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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.

9789176

199008

1

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

9

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

11

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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