Linköping University Medical Dissertations No. 1268

Interaction between insulin and IGF-I receptors in insulin sensitive and insulin resistant human cells and tissues

Karolina Bäck

Linköping University, Faculty of Health Sciences

Department of Clinical and Experimental Medicine

Division of Cell Biology

SE-581 85 Linköping, Sweden

Linköping 2011

© Karolina Bäck

ISBN 978-91-7393-042-0

ISSN 0345-0082

Published articles have been reprinted with the permission of the respective copyright

holder: Elsevier (Paper I ©2008), Elsevier (Paper II ©2011)

Printed by LiU-Tryck, Linköping, Sweden, 2011

An expert is a man who has made all the mistakes

which can be made in a very narrow field.

Niels Bohr (1885-1962)

Supervisor

Hans Arnqvist, Professor

Department of clinical and experimental medicine, Division of cell biology

Linköping University, Linköping, Sweden

Co-supervisor

Mats Söderström, Associate Professor

Department of clinical and experimental medicine, Division of cell biology

Linköping University, Linköping, Sweden

Opponent

Hans Tornqvist, Senior research physician

Astra Zeneca, Mölndal, Sweden

Commettee board

Åke Wasteson, Professor

Department of clinical and experimental medicine, Division of cell biology

Linköping University, Linköping, Sweden

Peter Bang, Professor

Department of clinical and experimental medicine, Division of women and child health

Linköping University, Linköping, Sweden

Gunnar Norstedt, Professor

Department of molecular medicine and surgery

Karolinska Institutet, Stockholm, Sweden

Abstract

Insulin and insulin-like growth factor I (IGF-I) are two related peptides with similar

structure. They mediate their effects by binding to their respective receptor, the insulin

receptor (IR) and the IGF-I receptor (IGF-IR) and induce intracellular signalling cascades

resulting in metabolic or mitogenic effects. The relative abundance of IR and IGF-IR is of

importance for the type of effect that is the outcome of the signal. There are few studies

investigating the relative receptor abundance and its effects in human cells and tissues.

In this thesis we wanted to study abundance and regulation of insulin and IGF-I

receptors in different human cells and tissues and examine the effects of variations in

insulin and IGF-I receptor abundance between different cells and tissues.

We examined IR and IGF-IR gene and protein expression and the effects of insulin and

IGF-I on receptor phosphorylation, DNA synthesis and glucose transport.

Our results show that there is a large variation in the distribution of IR and IGF-IR in

different human cells and tissues. Renal artery intima-media expressed predominantly

IGF-IR while in liver IR was the predominant receptor type.

Differentiation of human preadipocytes results in a change in relative expression of IGF-

IR to IR. Mature adipocytes express almost 10-fold more IR than IGF-IR while

preadipocytes express almost the same amounts of both receptors. Mature tissues, such

as liver, skeletal muscle, myometrium and renal artery intima-media, express

predominantly IR-B. Preadipocytes express IR-A and the expression of IR-B is induced

during differentiation.

We could show the presence of insulin/IGF-I hybrid receptors in preadipocytes but not

in mature adipocytes. Cultured endothelial cells express mostly IGF-IR and insulin/IGF-I

hybrid receptors and these cells respond mainly to IGF-I. Due to the large abundance of

IR mature adipocytes are sensitive to insulin but insensitive to IGF-I whereas

preadipocytes expressing equal amounts of both receptors respond to both insulin and

IGF-I. Insulin and IGF-I are only partial agonists to each other’s receptors in human

preadipocytes and adipocytes.

The overall results indicate that differential expression of IGF-IR and IR is a key

mechanism in regulation of growth and metabolism.

Table of contents

List of original papers .................................................................................................................................... 1

Abbreviations .................................................................................................................................................... 2

Introduction ....................................................................................................................................................... 4

The IGF system ............................................................................................................................................. 4

Insulin .......................................................................................................................................................... 4

IGF-I .............................................................................................................................................................. 5

IGF-II ............................................................................................................................................................ 5

IGFBP ........................................................................................................................................................... 6

IR and IGF-IR ............................................................................................................................................ 7

Insulin/IGF-I hybrid receptors .......................................................................................................... 8

Insulin receptor isoforms .................................................................................................................... 9

IGF-IIR ......................................................................................................................................................... 9

Receptor distribution ................................................................................................................................ 9

Receptor signalling .................................................................................................................................. 10

Insulin resistance, inflammation and diabetes ............................................................................. 11

Insulin and IGFs in cancer ..................................................................................................................... 12

Aims of the thesis ......................................................................................................................................... 14

Methodological aspects .............................................................................................................................. 16

Tissues, cells and culture ...................................................................................................................... 16

Gene expression analysis ...................................................................................................................... 16

Protein expression analysis ................................................................................................................. 17

Glucose transport measurements ...................................................................................................... 18

Results and discussion ............................................................................................................................... 20

Gene expression of IR and IGF-IR ...................................................................................................... 20

Gene expression of IR-A and IR-B ...................................................................................................... 21

Protein expression of IR and IGF-IR ................................................................................................. 21

Phosphorylation of IR and IGF-IR ...................................................................................................... 22

Hybrid receptors ...................................................................................................................................... 23

Characterisation of differentiation .................................................................................................... 23

Cultured vs. freshly isolated cells ...................................................................................................... 24

Mitogenic effects – DNA synthesis ..................................................................................................... 25

Metabolic effects – Glucose transport .............................................................................................. 25

Anti-inflammatory effects – E-selectin expression ..................................................................... 26

General discussion ....................................................................................................................................... 28

Differentiation ........................................................................................................................................... 28

Tissue- and cell-specific receptor expression ............................................................................... 28

Impact of differences in distribution of IGF-IR and IR on effects of insulin, IGF-I or IGF-

II ...................................................................................................................................................................... 29

The importance of hybrid receptors ................................................................................................. 30

Insulin receptor isoforms ..................................................................................................................... 30

Sensitivity and insensitivity to insulin and IGF-I ......................................................................... 31

Summary and conclusions ........................................................................................................................ 34

Tack .................................................................................................................................................................... 36

References ....................................................................................................................................................... 38

1

List of original papers

This thesis is based on the following papers, which will be referred to by their Roman

numerals.

I. Bäck K, Arnqvist HJ (2009). “Changes in insulin and IGF-I receptor expression

during differentiation of human preadipocytes”. Growth Horm IGF Res

19:101-111.

II. Bäck K, Brännmark C, Strålfors P, Arnqvist HJ (2011). ”Differential effects of

IGF-I, IGF-II and insulin in human preadipocytes and adipocytes - Role of

insulin and IGF-I receptors”. Mol Cell Endocrinol 339:130-135.

III. Bäck K, Islam R, Johansson GS, Chisalita SI, Arnqvist HJ. ”Role of insulin and

IGF-I receptors in human cardiac microvascular endothelial cells; metabolic,

mitogenic and anti-inflammatory effects”. In manuscript.

IV. Bäck K, Wahlström O, Kjølhede P, Sandström P, Gasslander T, Arnqvist HJ.

”Differential expression of insulin and IGF-I receptors in human tissues”. In

manuscript.

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Abbreviations

cDNA – complementary DNA

ELISA – enzyme-linked immunosorbent assay

ERK – extracellular-signal-regulated kinase

GH – growth hormone

GLUT – glucose transporter protein

HR – insulin/IGF-I hybrid receptor

HRP – horseradish peroxidase

HMVEC – human microvascular endothelial cells

HMVEC-C – cardiac human microvascular endothelial cells

HUVEC – human umbilical vein endothelial cells

IGF-I – insulin-like growth factor type I

IGF-II – insulin-like growth factor type II

IGF-IR – IGF-I receptor

IGFBP – insulin-like growth factor binding protein

IR – insulin receptor

IR-A – insulin receptor isoform A

IR – insulin receptor isoform B

IRS – insulin receptor substrate

MAPK – mitogen activated protein kinase

PI3K – phosphoinositide 3-kinase

SH2 – Src-homology-2

TNF-α – tumour necrosis factor α

VEGF – vascular endothelial growth factor

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4

Introduction

The IGF system

The IGF system is a complex system of ligands and receptors that control growth,

metabolism and reproduction (73).

Figure 1. Overview of the IGF system with polypeptides,

receptors and binding proteins (IGFBP).

Insulin

Insulin is the main regulator of glucose uptake and metabolism in the body. It increases

glucose uptake in muscle and fat by stimulating the translocation of GLUT4, a glucose

transporter protein, from the cytoplasm to the cell surface and inhibits glucose

production in the liver (78). Insulin also has several other functions. It stimulates

lipogenesis and glycogen and protein synthesis as well as cell growth and differentiation

(78).

The insulin molecule is secreted from the β-cells in the pancreas. It consists of two

peptide chains, A and B, and a third chain, the C-peptide, is cleaved off when proinsulin

becomes active insulin (51).

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Lack of insulin causes diabetes with high fasting and postprandial glucose levels,

elevated free fatty acid levels and dysregulation of several other vital processes (78).

Survival is not possible if there is a total lack of insulin.

IGF-I

Insulin-like growth factor-I (IGF-I) is closely related to insulin and has a similar

structure. The IGF-I molecule is mainly produced in the liver, but also expressed in

several different tissues (45). Growth hormone (GH) is the main regulator of IGF-I

expression the GH-sensitivity in the liver being modulated by insulin probably via

regulation of GH receptor expression (49). IGF-I is involved in cell proliferation,

migration, growth and apoptosis; processes implicated in tissue formation, bone growth

and brain development (33). These functions subsequently affect organism size and

longevity by various mechanisms. It is assumed that less than 1% of total circulating

IGF-I exists as free molecules, the rest being bound to specific binding proteins, IGFBPs

(45). This increases the bioavailability of IGF-I by increasing its half-life.

Silencing of the igf-1 gene in mice results in offspring born as dwarfs after deficient

embryonic growth and subsequently impaired growth and infertility (3). Loss of

functional IGF-I in humans leads to pre- and post-natal growth retardation and

sometimes developmental retardation and partial or total deafness (102).

Secretion of insulin is rapid and regulated in a sophisticated manner by several

mechanisms. In response to food intake insulin levels increase in the bloodstream within

minutes (65). There is a large inter-individual variation in plasma insulin levels due to

differences in insulin sensitivity the highest postprandial concentrations reached are

around 1 nmol/L (15). In contrast circulating IGF-I is slowly regulated, changes

occurring within hours (45). The concentration of free bioactive IGF-I is around10-10 -

10-9 M. IGF-I expression varies with age with a peak during puberty (55). In contrast to

insulin, IGF-I expression is regulated locally (23).

IGF-II

IGF-II is involved in prenatal growth. In rodents IGF-II is not expressed after birth,

whereas in humans it is expressed throughout life (73). It is important for embryonic

stem cell survival and renewal, and is also known to be involved in differentiation of

muscle, brain and brown adipose tissue in adults (14). The effects of IGF-II on cell

growth, survival, migration, and differentiation is mediated by the IGF-IR (17).

IGF-II is the most abundant IGF in humans with circulating levels that are 5-10 times

higher than circulating levels of IGF-I (45).

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Figure 2. Insulin, proinsulin, IGF-I and IGF-II.

IGFBP

The IGF binding proteins (IGFBP) function as regulators of IGF-I and IGF-II

bioavailability and there are 6 different forms. By forming IGFBP-IGF complexes the

half-lives of IGF-I and -II increase substantially (108). The binding proteins also

function as inhibitors of IGF action by preventing IGFs from binding to their receptors.

Evidence suggests that the IGFBPs may also have IGF-independent effects as regulators

of gene transcription, cell growth and apoptosis (69, 108).

The various IGFBPs have different functions and are expressed differently. For instance,

IGFBP-1 promotes the effects of IGFs when non-phosphorylated, and inhibits the effects

when phosphorylated (54). IGFBP-2 and -4 generally inhibit IGF action. Serum levels of

IGFBP-1 vary during the course of the day with low levels after meals; the expression of

IGFBP-1 being regulated by insulin. IGFBP-2, -3 and -5 expressions in serum vary with

age (108). The majority of circulating IGFs are bound to IGFBP-3 which is regulated by

growth hormone (55).

IGFBP regulation of IGFs in different tissues is very complex involving

autocrine/paracrine effects. Binding affinity is affected by IGFBP binding to extracellular

matrix, IGFBP phosphorylation and proteolytic cleavage by serine proteases (69, 73).

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IR and IGF-IR

Receptors for insulin and IGF-I are called the insulin receptor (IR) and the IGF-I receptor

(IGF-IR) respectively. They are both from the same family of tyrosine kinase receptors

and share an overall homology of more than 50% and 84% homology in the tyrosine

kinase domains (88). The receptors are located in the cell membrane as homodimers

with two αβ-subunits linked together by disulphide bonds (88). The α-subunits are

extracellular and contain the ligand-binding sites while the β-subunits are

transmembrane and contain the tyrosine kinase domains on the intracellular part (106).

The IGF-IR generally mediates mitogenic effects, and the IR metabolic effects (58). The

factors involved in determining the physiological response to receptor signalling are still

largely unknown. There is evidence for differences in the activation of intracellular

proteins involved in receptor signalling, receptor tyrosine kinase domains and also of

ligand dissociation rate that determine the signal outcome (11, 13, 29, 35, 79). The

receptor type and the number of receptors expressed on the cell surface could also affect

the cellular response (35, 58, 106).

IR and IGF-IR have a high affinity for their own ligand but can also bind the other ligand

with approximately 100-fold lower affinity (106). IGF-II has a 10-fold lower affinity for

the IGF-IR compared to IGF- I (21).

The IR α-subunit contains two different binding sites, often referred to as Site 1 and Site

2. The insulin molecule binds to the low affinity Site 1 on either α-subunit and then to

the high affinity Site 2 on the other α-subunit (105). The binding to both Site 1 and Site 2

is necessary for high affinity binding. Negative cooperativity occurs when a second

molecule binds to the alternate low affinity Site 1 reducing the affinity to the initial Site

1/Site 2 binding (28, 104, 105). IGF-I binding to the IGF-I receptor exhibits similar

patterns of negative cooperativity but only requires one α-subunit binding site for high

affinity binding (30, 105).

The importance of IR and IGF-IR may be illustrated by animal models. IR knockout mice

are normal size at birth but afterwards they quickly loose metabolic control (2). They

suffer from increased glucose levels despite highly increased insulin secretion, and β-cell

failure leading to death ketoacidosis within days. Lack of IR in humans results in severe

growth retardation at birth and no, or very little, postnatal weight gain (2, 73, 107).

Mice lacking the IGF-IR show extensive growth retardation and several abnormalities at

birth, and die within minutes due to respiratory failure as a result of undeveloped

diaphragmatic and intercostal muscles (73).

Mutations in human IGF-IR result in pre- and postnatal growth retardation (59, 103).

Other anomalies reported in patients with total or partial IGF-IR impairment are mental

retardation, delayed motor development, deafness and mildly impaired glucose

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tolerance. Whether these anomalies are a direct cause of impaired IGF-I signalling or

have other causes has yet to be determined. IGF-IR mutations have also been associated

with an increased lifespan (96).

Insulin/IGF-I hybrid receptors

The IR and the IGF-IR can form heterodimers with one αβ-subunit from each receptor

(71). These receptors are called insulin/IGF-I hybrid receptors (HR) and have a high

binding affinity for IGF-I and a low affinity for insulin thereby functioning as an IGF-IR

(57, 82, 94). The formation of hybrid receptors is believed to occur by random assembly

of IR and IGF-IR halves. This means that the less abundant receptor type will, to a large

extent, be incorporated into hybrid receptors (44). Subsequently, cells with an excess of

IGF-IR will have most IR as hybrid receptors, making the cells less sensitive to insulin.

The lower affinity of insulin to hybrid receptors can be explained by the differences in

binding mechanisms between insulin and IGF-I (105). As mentioned above, the insulin

molecule requires two binding sites for high affinity binding to the IR, whereas IGF-I can

bind to one site with high affinity. There is only one binding site on hybrid receptors

which allows IGF-I, but not insulin, to bind with high affinity.

Figure 3. IGF-I receptor, insulin/IGF-I hybrid receptor, insulin receptor isoforms

and the IGF-II receptor and their binding to insulin, IGF-I and IGF-II.

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Insulin receptor isoforms

The insulin receptor has two isoforms as a result of alternative splicing of exon 11 on the

IR gene (8). Isoform A (IR-A) lacks the 12 amino acids encoded by exon 11, while

isoform B (IR-B) contains this sequence. These 12 amino acids are located at the

carboxy-terminus of the receptor α-subunit, on the ligand-binding subunit of the

receptor (109). Insulin has a high binding affinity for both isoforms while IGF-I binds the

receptors with at least 100-fold lower affinity (32, 42). IGF-II binds IR-A with 10-fold

lower affinity than insulin whereas IGF-II binding to IR-B is 50-fold weaker than insulin

binding. Insulin/IGF-I hybrid receptors seem to have the same affinities for their ligands

regardless of which IR isoform is incorporated (8). The two isoforms display the same

ability to form HR with the IGF-IR (109) but there is evidence suggesting that the two

hybrid isoforms may have different functions (85).

Apart from the differences in structure the two IR isoforms also differ in their

localisation on the cell membrane, as well as intrinsic rate of internalisation and rate of

insulin association and dissociation (61, 100, 109).

There is also evidence of two IGF-IR isoforms. One is selectively expressed in foetal and

postnatal life and the other is expressed both pre- and postnatally in animals (4).The

two isoforms differ in the size of the β-subunit but little is known of the importance and

function of these two receptor variants.

IGF-IIR

The main function of the IGF-IIR, also called the cation-independent mannose-6-

phosphate receptor is believed to be regulation of circulating IGF-II and thereby its

binding to the IGF-IR (73). It may also have direct IGF-II effects including, for instance,

activation of the mitogen-activated protein kinase (MAPK) pathway and apoptosis (14).

Receptor distribution

Expression of IR and IGF-IR is tissue-specific with large differences in the abundance of

the two receptors between different tissues. Most data have been obtained by the ligand

binding technique and there are few studies that directly compare of IR and IGF-IR in

the same tissue. Ligand binding studies led to the detection of IR in isolated human

hepatocytes and liver. Adipose tissue and skeletal muscle expressed both IR and IGF-IR

IR being the most abundant receptor type in adipose tissue (6, 16, 38-40). The presence

of IR and IGF-IR in skeletal muscle and adipocytes has also been demonstrated using

Western blot and ELISA (26, 41, 95). Vascular endothelial cells and smooth muscle cells

have been shown to have more IGF-IR than IR, and these cells are also more sensitive to

IGF-I than to insulin (18-21, 53, 74).

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The expression of insulin/IGF-I hybrid receptors has been detected in many different

tissues. Results from our group show the presence of hybrid receptors in human

vascular endothelial cells and smooth muscle cells (18, 21, 53). There are also studies

showing hybrid receptors in human skeletal muscle and adipose tissue and several other

tissues (6, 39). The proportion of insulin/IGF-I hybrid receptors is altered in type 2

diabetes compared to normal subjects (40).

Alternative splicing of exon 11 on the IR gene is tissue-specific and it has been shown

that mature tissues such as liver, skeletal muscle and adipose tissue, express mostly IR-B

or equal amounts of both isoforms, whereas foetal and cancer tissues contain more IR-A

(8, 9, 84, 109). There is evidence suggesting that the relative expression of IR-B is

increased in patients with insulin resistance or type 2 diabetes compared to healthy

subjects, but results are not consistent (43, 85).

Receptor signalling

Ligand binding to the IR or IGF-IR results in a conformational change in the receptor

structure and subsequent autophosphorylation of the tyrosine kinase residues on the β-

subunit (35). This activates intracellular signalling pathways with different biological

responses. The signalling of IR and IGF-IR is extremely complex but may be summarised

in a few critical steps.

Autophosphorylation of the receptor β-subunit results in the recruitment and docking of

insulin receptor substrates (IRS) 1-6, where IRS1 and IRS2 are the most widely

distributed (98). The interaction between signalling molecules occurs via Src-homology-

2 (SH2) domains on the proteins involved (77). Phosphoinositide 3-kinase (PI3K) is

recruited to phosphorylated IRS via the SH2 domain and can then further activate

downstream proteins, including AKT/protein kinase B (PKB) (88). AKT/PKB will then

subsequently mediate metabolic effects via the phosphorylation of several other kinases,

signalling proteins and transcription factors, including the family of winged helix or

forkhead (FOX). Several of these transcription factors are involved in insulin action.

FOXO1 activates gluconeogenesis in the liver and inhibits adipocyte differentiation.

Insulin blocks the effects of FOXO1 via AKT/PKB (98).

The mitogenic pathway is activated by IRS interaction with the small G-protein Ras

which is followed by activation of the protein serine kinase Raf and the extracellular-

signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signalling

cascade (62, 88). This pathway is involved in cell growth, survival and differentiation

(98).

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It has been shown in L6 myotubes that IRS1 is more closely related to the regulation of

glucose transport while IRS2 is linked to the mitogenic pathway. The differences in

effects can be explained by differential ability to bind to other signalling proteins as well

as the IR (98).

Activation of the PI3K pathway via insulin in adipose tissue and skeletal muscle will

enhance glucose transport by regulating localisation of the glucose transporter GLUT4,

whereas in pancreatic β-cells PI3K signalling probably promotes β-cell survival (77).

Inhibition of PI3K will block almost all effects of the IR, such as stimulation of glucose

transport, synthesis of glycogen and lipids and adipocyte differentiation (98).

Figure 4. Signalling pathways for IGF-I and insulin.

Insulin resistance, inflammation and diabetes

Insulin resistance is defined as a state where target tissues show a decrease in

responsiveness to circulating levels of insulin (83). In the clinical setting insulin

resistance, which often precedes type 2 diabetes, is commonly assessed by glucose

clamp technique (31).

Both insulin resistance and type 2 diabetes are associated with inflammation and

atherosclerotic disease (25, 76). Over-nutrition causes oxidative stress and a pro-

inflammatory state (24). The expression of TNF-α together with other inflammatory

molecules is known to increase with increasing adipose depots (87). TNF-α inhibits

insulin signalling by affecting the kinase activity of signalling molecules (76). Under

normal conditions both insulin and IGF-I have anti-inflammatory properties (25, 97).

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In type 1 diabetes a decrease in insulin levels in the portal vein results in dysregulation

of the GH-IGF-IGFBP axis (10, 48, 99). Type 1 diabetes has been associated with hepatic

GH resistance and increased production of IGFBP-1 and -2 and reduced levels of IGFBP-3

resulting in decreased levels of circulating IGF-I.

Insulin and IGFs in cancer

The role of insulin, insulin analogues and the IGF system in cancer development and

progression has received much attention (93). High levels of IGF-I and IGF-II and low

levels of IGFBP-3 are associated with an increased relative risk for the development of

cancer in the breast, prostate, colon and lung (63). The IGF-IR is over-expressed in many

different cancers (63). Focus in cancer research has been on IGFs but insulin is also to a

high degree is involved in tumour cell proliferation and survival. In several different

malignancies there is an over-expression of IR-A compared to IR-B, which makes the

cells susceptible to, not only insulin, but also IGF-II (7). There is an association between

obesity, insulin resistance and type 2 diabetes and the risk of developing cancer since

high levels of insulin could stimulate the IR-A, which is over-expressed in cancer, and

also increase bioavailability of IGF-I by reducing levels of IGFBP-1 and -2 (43).

13

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Aims of the thesis

The aims of this thesis are to study abundance and regulation of insulin and IGF-I

receptors in different human cells and tissues, and to examine the effects of variations in

insulin and IGF-I receptor abundance between different cells.

We would particularly like to answers the following questions:

How does differentiation of human preadipocytes affect the distribution of

insulin receptors (IR) and IGF-I receptors (IGF-IR), and influence adipocyte

characteristics?

What impact do differences in distribution of IGF-IR and IR have on the effects of

insulin, IGF-I and IGF-II? Do insulin and IGF-I act as full or partial agonists to each

other’s receptors?

What is the expression of IR and IGF-IR in endothelial cells, and what is their role

in metabolic, mitogenic and inflammatory responses to insulin and IGF-I?

How is IR and IGF-IR expressed in classical insulin target tissues such as fat, liver

and muscle, and in non-classical insulin target tissues such as blood vessels and

smooth muscle?

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16

Methodological aspects

Tissues, cells and culture

In this project we have used both cultured human cells and isolated tissue biopsies for

our experiments. The use of human material instead of material from animals is

preferable since there are differences between humans and animals regarding, for

instance, intracellular signalling components (56).

In Papers I, II and IV, the material used came from tissue biopsy samples retrieved

during surgery. Although we used only “healthy tissue” patients undergoing surgery are

ipso facto not healthy individuals. How the state of the patient affects the tissues used is

not known, but should be considered as a possible confounder.

The adipose tissue used to isolate preadipocytes and adipocytes in Papers I and II came

mostly from abdominal subcutaneous fat depots. This tissue may not be representative

of all adipose tissue since there are variations in gene and protein expression in

adipocytes from different locations in the body. This is especially the case between

subcutaneous and visceral fat where studies have shown differences (80, 101). In Paper

I preadipocytes were differentiated towards mature adipocytes but we were never able

to reach completely differentiation since not all preadipocytes differentiated at the same

time. Furthermore, when the cells became mature they were no longer adherent and

were removed when the medium was changed and discarded. We therefore considered

these differentiated cells to be an intermediate stage between preadipocytes and mature

adipocytes. There are no available markers to characterise preadipocytes and we cannot

be completely certain that all cells in our population were preadipocytes. The majority

of the cells did, however, differentiate to some extent when we added a mixture of

substances known to induce differentiation in preadipocyte cell lines (81). It should be

noted that the experiments on preadipocytes and adipocytes were not primarily

performed to characterise adipocytes, but to characterise IR and IGF-IR in cells during

differentiation from one state to another.

In Paper IV we used whole tissue from renal artery intima-media, myometrium, skeletal

muscle and liver. Although these tissues consist mostly of endothelial cells, smooth

muscle cells, skeletal muscle cells and hepatocytes, they also contain stromal cells and

vasculature which could have affected the results to some extent.

Gene expression analysis

Real-time RT-PCR is used to examine gene expression in cells and tissues. RNA is

extracted from the cells and converted into cDNA using reverse transcriptase enzyme.

The cDNA is amplified in a polymerase chain reaction (PCR) using primers and probes

designed to match the gene being examined. Amplification of the gene is exponential

17

since it is doubled in each PCR cycle. A reporter and a quencher are attached to the

probe. When the DNA fragment is amplified the quencher is cleaved off from the probe

and the reporter can then emit a fluorescent signal corresponding to the amount of

cDNA amplified. By measuring how many cycles it takes for a sample to reach a certain

threshold value the gene expression is calculated. The value obtained is normalised to

another gene, often a housekeeping gene, to give a relative value of gene expression.

Comparing differences in threshold cycles is called the delta-Ct method, as described in

Applied Biosystems User Bulletin #2, and requires that the efficiency of the probes used

are the same. If this is not the case, a standard curve with known dilutions of a sample is

a better choice for comparison of different samples.

Real-time RT-PCR is a sensitive and informative method but the results may not reflect

the in vivo situation since not all mRNAs are translated to proteins.

Protein expression analysis

Enzyme-linked immunosorbent assay (ELISA) is a widely used method to detect and

quantify proteins, hormones and growth factors. There are some variations in the

technique but the basic principle of the ELISAs used in this project is that the protein we

wish to detect is allowed to bind to a plate coated with antibody. Another antibody is

used for detection. A substrate enzyme reacts with molecules coupled to the secondary

antibody and give rise to a colour change of the solution in the well. The intensity of the

colour can be measured as optical density and gives a measurement of how much

protein the sample contains. If the assay also contains a standard curve with known

concentrations of the protein we can obtain a quantitative value. When measuring total

IR and total IGF-IR in Papers III and IV, we used commercially available kits. The

sensitivity of the IGF-IR ELISA was in the range of 156-10000 pg/ml while the IR ELISA

was in the range of 1250-80000 pg/ml. According to our results IR were more abundant

than IGF-IR in all tissues and cells examined, in contrast to the real-time RT-PCR results.

Due to the varying sensitivity of the ELISAs, the results may not be directly comparable

and should be interpreted with caution.

Western blot is another method used to study protein expression. The proteins in the

lysate are separated on a gel by electrophoresis and then blotted onto a membrane. The

membrane is incubated first with an antibody against the protein investigated and then

a horseradish peroxidase-coupled (HRP) secondary antibody. The HRP can react with

other substances and give rise to chemiluminescence that can be measured by

autoradiography. In this project we often judged our gel results manually by looking at

them and estimating whether one band was stronger or weaker than another. It is of

course difficult to distinguish discrete band differences using this method. To obtain

18

quantitative values the gel patterns can be scanned and the intensity of the bands

measured with densitometry. Although this gives us a value of the intensity of the bands

and thereby an indication of the amount of protein present, it is still not totally accurate

since it is the person analysing the gel who decides where on the gel the band intensity

and background are to be measured.

Glucose transport measurements

Glucose transport was measured in this project using two different methods. The basic

principle of the two methods is the same. Cells are stimulated with hormone in the

presence of radioactive glucose. The cells are lysed and the amount of radioactive

glucose transported into the cell is measured. The factor that differs between the two

methods is the different forms of glucose used. Glucose transport into adipocytes was

measured using 2-deoxy-D-[1-3H]glucose, and in other cells we used D-[U-14C]-glucose.

2-deoxyglucose is a monosaccharide which is transported into the cell and

phosphorylated but not further metabolised, whereas D-glucose is transported into the

cell and metabolised.

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Results and discussion

Gene expression of IR and IGF-IR

The gene expression of IR and IGF-IR was measured in cells and tissues by real-time RT-

PCR. The results are presented as the ratio of IGF-IR to IR expression where the

expression of the IR gene is put to one and the IGF-IR expression is presented as fold

difference.

Table 1. Gene expression of IGF-I receptor:insulin receptor ratios in tissues and cells.

Tissue/cell type IGF-IR/IR ratio Paper no

Renal arteries 7.7 IV

Myometrium 1.8 IV

Skeletal muscle 0.7 IV

Liver 0.1 IV

HMVEC-C 120.3 III

HUVEC 19.9 III

Preadipocytes 0.9 I

Differentiated preadipocytes 0.3 I

Mature adipocytes 0.1 I

As seen in Table 1 endothelial cells express more IGF-IR than IR. This is in agreement

with previous results from our group showing high expression of IGF-IR and low

expression of IR in both micro- and macrovascular endothelial cells (19, 21, 53, 74).

In the whole tissues biopsy samples the order of magnitude for the IGF-IR/IR ratio was

renal artery intima-media > myometrium > skeletal muscle > liver. It must be taken into

consideration that the majority of the cells in the whole tissues used in these

experiments are of the same type, but there are also small amounts of other cells such as

stromal cells and vasculature. This may influence the results.

Differentiation of preadipocytes towards mature adipocytes increased the ratio of IR to

IGF-IR gene expression. To our knowledge this has not previously been shown in human

preadipocytes and adipocytes.

21

Gene expression of IR-A and IR-B

Real-time RT-PCR was used to measure the expression of the two IR isoforms in renal

artery intima-media, myometrium, skeletal muscle and liver (Paper IV) and in cultured

preadipocytes, differentiated preadipocytes and isolated mature adipocytes (Paper I).

Results are presented as the ratio between the two isoforms.

IR-B was the most abundant isoform in skeletal muscle (3.2 times more IR-B), liver (2.3)

and renal arteries (1.4). Myometrium expressed almost equal amounts (1.2). The results

obtained in skeletal muscle and liver are in agreement with previous studies (70, 84).

There are no previous studies on IR isoform expression in human arteries and uterus.

IR-A was the only IR isoform detected in preadipocytes and differentiated

preadipocytes. IR-B was detected in mature adipocytes but at lower levels than IR-A.

The expression of IR-A increased during differentiation. The high expression of IR-A

indicates a role for IGF-II since IGF-II has a high affinity for IR-A but not IR-B (32, 42).

Protein expression of IR and IGF-IR

Western blot was used to detect the presence or absence of IR and IGF-IR in HMVEC-C

(Paper III), myometrium, skeletal muscle and liver (Paper IV), preadipocytes (Papers I

and II), differentiated preadipocytes (Paper I) and mature adipocytes (Papers I and II).

Both IR and IGF-I were detected in HMVEC-C and also in preadipocytes and

differentiated preadipocytes but inconsistently in mature adipocytes. In myometrium,

skeletal muscle and liver IRs were demonstrated but we could not with certainty detect

IGF-IR.

IR and IGF-IR protein expression was also examined using receptor-specific ELISAs in

cultured HMVEC-C and HUVEC (Paper III), tissue biopsies from renal artery intima-

media, myometrium, skeletal muscle and liver (Paper IV) and cultured preadipocytes,

differentiated preadipocytes and isolated mature adipocytes (Paper I). The two receptor

ELISAs used in Papers III and IV had different sensitivities, the IGF-IR assay being almost

tenfold more sensitive than the IR assay. It is therefore difficult to compare the

expression of the two receptors and results should be interpreted with caution.

The amounts of IGF-IR in HMVEC-C and HUVEC were the same, 2.77 pg IGF-IR/µg total

protein and 2.86 IGF-IR/µg total protein respectively. IRs could not with certainty be

detected because of the lower sensitivity of the IR assay.

The IGF-IR content in renal arteries was 2.8 (2.7-4.0) pg IGF-IR/µg total protein, in

myometrium 1.9 (1.7-2.8) pg IGF-IR/µg total protein, in skeletal muscle 0.4 (0.3-0.6) pg

IGF-IR/µg total protein and in liver 0.6 (0.5-0.8) pg IGF-IR/µg total protein. IR was

measured to 18.8 (17.4-19.3) pg IR/µg total protein in renal arteries, 26.2 (14.6-35.5) pg

IR/µg total protein in myometrium, 17.5 (10.0-43.2) pg IR/µg total protein in skeletal

22

muscle and 87.6 (59.8-110.2) pg IR/µg total protein in liver. The order of magnitude of

IGF-IR:IR ratios would then be: renal arteries <myometrium<skeletal muscle<liver

(Table 2).

When inducing differentiation in preadipocytes the relative expression of IR to IGF-IR

increased several-fold and mature adipocytes contained 10 times more IR than IGF-IR

while in preadipocytes there were almost the same amounts of both receptors. To our

knowledge there have been no previous studies on IR and IGF-IR expression during

differentiation of human preadipocytes, but our results agree well with those obtained

from animal cell-lines (86, 92).

Table 2. Protein expression ratios of insulin receptor to IGF-I receptor in tissues when compared to the ratio in renal arteries.

Tissue type IR/IGF-IR ratio

compared to renal arteries

Renal arteries 1

Myometrium 2.6

Skeletal muscle 14.2

Liver 30.2

Phosphorylation of IR and IGF-IR

IR and IGF-IR phosphorylation was visualised using Western blot in HMVEC-C (Paper

III), preadipocytes (Papers I and II), differentiated preadipocytes (Paper I) and mature

adipocytes (Paper II).

In HMVEC-C the IGF-IR was phosphorylated by IGF-I 10-8 M and inconsistently by IGF-I

10-9 M. The IR was phosphorylated by IGF-I or insulin at 10-8 M and inconsistently by

IGF-I or insulin at 10-9 M.

In preadipocytes phosphorylation of the IGF-IR was stimulated by IGF-II 10-8 M, IGF-I

10-8 M and inconsistently by IGF-I 10-9 M, but not by insulin. IR was phosphorylated by

IGF-II 10-8 M, insulin 10-9-10-8 M and IGF-I 10-8 M. Both IR and IGF-IR were

phosphorylated by IGF-I 10-9 M in differentiated preadipocytes but we found no

activation of IR or IGF-IR by insulin.

With Western blot we could barely detect IGF-IR in mature adipocytes and we found no

phosphorylation of the IGF-IR. IR phosphorylation was stimulated by insulin 10-9-10-8 M,

IGF-II at 10-8-10-7 M or IGF-I 10-7 M and inconsistently by IGF-I 10-8 M.

23

A new ELISA-method was used to detect IR and IGF-IR phosphorylation in HMVEC-C and

HUVEC (Paper III). Cells were stimulated with insulin or IGF-I at 10-8 M and the results

were expressed as % of unstimulated controls. IGF-I stimulated phosphorylation of IGF-

IR in HUVEC and of IR and IGF-IR in HMVEC-C. Insulin had no effect on receptor

phosphorylation in either cell type.

Hybrid receptors

Presence of insulin/IGF-I hybrid receptors can be shown using immunoprecipitation

and immunoblotting. If bands appear on a SDS-PAGE gel after immunoprecipitating

samples with antibodies against the IGF-IR and immunoblotting with antibodies against

the IR, and vice versa, this provides evidence for hybrid receptors.

Hybrid receptors could be shown in samples from preadipocytes (Papers I and II),

mature adipocytes (Paper II) and in HMVEC-C (Paper III). The presence of insulin/IGF-I

hybrid receptors in HMVEC-C is in line with previous results from our group showing

hybrid receptors in HUVEC, human coronary artery endothelial cells and human dermal

microvascular endothelial cells (21, 53, 74).

There is a possibility that the presence of both IR and IGF-IR β-subunits on the same gel

is a result of unspecific binding of the antibody. However this has been investigated in

our previous work using the same methods and the same antibodies as we used herein.

We could show that after reducing the receptors with DTT no IR bands were detectable

when immunoblotting with IGF-IR antibody and vice versa or when using non-cross-

reactivity antibodies (52, 74). This indicates that co-precipitation was not due to cross-

reactivity of the antibodies.

Characterisation of differentiation

Preadipocytes used in Papers I and II were isolated from adipose tissue after mature

adipocytes had been removed. There are no known markers to characterise

preadipocytes and characterisation must therefore be performed with other methods.

Cell cultures were inspected visually in a microscope and were seen to have a fibroblast-

like shape before induction of differentiation. When a mix of substances known to cause

differentiation in preadipocytes (81) was added, the cells acquired a more rounded

shape and small lipid droplets were formed.

To further verify the presence of preadipocytes in our cultures we measured the gene

expression of molecules that are known to be expressed in mature adipocytes. Gene

expression of adiponectin, glucose transporter GLUT4 and growth hormone receptor all

increased during differentiation of the cells, with very low or undetectable levels in

preadipocytes and much higher levels in differentiated preadipocytes and mature

adipocytes.

24

Cultured vs. freshly isolated cells

The endothelial cell experiments in Paper III were performed on cultured cells. To

ensure that the culture environment did not alter expression of IR and IGF-IR thereby

influencing our results, we measured the gene expression of the two receptors in our

cultured HUVECs and compared the results with receptor gene expression in freshly

isolated HUVECs. No significant difference was found between the two different

conditions. This suggests that cultured cells are representative when studying insulin

and IGF-I effects on vascular cells.

Figure 5. (A) Effects of IGF-II (gray bars), insulin (white bars) and IGF-I (black bars) on the incorporation of [6-3H]-thymidine in human preadipocytes. Near confluent cells were stimulated with peptides in serum-free DMEM over night and then incubated with [6-3H]-thymidine for 3 h. Results are mean ± SE from 6 separate experiments with preadipocytes from different subjects. *p < 0.05 in comparison with unstimulated control. **p < 0.001 in comparison with unstimulated control. (B) Effects of insulin (white bars), insulin + IGF-I 10−8 M (gray bars) or insulin + IGF-I 10−7 M (black bars) on the incorporation of [6-3H]-thymidine in human preadipocytes. Near confluent cells were stimulated with peptides in serum-free DMEM over night and then incubated with [6-3H]-thymidine for 3 h. Results are mean ± SE from 6 separate experiments with preadipocytes from different subjects.

25

Mitogenic effects – DNA synthesis

Effects of insulin and IGF-I on thymidine incorporation into DNA was examined in

HMVEC-C (Paper III) and the effects of insulin, IGF-I (Paper I) and IGF-II (Paper II) was

studied in preadipocytes.

Compared to unstimulated controls IGF-I 10-8 M stimulated thymidine incorporation in

HMVEC-C. In preadipocytes thymidine incorporation was stimulated by IGF-II 10-8 M,

IGF-I 10-8 and 10-7 M and inconsistently by insulin 10-8 M. A tendency towards a stronger

effect with IGF-I than with insulin stimulation was noticed (Figure 5A). To further

investigate this stronger IGF-I effect we looked to see if the effect of IGF-I was additional

to the effect of insulin (Paper II). Cells were treated with insulin 10-8-10-5 M alone or

with added IGF-I at 10-8 or 10-7 M. Results show that when preadipocytes are stimulated

with both insulin and IGF-I 10-8 M the effect on thymidine incorporation is greater than

with insulin alone (Figure 5B). IGF-I thus has an additional effect compared to insulin.

Metabolic effects – Glucose transport

As a means of measuring of metabolic effects we examined the effects of insulin, IGF-I or

IGF-II on glucose transport. Accumulation of D-[U-14C]-glucose was measured in

HMVEC-C (Paper III) and preadipocytes (Papers I and II) and transport of 2-deoxy-D-[1-

3H]glucose was measured in mature adipocytes (Paper II).

In HMVEC-C glucose accumulation increased with increasing concentrations of insulin

or IGF-I but the effects of IGF-I were more pronounced. The results suggest that these

cells are more sensitive to IGF-I and this is supported by results obtained in dermal

microvascular endothelial cells (19).

Glucose accumulation in preadipocytes tended to be higher after stimulation with IGF-I

than with insulin while the EC50 values were approximately the same. In mature

adipocytes insulin stimulation of glucose transport was 100 times more sensitive to

insulin than to IGF-I or IGF-II. Previous results from other groups show similar effects of

insulin, IGF-I or IGF-II on glucose accumulation (89, 90).

It should be noted that the transport of glucose occurs with different glucose

transporters in different cell types. Endothelial cells and preadipocytes express GLUT1

only while transport in mature adipocytes is via the insulin-dependent glucose

transporter GLUT4 (47, 72).

26

Anti-inflammatory effects – E-selectin expression

We studied the possible anti-inflammatory effects of insulin and IGF-I by measuring the

expression of E-selectin protein in cultured HMVEC-C and HUVEC (Paper III). Cells were

stimulated with a peptide concentration of 10-8 M.

We found no significant changes in E-selectin expression after stimulation with insulin

or IGF-I in HMVEC-C and HUVEC. TNF-α at 5 ng/ml had a very pronounced effect on E-

selectin expression compared to unstimulated controls.

Possible suppressant effects of insulin or IGF-I were investigated by stimulating HMVEC-

C with TNF-α (10, 100, 1000, 10000 pg/ml) alone or with the addition of insulin or IGF-I.

No significant suppression was seen after the addition of insulin or IGF-I. The fact that

insulin lacked anti-inflammatory effects in our experiments may be explained by the

more potent pro-inflammatory effect of TNF-α. In human aortic endothelial cells TNF-α

inhibited IR phosphorylation by reducing IR expression (5).

27

28

General discussion

Differentiation

The ability to develop new fat cells from preadipocytes is of great importance when the

storage capacity of the existing fat cells is exceeded (46, 66). During differentiation the

ratio of IR to IGF-IR increased and adipocytes expressed approximately 10 times more

IR than IGF-IR in contrast to preadipocytes where IR and IGF-IR were expressed almost

equally. Experiments in preadipocytes from brown adipose tissue, where the expression

of either IR or IGF-IR is silenced, reveal that IR and IGF-IR have different roles in

adipocyte differentiation regarding the activation of various signalling molecules (37). In

these cells signal failure in one receptor type cannot be compensated for by signalling

through the other receptor. The low expression of IGF-IR and hybrid receptors in

mature adipocytes might be due to the fact that the fully growth arrested mature

adipocyte is no longer required to propagate signals via IGF-I, but only metabolic signals

via the IR (68).

We found no difference in the IGF-IR:IR ratio in freshly isolated compared to cultured

endothelial cells (HUVEC). This indicates that alteration in receptor expression and the

IGF-IR:IR ratio during differentiation is not a general phenomenon but related to the

type of cell that is developed during the differentiation process.

Tissue- and cell-specific receptor expression

Our aim was to study the distribution of IR and IGF-IR in different human cells and

tissues using modern techniques. The results presented in this thesis show a great

diversity in the distribution of IR and IGF-IR in different human tissues and cells. Since

these tissues and cells all have distinct properties it is likely that distribution of the

receptors is of great importance. Looking at gene expression in isolated cells we found a

large difference in the IGF-IR:IR ratio between endothelial cells and adipocytes. The IGF-

IR:IR ratio was 19.9 and 120.3 in HUVEC and HMVEC-C respectively, whereas it was 0.1

in mature adipocytes. These results should be interpreted with caution since gene

expression may not reflect protein expression. Using Western blot, IGF-IR protein could

clearly be demonstrated in endothelial cells but not in adipocytes whereas IR protein

could be demonstrated in both endothelial cells and adipocytes. Using ligand binding,

specific binding of both insulin and IGF-I was detected in endothelial cells (19). The

specific binding of IGF-I exceeded the specific binding of insulin 2-fold or more. It should

be mentioned that ligand binding of IGF-I includes binding to insulin/IGF-IR as well as

pure IGF-IR. In adipocytes there is a high specific binding of insulin but no binding of

IGF-I (12). Receptor protein can be quantitatively measured in cell lysates with ELISA.

With this technique we could measure IGF-IR in endothelial cells but IR protein content

29

was below the sensitivity of the assay. In mature adipocytes both IR and IGF-IR were

detected with IR being 10 times more abundant. Ligand binding is a classical method for

detecting receptors and has been used extensively. When applied on whole cells, ligand

binding measures the amount of receptors on the cell surface. ELISA measures the total

amount of receptors, including intracellular fractions.

Ligand binding is easy to apply to cultured or isolated cells but is not suitable for use on

whole tissues. Using ELISA we found the highest levels of IGF-IR in renal arteries and

myometrium and the highest levels of IR in liver. The ELISA results obtained in the assay

is normalised to total protein. The amount of cellular protein may therefore affect the

absolute values (large amounts of contractile proteins in skeletal muscle, for example).

To avoid this we calculated the ratio of IGF-IR to IR protein. As mentioned above, in

whole tissues the order of magnitude for the IGF-IR/IR ratio was renal artery intima-

media > myometrium > skeletal muscle > liver.

Liver, adipose tissue and skeletal muscle are classical insulin target tissues. We found

the highest IR/IGF-IR ratio in liver. Previous results suggest that isolated mature

hepatocytes express IR but not IGF-IR (16). In mature adipocytes there are IR and no or

few IGF-IR. Skeletal muscle contains IR but also a considerable amount of IGF-IR (95).

Mature liver and adipose cells are terminally differentiated and have key roles in glucose

and fat metabolism (60, 78). Since there are no or few IGF-IR in mature adipocytes and

hepatocytes they are probably not dependent on IGF-I for their function. This is also

born by the fact that IGF-I has no effect on lipolysis or lipogenesis (110). Skeletal

muscles express both IR and IGF-IR and are involved in glucose homeostasis (22) and

account for a major part of glucose uptake in response to insulin. However, besides

being involved in metabolic regulation skeletal muscle has other important roles

including support and movement of the body. IGF-I has anabolic effects which are

important for skeletal muscle function (34, 50).

Impact of differences in distribution of IGF-IR and IR on effects of insulin, IGF-I or

IGF-II

To observe the effects of insulin or IGF-I on target cells, a certain amount of receptors

are needed. The lack of IGF-I effect at 10-10-10-9 M on glucose transport in mature

adipocytes reflects the lack of IGF-I receptors. The stimulation of glucose transport in

mature adipocytes was many times more sensitive to insulin than IGF-I, which can be

explained by the fact that mature adipocytes have IR but not IGF-IR. In preadipocytes

where IR and IGF-IR were expressed almost equally, both DNA synthesis and glucose

transport were equally sensitive to IGF-I and to insulin stimulation. Compared to

30

preadipocytes mature adipocytes were about ten times more sensitive to the effect of

insulin on glucose metabolism. Although different mechanisms for glucose transport

may influence our results the large difference observed probably reflects a difference in

the abundance of insulin receptors.

In HMVEC-C and HUVEC the gene expression of IGF-IR was several-fold higher than the

expression of IR. The IR was demonstrated using Western blot, but with some

uncertainty using ELISA. The IGF-IR could be detected with both methods. Using

immunoprecipitation we could demonstrate activation of IR and IGF-IR. However only

IGF-I elicited significant effects on glucose accumulation and thymidine incorporation

suggesting that the insulin signal was too weak to exert biological effects.

The importance of hybrid receptors

When both IR and IGF-IR are expressed on the same cell, this provides the basis for

formation of insulin/IGF-I hybrid receptors. We have shown the presence of hybrid

receptors in preadipocytes and endothelial cells. Previous results from our group

demonstrate hybrid receptors in other types of endothelial cells and also in vascular

smooth muscle cells (18, 20, 21, 53, 74). Hybrid receptors have also been shown in other

human tissues (6, 39). The incorporation of IR and IGF-IR into hybrid receptors may be

important in several different ways. Since they have a high affinity for IGF-I but not for

insulin, IR incorporated into hybrid receptors cannot respond to physiological levels of

insulin. On the other hand, IGF-I can trans-phosphorylate the IR β-subunit incorporated

into hybrids. It is proposed that these are involved in determining the level of insulin

resistance of cells by sequestering IR into insulin/IGF-I hybrid receptors (40).

In both preadipocytes and endothelial cells we found evidence for trans-

phosphorylation of IR β-subunits by IGF-I. The presence of hybrid receptors may have

contributed to the lack of insulin effects in the endothelial cells. It has been shown that

deletion of the IGF-IR can increase insulin sensitivity. When siRNA was used to reduce

the expression of IGF-IR in HUVEC or rat vascular smooth muscle cells, hybrid receptor

expression decreased and there was an increase in insulin-stimulated effects such as

phosphorylation of IR and downstream signalling molecules (1, 36).

Insulin receptor isoforms

IGF-II has a high affinity for IR-A and also for IGF-IR and insulin/IGF-I hybrid receptors

(21, 32, 42). Our results show that preadipocytes express only IR-A and it therefore led

us to believe that IGF-II might have effects on these cells. IGF-II was able to activate IR,

IGF-IR and hybrid receptors in these cells and also stimulate DNA synthesis. In the

circulation IGF-II is present in higher concentrations than IGF-I (45). It is therefore likely

31

that IGF-II has important functions in the human body, although still little is known

about its significance. Because of IGF-IIs high affinity for IR-A it probably is at least

partially responsible for the growth promoting effects mediated by IR-A (42).

A difference in the signalling properties of hybrid receptors containing IR-A compared to

hybrids containing IR-B was proposed by Pandini et al (75) while Slaaby et al could find

no difference between the two splice variants (91). Whether or not the IR isoforms have

differential effects when incorporated into hybrids needs further investigation.

Sensitivity and insensitivity to insulin and IGF-I

At concentrations attained in the circulation in vivo, insulin and IGF-I probably only

activate their cognate receptors. However due to their ability to cross-react with each

other’s receptors at supraphysiological concentrations (64, 74) it is possible to obtain

effects in vitro which do not occur in vivo which has created confusion. In this context it

should be pointed out that even if insulin and IGF-I cross-react with each other’s

receptors they are only partial agonists as shown in Paper II. Due to the distribution of

IR and IGF-IR cells can be sensitive to IGF-I and insensitive to insulin as endothelial cells,

sensitive to both IGF-I and insulin as preadipocytes and insensitive to IGF-I and sensitive

to insulin as mature adipocytes. This is schematically shown in Figure 6. Sensitive in this

sense means that the cells are responsive to insulin and IGF-I in vivo.

In the introduction it was mentioned that insulin resistance is defined as a state where

target tissues have decreased responsiveness to circulating levels of insulin, but this

does not mean that the target tissue is insensitive to insulin, i.e. does not respond to

insulin. Insulin resistance is commonly assumed to be due to post-receptor mechanisms

(27, 67). As discussed above receptor expression may also influence the

sensitivity/resistance to insulin and IGF-I.

32

Figure 6. Proposed model for insulin sensitivity or insulin insensitivity in tissues with high abundance of IGF-I receptors and low abundance of insulin receptors, high abundance of insulin receptors and low abundance of IGF-I receptors or similar relative amounts of insulin receptors and IGF-I receptors.

33

34

Summary and conclusions

Both IR and IGF-IR are present in human preadipocytes. Differentiation of

preadipocytes to mature adipocytes increases the ratio of IR to IGF-IR expression.

During differentiation human preadipocytes acquire characteristics of mature

adipocytes such as adiponectin, GLUT4 and growth hormone receptor

expression.

Adipocytes, expressing mostly IR, are sensitive to insulin but not to IGF-I whereas

endothelial cells, where IGF-IR are more abundant, are sensitive to IGF-I but not

to insulin. Preadipocytes expressing equal amounts of both receptors respond to

both insulin and IGF-I.

Insulin and IGF-I are only partial agonists to each other’s receptors in human

preadipocytes and adipocytes.

Insulin/IGF-I hybrid receptors are present and function in preadipocytes and

human cardiac microvascular endothelial cells.

There is a large variation in the relative abundance of IR and IGF-IR in human

tissues, the extremes being renal artery intima-media with predominantly IGF-IR

and liver with predominantly IR.

Mature tissues express predominantly IR-B. Preadipocytes express IR-A and the

expression of IR-B is induced during differentiation.

The overall results indicate that differential expression of IGF-IR and IR is a key

mechanism in regulation of growth and metabolism.

35

36

Tack

Det finns många människor som på olika sätt varit delaktiga i arbetet med den här

avhandlingen och min tid på Cellbiologen. Några människor förtjänar ett alldeles

speciellt tack.

Hans, för att jag fick bli doktorand i din grupp. Det är ett stort privilegium att få jobba

med någon som är så kunnig som du. Jag är tacksam för allt jag lärt mig och hoppas att

jag får mycket användning för det i framtiden.

Mats, på den korta tid vi har hunnit jobba tillsammans har jag ändå lärt mig väldigt

mycket. Ditt kunnande sträcker sig längre än vi nått tidigare och det har varit till stor

hjälp.

Anna-Kristina. Det har varit helt ovärderligt att ha dig i labbet, att alltid ha någon att

fråga när man undrar över något, eller någon som räddar en i laborativa krissituationer.

Tack för att du ställt upp som bollplank, extramamma och vän.

Git, Simona och Ellinor. Vi har haft så mycket roligt tillsammans och jag uppskattar all

hjälp, och alla tips och idéer jag fått av er.

Tack också till andra som förekommit på labbet: Marloes, Erika, Nosheen, Sakari och

Rakibul

Alla på plan 10 genom åren som fått mig att trivas så bra.

Särskilt vill jag tacka

Anneli för att du alltid är så glad och för allt du fixat för att vi skulle ha det trevligt på

jobbet.

Peter P och Lotta för stöd och uppmuntran.

Camilla, för den trevliga stämning du sprider och för att du verkar ha (åtminstone lite)

koll på allt.

Johan, Louise, Eva Å och Ing-Marie för många roliga luncher och fikastunder och även

en hel del intressanta jobbdiskussioner.

Anna L och Lena C, förutom att vi har det så trevligt när vi fikar tillsammans så är det

väldigt lyxigt att ha er till hjälp och att alltid ha ren och steril utrustning att jobba med.

IT-killarna, när datorn går sönder är det inte så dumt att ha experter i närheten.

37

Mina kollegor på plan 12, Åsa, Anita och Cissi, som alltid så snällt ställt upp och fixat

patientmaterial och svar på diverse frågor.

Siri, våra fikastunder och luncher har varit ett trevligt avbrott i arbetet.

Ia, din positiva inställning smittar av sig. Tack för gott samarbete.

Peter Strålfors för alla råd och all uppmuntran. Det var roligt att få samarbeta med er.

Gunnar Kratz och hans grupp för att ni hjälpte mig komma igång med odling och

differentiering av preadipocyter. Särskilt tack till Johan Junker och Kristina Briheim.

Ola Wahlström, Preben Kjölhede, Per Sandström och Thomas Gasslander, för att ni

visat intresse för vår forskning och ställt upp och fixat vävnadsmaterialet vi behövde.

Åsa Schippert och Annette Molbaek som alltid ställer upp och svarar på frågor och

visar apparater som man inte förstår sig på.

Alla trevliga människor på IKEs kansli som alltid är så hjälpsamma.

Alla snälla läkare och sköterskor på kirurgen och personal på förlossningen som

hjälpt till att ordna patientmaterial.

Mina vänner. Om det inte hände roliga saker på fritiden skulle man aldrig orka med

jobbet. Ett extra tack till Emma som alltid är den som får lyssna på mitt gnäll och mina

problem. Det är otroligt skönt att ha någon att älta och skvallra med.

Mamma, pappa, Kristoffer, Kristin och Henrik, jag är lyckligt lottad över att ha en sån

härlig familj. Det är få gånger som jag skrattar lika mycket som när vi ses.

Ove, Ulla-Britt, Ann och Fredrik, Mats och Johanna, för att jag fått bli en del av er

glada familj. Och tack för all hjälp och barnpassning.

Pär, min stora kärlek. Det finns ingen bättre att leva sitt liv tillsammans med. Jag är så

glad att jag har dig.

Och sist men inte minst, Olivia, ljuset i mitt liv.

38

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