse events that 
have occurred are respiratory infec9ons as is expected in cys9c ﬁbrosis. The 
inves9gators are all pediatric endocrinologists, well experienced with insulin 
administra9on and management of hypoglycemia.  Eﬃcacy data will not be 
reviewed un9l study end. No condi9ons have been iden9ﬁed that would trigger 
an immediate suspension of the research. 
17.
Provisions to Protect the Privacy Interests of Par9cipants 
17.1. Protec9ng Privacy: Your records will be kept private. Dr. Moran will keep the 
study data and your name on her password-protected desktop computer 
kept in her secure UM oﬃce.  No one outside of Dr. Moran and the 
research team will have access to your name or iden9fying informa9on. All 
samples sent to the Mayo Clinic will not have any informa9on from which 
you could be iden9ﬁed. In any publica9ons or presenta9ons, we will not 
include any informa9on that will make it possible to iden9fy you as a 
 
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VERSION DATE: 28 June 2021
subject. Your record for the study may, however, be reviewed by 
departments at the University with appropriate regulatory oversight.  To 
these extents, conﬁden9ality is not absolute. 
17.2. Access to Par9cipants: Research staﬀ may have to look through medical 
records if an enrolled subject becomes hospitalized or starts any new 
medica9on.  This is a way that the research staﬀ can document what is 
happening and know how to proceed. 
18.
Compensa9on for Research-Related Injury 
18.1. Compensa9on for Research-Related Injury: In the event that this research 
ac9vity results in an injury, treatment will be available, including ﬁrst aid, 
emergency treatment and follow-up care as needed. Care for such injuries 
will be billed in the ordinary manner to you or your insurance company. 
18.2. Contract Language:  In the event that this research ac9vity results in an 
injury, treatment will be available, including ﬁrst aid, emergency treatment 
and follow-up care as needed. Care for such injuries will be billed in the 
ordinary manner to you or your insurance company. If you think that you 
have suﬀered a research related injury, let the study physicians know right 
away. 
19.
Consent Process 
19.1. Consent Process (when consent will be obtained):  
• Dr. Moran will have the original discussion with par9cipants over 
the phone or face-to-face in clinic and will then send/give a copy 
of the consent to read. She will then follow up with them in 
person or on the phone to assess their interest level and answer 
any ques9ons. Interested pa9ents will then be consented in clinic 
by Dr. Moran and given a demonstra9on of the dummy injec9on 
for insulin. This pre-visit will occur within 3 months of the baseline 
visit. 
•  What is the purpose of this study? 
• What if you decide you don't want to do this study 
anymore? 
• What are the side eﬀects? 
• What is the beneﬁt to you? 
• It will be made clear during recruitment and informed consent 
process that this is a voluntary study. Each 9me the par9cipant is 
scheduled, they will be made aware again that their par9cipa9on 
 
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is voluntary and no rela9ons will be changed if you decide you no 
longer want to par9cipate. 
19.2. Par9cipants Who Are Not Yet Adults (infants, children, teenagers under 18 
years of age): 
•
Given the universal prevalence of insulin insuﬃciency in CF, the high 
life9me risk of developing diabetes, the clinical impact of insulin 
insuﬃciency on protein catabolism and survival in CF, and the cri9cal 
importance of maintaining body weight and LBM in this popula9on, 
there is an urgent need to determine whether insulin replacement 
therapy should be ins9tuted for anabolic purposes prior to the actual 
onset of diabetes and, if so, to ascertain the op9mal regimen. The 
current protocol describes a double-blind, placebo-controlled trial to 
determine whether insulin therapy improves protein catabolism in 
youth with CF and abnormal glucose tolerance, and to explore 
diﬀerences in eﬃcacy between mul9ple daily pre-meal insulin dosing 
(as is currently standard for early CFRD) versus a more convenient 
once daily basal insulin dose (as has been used in small uncontrolled 
pilot studies). The ﬁndings of this study will provide a mechanis9c 
ra9onale for ins9tu9ng insulin in youth with CF and pre-diabetes, and 
will inform both research studies and clinical prac9ce as to the best 
regimen for insulin delivery in this popula9on. 
•
Both parents must give their permission unless one parent is 
deceased, unknown, incompetent, or not reasonably available, 
or when only one parent has legal responsibility for the care 
and custody of the child.  
•
The researcher conﬁrms that the minor subject ages 8-17 may choose 
not to par9cipate and his/her dissent will be honored. 
•
The child ages 8-17 will sign an assent form which will be kept along 
with the parental consent.  If the subject turns 18 before comple9on 
of the study, they will be reconsented and sign a main consent form to 
be kept in their ﬁle. 
•
When enrolling minors, if there is more than one custodial 
parent or guardian, both must provide consent whenever 
possible.  Documenta9on should demonstrate the informed 
consent process for each and the consent forms should be 
signed by both (if applicable) prior to any study procedure. 
The en9re process must be completed, including the return of 
the second parent’s signed consent document for signature by 
the person obtaining consent, prior to ini9a9ng any study 
procedures.  
 
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       Ideally, both custodial parents or guardians would be present in 
person at a study visit.  However, if one parent is unable to a?end a 
visit in person the signature can be obtained on a remote basis.   In 
general, this is done by providing the study materials (handbook, 
consent forms) to the parent and then speaking to the second parent 
by phone to provide fully informed consent.  
Afer discussing the study and answering ques9ons, the signed 
consent form can be returned (sent via mail, email, or fax) or brought 
to the study visit by the other responsible parent.   If the signature 
dates are diﬀerent (which is likely), an explana9on that the remote 
signature was collected oﬀ site should be noted in the source 
document. If, in rare cases, one of the custodial parents is not 
available despite eﬀorts to reach them (e.g. ac9ve duty), this should 
be documented in a note to ﬁle. 
19.3 Non-English Speaking Par9cipants: We have a translated parental consent form for 
those pa9ents that have Spanish speaking parents to abide by our unexpected 
encounter that we will have used the short form on.  
20.
Secng 
20.1. Research Sites:  
•
Dr. Moran maintains the CF OGTT Database and follows these pa9ents 
clinically and will iden9fy poten9al subjects through this. 
•
Research procedures will take place at the University of Minnesota 
Health Clinical Research Unit (M Health CRU) as well as at the 
Delaware Clinical Research Unit (DCRU). 
•
School-aged par9cipants who are randomized into the pre-meal 
insulin arm will require the school to assist in the proper storage and 
overseeing the administra9on of the required insulin dosage. 
21.
Mul9-Site Research 
21.1. Study-Wide Number of Par9cipants: 100-- number is high in case of drop 
outs, especially due to acute illnesses which are common in CF pa9ents 
21.2. Study-Wide Recruitment Methods:  N/A 
21.3. Study-Wide Recruitment Materials: N/A 
22.
Resources Available 
 
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Resources Available: We have a team of 8 research coordinators to do research for this 
research. We have one full 9me dedicated coordinator, regulatory specialist, and lab 
coordinator to assist in carrying out this protocol. We have access to the cys9c ﬁbrosis 
registry and colleagues to help us recruit poten9al par9cipants. All of the research visits 
will take place at the University of Minnesota research units where they have all of the 
necessary equipment and staﬀ to carry out the visits.   
23.
References 
1. 
De Luca F, Arrigo T, Con9 Nibali S, Sferlazzas C, Gigante A, DiCesare E, Cucino?a D. 
1991. Insulin Secre9on, Glycosylated Haemoglobin and Islet Cell An9bodies in 
Cys9c Fibrosis Children and Adolescents with Diﬀerent Degrees of Glucose 
Tolerance. Horm Metab Res 23: 495-8 
2. 
Hamdi I, Green M, Shneerson JM, Palmer DR, Hales CN. 1993. Proinsulin, 
proinsulin intermediate and insulin in CF. Clin Endocrinol 39: 21-6 
3. 
Lanng S, Thorsteinsson B, Roder ME, Orskov C, Holst J, Nerup J, Koch C. 1993. 
Pancreas and gut hormone responses to oral glucose and intravenous glucagon in 
cys9c ﬁbrosis pa9ents with normal, impaired, and diabe9c glucose tolerance. 
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4. 
Costa M, Potvin S, Hammana I, Malet A, Berthiaume Y, Jeanneret A, Lavoie A, 
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Hameed S, Morton J, Jaﬀe A, Field P, Belessis Y, Yoong T, Katz T, Verge C. 2010. 
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Schiaﬃni R, Brufani C, Russo B, Fin9ni D, Migliaccio A, Pecorelli L, Bizzarri C, 
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8. 
Ode KL, Frohnert  B, Laguna T, Phillips J, Holmes B, Regelmann W, Thomas W, 
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9. 
Koch D, Rainisio M, Madessani U, Harms HK, Hodson ME, Mastella G, McKenzie 
SG, Navarro J, Strandvik B. 2001. Presence of cys9c ﬁbrosis-related diabetes 
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10. 
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VERSION DATE: 28 June 2021
11. 
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diabetes mellitus on clinical status in pa9ents with cys9c ﬁbrosis. Eur J Pediatr 
151: 684-7 
15. 
Milla CE, Warwick WJ, Moran A. 2001. Trends in pulmonary func9on in cys9c 
ﬁbrosis pa9ents correlate with the degree of glucose intolerance at baseline. Am 
J Resp Crit Care Med 162: 891-5 
16. 
Bismuth E, Laborde K, Taupin P, Velho G, Ribault V, Jennane F, Grasset E, Sermet I, 
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Suratwala D, Chan JSH, Kelly A, Meltzer LJ, Gallagher PR, Traylor J, Rubenstein RC, 
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18. 
Moran A, Milla C, DuCret R, Nair KS. 2001. Protein metabolism in clinically stable 
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21. 
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24. 
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VERSION DATE: 28 June 2021
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26. 
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Demitriadou A. 2000. Islet autoan9bodies and insulin dependent diabetes 
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PB. 2005. Changes in response to insulin and the eﬀects of varying glucose 
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Clin Nutr 81: 421-6 
28. 
Rolon MA, Benali K, Munck A, Navarro J, Clement A, Tubina-Ruﬁ N, Paul C, Polzk 
M. 2001. CFRD: Clinical impact of prediabetes and eﬀects of insulin therapy. Acta 
Paediatr 90: 860-7 
29. 
Mozzillo E, Franzese A, Valerio G, Sepe A, De Simone I, Mazzarella G, Ferri P, Raia 
V. 2009. One year glargine treatment can improve the course of lung disease in 
children and adolescents with cys9c ﬁbrosis and early glucose derangements. 
Pediatric Diabetes 10: 162-7 
30. 
Bizzarri C, Lucidi V, Ciampalini P, Bella S, Russo B, Cappa M. 2006. Clinical eﬀects 
of early treatment with insulin glargine in pa9ents with cys9c ﬁbrosis and 
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