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On Meal Effects and DPP-4 Inhibition Islet- and Incretin Hormones in Health and Type 2Diabetes
Alsalim, Wathik
2020
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Citation for published version (APA):Alsalim, W. (2020). On Meal Effects and DPP-4 Inhibition Islet- and Incretin Hormones in Health and Type 2Diabetes. Lund: Lund University, Faculty of Medicine.
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WA
THIK
ALSA
LIM
O
n Meal E
ffects and DPP-4 Inhibition Islet- and Incretin H
ormones in H
ealth and Type 2 D
iabetes 2020:39
Department of Clinical Sciences
Lund University, Faculty of Medicine Doctoral Dissertation Series 2020:39
ISBN 978-91-7619-900-8ISSN 1652-8220
On Meal Effects and DPP-4 Inhibition Islet- and Incretin Hormones in Health and Type 2 DiabetesWATHIK ALSALIM
DEPARTMENT OF CLINICAL SCIENCES | LUND UNIVERSITY
About the author
Wathik Alsalim completed his medical training at Baghdad College of Medicine, Baghdad University, Iraq, in 1999. He is currently working as a senior registrar in endocrinology at Skåne University Hospital, Sweden. He conducted his doctoral research at the Department of Clinical Sciences, Faculty of Medicine, Lund University, Sweden. His PhD thesis focuses on the effects of meals on the regulation of glycaemia, and islet and incretin hormones in health and type 2 diabetes.
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On Meal Effects and DPP-4 Inhibition Islet- and Incretin
Hormones in Health and Type 2 Diabetes
2
3
On Meal Effects and DPP-4 Inhibition
Islet- and Incretin Hormones in Health
and Type 2 Diabetes
Wathik Alsalim
DOCTORAL DISSERTATION
which, by due permission of the Faculty of Medicine, Lund University, Sweden,
will be defended at Segerfalksalen, BMC, Sölvegatan 19, Lund.
16th May 2020 at 09:00
Faculty opponent
Professor Erik Näslund Clinical Sciences, Danderyd Hospital. Karolinska Institute
4
Organization
LUND UNIVERSITY
Document name
DOCTORAL DISSERTATION
DEPARTMENT OF CLINICAL SCIENCES, LUND, FACULTY OF MEDICINE
Date of issue: 2020-05-16
Author: Wathik Alsalim Sponsoring organization
Title: On Meal Effects and DPP-4 Inhibition Islet- and incretin hormones in health and type 2 diabetes
Abstract:
Meal composition and size are crucial in the regulation of glycaemia and islet hormones in both healthy subjects and those with type 2 diabetes (T2D). Incretin hormones, glucagon-like peptide-1 and glucose-dependent insulinotropic poly-peptide, are released after meal intake. They regulate postprandial glycaemia by stimulation of insulin secretion. Incretin hormones are rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP-4). The effect of incretin hormones is reduced in T2D. DPP-4 inhibitors stimulate insulin secretion in the presence of hyperglycaemia. It has not been established whether DPP-4 inhibitors affect postprandial glycaemia or islet hormones following meal ingestion in normoglycaemic conditions is not established.
The aim of the research presented in this thesis was to characterize the effect of meal composition, size and timing with or without DPP-4 inhibition on the response of plasma glucose and islet and incretin hormones in healthy and T2D subjects.
The studies were single-centre studies, and included both men and women, with and without T2D. Meals of different composition and size were ingested with or without the intake of DPP-4 inhibitors.
The most important findings were as follows.
1. In healthy subjects, increasing the caloric content of the meal induced adaptive stimulation of incretin hormones and beta-cell function to maintain similar plasma glucose levels.
2. The intake of a mixed meal reduced the increase in plasma glucose and stimulated beta-cell function and incretin hormone secretion in both healthy and drug naïve and well-controlled T2D subjects, compared to the intake of individual macronutrients.
3. DPP-4 inhibition reduced plasma glucose after separate macronutrient intake in healthy subjects, and after mixed meal ingestion in both healthy and drug naïve and well-controlled T2D subjects.
4. Three different DPP-4 inhibitors (sitagliptin, saxagliptin and vildagliptin) similarly elevated the level of intact incretin hormones and suppressed incretin hormone secretion throughout the day. DPP-4 inhibitors also suppressed glycaemia and stimulated beta-cell function after meal intake in metformin-treated T2D subjectsthroughout the day.
In conclusion, new knowledge has been gained on the dynamic regulation of glucose homeostasis, islet and incretin hormone responses in healthy subjects and T2D subjects following meal ingestion with or without DPP-4 inhibition.
.
Key words: Type 2 diabetes, healthy subjects, meal composition, GLP-1, GIP, DPP-4 inhibition
Classification system and/or index terms (if any)
Supplementary bibliographical information Language: English
ISSN and key title: 1652-8220 ISBN: 978-91-7619-900-8
Recipient’s notes Number of pages:70 Price
Security classification
I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all reference sources permission to publish and disseminate the abstract of the above-mentioned dissertation.
Signature Date 2020-03-22
5
On Meal Effects and DPP-4 Inhibition
Islet- and Incretin Hormones in Health
and Type 2 Diabetes
Wathik Alsalim
6
Cover layout: Frida Nilsson:
Copyright pp 1-70 Wathik Alsalim
Paper 1 © Diabetes Obes Metab, 15: 531-7, 2013
Paper 2 © J Clin Endocrinol Metab, 100: 561-8, 2015
Paper 3 © Diabetes Obes Metab, 18: 24-33, 2016
Paper 4 © Diabetes Obes Metab, 20: 1080-1085, 2018
Paper 5 © Diabetes Obes Metab, 22: 590-598, 2020
Faculty of Medicine
Department of Clinical Sciences
Lund University
ISBN 978-91-7619-900-8
ISSN 1652-8220
Printed in Sweden by Media-Tryck, Lund University
Lund 2020
7
To my family,
Rana, Yasmin and Linn
قل لمن يدعي في العلم فلسفة حفظت شيئا وغابت عنك اشياء
”Tell those who claim philosophy in science,
you have preserved something but you may have missed other things”
ابو نواسAbu Nuwas, (757-815), Baghdad, Iraq
8
Contents
Abbreviations ...............................................................................................10
Abstract ........................................................................................................11
Populärvetenskaplig sammanfattning...........................................................12
13................................... (Scientific summary in Arabic) الملخص العلمي لهذه الرسالة
List of Papers ................................................................................................16
Related papers not included in this thesis ....................................................16
Introduction ..........................................................................................................17
Background ...........................................................................................................18
Regulation of postprandial glycaemia ..........................................................18
The role of nutrients in the regulation of postprandial glycaemia ................19 Meal composition ................................................................................19 Meal size ..............................................................................................20
Incretin hormone biosynthesis and secretion ...............................................20
Mechanisms whereby macronutrients regulate incretin hormone secretion 22 Carbohydrate sensing ..........................................................................22 Lipid sensing .......................................................................................22 Protein sensing.....................................................................................22
Incretin hormone degradation ......................................................................23
GLP-1 and GIP receptors .............................................................................23
The physiological role of incretin hormones ................................................24 Pancreatic effects of incretin hormones ...............................................24 Extrapancreatic effects of incretin hormones ......................................25
Incretin hormones in T2D ............................................................................25
Treatment of T2D .........................................................................................26
Incretin-based therapy ..................................................................................27 Pancreatic effects of incretin-based therapy ........................................27 Extra-pancreatic effects of incretin-based therapy ..............................29
Incretin-based therapy in type 1 diabetes .....................................................30
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Rationale and the Aims of the Research .............................................................31
Subjects and Methods ..........................................................................................32
Study design, medication and meal composition .........................................32
Study populations .........................................................................................34
Ethics and good clinical practices ................................................................35
Power calculation .........................................................................................35
Laboratory measurements ............................................................................35
Assessment of beta-cell function ..................................................................36
Calculations ..................................................................................................36
Statistical analysis ........................................................................................37
Results ....................................................................................................................38
Part 1: Effect of the meal on glycaemia and the islet and incretin
hormones ......................................................................................................38
Part 2: DPP-4 inhibition and meal composition: subgroup analysis ............40 Meal composition in healthy subjects and drug-naïve subjects
with T2D ..............................................................................................40 DPP-4 inhibition in healthy subjects and drug-naïve subjects
with T2D ..............................................................................................41 DPP-4 inhibition in drug-naïve T2D subjects and
metformin-treated T2D subjects ..........................................................42
Main findings ...............................................................................................43
Discussion ..............................................................................................................44
The effects of meal composition ..................................................................44
The effects of meal size ................................................................................46
The effect of DPP-4 inhibition .....................................................................47
Strengths and limitations of this work ..........................................................49
Concluding Remarks and Future Perspectives ..................................................50
Clinical significance ..............................................................................................51
Acknowledgements ...............................................................................................52
References .............................................................................................................54
10
Abbreviations
ADA American Diabetes Association
AEs Adverse Events
AUC Area Under the Curve
BMI Body Mass Index
cAMP cyclic Adenosine Monophosphate
CNS Central Nervous System
DPP-4 Dipeptidyl Peptidase-4
EASD European Association for the Study of Diabetes
ELISA Enzyme linked immunosorbent assay
GIP Glucose-Dependent Insulinotropic Polypeptide
GIPR GIP Receptor
GLP-1 Glucagon-Like Peptide-1
GLP-1 RAs GLP-1 Receptor Agonists
GPCRs G Protein-Coupled Receptors
HbA1c Glycated Hemoglobin
IGT Impaired Glucose Tolerance
ISR Insulin Secretion Rate
MACE Major Adverse Cardiovascular Events
RIA Radioimmunoassay
SGLT-1 Sodium-Glucose co-Transporter-1
T1D Type 1 Diabetes
T2D Type 2 Diabetes
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Abstract
Meal composition and size are crucial in the regulation of glycaemia and islet
hormones in both healthy subjects and those with type 2 diabetes (T2D). Incretin
hormones, glucagon-like peptide-1 and glucose-dependent insulinotropic poly-
peptide, are released after meal intake. They regulate postprandial glycaemia by
stimulation of insulin secretion. Incretin hormones are rapidly degraded by the
enzyme dipeptidyl peptidase-4 (DPP-4). The effect of incretin hormones is reduced
in T2D. DPP-4 inhibitors stimulate insulin secretion in the presence of
hyperglycaemia. It has not been established whether DPP-4 inhibitors affect
postprandial glycaemia or islet hormones following meal ingestion in
normoglycaemic conditions is not established.
The aim of the research presented in this thesis was to characterize the effect of meal
composition, size and timing with or without DPP-4 inhibition on the response of
plasma glucose and islet and incretin hormones in healthy and T2D subjects. The
studies were single-centre studies, and included both men and women, with and
without T2D. Meals of different composition and size were ingested with or without
the intake of DPP-4 inhibitors.
The most important findings were as follows.
1. In healthy subjects, increasing the caloric content of the meal induced
adaptive stimulation of incretin hormones and beta-cell function to maintain
similar plasma glucose levels.
2. The intake of a mixed meal reduced the increase in plasma glucose and
stimulated beta-cell function and incretin hormone secretion in both healthy
and drug naïve and well-controlled T2D subjects, compared to the intake of
individual macronutrients.
3. DPP-4 inhibition reduced plasma glucose after separate macronutrient
intake in healthy subjects, and after mixed meal ingestion in both healthy
and drug naïve and well-controlled T2D subjects.
4. Three different DPP-4 inhibitors (sitagliptin, saxagliptin and vildagliptin)
similarly elevated the level of intact incretin hormones and suppressed
incretin hormone secretion throughout the day. DPP-4 inhibitors also
suppressed glycaemia and stimulated beta-cell function after meal intake in
metformin-treated T2D subjects throughout the day.
In conclusion, new knowledge has been gained on the dynamic regulation of glucose
homeostasis, islet and incretin hormone responses in healthy subjects and T2D
subjects following meal ingestion with or without DPP-4 inhibition.
12
Populärvetenskaplig sammanfattning
Tarmhormoner har betydelse för reglering av blodsocker och insulin samt
mättnadskänslan efter maten. Inkretinhormoner: Glukagon-like peptid -1 (GLP-1) och
glukosberoende insulinfrisättande polypeptid (GIP) är bland de viktigaste hormonerna
involverade i dessa mekanismer. Dessa hormoner frisätts från tarmen efter måltid och
påverkar bland annat insulinkoncentrationerna i blodet. Det är bekräftat att GLP-1 och
GIP-effekten är nedsatt hos patienter med diabetes typ 2 (T2D). DPP-4 hämmare är en
läkemedelsform som hämmar nedbrytningen av GLP-1 och GIP vilket ökar deras
koncentrationen i blodet.
Måltidssammansättningen och storleken på måltiden är också avgörande för insöndring
av GLP-1 och GIP. Kunskapen om hur dessa hormoner påverkas av olika
måltidssammansättningar och måltidsstorleken är viktig för att kunna ge individer med
T2D anpassade kostråd.
Således är det övergripande syftet med de vetenskapliga studierna som är presenterade
i denna avhandling att karaktärisera responsen av blodsocker, insulin och
inkretinhormoner efter intag av måltider hos friska försökspersoner och personer med
välkontrollerad T2D med fokus på effekten av måltidsammansättning, storlek och
tidpunkten för måltidsintaget även tillsammans med intag av DPP-4-hämmare. De mest
viktiga resultaten av de vetenskapliga studierna är:
1. Gradvis ökning av kaloriinnehållet av lunchmåltiden hos friska individer
medför en anpassad ökning av inkretinhormoner och insulinfrisättningen för att
bibehålla identiska blodsockernivåer.
2. Intag av en blandad måltid som innehåller kolhydrater, protein och fett hos
friska personer och personer med T2D minskar ökningen av blodsocker,
förbättrar insulinfrisättningen och stimulerar inkretinhormonernas sekretion
jämfört med intaget av enskilda makronäringsämnen.
3. DPP-4-hämningen sänker blodsocker, både efter separat intag av varje
makronäringsämne (friska försökspersoner) och efter blandad måltid (friska
och personer med T2D).
4. DPP-4-hämmare har ihållande effekt på blodsockernivåer, insulinfrisättningar
och inkretinhormoner under dagen.
5. Det fanns inga skillnader i dessa effekter mellan de välkända DPP-4 hämmare
som finns på marknaden (sitagliptin, saxagliptin och vildagliptin).
Sammanfattningsvis ger denna avhandling en detaljerad kunskap om hur
kostsammansättningen och DPP-4-hämmare påverkar blodsockret hos friska personer
och personer med T2D. Därför anses att denna kunskap kan vara användbar vid
kostrådgivningen av sjukvårdspersonal under deras dagliga arbete med personer med
T2D.
13
(Scientific summary in Arabic)العلمي لهذه الرسالة الملخص
ان مرض السكري من النمط الثاني اصبح من االمراض المنتشرة في العالم اجمع . من اهم العوامل التي تؤدي
وعدم التنظيم في اوقات األكل ، وخمول في النشاط البدني والعوامل صابه بهذا المرض هي زيادة الوزن،الى اال
السكر في الدم وبهذا ةؤدي الى ارتفاع نسبت ا البنكرياس في افراز االنسولينية . ان عدم قدرة خاليالوراث
يتميز النمط الثاني من السكري باختالفه عن النمط األول من حيث وجود مقاومة مضادة بداء السكري. ةاالصاب
نسجة الجسم بالت األنسولين الموجودة في اكما أن مستق ،ة إفراز األنسولينلمفعول األنسولين باإلضافة إلى قل
تكون مصحوبة بارتفاع مستويات األنسولين في للمرض المراحل األولىان .لينألنسولمقاومة تكون المختلفة
% من المرضى المصابين 55يعاني نكرياس.الب غدة تقل كفاءة إفراز األنسولين من المرضكلما تطور .الدم
% من المسنين يعانون 20توجد عوامل أخرى مثل التقدم بالعمر، فحوالي من السمنة. بالنمط الثاني من السكري
.من السكري في أمريكا الشمالية
ناول الطعام. الهرمونات المعوية مهمة لتنظيم السكر واألنسولين في الدم وكذلك الشعور بالشبع بعد تان
نسولين ( وبولي ببتيد المفرز لالGLP-1) 1-(: الببتيد الشبيه بالجلوكاجون Incretinهرمونات االنكرتين )
تفرز هذه الهرمونات . ( من بين الهرمونات الرئيسية المشاركة في هذه اآللياتGIPالمعتمد على الجلوكوز )
معدل ىيطر علسمستويات األنسولين في الدم. بهذه الطريقة ت من األمعاء بعد الوجبة الغذائية وتؤثر على تنظيم
في مرض السكر من النمط GIPو GLP-1تأكد ان فعالية التم وث العلميةحمن خالل الب السكر في الدم.
منخفظة جدا وهذا بدوره يؤدي الى انخفاظ في االنسولين المفروز وارتفاع في نسبة السكر في الدم بعد الثاني
-DPP.4الطعام. تتحلل هرمونات االنكرتين بسرعة الى هرمونات غير فعالة بواسطة انزيم يدعى تناول
وفعاليتها مما يزيد من تركيزها GIPو GLP-1هي شكل من أشكال األدوية التي تمنع تحلل DPP-4مثبطات
في دولة السويد على في العالم اجراء اول دراسة علمية 2000في عام تفي الدم. تم في خفظ نسبة السكر
تتفي عالج مرض السكر من النمط الثاني. ان هذه الدراسة وما تالها من دراسات اثب DPP-4فعالية مثبطات
DPP-4مثبطات تاصبح 2006ان العالج بهذه العقاقير هو فعال لخفظ نسبة السكر في الدم. لذلك ومنذ عام
.جزء مهم في عالج هذا الداء
معرفة كيف تتأثر هذه الهرمونات ان .GIPو GLP-1وحجم الوجبة الغذائية مهمان أيضا في افراز مكونات
السكري ة المختلفة مهم للكادرالطبي المتخصص في تزويد افراد مرضى يبتركيب وحجم الوجبات الغذائ
بنصائح غذائية جيدة لتجنب ارتفاع السكر في الدم عند االكل.
،ف ومعرفة دقيقة في استجابة السكروص يذه الرسالة والبحوث المقدمة فيها هإن الهدف العام من ه
، وهرمونات االنكرتين في الدم بعد الوجبات الغذائية لالشخاص األصحاء واألفراد الذين يعانون واألنسولين
لها. وقمنا وحجم الوجبة الغذائية ووقت تناو نوعية، مع التركيز على تأثير مرض السكري من النمط الثانيمن
ضاي ضبعد االكل واي DPP-4ثير مثبطات في فحص مدى فعالية وتا ا ة المثالية يمعرفة ما هي الوجبة الغذائ ا
:هذا البحث اثبتتان نتائج كما. في هذا السياق
تؤدي وجبة الغداء لدى األفراد األصحاءالزيادة التدريجية في محتوى السعرات الحرارية في ان
ل هرمونات االنكرتين وإفراز األنسولين للحفاظ على مستويات السكر في الدم متطابقة إلى زيادة معد
الغذائية. هذه الوجباتوغير معتمدة على حجم
تناول وجبة متعددة المكونات تحتوي على الكربوهيدرات والبروتين والدهون لدى األفراد األصحاء
يقلل من نسبة السكر في الدم ويحسن الثانيمرض السكري من النمط واألشخاص الذين يعانون من
ة كل عل حدة .يإفراز األنسولين ويحفز إفراز هرمونات االنكرتين مقارنة بتناول المكونات الغذائ
14
مثبطات عقاقيرتعملDPP-4 بعد تناول وجبة تحتوي على على خفض نسبة السكر في الدم ،
صحاء بدون زيادة في افراز هرمون االنسولينالكربوهيدرات اوالبروتين اوالدهون لدى األفراد األ
ىعل تعمل DPP-4مثبطات ان ا ضزيادة افراز هرمونات االنكرتين. واي ىثيرها علأمن خالل ت و
األصحاء عند ) تنوعةمالالغذائية ةالسكر في الدم بعد الوجب خفضزيادة فعالية افراز االنسولين و
(.النمط الثانيالسكري من مرض واألشخاص الذين يعانون من
مثبطات عقاقير تأثيرانDPP-4 وإفرازات مستويات السكر في الدم على رالنها لخال دائم ،
.مرض السكرياألنسولين وهرمونات االنكرتين عند األشخاص الذين يعانون من
مثبطات ان فعاليةDPP-4 المتداولة(sitagliptin ،saxagliptin وvildagliptin) متساوية
.وبدون وجود فروق ملحوظة
على DPP-4معرفة تفصيلية ودقيقة بكيفية تأثير التركيب الغذائي ومثبطات بهذه الرسالة تزودناباختصار ،
. لذلك أن هذه مرض السكرينسبة السكر في الدم لدى األشخاص األصحاء واألشخاص الذين يعانون من
ألخصائيي الرعاية الصحية أثناء عملهم اليومي مع األشخاص الذين النصائح الغذائية تعززيمكن أن النتائج
.مرض السكري من النمط الثانييعانون من
15
ارتقي م شموخا وانا الي وما كنت ورونق شمس لكتابلهذا ا كانما لوالكم
:مسكنهم من في القلب الى
ولين وياسمين رنا
بمطمح من يحاول أن يسوداأرى مستقبل األيام أولى
فما بلغ المقاصد غير ساع يردد في غد نظرا سديدا
ه وجه عزمك نحو آت وال تلفت إلى الماضين جيدا فوج
(1945 – 1875معروف عبد الغني الرصافي )
16
List of Papers
This thesis is based on the following papers, which will be referred to in the text by
their roman numerals.
I. Ohlsson L, Alsalim W, Carr RD, Tura A, Pacini G, Mari A & Ahrén B
Glucose-lowering effect of the DPP-4 inhibitor sitagliptin after glucose and
non-glucose macronutrient ingestion in non-diabetic subjects Diabetes
Obes Metab, 2013: 15(6): p. 531-7
II. Alsalim W, Omar B, Pacini G, Bizzotto R, Mari A & Ahrén B Incretin and
islet hormone responses to meals of increasing size in healthy subjects . J
Clin Endocrinol Metab, 2015: 100(2): p. 561-8
III. Alsalim W, Tura A, Pacini G, Omar B, Bizzotto R, Mari A & Ahrén B
Mixed meal ingestion diminishes glucose excursion in comparison with
glucose ingestion via several adaptive mechanisms in people with and
without type 2 diabetes Diabetes Obes Metab, 2016: 18(1): p. 24-33
IV. Alsalim W, Göransson O, Carr RD, Bizzotto R, Tura A, Pacini G, Mari A
&Ahrén B . Effect of single-dose DPP-4 inhibitor sitagliptin on beta-cell
function and incretin hormone secretion after meal ingestion in healthy
volunteers and drug-naïve, well-controlled type 2 diabetes subjects.
Diabetes Obes Metab, 2018: 20(4): p. 1080-1085
V. Alsalim W, Göransson O, Tura A, Pacini G, Mari A & Ahrén B Persistent
whole-day meal effects of three dipeptidyl peptidase-4 inhibitors on
glycaemia and hormonal responses in metformin-treated type 2 diabetes.
Diabetes Obes Metab, 2020: 22(4): p. 590-598
Related papers not included in this thesis
1. Alsalim, W. and Ahren B. Insulin and incretin hormone responses to rapid
versus slow ingestion of a standardized solid breakfast in healthy subjects.
Endocrinol Diabetes Metab, 2019. 2(2): p. e00056
2. Alsalim, W., Persson M. and Ahren B. Different glucagon effects during DPP-
4 inhibition versus SGLT-2 inhibition in metformin-treated type 2 diabetes
patients. Diabetes Obes Metab, 2018. 20(7): p. 1652-1658
3. Alsalim, W., Al-Hakeem, S., Elf, J, and Erfurth EM. Apoplexy of an
adrenocorticotrophic pituitary macroadenoma after treatment of acute
myocardial infarction. Clin Case Rep J, (accepted article, Mars 2020)
17
Introduction
The type of nutrition plays an essential role in the control of postprandial glycaemia.
Medical nutrition therapy and an increase in physical activity are considered the
first-line treatment of type 2 diabetes (T2D) in combination with glucose-lowering
therapies [1]. The American Diabetes Association (ADA) and the European
Association for the Study of Diabetes (EASD) recommend daily nutrition consisting
of all macronutrients in each meal [2,3]. It is also recommended that the
macronutrient distribution of the meal be individualized depending on the metabolic
goals and eating habits of the people with T2D.
Little is known on how the different macronutrients in a meal regulate postprandial
glycaemia and islet hormone secretion [2]. Furthermore, most of the studies
presented to date have investigated the effects of meal composition and meal size
on glycaemia and islet hormone secretion only after breakfast, which may not be
representative of the day-to-day eating patterns of most people [4-8].
It is also known that the postprandial glycaemic response to the ingestion of a
specific meal is different in the morning than in the afternoon [9]. It is not known
whether all the macronutrients in a meal are essential in regulating postprandial
glycaemia and islet hormone response. Neither are the effects known of meal size,
or the time of day at which it is ingested. Such knowledge is required for clinical
professionals in their daily practice when treating people with T2D.
It is thus important to understand how meal composition, size and timing affect
postprandial glycaemia and islet hormones in health and T2D. Furthermore, it is
essential to investigate the effect of meal composition in combination with glucose-
lowering medication such as dipeptidyl peptidase-4 (DPP-4) enzyme inhibitors on
the regulation of postprandial glycaemia and islet hormone secretion.
The focus of work presented in this thesis was to investigate the effects of meal
composition, size and timing with or without intake DPP-4 inhibition on glucose
homeostasis and islet and incretin hormone response in healthy subjects and subjects
with T2D.
18
Background
Following a meal, glucagon-like peptide-1 (GLP-1) and glucose-dependent
insulinotropic polypeptide (GIP) are released from the enteroendocrine cells, which
are responsible for the stimulation of insulin secretion. It is established that the
insulin response to oral glucose intake is a factor 2-3 higher than after intravenous
glucose infusion when the same plasma glucose levels is elicited in healthy subjects
[10]. This difference in insulin secretion between oral and intravenous glucose
administration is known as the incretin effect [10]. Interestingly, a similar effect is
seen when comparing insulin secretion after oral lipid or protein ingestion with
intravenous lipid or amino acid infusion. This indicates that the incretin effect is not
only glucose-dependent [11,12].
The postprandial hyperglycaemia in T2D may be caused by a reduction in the beta-
cell function, which results in a reduction in insulin secretion in response to the
ingested nutrients [13,14]. In addition, the incretin effect, i.e. the postprandial
augmentation of insulin secretion by GLP-1 and GIP, is impaired in T2D. The
postprandial insulin secretion in response to the incretin hormones has been
estimated to be < 20% in T2D [15-17]. In line with beta-cell dysfunction, an increase
in insulin resistance and inappropriate glucagon secretion also contribute to the
increase in postprandial hyperglycaemia in T2D [18,19].
Incretin-based therapy has been developed for the treatment of T2D. DPP-4
inhibition and GLP-1 receptor agonists (GLP-1RAs) are established glucose-
lowering therapies in T2D, which increase insulin secretion and suppress glucagon
secretion [20,21].
Regulation of postprandial glycaemia
Glucose excursion after the ingestion of a meal is similar in healthy subjects with
normal glucose tolerance, regardless of the composition and size of the meal [22-
24]. However, the insulin response to the ingested meal varies. Thus, the regulation
of glucose homeostasis depends on the interaction between insulin sensitivity and
beta-cell function after a meal [25]. Under normal conditions, glucose homeostasis
is regulated by the feedback loop between insulin sensitivity and beta-cell function
so as to maintain normal plasma glucose levels [25]. When there is an increase in
insulin resistance, as in obesity, insulin secretion is enhanced to maintain normal
19
plasma glucose levels after a meal [26]. When the beta-cell response to the
continuous increase in insulin demand is reduced, hyperglycaemia ensues. Thus, a
progressive increase in insulin resistance is considered an essential risk factor in the
development of T2D in association with continued progression in beta-cell
dysfunction [27,28].
The role of nutrients in the regulation of postprandial glycaemia
According to Nordic nutritional recommendations, the total energy (E%) intake
during one day for a healthy adult should be: 45-60 E% from carbohydrates, 10-20
E% from protein and 25-40 E% from fat [29]. Although many studies have
attempted to identify the optimal combination of macronutrients for those with
diabetes, there is still no ideal meal composition. Therefore, the composition of
macronutrients in meals may be individualized for those with T2D [30,31].
Considering the above, it is important to investigate the impact of meal composition
and size on the regulation of glycaemia in physiology and pathophysiology (subjects
with T2D).
Meal composition
The choice of meal plays an important role in the regulation of postprandial
glycaemia in health and T2D. For example, the intake of a protein- or fat-enriched
meal before a carbohydrate-enriched meal suppresses postprandial glucose excur-
sion compared to the ingestion of a carbohydrate-enriched meal alone [32-35]. This
is due to the effects of protein and fat on the reduction in the gastric emptying and
glucose absorption. Furthermore, protein and fat affect the stimulation of insulin
secretion through the stimulation of incretin hormones [6]. Interestingly, these
effects seem to be similar in healthy subjects, subjects with impaired glucose
tolerance (IGT), and subjects with well-controlled T2D [36].
Ingestion of DPP-4 inhibition before the ingestion of a protein-rich meal enhances
the glucose-lowering effect of DPP-4 inhibition compared to the ingestion of a fat-
rich meal [37,38]. This indicates that tailoring the composition of a meal is important
as a complement to glucose-lowering drugs in the management of T2D. The
postprandial response also varies depending on the type and amounts of
macronutrients in the meal [31,39].
Although all macronutrients are involved in the regulation of postprandial glucose
after meal ingestion, it is necessary to understand the response of postprandial
glycaemia and islet function to the intake of individual macronutrients (glucose,
protein and fat) alone and in combination in a mixed meal. Furthermore, it is
important to investigate the impact of DPP-4 inhibition on postprandial glycaemia
20
and the secretion of islet hormones throughout the day in healthy subjects and T2D
subjects after the ingestion of meals with different compositions.
Meal size
In addition to meal composition, it has been suggested that meal size affects the
postprandial glycaemic response. An increase in the oral glucose load has a minor
impact on postprandial glycaemia in healthy subjects [22]. This is partly due to the
dose-dependent increase in insulin secretion through the enhancement of the
incretin effect after oral glucose ingestion [15]. In contrast, the incretin contribution
seems to have a minor impact on insulin secretion following an increase in the
protein load. One possible explanation of this is a slight increase in glucose levels,
which may not be sufficient to stimulate insulin secretion after oral protein ingestion
[40].
It has been reported that an increase in the meal size at breakfast, from 260 kcal to
520 kcal, elicited a caloric-dependent increase in incretin hormone secretion in
obese subjects [7]. This gave rise to high insulin secretion after the larger meal.
However, the postprandial glucose levels were similar after the intake of both meals.
As it has been found that there is a diurnal variation in the postprandial glycaemic
response after meal ingestion [9]. Therefore, it is important to ascertain whether an
increase in meal size in everyday life (e.g., lunchtime) in healthy subjects has a
similar impact on incretin and islet hormone secretion as breakfast.
Incretin hormone biosynthesis and secretion
Following the intake of a meal, GLP-1 and GIP are released. Their concentrations
initially increase in the portal circulation, and then in the systemic circulation within
a couple of minutes [6,12,41]. This increase in both GLP-1 and GIP is sustained
several hours after a meal [12,42].
The release of incretin hormones is dependent on the direct stimulation of endocrine
cells in the gastrointestinal tract by nutrients. The so-called open-type morphology
of these cells promotes their ability to sense nutrients quickly in the gastrointestinal
lumen [43,44]. K cells are the endocrine cells responsible for the release of GIP.
They are mostly found in the proximal small intestine. GLP-1, in contrast, is
released by the L cells, which are predominantly located in the distal small intestine
and the colon [43].
GIP was identified in 1969 [45]. Initially, it was shown that GIP inhibited gastric
acid secretion, hence its name, “gastric inhibitory peptide”. However, later studies
showed that GIP is involved in the stimulation of insulin secretion after meal
ingestion [46,47], and it was therefore renamed “glucose-dependent insulinotropic
21
polypeptide”. The active (intact) GIP (1-42) is a peptide produced by the K cells
from the pro-GIP peptide (Figure 1) [48].
GLP-1 was identified as a peptide derived from the proglucagon peptide produced
by L cells in the 1980s. GLP-1 is initially cleaved from proglucagon as GLP-1 (1-
37) and GLP-1 (1-36) NH2. These forms are not biologically active until they are
produced from their full-length precursors by the action of prohormone convertase
1/3 to either GLP-1 (7-37) or GLP-1 (7-36) NH2. The latter represents the dominant
circulating form of intact (active) plasma GLP-1 [49-51]. The incretin hormone
secretion (total GIP and total GLP-1) from the K and L cells is determined by
measuring the levels of both the active and inactive forms of the incretin hormones
(Figure 1).
It has been suggested that the incretin hormones secretion is inhibited through a
feedback loop by an increase in their intact levels of incretin hormones after meal
ingestion [52,53]. Whether this feedback mechanism is exerted after each meal
throughout the day, and whether incretin-based therapy has an impact on the
regulation of incretin hormone secretion, has not yet been thoroughly explored.
Figure 1. Secretion and metabolism of proGIP to active GIP in the K cells and proglucagon to the active form of GLP-1 in the L cells after meal intake. The effect of active GLP-1 and GIP on the islet hormones and the degradation of active GIP (1-42), GLP-1 (7-36)NH2 and GLP-1 (7-37) are also shown. DPP-4 converts active GLP-1 and GIP to inactive GLP-1 (9-36)NH2, GLP-1 (9-37) and GIP (3-42) in vivo. DPP-4 inhibitors prevent the degradation of active GIP and GLP-1.
22
Mechanisms whereby macronutrients regulate incretin hormone
secretion
The mechanisms by which macronutrients stimulate GLP-1 and GIP secretion
differ. The mechanisms thought to be behind the secretion of GLP-1 and GIP after
the ingestion of carbohydrates, lipids and protein, based mainly on animal studies,
are described below. However, clinical studies have not been able to confirm the
involvement of these pathways in incretin hormone secretion [54,55]. Therefore, the
definitive mechanism behind GLP-1 and GIP secretion in humans following
macronutrient intake is still unclear.
Carbohydrate sensing
Glucose intake triggers the release of GLP-1 and GIP [56]. Glucose uptake in K and
L cells through a sodium-glucose co-transporter-1 (SGLT-1) activates several
intracellular pathways, which trigger the electrical activity of the cell membranes.
As a consequence, K and L cells release GIP and GLP-1 [57]. The lack of release of
GLP-1 and GIP in SGLT-1 knockout mice after oral glucose load supports the
suggestion that SGLT-1 is involved in the release of GLP-1 and GIP [58]. It has also
been suggested that activation of the sweet taste receptors in the mouth is also
involved in incretin hormone release after carbohydrate ingestion [59].
Lipid sensing
Fat ingestion directly stimulates GLP-1 and GIP secretion [6,60-62]. The mechanism
behind incretin hormone secretion after fat ingestion involves stimulation of G
protein-coupled receptors (GPCRs) such as GPR40, GPR120 and GPR119 [63-66].
The long- and medium-chain fatty acids stimulate GPR120 and GPR40, which leads
to an increase in intracellular calcium and the release of incretin hormones from the
enteroendocrine cells [67]. Triglycerides stimulate cyclic adenosine monophosphate
(cAMP) which in turn results in peptide secretion [68,69].
Protein sensing
Protein ingestion also enhances GLP-1 and GIP secretion [6,41,53,56,70]. However,
the mechanism underlying the effect of amino acids on endocrine cells in the release
of incretin hormones is still not well established. It has been suggested that amino
acids are involved in both the electrical activation of the K and L cell membranes
and in the stimulation of GPCRs [71,72].
23
Incretin hormone degradation
It has been shown through intravenous administration of low concentrations of
GLP-1, that its degradation occurs within 2-5 minutes [73]. It has also been estab-
lished that peptides such as GLP-1 and GIP undergo cleavage at the N-terminus by
the action of DPP-4 after meal ingestion (Figure 1) [49,73-75].
The degradation of GLP-1 and GIP occurs directly by DPP-4 after their release in
the circulation [49,76]. In fact, more than 50% of the biologically active GLP-1 and
GIP is already degraded before they enter the portal circulation [77]. Therefore, only
10-15% of the active GLP-1 and GIP reaches the systemic circulation [78]. Intact
GLP-1 (7-36) NH2, intact GLP-1 (7-37) and intact GIP (1-42) undergo degradation
into inactive forms of GLP ((9-36) NH2 and 9-37) and GIP (3-42) (Figure 1)
[73,74,79]. Animal studies have suggested that an enzyme other than DPP-4 can also
degrade GLP-1 and GIP, namely, neprilysin [80]. However, the development of
neprilysin inhibition as a strategy to prolong the insulinotropic effect of incretin
hormones has been stopped because of the high risk of severe adverse events (AEs)
[80].
DPP-4 is also known as T-cell antigen CD26, and the protein is related to the prolyl
oligopeptidase family. The DPP family also consists of fibroblast activation proteins
DPP-8 and DPP-9. All these (except DPP-4) proteins are distributed and
enzymatically active intracellularly [81]. DPP-4, in contrast, is predominantly
extracellular and widely produced and distributed in different types of tissues, e.g.,
kidney hepatocytes, spleen, lungs, the brush-border of the intestinal membranes,
mammary glands, skin, adrenal glands and the endothelial cells of blood vessels
[82,83].
DPP-4 is also involved in the degradation of other gastrointestinal peptides such as
peptide YY and oxyntomodulin. Whether DPP-4 inhibition enhances the metabolic
effect of peptide YY or oxyntomodulin is not yet known [81].
GLP-1 and GIP receptors
GLP-1 and GIP receptors belong to the same family, so-called GPCRs [84]. GLP-
1R is widely distributed in different types of tissue in the body, such as the alpha,
beta and delta cells of the pancreas, the lungs, heart, kidneys, stomach, intestine,
pituitary gland and several regions in the central nervous system (CNS) [85-87].
GIPR is also expressed in different types of tissue, i.e., the alpha and beta cells of
the pancreas, adipose tissue, bone and CNS [88-90]. The N-terminal domain of
GIPRs has a high capacity for GIP binding. The overall mechanism of action of
24
GLP-1 and GIP depends on the elevation of the intracellular cAMP [77]. These
mechanisms mediate the effect of the incretin hormones on the target organs.
The physiological role of incretin hormones
Pancreatic effects of incretin hormones
Beta cells
The stimulation of insulin secretion from the beta cells is a glucose-dependent
pathway. GLP-1 and GIP stimulate GLP-1R and GIPR on the beta-cell membranes,
respectively. This results through several intracellular pathways in elevation of
cAMP level, which in turn can have an influence on the closure of the ATP-
dependent potassium channel, voltage-gated calcium channels, and on the process
of exocytosis. The fact that incretin hormones alone (i.e., in the absence of an
elevated plasma glucose levels) cannot close the ATP-dependent potassium channel
at low glucose concentrations, or when elevated glucose concentrations return to
lower values, explains why their action on insulin secretion is so explicitly glucose-
dependent [77,91,92]. Therefore, incretin-stimulated insulin secretion is triggered
when the plasma glucose level is higher than about 4.0 mmol/l. In contrast, the
insulinotropic effect of incretin hormones is lost when the plasma glucose level is
less than about 4.0 mmol/l. This applies even at supra-physiological levels of
incretin hormones (Figure 1) [92].
Alpha cells
The regulation of glucagon secretion from the alpha cells by incretin hormones is
more complicated and less clear. In the presence of hyperglycaemia, GLP-1 inhibits
glucagon secretion through its stimulation of insulin and somatostatin secretion
from beta and delta cells after meal ingestion. Increases in both insulin and
somatostatin secretion lead to inhibition of glucagon secretion [93,94]. In contrast,
under hypoglycaemia, the GLP-1 inhibitory effect on glucagon seems to be
completely lost. Therefore, incretin-based therapy is highly suitable for those with
T2D and a high risk of hypoglycaemia (Figure 1) [95-97].
In contrast to GLP-1, GIP is known to stimulate glucagon secretion, in particular at
glucose levels less than 4.0 mmol/l in both healthy subjects and those with T2D [98].
Intravenous administration of glucose suppresses glucagon dose-dependently,
compared to the oral glucose load of an isoglycaemic clamp, in both healthy subjects
and those with T2D. This is believed to be a consequence of GIP secretion after oral
glucose ingestion (Figure 1) [99,100].
25
Extrapancreatic effects of incretin hormones
Effects on the gastrointestinal tract and liver
GLP-1, but not GIP, delays gastric emptying after meal ingestion. This causes a
delay in the absorption of nutrients from the intestinal lumen, which results in the
suppression of postprandial plasma glucose excursion [101,102]. GLP-1, but not
GIP, also reduces gastrointestinal motility and pancreatic exocrine secretion [103].
GLP-1 suppresses hepatic glucose production and thus glycaemia in humans, while
the effect of GIP on the liver is so far unclear [104].
Effects on the cardiovascular system
GLP-1 improves the endothelial function in both healthy subjects and subjects with
T2D during the hyperglycaemic clamp [105]. The infusion of CLP-1 has also been
reported to improve cardiac output after an acute myocardial infarction in animal
studies [105,106]. Collectively, these findings show that GLP-1 has cardioprotective
effects, while the effect of GIP on the cardiovascular system is still unclear [107].
Effects on the other tissues and organs
GLP-1 crosses the blood-brain barrier and stimulates GLP-1R, mainly in the hypo-
thalamus. Accordingly, GLP-1 increases satiety and reduces appetite [108]. The
effects of GIP in the CNS are less clear in humans, despite the presence of GIPR
[69,77,108].
The effect of GLP-1 and GIP on adipose tissue in humans remains unclear [109].
However, based on animal studies, it has been proposed that GIP triggers fat storage
[102]. The physiological effects of GLP-1 and GIP on human skeletal muscle is also
still unclear. However, it has been demonstrated that GLP-1 increases glucose
uptake in mouse muscle [77].
GIP and GLP-1 have been reported to have an osteogenic effect in animal studies
[110,111]. However, treatment with GLP-1RAs has not been found to improve bone
formation in humans [112-115].
Physiological increase in the levels of GLP-1, but not GIP, have been shown to
increase natriuresis [69,116]. Therefore, incretin-based therapy appears to be
beneficial in those with nephropathy-related complications due to T2D [117-119].
Therefore, the ADA/EASD guidelines recommend incretin-based therapy in
patients with T2D and chronic kidney diseases [1].
Incretin hormones in T2D
Regarding the effect of incretin hormones on the islet cell function in healthy indi-
viduals, it is of considerable interest to study the role of incretin hormones in the
26
pathophysiology of T2D. The secretion of incretin hormones and their insulino-
tropic effects in T2D are discussed below.
Incretin hormone secretion in T2D
Meal and macronutrient ingestion stimulate incretin hormone secretion in T2D.
Initial studies reported a decrease in GIP secretion in response to a mixed meal and
glucose ingestion in subjects with T2D, compared to healthy controls [120,121].
Similar results have been obtained regarding GLP-1 secretion [122,123]. However,
many other studies performed later on incretin hormone secretion demonstrated that
there was no major defect in incretin hormone secretion in T2D, and this has been
confirmed in meta-analyses [124,125]. It is therefore believed that there is no defect
in nutrient-induced incretin hormone secretion in T2D, compared to healthy
subjects.
Is the reduction in the incretin effect a primary cause of T2D?
While the secretion of incretin hormones is normal, the insulinotropic effect of
incretin hormones is reduced or absent in patients with T2D [126]. A fundamental
question is thus whether a defect in the incretin effect is a primary cause of the
development of T2D, or whether the deficiency in the insulinotropic effect of the
incretin hormones is a consequence of the disease. Women with previous gestational
diabetes, first-degree relatives of those with T2D, and subjects with chronic
pancreatitis have been found to have an intact incretin effect [126-128]. Thus, a
defect in the incretin effect is considered a secondary phenomenon resulting from
the progression of T2D. Furthermore, studies have shown that the administration of
exogenous GLP-1 could partially restore the insulinotropic effect of GLP-1. In
contrast, it has been reported that the administration of exogenous GIP could not
restore the insulinotropic effect of GIP in subjects with T2D [93,129,130].
Treatment of T2D
Type 2 diabetes is associated with an increase in insulin resistance, inadequate
pancreatic endocrine hormone secretion in the fasting state and in response to meal
ingestion [125]. It is therefore crucial to target these pathophysiological processes in
the treatment of T2D to enhance beta-cell function, increase insulin sensitivity, and
reduce glucagon secretion (Table 1) [131].
The goals of T2D management are to optimise the patient’s quality of life, and to
prevent or reduce related complications with a low risk of hypoglycaemia. This
requires multifactorial intervention including regular control of glycaemia, blood
pressure and lipids [132]. It has therefore been suggested that the economic cost of
managing the complications associated with T2D is considerably higher than the
cost of T2D drugs [133-135].
27
According to the ADA/EASD guidelines for the treatment of T2D, medical profes-
sionals should individualize the glycated haemoglobin (HbA1c) target and take
cardiovascular or chronic kidney disease into consideration when they decide which
kind of therapy to apply [1].
Lifestyle modification and treatment with metformin are recommended as first-line
therapy for those with T2D [1]. Changes in eating patterns, meal size and meal
composition may have a beneficial effect on the glycaemic target [136,137]. An
increase in regular physical activity is also useful in the reduction of HbA1c
[138,139].
The reduction of overweight and obesity in subjects with T2D is recommended.
Treatment with GLP-1RAs induces weight loss and improves glycaemia [140].
Moreover, in patients with T2D and severe obesity (body mass index > 40 kg/m2)
bariatric surgery may be considered appropriate in the treatment of T2D [1]. In fact,
bariatric surgery improves metabolic control, and disease remission is frequently
seen several years after surgery [141,142].
Furthermore, in the case of established cardiovascular events or chronic kidney
disease, treatment with sodium-glucose co-transporter 2 (SGLT-2) inhibitors or
incretin-based therapy (mainly GLP-1RAs) may be considered [1].
Table 1. Approaches for the treatment of T2D, depending on its pathophysiology and the available therapies.
Islet dysfunction Insulin resistance Other targets Comments
Insulin therapy
Sulphonylureas
Bariatric surgery
GLP-1RAs
DPP-4 inhibitors
Lifestyle modification
Metformin
Thiazolidinedione
Bariatric surgery
α-glucosidase inhibitors → Reduces intestinal glucose absorption
SGLT-2 inhibitors → Increases renal glucose excretion
GLP-1RAs → Delays gastric emptying and reduces appetite
Incretin-based therapy
Pancreatic effects of incretin-based therapy
Incretin-based therapy reduces glycaemia through the stimulation of insulin secre-
tion and the suppression of glucagon secretion after meal ingestion. This therapy is
also associated with a low risk of hypoglycaemia [21]. There are two types of
incretin therapy: DPP-4 inhibitors and GLP-1RAs [143]. In contrast, GIP receptor
agonists have not proved a suitable candidate in the treatment of T2D, since the
exogenous administration of GIP was not found to induce glucose-dependent insulin
secretion [93,144]. This could be due to the reduced response of the beta cells to GIP
in T2D [145].
28
GLP-1RAs directly stimulate GLP-1R through a supraphysiological increase in the
concentrations of ligands, which in turn stimulate GLP-1R on the target tissues
[146]. In contrast, DPP-4 inhibitors prevent the inactivation of endogenous incretin
hormones. Thus, the GLP-1RAs and DPP-4 inhibitors exhibit different pharmaco-
kinetics, efficiency and tolerability [118,147].
It has been known since the early 1990s that the degradation of the incretin
hormones is caused by the DPP-4 enzyme [79]. DPP-4 inhibitors were thus rapidly
developed as a therapeutic glucose-lowering agent in the treatment of T2D. In fact,
the first randomized controlled trial on the treatment of T2D with DPP-4 inhibitors
was carried out in Sweden in 2000 [148]. This study showed that four weeks of
treatment of drug-naïve T2D subjects with a DPP-4 inhibitor reduced glycaemia
significantly, compared to the placebo. Noawayds are DPP-4 inhibitors widely used
in the management of T2D. Several DPP-4 inhibitors exist (Table 2) [149,150], and
although they are all small, orally active molecules, they differ in their chemical
structure, enzyme binding characteristics and pharmacokinetics [151]. In a meta-
analysis of DPP-4 inhibitor studies, these differences were not found to be essential
for their long-term metabolic effect [147]. However, it has not been ascertained
whether these DPP-4 inhibitors differ in their impact on postprandial islet hormone
secretion after meal ingestion throughout the day.
In clinical praxis, treatment with DPP-4 inhibitors is recommended as an add-on
therapy to other glucose-lowering treatments to achieve glycaemic control [1].
Monotherapy with DPP-4 inhibitors may be preferred if the patient cannot tolerate
other glucose-lowering medications. DPP-4 inhibitors can also be used as a mono-
therapy in subjects with T2D who have previously undergone bariatric surgery due
to their high tolerability to DPP-4 inhibitors, compared to other glucose-lowering
therapies [152].
GLP-1RAs have emerged in parallel with DPP-4 inhibitors, and the first GLP-1RA
approved for the treatment of T2D was exenatide (Table 2) [143,153,154]. Like DPP-
4 inhibitors, GLP-1RAs differ in their molecular and kinetic characteristics. GLP-
1RAs are classified into short-acting (administrated once daily: exenatide,
lixisenatide and liraglutide) and long-acting (administrated once weekly: exenatide,
dulaglutide and semaglutide). They differ in their efficacy and tolerability
depending on their classification (Table 2) [81,118,155].
It has been established that treatment with DPP-4 inhibitors or GLP-1RAs is
associated with a lower risk of AEs than other antidiabetic therapies [156].
Compared with treatment with GLP-1RAs, treatment with DPP-4 inhibitors has a
lower risk of gastrointestinal AEs, it is easily administered (orally), and the cost is
lower. However, treatment with GLP-1RAs induces weight loss and is more
effective in the reduction of HbA1c, and fasting and postprandial blood glucose
levels (Table 2) [145].
29
Extra-pancreatic effects of incretin-based therapy
The cardiovascular system
Following requirement by the American Food and Drug Administration that the risk
of major adverse cardiovascular events (MACE), i.e. nonfatal stroke, nonfatal
myocardial infarction and cardiovascular death, be declared in new diabetes
therapies, extensive prospective cardiovascular randomized control studies have
been undertaken, demonstrating that treatment with incretin-based therapy is safe in
those with T2D [157-159]. Moreover, treatment with GLP-1RAs (liraglutide or
semaglutide) has been shown to reduce MACE [160,161]. Based on the findings of
these studies, guidelines for the management of T2D have been changed by the
ADA/EASD and GLP-1RAs are recommended to the subjects with T2D and
cardiovascular disease independent on their HbA1c [1].
In clinical praxis, treatment with liraglutide or semaglutide could be combined with
other forms of glucose-lowering therapy in those with T2D and cardiovascular
events, even if they have good metabolic control [162].
Obesity
GLP-lRAs (liraglutide 3.0 mg once daily) have been considered as a novel form of
treatment for those with obesity. A weight loss of ~6 kg over 53 weeks has been
reported, compared to the placebo [163]. Treatment with GLP-1RAs is also bene-
ficial in weight reduction in subjects with obesity caused by hypothalamic-related
disease such as craniopharyngioma [164].
The liver and pancreatic safety
Patients with T2D have an increased risk of non-alcoholic fatty liver disease
(NAFLD), which in turn increases the risk of liver cirrhosis [165]. Incretin-based
therapy has been shown to have a beneficial effect in reducing hepatic steatosis and
fibrosis in patients with T2D and NAFLD [166-168].
T2D is associated with an increased risk of acute and chronic pancreatitis, and thus
increased levels of serum lipase or amylase [169,170]. It has been reported that
treatment with incretin-based therapy may increase levels of lipase and amylase
[171-173], while other studies found that treatment with incretin-based therapy was
not associated with an increased risk of pancreatitis [173,174].
From a clinical point of view, it has been widely assumed that incretin-based therapy
should be avoided in subjects with T2D with a history of pancreatitis [173,175,176].
30
Table 2. Recommended dosage, administration and effects of commonly used DPP-4 inhibitors and GLP-1RAs.
DPP-4 inhibitors GLP-1RAs
Drug Sitagliptin (100 mg QD1)
Vildagliptin (50 mg BID)
Saxagliptin (5 mg QD)
Linagliptin (5 mg QD)
Alogliptin (25 mg QD)
Short-acting GLP-1RAs
Exenatide (10 μg BID2)
Lixisenatide (20 μg QD)
Liraglutide (1.8 mg QD)
Long-acting GLP-1RAs
Exenatide (2 mg QW3)
Dulaglutide (1.5 mg QW)
Semaglutide (1.0 mg QW)
Semaglutide (14 mg QD)
Administration Oral Subcutaneously injection
Oral (semaglutide (14 mg))
Main antihyperglycaemic effects Enhances insulin secretion (glucose-dependent)
Suppresses glucagon
(glucose-dependent)
Enhances insulin secretion (glucose-dependent)
Suppresses glucagon
(glucose-dependent)
Reduces gastric emptying
(short-acting GLP-1RAs)
Effects on HbA1c Improves HbA1c level
(~5-10 mmol/mol)
Improves HbA1c level
(~10-15 mmol/mol)
Effects on appetite and body weight Neutral Reduced
Effects on
gastric emptying
Neutral Delayed
Main AEs Well tolerated Gastrointestinal side effects (nausea, vomiting and diarrhoea)
Risk of hypoglycaemia Low Low
*1Once daily; 2Twice daily; 3Once weekly
Incretin-based therapy in type 1 diabetes
Treatment with incretin-based therapy is still not approved for people with type 1
diabetes (T1D). However, some individuals with T1D develop insulin resistance
and weight gain [177]. It is also known that T1D is associated with
hyperglucagonaemia [178]. Bearing in mind the effect of incretin hormones on the
suppression of glucagon secretion and weight loss, the introduction of incretin-
based therapy for the treatment of T1D has attracted considerable interest. Incretin-
based therapy is associated with low risks of hypoglycaemia and hyperglycaemia,
including ketoacidosis, in patients with T1D, but GLP-1RAs increase the risk of
gastrointestinal AEs [179,180]. However, GLP-1RAs, but not DPP-4 inhibitors,
improve glycaemia and reduce weight [181,182]. It is still not clear whether incretin-
based therapy has beneficial effects on cardiovascular events in people with T1D.
31
Rationale and the Aims of the Research
Despite the fact that much research has been carried out into T2D and its treatment,
it is essential to improve our knowledge on the effects of meal composition, timing,
and size on the regulation of glycaemia in healthy subjects and subjects with T2D,
in particular those taking glucose-lowering agents, such as DPP-4 inhibitors.
Understanding the mechanisms of glycaemic control after meal ingestion is of
importance in the clinical management of T2D.
The overall aim of the research presented in this thesis was to investigate the
response of plasma glucose and islet and incretin hormones after meal ingestion in
healthy subjects and those with well-controlled T2D, with regard to the effect of
meal composition, size and timing, as well as the impact of acute DPP-4 inhibition
(Figure 2).
Figure 2. Overview of the study aims.
32
Subjects and Methods
Study design, medication and meal composition
The studies presented in this thesis were designed as single-centre and open-label
studies. Healthy and T2D Caucasian subjects of both genders were invited to take
part in the studies through advertisements in newspapers and public places. Partici-
pants were also enrolled in the studies through direct collaboration with the several
diabetes nurses working in primary healthcare units. The studies were carried out at
the Clinical Research Centre of Skåne University Hospital in Lund.
DPP-4 inhibitors sitagliptin (100 mg), Papers I and IV, and sitagliptin (100 mg),
saxagliptin (5 mg) or vildagliptin (50 twice daily) Paper V), or PBO were given
after overnight fasting and 30 min before the meal test (Figure 3). paracetamol (1.5
g) was given together with the meal for the assessment of gastric emptying.
Figure 3. Overview of the study design. SITA = Sitagliptin; VILDA = Vildagliptin; SAXA = Saxagliptin; PBO = Placebo
33
The tests were performed at different times of the day in cross-over (each participant
underwent all tests) and randomized order. The caloric contents of the meal test in
each study is summarized in Figure 4.
Figure 4. Overview of the caloric contents of the meals served in each study. MMT = Mixed meal test
Paper I
The subjects consumed either 2 g protein mixture (ISO Whey)/kg, 0.9 g olive oil/kg,
2 g glucose/kg body weight on three different occasions (Figure 4).
Paper II
Participants were given a standardized mixed meal at breakfast time with a caloric
content of 524 kcal. At lunchtime, a meal based on sirloin steak was served, with
caloric contents of 511 kcal, 743 kcal or 1034 kcal, on three different occasions. All
three meals had the same nutrient composition (18% protein, 32% fat and 50%
carbohydrates) (Figure 4).
Paper III
Subjects either ingested one of the macronutrients alone (glucose (330 kcal), protein
mixture (110 kcal, ISO Whey protein) or fat emulsion (110 kcal)), or the same
macronutrients mixed together as a 550 kcal meal (glucose 330 kcal, protein 110
kcal and fat 110 kcal). The proportions of the macronutrients were: 20% protein,
20% fat, and 60% carbohydrates (Figure 4).
Paper IV
Subjects were served a standardized mixed meal of 525 kcal containing 20%
protein, 20 % fat and 60% carbohydrates (Figure 4).
34
Paper V
Participants ingested a standard breakfast of 525 kcal containing 20% protein, 20%
fat and 60% carbohydrates. At lunchtime, they ingested a meal of 780 kcal (protein
40%, fat 20% and carbohydrates 40%) and at dinnertime, a meal of 560 kcal (protein
25%, fat 35% and carbohydrate 40%) (Figure 4).
Study populations
The details of the study populations are consisting of healthy subjects and subjects
with T2D. The baseline characteristics (at the time of inclusion) are summarized in
Tables 3 and 4.
Table 3. The demographic and baseline characteristics of the healthy subjects who participated in the studies described in Papers I, II, III and IV.
Healthy subjects
Papers I and IV Paper II Paper III
Number (males/females) 12 (12/0) 24 (12/12) 18 (11/7)
Age (years) 22 ± 1 25 ± 2 62 ± 5
BMI (kg/m2) 22 ±1 22 ±1 25 ± 2
Weight ( kg) 72.2 ±5.7 67.0 ± 9.0 76 ± 11
HbA1c (mmol/mmol) ND 31.1 ± 5.0 37.7 ± 3.7
HbA1c (%) ND 5.0 ± 0.2 5.7 ±1.4
Fasting blood glucose (mmol/l) 4.5 ± 0.6 4.2 ± 0.7 5.5 ± 0.7
Mean ± SD are given. ND = Not determined.
Table 4. The demographic and baseline characteristics of the subjects with T2D who participated in the studies described in Papers III, IV and V.
T2D subjects
Paper III Drug-naïve
Paper IV
Drug-naïve
Paper V
Metformin-treated
Number (males/females) 18 (13/5) 12 (12/0) 24 (12/12)
Age (years) 63 ± 5 65 ± 0.6 63 ± 6
BMI (kg/m2) 27 ± 4 28 ± 3 31 ± 0.5
Weight (kg) 76 ± 11 90 ±10 90 ± 18
HbA1c (mmol/mmol) 42.2±5.5 43.0 ± 7.0 44.7 ± 6.0
HbA1c (%) 6.1± 1.6 6.2 ± 1.7 6.2 ± 6.0
Diabetes duration (years) 3 ± 2 4 ± 1 4 ± 1
Fasting blood glucose (mmol/l) 7.0 ± 1.0 7.3 ± 0.9 7.1 ± 1.2
Mean ± SD are given.
35
Ethics and good clinical practices
The studies were approved by the Ethical Review Board in Lund, Sweden, and the
Swedish Medical Product Agency (Papers I, IV and V). The studies were registered
in the clinicaltrials.gov and clinicaltrialsregister.eu databases. The details of the
registry numbers are given separately in each publication.
All the participants gave their written informed consent. After each study had been
completed and the results published, the participants received a scientific summary
providing information on the results and the conclusions of the study. All the studies
were monitored by an independent monitor and were conducted using good clinical
practice and good laboratory practice, and followed the Declaration of Helsinki.
Power calculation
The minimal sample size required to provide >80% chance of revealing a statistical
difference with a probability of 95% was calculated for each study. An additional
four subjects were recruited to ensure reliable statistics. With the sample size of
each study, a power analysis is used, aiming at showing a 30% difference between
groups, which was considered as relevant to investigate the primary endpoint of the
studies.
Laboratory measurements
The participants were provided with an antecubital vein catheter. Blood samples
were collected at each time point during the tests in chilled tubes containing EDTA
(7.4 mmol/l). Samples for the determination of GIP and GLP-1 were collected in
chilled tubes containing EDTA and Diprotin A. All samples were immediately
centrifuged and Plasma was frozen until analysis.
Glucose was determined by a colorimetric method using the glucose oxidase, which
catalyses the glucose in the plasma.
Insulin was analysed with radioimmunoassay (RIA) (Paper I) or enzyme linked
immunosorbent assay (ELISA) (Papers II-V).
C-peptide was determined with RIA (Paper I) or ELISA (Papers II-V).
Intact GLP-1 was determined with RIA (Paper I) or ELISA (Papers II, IV and
V). Both assays are N-terminally directed.
Intact GIP was measured with ELISA. The assay is N-terminally directed.
36
Total GLP-1 was determined with ELISA. The assay is C-terminally directed to
different parts of the molecules and cross-reacts completely with GLP-1 (7-36) and
GLP-1 (9-37).
Total GIP was determined with ELISA. The assay is C-terminally directed to
different parts of the molecule and cross-reacts completely with GIP (1-42) and GIP
(3-42).
Glucagon was determined with RIA (Papers I, II, IV and V) or ELISA (Paper
III).
Paracetamol levels in the plasma were analysed using the colorimetric assay.
Assessment of beta-cell function
The dynamic beta-cell function is dependent on the response of the beta cells to the
change in the plasma glucose level following the ingestion of a meal. A
mathematical model for the estimation of insulin secretion has been developed by
Mari et al. [183,184]. This model is based on the dose response relationship between
plasma glucose levels and insulin secretion rate (ISR), calculated using
deconvolution of the C-peptide [185]. The slope of the dose response curve
represents the beta-cell glucose sensitivity. Potentiation is the ability of the ingested
meal to enhance (potentiation >1) or inhibit (potentiation <1) insulin secretion
during the test. Potentiation is affected by several factors, including incretin
hormones, and is a measure of the insulin secretion rate during the test period [186]. This model provides values of the insulin secretion rate every 5 minutes.
Calculations
Beta-cell function was determined using the ratio of the the area under the curve
(AUC)ISR or C-peptide to the AUCglucose (i.e. the insulinogenic index) [27]. Beta-cell dose
response was used to evaluate the effects of the nutrients on the relationship between
insulin secretion and glucose levels.
Insulin clearance was calculated by dividing the AUC for total insulin secretion by
the AUC for insulin concentration. To evaluate the effect of the meal or individual
macronutrients on the pancreatic insulin release in the portal vein, and before insulin
undergoes metabolism in the liver, hepatic insulin extraction was also calculated
from the C-peptide and insulin values [187]. Insulin sensitivity was assessed by oral
glucose insulin sensitivity index (OGIS) from the plasma glucose and insulin
responses in the mixed meal and in the glucose test [188]. Insulin sensitivity was
37
also determined using an extension of the QUICKI method. This makes it possible
to calculate insulin sensitivity also after fat and protein ingestion [189].
Statistical analysis
Data are presented as means ± SEM, unless otherwise stated. The differences
between groups in the original publications were based on calculations of the AUC
for each variable. A paired t-test (Papers I, III and IV) and ANOVA (Papers II
and V) were used for comparisons. New data analysis was performed using the
original data to obtain the differences in the percentage changes in the AUC of the
variables investigated. These results are presented in part 2 of the results.
Statistical differences between groups were determined by a multiple comparisons.
A p-value of < 0.05 indicates that there is a 95% probability that there is a
statistically significant difference between the values being compared. IBM SPSS
statistics and GraphPad Prism software were used to analyse the data.
38
Results
Part 1: Effect of the meal on glycaemia and the islet and incretin
hormones
The important results of this work are summarized below. Further details can be
found in each publication. Increasing the meal size significantly increased the levels
of insulin and intact GLP-1 and GIP, and stimulated beta-cell function in healthy
subjects, as can be seen from the results presented in Table 5. The effects on islet
and incretin hormone secretion resulted in the maintenance of glucose levels over
the entire study period after the ingestion of all three meals (Paper II).
The ingestion of a meal including all the macronutrients counteracted the increase
in glycaemia, and stimulated beta-cell function and total GIP levels, compared to
the ingestion of glucose alone. The total GLP-1 levels increased significantly after
meal ingestion, compared to glucose intake alone, in subjects with T2D, but not in
healthy subjects (Paper III).
In healthy subjects, an acute dose of a DPP-4 inhibitor reduced glycaemia, increased
levels of intact GLP-1, and suppressed glucagon after the separate intake of glucose,
fat or protein (Paper I). Further DPP-4 inhibition reduced glycaemia, stimulated
beta-cell function, and increased levels of intact GLP-1 and GIP after meal ingestion
in both healthy subjects and subjects with T2D (Paper IV).
The effects of DPP-4 inhibition (using sitagliptin, vildagliptin or saxagliptin)
(Figure 3) after the ingestion of breakfast, lunch and dinner on glycaemia and islet
and incretin hormones persisted throughout the day. These included a reduction in
glycaemia, stimulation of beta-cell function and an increase in intact incretin
hormones, as well as suppression of glucagon and incretin hormone secretion in
subjects with metformin-treated T2D (Paper V).
39
Ta
ble
5.
Ove
rvie
w o
f th
e r
esults o
f th
e s
tudie
s p
resen
ted in t
he
origin
al p
ublic
atio
ns
Pap
er
Meal s
ize
M
eal c
om
po
sit
ion
D
PP
-4 i
nh
ibit
ion
, m
eal
co
mp
osit
ion
an
d m
eal
tim
ing
The e
ffect
of
incre
asin
g
mix
ed m
eal
siz
e
511
74
3
103
4 k
cal
The e
ffect
of
a m
ixe
d m
eal
vs. glu
cose in
ge
stio
n
alo
ne
The e
ffect
of
DP
P-4
inhib
itio
n
afte
r in
gestion
of
glu
cose, fa
t or
pro
tein
, com
pare
d t
o t
he
pla
ce
bo
The e
ffect
of
DP
P-4
inhib
itio
n
afte
r m
ixe
d m
eal in
gestio
n,
com
pa
red
to t
he p
lace
bo
The e
ffect
of
DP
P-4
inhib
itio
n
over
the w
hole
da
y a
fte
r m
ixed
m
eal in
gestion,
com
pare
d t
o
the p
laceb
o
II2
III3
I1
IV
4
V5
H
ealth
y
Health
y
Dru
g-n
aïv
e
T2D
H
ealth
y
Health
y
Dru
g-n
aïv
e T
2D
M
etf
orm
in-t
reate
d T
2D
Glu
co
se
ns
↓
↓
↓
↓
↓
↓
Ins
uli
n
↑
↑
↑
ns
ns
ns
ns
Beta
-cell
fun
cti
on
↑
↑
↑
ns
↑
↑
↑
Glu
ca
go
n
ns
↑
↑
↓
ns
ns
↓
Inta
ct
GL
P-1
↑
ND
N
D
↑
↑
↑
↑
Inta
ct
GIP
↑
ND
N
D
ND
↑
↑
↑
To
tal
GL
P-1
N
D
ns
↑
ND
ns
ns
↓
To
tal
GIP
N
D
↑
↑
ND
ns
↓
↓
1P
ap
er
I: A
UC
12
0 m
in f
or
pla
sm
a g
lucose,
an
d isle
t a
nd
incre
tin h
orm
ones a
fter
inta
ke o
f glu
cose,
fat
or
pro
tein
in
12
he
alth
y s
ubje
cts
giv
en 1
00 m
g s
itaglip
tin, com
pare
d t
o t
he
pla
ce
bo
. 2P
ap
er
II: A
UC
18
0 m
in f
or
pla
sm
a g
lucose,
an
d isle
t a
nd
incre
tin h
orm
ones a
fter
inta
ke o
f 5
11 k
cal, 7
43 k
cal o
r 103
4 k
cal m
ixe
d m
eal in
24 h
ealth
y s
ubje
cts
. T
he a
rro
ws d
enote
a s
ignific
ant in
cre
ase (
↑)
or
decre
ase (
↓)
wh
en incre
asin
g t
he m
eal siz
e f
rom
511 t
o 7
43
kcal or
from
74
3 t
o 1
03
4 k
cal.
3P
ap
er
III: A
UC
180 m
in f
or
pla
sm
a g
lucose,
and isle
t and
incre
tin h
orm
ones a
fter
inta
ke o
f m
ixe
d m
eal vs.
glu
cose in 1
8 h
ealth
y s
ubje
cts
and 1
8 s
ubje
cts
with T
2D
. 4P
ap
er
IV: A
UC
300
min
for
pla
sm
a g
lucose,
insulin
an
d isle
t a
nd incre
tin
ho
rmone
s a
fter
mix
ed m
eal in
ge
stio
n in 1
2 h
ealth
y s
ubje
cts
and
12 s
ubje
cts
with d
rug
-na
ïve T
2D
, giv
en
100
mg s
itaglip
tin o
r pla
ceb
o.
5P
ap
er
V:
AU
C 7
80 m
in fo
r pla
sm
a g
lucose,
and isle
t and
incre
tin h
orm
ones a
fter
ing
estion o
f bre
akfa
st, lunch
and
din
ne
r, w
ith p
rio
r a
dm
inis
tration o
f sitaglip
tin (
100 m
g),
vild
aglip
tin
(5
0 m
g t
wic
e d
aily
.),
sa
xaglip
tin (
5 m
g)
or
pla
cebo
in 2
4 s
ubje
cts
with m
etf
orm
in-t
reate
d T
2D
. E
ach s
tud
y p
art
icip
ant re
ceiv
ed
DP
P-4
in
hib
itors
on d
iffe
rent a
nd
sep
ara
te o
cca
sio
ns,
and
th
e r
esults p
resente
d h
ere
re
pre
se
nt th
e m
ean o
f th
e r
esults f
or
all
thre
e D
PP
-4 inhib
itors
co
mpare
d t
o t
he p
laceb
o.
Beta
-cell
functio
n e
stim
ate
d u
sin
g b
eta
-cell
dose r
espo
nse
and
insulin
se
cre
tio
n r
ate
(P
ap
ers
I, IV
), a
nd insulin
oge
nic
inde
x (
Pa
pers
II,
II
an
d V
).
ns =
Not sig
nific
an
t; N
D =
Not
dete
rmin
ed
40
Part 2: DPP-4 inhibition and meal composition: subgroup analysis
The data presented in the original papers were further analysed to investigate
whether different groups of participants, healthy, drug-naïve and metformin-treated
subjects with T2D, responded differently to DPP-4 inhibition or changes in meal
composition. The data presented in Papers III and IV were re-analysed to
determine the impact of meal composition and DPP-4 inhibition after the ingestion
of mixed meal, in healthy subjects compared to subjects with T2D, in terms of the
levels of plasma glucose and islet and incretin hormones.
A subgroup analysis was also performed based on data from Papers IV and V in
order to investigate the effect of acute DPP-4 inhibition after mixed meal ingestion
in T2D, drug-naïve, and metformin-treated subjects.
Meal composition in healthy subjects and drug-naïve subjects with T2D
Figure 5 summarises the results obtained from the re-analysis of the data presented
in Paper III in terms of a percentage increase or decrease after the ingestion of a
mixed meal, in relation to the results obtained following the intake of glucose alone
(set to 0%), in each treated group. Although differences were found in the mean
percentage changes in subjects with T2D, compared to the healthy subjects, in
plasma glucose (+ 0.5%), beta-cell function (+8%), intact GLP-1 (-5%), intact GIP
(-32%), total GLP-1 (-10%), total GIP (+15%) and glucagon (-3.6%), we found no
significant differences in the effects between the two groups.
Figure 5. Comparison of the effects of a mixed meal vs. the ingestion of glucose alone (0%) in healthy subjects and drug-naïve subjects with T2D, on plasma glucose, beta-cell function, incretin hormones and glucagon. Data are means ± SEM. (Data from Paper III)
41
DPP-4 inhibition in healthy subjects and drug-naïve subjects with T2D
Figure 6 shows a summary the results obtained from the re-analysis of the data
presented in Paper IV, comparing the effect of sitagliptin after meal ingestion, in
healthy subjects and drug-naïve subjects with T2D. The effect on intact GIP (+85%)
was higher in healthy subjects than in subjects with T2D. However, no significant
differences were found in the effect of sitagliptin on plasma glucose, beta-cell
function, intact GLP-1, glucagon, or incretin hormone secretion when comparing
healthy subjects with the drug-naïve subjects with T2D.
Figure 6. The effect of sitagliptin (100 mg) in healthy subjects and drug-naïve subjects with T2D after meal ingestion. The values obtained with placebo were set to 0% in each group. ** Indicates a significant difference (p<0.001). The values given are means ± SEM. (Data from Paper IV)
42
DPP-4 inhibition in drug-naïve T2D subjects and metformin-treated T2D
subjects
Drug-naïve T2D subjects (Paper IV) and metformin-treated T2D subjects (Paper
V) were given the same meal (breakfast). Therefore, the results obtained from the
re-analysis of the data after the ingestion of sitagliptin in Paper IV and Paper V
were presented. The effects of sitagliptin on plasma glucose and islet and incretin
hormones were not significantly different between the two groups (Figure 7).
Figure 7. The effects of sitagliptin (100 mg) in drug-naïve subjects with T2D and metformin-treated T2D subjects after meal ingestion. The values obtained with placebo were set to 0% in each group. The values given are means ± SEM. (Data from Papers IV and V)
43
Main findings
Ingesting a mixed meal of fat, protein and glucose prevents an increase in
plasma glucose, and stimulates beta-cell function and incretin hormone secre-
tion, compared to the intake of glucose alone, in both healthy subjects and
subjects with T2D.
Increasing the caloric load of a meal at lunchtime in healthy subjects leads to a
significant increase in the levels of insulin and intact incretin hormones, and
stimulated beta-cell function, which resulted in a similar (non-significant)
plasma glucose excursion over the study period. There is no significant
difference in the effect of the meal ingestion compared to the glucose intake on
glycaemia and islet hormones between healthy and T2D subjects.
DPP-4 inhibition causes a reduction in plasma glucose levels after the separate
ingestion of each macronutrient in healthy subjects, and after mixed meal
ingestion in healthy and T2D subjects. The impact of DPP-4 inhibition on the
intact GIP is significantly higher in healthy subjects than in subjects with T2D.
DPP-4 inhibition has a persistent glucose-lowering effect, stimulates beta-cell
function and levels of intact incretin hormones throughout the day. The incretin
hormones secretion (total GLP-1 and total GIP) is reduced throughout the day
after intake of DPP-4 inhibition. The effects of DPP-4 inhibitors on glycaemia
and islet and incretin hormones are similar in drug-naïve subjects with T2D and
metformin-treated T2D subjects after meal ingestion.
44
Discussion
The effects of meal composition
It is known that glucose intake increases the secretion of incretin hormones and their
insulinotropic effect [56,190]. The findings presented in this thesis (Papers I and
III, Table 5) confirm this. In addition, the ingestion of fat or protein also stimulates
the secretion of incretin hormones, which is in line with previous results [6,41].
Furthermore, DPP-4 inhibition (using sitagliptin) considerably increases the level
of intact GLP-1 after the separate intake of glucose, protein or fat (Paper I). This,
in turn, reduced plasma glucose levels, but without increasing insulin secretion.
Beta-cell function was assessed using a model developed by Mari et al. [183], which
is based on mathematical calculations of the relationship between the change in
plasma glucose levels (dose response) and the dynamic response of insulin
secretion, i.e. the potentiation factors that drive insulin secretion.
The reduction in plasma glucose levels with unchanged plasma insulin levels after
the ingestion of individual macronutrients in combination with DPP-4 inhibition
could be explained by the increase in beta-cell glucose sensitivity (beta-cell
function) (Paper I) [54]. However, the beta-cell function is not increased after
ingestion of DPP-4 inhibition compared to the placebo (Paper I). In fact, the change
in plasma glucose levels was small after the ingestion of individual macronutrients
compared to the placebo in healthy subjects, which could explain the lack of beta-
cell stimulation by DPP-4 inhibition (Paper I). In contrast, DPP-4 inhibition in
subjects with prediabetes (impaired fasting glucose levels or impaired IGT)
suppresses postprandial glycaemia resulting in unchanged plasma insulin levels.
This is due to stimulation of beta-cell glucose sensitivity after DPP-4 inhibition,
which could explain why the effect of DPP-4 inhibition on beta-cell function is
glucose-dependent [191,192]. This also indicates that the insulinotropic effect of
incretin hormones and stimulating beta-cell function, is glucose-dependent, and this
effect is more apparent at glucose levels >4.0 mmol/l [92].
The rate of gastric emptying could affect the regulation of postprandial glycaemia
after macronutrient or mixed meal ingestion in both healthy and T2D subjects
[193,194]. This could be the result of the stimulation of incretin hormones after meal
ingestion which in turn reduces the rate of gastric emptying, and thus inhibition of
the postprandial glucose excursion [6,195].
45
The paracetamol test was used to measure the gastric emptying (Papers I-IV). This
method is easy to perform and non-invasive. Paracetamol is absorbed in the
duodenum, but not in the stomach. The level of paracetamol in the plasma therefore,
provides a measure of the rate of gastric emptying [196,197]. However, the
assessment of gastric emptying using MRI or ultrasonography could be more
reliable [194,198].
The lack of change in the rate of gastric emptying, insulin sensitivity and insulin
clearance confirms that these mechanisms are not involved in the glucose-lowering
effects of sitagliptin in healthy subjects after the separate ingestion of each
macronutrient [199,200]. The findings presented in Paper I indicate that all the
macronutrients are required in a mixed meal for the stimulation of beta-cell function
in healthy subjects.
The intake of a mixed meal including all macronutrients significantly reduced the
increase in postprandial glycaemia compared to the ingestion of the same amount of
glucose without fat and protein, in both healthy subjects and subjects with T2D
(Paper III). This is believed to be the result of increased incretin hormone secretion,
stimulated beta-cell function, and a decrease in insulin clearance. The ingestion of
all macronutrients together in a mixed meal delayed gastric emptying in both healthy
subjects and subjects with T2D, and could also be involved in the regulation of
postprandial glycaemia.
Several studies have reported impaired incretin hormone secretion following either
oral glucose or mixed meal ingestion in subjects with T2D, compared to healthy
subjects [122,201], while no such differences were found in other studies [124,125].
The study populations in Paper III were of subjects with well-controlled T2D. As
can be seen from Figure 5, similar effects of the mixed meal intake on glucose
homeostasis and islet and incretin hormones were seen in healthy subjects and
subjects with T2D. This could be due to several factors, such as the relatively short
duration of T2D (~3 years) and the lack of glucose-lowering medication such as
metformin, which could in turn explain why the T2D subjects had preserved beta-
cell function [202]. Also, the composition of the meal (60% carbohydrate, 20%
protein and 20% fat) may explain the similarity responses of the healthy subjects
and the T2D subjects. Furthermore, it has been suggested that both protein and fat
inhibit the activity of DPP-4 locally in the lumen of the gastrointestinal tract, thereby
increasing the effect of incretin hormones on beta-cell function [203].
Thus, the macronutrient composition of a meal is of importance for the regulation
of plasma glucose through its effect on islet and incretin hormone secretion. These
results support recommendations concerning nutritional therapy for T2D subjects
that a meal should include all macronutrients [31].
46
The effects of meal size
An increase in glucose load is known to have little impact on postprandial glycaemia
in healthy subjects [8]. However, an increase in the caloric load of breakfast has
been found to induce a dose-dependent increase in the secretion of incretin and
insulin hormones in healthy obese subjects, resulting in a similar glucose excursion,
regardless of meal size [7].
It was found that increasing the meal size but with the similar proportions of energy
from carbohydrates, protein and fat elicited caloric-dependent stimulation of GLP-
1 and GIP, resulting in the stimulation of beta-cell function (Paper II, Table 5). The
regulation of postprandial glycaemia may also be explained by the role of incretin
hormones in the reduction of insulin clearance after an increase in the caloric load
of the meal [204,205]. Also, a large meal (1034 kcal) induces rapid gastric emptying.
This is an interesting physiological effect of the gut after a large meal ingestion,
which promotes the rapid distribution of nutrients to the distal part of the intestinal
lumen (ileum). As the ileum is rich in L cells, an increase in GLP-1 is expected soon
thereafter. This rapid increase in GLP-1 causes a delay in gastric emptying, which
in turn inhibits plasma glucose levels by a delay in the intestinal glucose absorption.
This physiological phenomenon is known as “the ileal brake” [101,103]. The
combination of these two effects could explain the identical glucose excursions seen
after an increase in meal size.
The nutritional energy in the meals in the various studies (Papers II, III and IV)
were similar (carbohydrates ~60E%, protein ~20E% and fat ~20E%). However,
these meals differed in the type and texture of the macronutrients (liquid meal,
Paper III, and solid meals, Papers II and IV). However, an interesting observation
concerning healthy subjects was the sustained increase in GIP rather than GLP-1
after meal ingestion throughout the studies, which is in line with previous reports
[206]. A possible explanation of the difference between GIP and GLP-1 secretion
could be the location of the K cells in the proximal small intestine, resulting in
earlier stimulation by nutrients to release GIP, compared to L cells, which are
located in the distal gut. The high levels of GIP after meal ingestion in healthy
subjects suggest that GIP is the most important incretin hormone in the regulation
of glucose homeostasis [207], while GLP-1 may act as a complement to GIP in
mediating the incretin effect [69].
In summary, an increase in the caloric content of the ingested meal at lunchtime in
healthy subjects induced a caloric-dependent stimulation of incretin hormone
secretion and thus beta-cell function regulating glucose homeostasis. This reflects
the importance of the meal size on the response of incretin hormones and their
impact on islet function.
47
These findings, together with those above regarding meal composition, indicate that
the postprandial glycaemic response is dependent on the balanced distribution of
macronutrients in the meal and the meal size.
The effect of DPP-4 inhibition
According to the results presented above, DPP-4 inhibition does not stimulate beta-
cell function after separate ingestion of each macronutrient (glucose, fat or protein)
in healthy subjects (Paper I). After the administration of sitagliptin and the
ingestion of separate macronutrients, plasma glucose levels were low compared to
the placebo. On the other hand, DPP-4 inhibition reduces postprandial glycaemia
and stimulates beta-cell function after the ingestion of a mixed meal in healthy
subjects (Paper IV), compared to the placebo. These findings could indicate that
the effect of DPP-4 inhibition on beta-cell function is glucose-dependent [53,118].
The data presented in Paper IV were used to compare the effect of DPP-4 inhibition
after mixed meal ingestion in healthy subjects and those with T2D (Figure 6). It was
found that DPP-4 inhibition (with sitagliptin) before a mixed meal had a similar
effect on incretin hormone secretion, beta-cell function and glucose homeostasis in
healthy subjects and subjects with T2D. The T2D subjects had a short duration of
T2D, were drug-naïve and had well-controlled T2D (HbA1c ~43 mmol/mol). It thus
appears that the early instigation of DPP-4 inhibition could regulate glucose
homeostasis in T2D subjects.
It was also found that the effect of DPP-4 inhibition on intact GIP was significantly
higher in healthy subjects than in those with T2D. This result could be explained
that the glucose-lowering effect of DPP-4 inhibition is probably mediated through
the GIP after meal ingestion in healthy subjects [207].
T2D is associated with hyperglucagonaemia. Therefore, the suppression of gluca-
gon levels is considered important in the treatment of T2D [14]. The effect of DPP-
4 inhibition on glucagon was found to be similar in healthy subjects and subjects
with T2D (Figure 6). A possible explanation of this could be that the glucagon-
suppressive effect of DPP-4 inhibitors is restricted to those with high glucose levels
[92]. Also, the effect of DPP-4 inhibition on the GIP is higher in healthy subjects
than those with T2D. This could in turn stimulate the glucagon levels since the
plasma glucose levels are lower in healthy subjects compared to those with T2D
[99,100].
Based on these findings, it is concluded that the effect of DPP-4 inhibition on the
islet hormones is glucose-dependent in healthy subjects and subjects with well-
controlled T2D.
48
The effects of three different DPP-4 inhibitors (sitagliptin, vildagliptin and
saxagliptin) on plasma glucose, and islet and incretin hormone secretion after the
ingestion of breakfast, lunch and dinner, were investigated in metformin-treated
subjects with T2D (Paper V, Table 5). It was found that DPP-4 inhibitors had a
similar and persistent effect on the suppression of glycaemia, the stimulation of
beta-cell function and the suppression of glucagon secretion. It could be that these
effects are mediated through the persistent stimulation of intact incretin hormones
after meal ingestion throughout the day [149]. The present findings also support the
previous suggestion that the secretion of incretin hormones is controlled by
feedback mechanisms acting on the K and L cells, i.e. an increase in the levels of
intact incretin hormones suppresses their secretion (total level of incretin
hormones). Furthermore, it seen that this feedback continues throughout the day
after each meal ingestion [208].
Data presented in Papers IV and V were combined to compare the effect of DPP-4
inhibition in drug-naïve and metformin-treated T2D subjects after mixed meal
ingestion (breakfast) (Figure 7). DPP-4 inhibition was found to have a similar effect
on plasma glucose, beta-cell function, incretin hormone secretion and glucagon in
both groups. These results indicate that the DPP-4 inhibitory effect on glycaemia
and islet and incretin function may be independent of metformin therapy. However,
this would be somewhat surprising as it is known that metformin also stimulates
GLP-1 [209,210]. Therefore, combined therapy with metformin and DPP-4
inhibition could reinforce the effect of incretin hormones on islet function. It has
been suggested that metformin reduces DPP-4 activity and thereby prevents the
degradation of the intact incretin hormones. On the other hand, the degree of DPP-
4 inhibition achieved by DPP-4 inhibitors is > 80%, and the impact of metformin
on the degradation of DPP-4 after meal ingestion can be considered small in this
context [149,211]. In contrast, long-term treatment of T2D with DPP-4 inhibitors in
addition to metformin has been reported to improve glycaemia and insulin-secretion
compared to monotherapy with DPP-4 inhibitors [212].
Based on these findings, it can be concluded that the effects of DPP-4 inhibition on
glycaemia, and islet and incretin hormone responses are similar in well-controlled,
drug-naïve and metformin-treated subjects with T2D.
It is known that there are no differences in the effect of these three different DPP-
inhibitors, therefore treatment with DPP-4 inhibition can be individualized. Some
people with T2D may find it more convenient to take one tablet of sitagliptin or
saxagliptin daily, rather than vildagliptin, which must be taken twice daily.
Furthermore, liver function should be monitored in people taking vildagliptin
compared to those taking sitagliptin or saxagliptin [213].
49
Strengths and limitations of this work
One of the strengths of this research is the design of the five studies included. The
study populations were similar regarding their glycaemic control, which allows
comparison between the studies. Furthermore, the studies had a cross-over design
which means that each participant in each study received all tests, which provides a
large data cohort, making the findings and conclusions more robust. The results and
outcomes can also be generalised as the study populations consisted of subjects who
were healthy or had well-controlled T2D, and included both lean and overweight,
young and old, and men and women.
The meal tests were performed by the same research nurse, which means that the
procedure was standardized. The T2D subjects had well-glycaemic control and a
relatively short duration of diabetes, which makes it possible to compare their
responses to meal ingestion with those of healthy subjects.
Beta-cell function was estimated by the model developed by Mari et al. [183,184].
This model characterizes the response of the beta-cell function over the study
period. Furthermore, the levels of intact incretin hormones were measured to eval-
uate their effect on the islet hormones and the total levels of incretin hormones, in
order to characterize the impact of the ingested meals and DPP-4 inhibition on
incretin hormone secretion.
On the other hand, including only well-controlled subjects with T2D can also be
considered a limitation, as the results cannot be applied to patients with a long
duration of T2D or poor metabolic control T2D with elevated HbA1c levels.
It is known that insulin response is higher after the ingestion of a solid meal than
after a meal in liquid form [214]. Some of the meals in these studies (Papers I and
III) were given in liquid form, which might be considered a limitation as meals are
normally solid [2]. However, it was important to administer exact amounts of the
macronutrients in each meal test, and therefore, a liquid form was chosen.
The single-day study approach adopted in all the studies described in this thesis
might be considered a limitation, as no conclusions can be drawn on the day-to-day
variations of the meal effects or the long-term effects of DPP-4 treatment.
50
Concluding Remarks and Future Perspectives
Novel results regarding the characterization of the metabolic response to mixed
meal ingestion have been presented in this thesis. Firstly, it is shown that including
all macronutrients in a meal results in better regulation of postprandial glycaemia
than the ingestion of each macronutrient separately. These findings were similar in
healthy and well-controlled T2D subjects. However, these findings should be
confirmed using a solid meal in future studies, instead of the liquid meal used in the
present studies.
It is also demonstrated that an increase in the meal size induces an adaptive
metabolic response of the islet and incretin hormone responses to prevent post-
prandial hyperglycaemia. As the metabolic response to the ingested meal was
similar in healthy and well-controlled T2D subjects, it would be of interest to
perform a comparative study in dysregulated T2D subjects, and patients with a long
duration of the disease. Such investigations would help to further evaluate the islet
and incretin hormone responses and the degree of deterioration in beta-cell function.
Furthermore, it is found that a single dose of a DPP-4 inhibitor in drug-naïve T2D
subjects with well-controlled glycaemia regulates glucose homeostasis through its
effects on islet function, resulting in a reduction in postprandial glycaemia to levels
similar to those in healthy subjects. However, T2D results in a gradual decrease in
beta-cell function [215]. Therefore, more studies are required to investigate the effect
of DPP-4 inhibition on glucose homeostasis and islet function in different stages of
the disease.
Moreover, it is established that the effect of DPP-4 inhibition on islet and incretin
hormone responses persists throughout the day in metformin-treated and well-
controlled T2D subjects. It has also been shown that the effects of DPP-4 inhibition
are independent of metformin treatment. This could be further investigated in the
future, in subjects with T2D treated with other antidiabetic medications such as
SGLT-2 inhibitors or insulin.
51
Clinical significance
The most fundamental aspect of diabetes care is dietary intervention. It is important
to modify eating patterns in subjects with T2D in order to regulate glycaemia.
However, there is no “one size fits all” recommendation.
This thesis provides new knowledge on the dynamic regulation of glucose
homeostasis by islet and incretin hormones following meal ingestion, which can be
used to improve advice on nutrition for those with T2D.
Based on the findings of this work, it is recommended that each meal should consist
of a combination of carbohydrates, protein and fat. Furthermore, a suitable energy
distribution of the meal is suggested to be 50-60 E% carbohydrates, 20 E% protein
and 20 E% fat. The results presented in this thesis are primarily of use to dietitians,
diabetes nurses and medical doctors involved in primary healthcare of patients with
T2D.
Previous studies have shown that oral antidiabetic treatment as monotherapy does
not provide complete normalization of glycaemia in the long term. It is also essential
to target postprandial hyperglycaemia to prevent the complications associated with
T2D [216]. A single dose of a DPP-4 inhibitor has been found to suppress
postprandial glycaemia, stimulate beta-cell function, and reduce glucagon in
subjects with well-controlled T2D (HbA1c ~43 mmol/mol). Therefore, the
introduction of DPP-4 inhibition as early as possible appears to be beneficial in the
medical treatment of patients with T2D.
Also, concerning the elderly subjects with T2D who were participating in these
studies, it has been found that DPP-4 inhibition reduces glycaemia in a similar
degree as those younger people with T2D and the treatment not associated with any
AEs. Thus monotherapy with DPP-4 inhibitors could be suitable for the treatment
of elderly people with well-control T2D.
Management of patients with T2D who have previously undergone bariatric surgery
is considered challenging by clinical professionals. This is due to the increased risk
of AEs when using conventional antidiabetic therapies such as metformin or
sulfonylureas [152]. As DPP-4 inhibitors appear to be efficient in the suppression of
postprandial hyperglycaemia their use may be beneficial in the treatment of T2D
patients who have undergone bariatric surgery.
52
Acknowledgements
I would like to begin by expressing my sincere gratitude to everyone who
contributed to the studies presented in this thesis. Without your time and
commitment, this work would not have been possible. I would like to express my
special thanks to the following people.
My supervisor, Professor Bo Ahrén, thank you for guiding me through this work,
for being available for discussions even when our meetings took place at the
weekend, early in the morning, or late in the evening. It was an honour to work with
you and to be your last PhD student. I hope I have made you proud.
My co-supervisor, Olga Göransson, thank you for your valuable feedback. Thanks
also to my former co-supervisors, Lena Ohlsson and Bilal Omar for your help and
fruitful discussions at the beginning of my PhD journey.
My co-authors, Richard Carr, Andrea Mari, Giovanni Pacini, Andrea Tura and
Roberto Bizzotto, thank you for sharing your professional experience with me, and
for taking time out for EASD conferences, and my phone calls and emails.
Bertil Nilsson and Gustav Dahl, research nurses, thank you for your valuable
contributions, Kristina Andersson for help with laboratory analysis, and all the
staff at the BMC for nice fika.
I would like to express my special appreciation and thanks to Magnus Löndahl,
Head of the Department of Endocrinology, Skane University Hospital in Lund, for
his patience and support.
My clinical mentors, Sven Karlsson, Stig Waldemarsson, Viveca Engblom,
Bengt Hallengren and Erik Waldenström, for excellent clinical support and for
sharing their knowledge.
My colleague and friend, Sigridur Fjalldal, it is difficult to find the words to
describe how grateful I am for your help and input in the writing of this thesis. I will
never forget your support and encouragement when my self-confidence was at rock
bottom. I look forward to visiting you in Iceland soon.
My colleagues: Ola Lindgren, for introducing me to this field of research, Karin
Filipsson, Karin Olsson, Ulrika Moll, Henrik Borg and Katarina Fagher, for
their warm friendship and fun moments after work, and all my other colleagues for
creating such a brilliant environment at the clinic.
53
Our friends, Muhanad, Dalia, Baha and Hadeel, for your true friendship, support
and help when needed. We look forward to the celebrations, even if they are not
going to be held as planned; as we say in Arabic “ Every cloud“ ,“ كل تأخيرة فيها خيرة
has a silver lining”. Thank you for lovely dinners and all the laughter.
Selwan Khamisi, my friend and brother since childhood, we have survived many
challenges together over the years. Thank you, and I wish you good luck with your
own PhD thesis.
My friends, Jaber Al Hashhoush, Elke Peters, Bassam Hazem, Samir Al-
Hakeem, Edmund Gazala, Petros Katsogiannos and Nawras Altaie, for your
support and encouragement. Hamid Al Haidar, for help with the Sumerian cover
art, which represents our culture and roots, Yahya Jany, my brother-in-law, for
providing me with excellent feedback and helping me with the English language,
structure and finishing touches to this thesis.
My parents Salim and Halima, for fostering the love of knowledge in me, and for
your prayers that kept me going. My siblings, Ibihage, Resha, and Suhiel, my
uncle, Hikmet and my cousins, thank you for believing in me. My parents-in-law,
Basim and Rajiha, thank you for your support, cheerleading and your delicious
dinners when we needed them most. Thank you to my mother-in-law for correcting
the scientific summary in Arabic. To my brothers-in-law, thank you for your
support.
My firstborn daughter, Yasmin, thank you for your calming words, your viola
playing and delicious cookies and cups of tea. My second-born daughter, Linn,
thank you for your “energy hugs” and beautiful viola playing when I needed to relax.
I will never forget you saying, “ Pappa, ge aldrig upp”. I hope you both realize that
if you have the patience, you will reach your goals.
Last but not least, my beloved wife, Rana, who has been incredibly supportive of
me throughout this entire process. You have made countless sacrifices to help me
reach this point. It is difficult to find the words to describe how grateful I am for
your support and patience.
My wonderful three girls, Rana, Yasmin and Linn, thank you for your endless
positive energy. You have contributed to this work more than anyone will realize.
These final words were written when practically the whole world is lockdown due
to the coronavirus (COVID-19) pandemic. We must remain optimistic, focusing on
positive thoughts and the belief that science will help us to get through this crisis.
“Where there’s a will, there’s a way”.
Funding: This work was supported by the Medical Faculty, Lund University,
Sweden and The Swedish Research Council, Region Skåne.
54
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WA
THIK
ALSA
LIM
O
n Meal E
ffects and DPP-4 Inhibition Islet- and Incretin H
ormones in H
ealth and Type 2 D
iabetes 2020:39
Department of Clinical Sciences
Lund University, Faculty of Medicine Doctoral Dissertation Series 2020:39
ISBN 978-91-7619-900-8ISSN 1652-8220
On Meal Effects and DPP-4 Inhibition Islet- and Incretin Hormones in Health and Type 2 DiabetesWATHIK ALSALIM
DEPARTMENT OF CLINICAL SCIENCES | LUND UNIVERSITY
About the author
Wathik Alsalim completed his medical training at Baghdad College of Medicine, Baghdad University, Iraq, in 1999. He is currently working as a senior registrar in endocrinology at Skåne University Hospital, Sweden. He conducted his doctoral research at the Department of Clinical Sciences, Faculty of Medicine, Lund University, Sweden. His PhD thesis focuses on the effects of meals on the regulation of glycaemia, and islet and incretin hormones in health and type 2 diabetes.
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