The Effect of Pancreatic Insufficiency on Hypoglycemia and Glucagon Response in Children With Cystic Fibrosis
Conditions: Cystic Fibrosis, Hypoglycemia, Glucagon Deficiency, Pancreatic Insufficiency
Sponsor: Marmara University

 
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THE EFFECT OF PANCREATIC INSUFFICIENCY ON HYPOGLYCEMIA 
AND GLUCAGON RESPONSE IN CHILDREN WITH CYSTIC FIBROSIS 
 
Short Running Title: Hypoglycemia and Glucagon Response in CF 
 
Belma Haliloglu, MD,PhD 1,2 
Tuba Seven Menevse, MD 1 
Seda Gulec Yilmaz, PhD 2 
Tuba Akdeniz, PhD 2 
Busra Gurpinar Tosun, MD 1 
Serap Demircioglu, MD 1 
Tulay Guran, MD 1 
Yasemin Gokdemir, MD 3 
Ela Erdem, MD 3 
Bulent Karadag, MD 3 
Turgay Isbir, MD, PhD 2 
Abdullah Bereket, MD 1 
 
1 Division of Pediatric Endocrinology and Diabetes, Department of Child Health and 
Disease, Marmara University School of Medicine, Istanbul, Turkey 
2 Department of Molecular Medicine, School of Health Science, Yeditepe University, 
Istanbul, Turkey 
3 Division of Pediatric Pulmonogy, Department of Child Health and Disease, Marmara 
University School of Medicine, Istanbul, Turkey 
 
Corresponding author: Belma Haliloglu, belma.haliloglu@marmara.edu.tr 
                                                                               belmahaliloglu26@hotmail.com 
                                                                      
 
Date of document: 30/12/2022 
 
 

 
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STUDY PROTOCOL 
 
1.Introduction 
The exact underlying mechanism of hypoglycemia in CF is still unknown. Some recent 
studies support the delayed and prolonged insulin secretion and impaired 
counterregulatory hormone response as the reason of reactive hypoglycemia, whereas 
the others argued an additive effect of an intrinsic factor. However, the weakness of 
these limited studies is that nearly all of them included CF patients who had pancreatic 
insufficiency (PI) and could not reveal the mechanism of hypoglycemia seen in those 
without PI.  In addition, there were no healthy controls for comparison of glucagon 
secretion in CF patients with hypoglycemia. Moreover, the studies that evaluate the role 
of glucagon in hypoglycemic CF patients were performed in hypoglycemic adult 
patients with abnormal glucose tolerance (AGT) and the delayed and prolonged insulin 
release is expected to be more likely as the reason of hypoglycemia in this setting. 
Previously, the investigators had demonstrated isolated hypoglycemia in some of the 
pediatric CF patients during OGTT. In this study, they further evaluated possible 
mechanisms of hypoglycemia. The investigators hypothesized that the mechanism of 
isolated hypoglycemia might be different from hypoglycemia seen in patients with 
AGT. Furthermore, they investigated the role of pancreatic insufficiency in 
hypoglycemia of CF patients by analyzing glucose, insulin and glucagon response to a 
glucose load in CF patients with an without PI.    
 
2. Methods 
2.1. Participants  

 
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Children and adolescents aged 10-18 years with CF who were regularly followed-up at 
the Pediatric Pulmonology and Endocrinology Units, between January 2020 and 
December 2020 were invited to participate in the study before their annual OGTT. 
Pancreatic insufficiency (PI) was defined by need for enzyme replacement therapy 
based on clinical symptoms or fecal elastase level (<200 µg/g). Individuals using 
corticosteroid therapy, those who had acute exacerbation in the last 3 months or 
previously diagnosed with diabetes were not eligible. Height, weight, BMI, FEV1, 
genetics and the presence of PI of the participants were recorded.  
The control group included age-matched healthy, non-diabetic siblings of patients with 
type 1 diabetes who were followed-up at our clinic. All the controls were negative for β-
cell autoantibodies (anti-glutamic acid decarboxylase, islet cell antibody, and insulin 
antibody). The study protocol was approved by the Marmara University Ethics 
Committee (no:09.2019.933), and written informed consent was obtained from the 
parents and the participants. 
2.2. Procedures  
A 3-h OGTT was performed in the morning following overnight fasting of ≥8 h. All 
participants (CF patients and controls) received oral glucose solution (1.75 g/kg; max: 
75 g). Blood samples were collected at 0, 30, 60, 90, 120, 150 and 180 min for glucose 
and insulin, 0, 60, 120,150 and 180.min for glucagon measurement. HbA1c and CRP 
levels at baseline and cortisol levels at 0-180.min were also measured to assess the 
status of inflammation and body stress.  
OGTT results were categorized into different subgroups of glucose tolerance according 
to ISPAD guidelines (12). Subjects who had INDET (Indeterminate Glucose Tolerance) 
or IGT (Impaired Glucose Tolerance) without hypoglycemia on OGTT were classified 

 
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as Abnormal Glucose Tolerance (AGT). According to the IHSG position statement, 
hypoglycemia was defined in the presence of any venous glucose level is lower than 70 
mg/dL (13). The terminology of “isolated hypoglycemia” (IsoHypo) was used for the 
participants who had hypoglycemia without AGT or CFRD and “Hypo+AGT” was used 
for the participants who had hypoglycemia with AGT. After the first analyses, the 
participants with IsoHypo and NGT were also sub-classified according to the presence 
of PI to evaluate the effect of PI on hypoglycemia (Figure-1). 
2.3. Plasma Glucose, Insulin, Cortisol and Glucagon Analysis  
Blood samples of the participants were collected in EDTA tubes and centrifuged 
immediately after collection. Glucose, insulin, cortisol and CRP were measured in the 
same day in the laboratory at Marmara University Hospital.  Glucose was measured by 
glucose hexokinase method (Cobas c701/702, Roche). Insulin and cortisol were 
measured by ECLIA (Cobas e801 Roche and e601 Roche, respectively. For glucagon 
samples, plasma was separated after centrifugation, and stored at 4°C until the OGTT 
was ended (180 min). Following the completion of OGTT, all glucagon samples were 
immediately stored at -80°C prior to shipment to the laboratory at Yeditepe University 
where glucagon assay was performed.  
Plasma glucagon was measured by using a direct sandwich ELISA technique (Mercodia 
Glucagon ELISA, cat. no. 10-1271-01, lot no. 29870, Uppsala, Sweden) with standard 
manufacturer’s protocol. Optical density  (OD) values of the samples were read in 
ELISA plate reader at 450 nm wavelengths (EPOCH-Spectrophotometer, BioTek, 
USA). The concentrations of the samples were calculated according to the standard 
curve drawn based on the OD of the manufacturer’s standards with known 
concentrations. Each sample was measured in two different wells for double-check. 

 
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Commercial controls pools with low, intermediate and high glucagon concentrations 
routinely assayed as samples. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
6 
THE EFFECT OF PANCREATIC INSUFFICIENCY ON HYPOGLYCEMIA 
AND GLUCAGON RESPONSE IN CHILDREN WITH CYSTIC FIBROSIS 
 
Short Running Title: Hypoglycemia and Glucagon Response in CF 
 
Belma Haliloglu, MD,PhD 1,2 
Tuba Seven Menevse, MD 1 
Seda Gulec Yilmaz, PhD 2 
Tuba Akdeniz, PhD 2 
Busra Gurpinar Tosun, MD 1 
Serap Demircioglu, MD 1 
Tulay Guran, MD 1 
Yasemin Gokdemir, MD 3 
Ela Erdem, MD 3 
Bulent Karadag, MD 3 
Turgay Isbir, MD, PhD 2 
Abdullah Bereket, MD 1 
 
1 Division of Pediatric Endocrinology and Diabetes, Department of Child Health and 
Disease, Marmara University School of Medicine, Istanbul, Turkey 
2 Department of Molecular Medicine, School of Health Science, Yeditepe University, 
Istanbul, Turkey 
3 Division of Pediatric Pulmonogy, Department of Child Health and Disease, Marmara 
University School of Medicine, Istanbul, Turkey 
 
Corresponding author: Belma Haliloglu, belma.haliloglu@marmara.edu.tr 
                                                                               belmahaliloglu26@hotmail.com 
                                                                      
 
 
 
 

 
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STATISTICAL ANALYSIS METHOD 
All analyses were carried out using GraphPad Prism® V5.0 software (GraphPad 
Software Inc., San Diego, California, USA). Variables are summarized for each group 
using descriptive statistics as percentages and mean±SD. Pairwise comparisons were 
performed using the Student T test or the Mann-Whitney U test depending on the data's 
normality. Kolmogorov-Smirnov test was used to evaluate of the data normality. Data 
were expressed as mean and standard error or deviation, which was stated. Statistical 
significance was set at p < 0.05.  
 
 
 
 
 
 
 
 
 
 
 
 
 

 
8 
THE EFFECT OF PANCREATIC INSUFFICIENCY ON HYPOGLYCEMIA 
AND GLUCAGON RESPONSE IN CHILDREN WITH CYSTIC FIBROSIS 
 
Short Running Title: Hypoglycemia and Glucagon Response in CF 
 
Belma Haliloglu, MD,PhD 1,2 
Tuba Seven Menevse, MD 1 
Seda Gulec Yilmaz, PhD 2 
Tuba Akdeniz, PhD 2 
Busra Gurpinar Tosun, MD 1 
Serap Demircioglu, MD 1 
Tulay Guran, MD 1 
Yasemin Gokdemir, MD 3 
Ela Erdem, MD 3 
Bulent Karadag, MD 3 
Turgay Isbir, MD, PhD 2 
Abdullah Bereket, MD 1 
 
1 Division of Pediatric Endocrinology and Diabetes, Department of Child Health and 
Disease, Marmara University School of Medicine, Istanbul, Turkey 
2 Department of Molecular Medicine, School of Health Science, Yeditepe University, 
Istanbul, Turkey 
3 Division of Pediatric Pulmonogy, Department of Child Health and Disease, Marmara 
University School of Medicine, Istanbul, Turkey 
 
Corresponding author: Belma Haliloglu, belma.haliloglu@marmara.edu.tr 
                                                                               belmahaliloglu26@hotmail.com 
                                                                      
 
 
 
 

 
9 
INFORMED CONSENT FORM 
 
Title of the Study: The Effect Of Pancreatic Insufficiency On Hypoglycemia And 
Glucagon Response In Children With Cystic Fibrosis 
 
Principal Investigators  
Tuba Seven Menevse, MD 
Department of Pediatric Endocrinology and Diabetes 
Basibuyuk Mah. Marmara University Hospital, 34854, Maltepe-İstanbul / Turkey 
+90542 553 8337 
tuba.seven@hotmail.com 
 
Belma Haliloglu, MD,PhD 
Department of Pediatric Endocrinology and Diabetes 
Basibuyuk Mah. Marmara University Hospital, 34854, Maltepe-İstanbul / Turkey 
+90505 267 0197 
belma.haliloglu@marmara.edu.tr 
PURPOSE OF STUDY 
You are being asked to take part in a research study. Before you decide to participate in 
this study, it is important that you understand why the research is being done and what 
it will involve. Please read the following information carefully. Please ask the 
researcher if there is anything that is not clear or if you need more information. 
Hypoglycemia can be seen in patients with Cystic Fibrosis (CF). In these patients, the 
production and secretion of insulin hormone is diminished and dysregulated. However, 
the role and effect of glucagon in hypoglycemic patients with cystic fibrosis is still 
unknown. In this study, we aimed to investigate the frequency and etiopathogenesis of 
hypoglycemia in CF. 
 
STUDY PROCEDURES 
Oral glucose Tolerance Test (OGTT) is performed annually in all patients with Cystic 
Fibrosis. You will be also performed the OGTT test today, which should be performed 

 
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routinely. During OGTT, extra blood samples at the 0-60-120-150-180.min for 
glucagon analysis and at 0.min for CRP will be obtained in addition to the normal blood 
sampling. No additional procedure will be made for this study. 
 
RISKS 
In this study, no alternative treatment will be implemented and no additional procedure 
will be made. Therefore, there is no risk to you and does not cause any discomfort. You 
may decline to answer any or all questions and you may terminate your involvement at 
any time if you choose. 
 
BENEFITS 
There will be no direct benefit to you for your participation in this study. However, we 
hope that the information obtained from this study may help to understand the reason of 
hypoglycemia in CF. 
 
CONFIDENTIALITY 
Your data and results in this study will be anonymous. Every effort will be made by the 
researcher to preserve your confidentiality including the following:  
ü Assigning code numbers for participants that will be used on all research notes and 
documents 
ü Keeping notes and any other identifying participant information in a locked file 
cabinet and in a private computer of the researcher. 
Participant data will be kept confidential except in cases where the researcher is legally 
obligated to report specific incidents.  
 
CONTACT INFORMATION  
If you have questions at any time about this study, or you experience adverse effects as 
the result of participating in this study, you may contact the researcher whose contact 
information is provided on the first page. If you have questions regarding your rights as 
a research participant, or if problems arise which you do not feel you can discuss with 

 
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the Primary Investigator, please contact the Department of Pediatric Endocrinology and 
Diabetes in Marmara University Hospital, 34854, Maltepe-İstanbul / Turkey 
VOLUNTARY PARTICIPATION 
Your participation in this study is voluntary. It is up to you to decide whether or not to 
take part in this study. If you decide to take part in this study, you will be asked to sign 
a consent form. After you sign the consent form, you are still free to withdraw at any 
time and without giving a reason. Withdrawing from this study will not affect the 
relationship you have, if any, with the researcher. If you withdraw from the study before 
data collection is completed, your data will be returned to you or destroyed.  
 
 
CONSENT 
 
I have read and I understand the provided information and have had the opportunity to 
ask questions. I understand that my participation is voluntary and that I am free to 
withdraw at any time, without giving a reason and without cost. I understand that I will 
be given a copy of this consent form. I voluntarily agree to take part in this study.  
 
 
Participant's signature ______________________________ Date __________  
 
 
 
Investigator's signature _____________________________ Date __________