1. health and fitness
2. disease
3. diabetes

Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 945
_____________________________
_____________________________
Pulsatile Insulin Release
from Single Islets of Langerhans
BY
JOHANNA WESTERLUND
ACTA UNIVERSITATIS UPSALIENSIS
UPPSALA 2000
Dissertation for the Degree of Ph.D., Faculty of Medicine presented at Uppsala
University in 2000
ABSTRACT
Westerlund, J. 2000. Pulsatile insulin release from single islets of Langerhans. Acta
Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from
the Faculty of Medicine 945. 49 pp. Uppsala. ISBN 91-554-4782-1
Insulin release from single islets of Langerhans is pulsatile. The secretory activities of
the islets in the pancreas are coordinated resulting in plasma insulin oscillations.
Nutrients amplitude-regulate the insulin pulses without influencing their frequency.
Diabetic patients show an abnormal plasma insulin pattern, but the cause of the
disturbance remains to be elucidated. In the present thesis the influence of the
cytoplasmic calcium concentration ([Ca2+]i) and cell metabolism on pulsatile insulin
release was examined in single islets of Langerhans from ob/ob-mice. Glucose
stimulation of insulin release involves closure of ATP-sensitive K+ channels
(KATP channels), depolarization, and Ca2+ influx in ß-cells. In the presence of 11 mM
glucose, pulsatile insulin secretion occurs in synchrony with oscillations in [Ca2+]i.
When [Ca2+]i is low and stable, e.g. under basal conditions, low amplitude insulin
pulses are still observed. When [Ca2+]i is elevated and non-oscillating, e.g. when the
ß-cells are depolarized by potassium, high amplitude insulin pulses are observed. The
frequency of the insulin pulses under these conditions is similar to that observed when
[Ca2+]i oscillations are present. By permanently opening or closing the KATP channels
with diazoxide or tolbutamide, respectively, it was investigated if glucose can
modulate pulsatile insulin secretion when it does not influence the channel activity.
Under these conditions, [Ca2+]i remained stable whereas the amplitude of the insulin
pulses increased with sugar stimulation without change in the frequency. Metabolic
inhibition blunted but did not prevent the insulin pulses. The results indicate that
oscillations in metabolism can generate pulsatile insulin release when [Ca2+]i is stable.
However, under physiological conditions, pulsatile secretion is driven by oscillations
in metabolism and [Ca2+]i, acting in synergy.
Key words: islets of Langerhans, insulin, ELISA, cytoplasmic calcium, oscillations,
type 2 diabetes, glucose, diazoxide, potassium, tolbutamide, DNP, antimycin A,
iodoacetamide, microfluorometry, fura-2, KATP channels.
Johanna Westerlund, Department of Medical Cell Biology, Uppsala University,
Biomedicum, Box 571, SE-751 23 Uppsala, Sweden
© Johanna Westerlund 2000
ISSN 0282-7476
ISBN 91-554-4782-1
Printed in Sweden by University Printers, Ekonomikum, Uppsala 2000
REPORTS CONSTITUTING THE THESIS
The thesis is based on the following papers, which will be referred to by their Roman
numerals:
I.
Westerlund, J., Hellman, B., Bergsten, P. (1996) Pulsatile insulin release from
mouse islets occurs in the absence of stimulated entry of Ca2+. J. Clin. Invest.
97:1860-1863.
II.
Westerlund, J., Gylfe, E., Bergsten, P. (1997) Pulsatile insulin release from
pancreatic islets with non-oscillatory elevation of cytoplasmic Ca2+. J. Clin
Invest. 100:2547-2551.
III.
Westerlund, J., Ortsäter, H., Palm, F., Sundsten, T., Bergsten, P. (2000)
Glucose-regulated pulsatile insulin release from mouse islets via the
KATP channel-independent pathway. Manuscript
IV.
Westerlund, J., Bergsten, P. (2000) Glucose metabolism and pulsatile insulin
release from isolated islets. Manuscript
4
CONTENTS
ABSTRACT..............................................................................................................................................2
REPORTS CONSTITUTING THE THESIS .......................................................................................4
CONTENTS .............................................................................................................................................5
LIST OF ABBREVIATIONS .................................................................................................................6
INTRODUCTION ...................................................................................................................................7
INSULIN OSCILLATIONS IN VIVO .............................................................................................................7
Disturbances in glucose metabolism causing diabetes....................................................................9
STIMULUS-SECRETION COUPLING OF THE ß-CELL.................................................................................10
Glucose metabolism of the ß-cell ...................................................................................................10
Insulin release and ionic fluxes......................................................................................................12
Origin of insulin pulses ..................................................................................................................13
AIMS.......................................................................................................................................................15
METHODS.............................................................................................................................................16
ISLETS OF LANGERHANS ......................................................................................................................16
The ob/ob-mouse ............................................................................................................................16
Paracrinology of the islet of Langerhans.......................................................................................16
Preparation of islets of Langerhans...............................................................................................17
EXPERIMENTAL DESIGN OF THE INSULIN MEASUREMENTS...................................................................18
Perifusion system ...........................................................................................................................18
Enzyme-linked immuno-sorbent assay (ELISA) .............................................................................20
EXPERIMENTAL DESIGN OF THE [Ca2+]i MEASUREMENTS .....................................................................21
Perifusion system ...........................................................................................................................21
Recordings of [Ca2+]i .....................................................................................................................22
DATA ANALYSIS ..................................................................................................................................22
RESULTS AND DISCUSSION ............................................................................................................23
PULSATILE INSULIN RELEASE AT STABLE [Ca2+]i (PAPERS I AND II).....................................................23
Insulin release at low [Ca2+]i.........................................................................................................23
Insulin release at high [Ca2+]i .......................................................................................................24
PULSATILE INSULIN RELEASE INDEPENDENT OF KATP CHANNEL ACTIVITY (PAPERS I, II AND III) .......26
PULSATILE INSULIN RELEASE AND GLUCOSE METABOLISM (PAPER IV)...............................................28
CONCLUSIONS ....................................................................................................................................30
SAMMANFATTNING PÅ SVENSKA................................................................................................31
ACKNOWLEDGEMENTS ..................................................................................................................34
REFERENCES ......................................................................................................................................36
APPENDIX: PAPER I – IV ..................................................................................................................49
5
LIST OF ABBREVIATIONS
ADP
adenosine 5’-diphosphate
ATP
adenosine 5’-triphosphate
BSA
bovine serum albumin
C
Celsius
[Ca2+]i
cytoplasmic Ca2+ concentration
cAMP
cyclic adenosine 3’5’-monophosphate
DNP
2, 4-dinitrophenol
ELISA
enzyme-linked immuno-sorbent assay
GLUT
glucose transporter
HEPES
N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic)-acid
HNF
hepatocyte nuclear factor
5-HT
5-hydroxytryptamine
IAA
iodoacetamide
KATP channel
ATP-sensitive potassium channel
MIDD
maternally inherited diabetes and deafness
MODY
maturity onset diabetes of the young
ob
the obesity gene
PFK
phosphofructokinase
PP
pancreatic polypeptide
SEM
standard error of the mean
6
INTRODUCTION
Insulin oscillations in vivo
Many hormones display regular variations in their blood concentration. The
periodicity of these hormonal oscillations varies from minutes to days (Takebe et al.,
1969; Goodner et al., 1977; Vagnucci, 1979; Carandente et al., 1987; Veldhuis et al.,
1987). Some rhythms have traditionally been ascribed a certain periodicity such as the
monthly rhythm of luteinizing hormone and follicular stimulating hormone and the
daily rhythms of cortisol and growth hormone (Liddle, 1966; Takebe et al., 1969;
Carandente et al., 1987). With the development of more sensitive hormone assays and
in vitro studies of hormone-producing cells more complex kinetics of hormone release
have been demonstrated (Simon et al., 1987; Bergsten & Hellman, 1993b; Bergendahl
et al., 1996).
The discovery of insulin oscillations with a periodicity of 10 – 15 min in the
blood of monkeys was made over two decades ago (Goodner et al., 1977). Soon after
plasma insulin oscillations were also observed in humans (Lang et al., 1979).
Indications of how these oscillations are regulated were obtained from humans
infused with glucose (Matthews et al., 1983a) or given tolbutamide (Lang et al., 1981;
Porksen et al., 1996). Such stimulation resulted in increased amplitude of the
oscillations without change in the frequency. In blood collected from healthy subjects
insulin displays oscillations with a mean period of approximately 14 minutes
(Matthews et al., 1983a). The amplitude of the oscillations accounts for 40% of
maximum levels during glucose stimulation. In a canine model with portal blood
sampling the amplitude of the oscillations accounted for 70 % of the maximum and
the periodicity was 5-10 minutes (Porksen et al., 1995). Differences in species may
explain the discrepancy between the oscillations in portal and peripheral blood.
7
However, the differences in the oscillatory pattern are explicable since the portal vein
drains the pancreas and the liver, which extracts almost 50 % of the hormone from the
portal blood (Chap et al., 1987). This reduction in the circulating insulin concentration
dampens some of the smaller peaks in the peripheral blood probably to such an extent
that they are no longer detected (Porksen et al., 1995).
When the plasma insulin concentrations were examined in type 2 diabetics
(Lang et al., 1981) and close relatives of patients with the disease (O'Rahilly et al.,
1988), disturbed insulin patterns with irregular oscillations were detected in both
groups. The importance of regular variations of the insulin concentration has been
evaluated in studies where insulin was either administered continuously or in pulses.
When the endogenous insulin production was suppressed by somatostatin in normal
subjects, pulsatile insulin delivery had a greater hypoglycemic effect than continuos
infusion in most (Matthews et al., 1983b; Schmitz et al., 1986; Paolisso et al., 1988a;
Paolisso et al., 1988b) but not all (Verdin et al., 1984; Kerner et al., 1988) studies. The
discrepancy in results may be related both to differences in the duration of the insulin
administration and the prevailing glucagon concentration, which is important for the
efficacy of the pulsatile delivery of insulin (Paolisso et al., 1988a). In diabetic patients
pulsatile is superior to continuos insulin delivery (Bratusch-Marrain et al., 1986;
Paolisso et al., 1988a). In one study 40 % less insulin was required with pulsatile
delivery (Bratusch-Marrain et al., 1986). The greater hypoglycemic action of pulsatile
delivery is probably related to increased expression of insulin receptors on target cells.
When hepatocytes were either perifused with a constant or a varying insulin
concentration, the receptor expression was significantly higher when simulating
oscillations similar to those in plasma (Goodner et al., 1988). A disturbed plasma
8
oscillatory pattern, like in type 2 diabetes (Lang et al., 1981), can be expected to be
associated with insulin resistance due to down regulation of its receptors.
Disturbances in glucose metabolism causing diabetes
Altered plasma insulin oscillations with decreased amplitudes have been
detected in patients suffering from different subtypes of type 2 diabetes. Maturity
onset diabetes of the young, type 2 (MODY2) -patients with mutations in their
glucokinase gene have such a diminished amplitude of the plasma insulin oscillations
(Froguel et al., 1992; Byrne et al., 1994). Glycogenosis type VII or Tarui’s disease is
related to mutations in the muscle-type of the phosphofructokinase (PFK-M) gene
(Tarui et al., 1980; Vora et al., 1980) which is the physiologically activated form in
islets of Langerhans (Yaney et al., 1995). Such mutations have also been found to
result in alterations in insulin secretion (Ristow et al., 1997) leading to irregular
insulin oscillations from the affected subjects (Ristow et al., 1999). In maternally
inherited diabetes and deafness (MIDD) a mutation in the mitochondrial genome
coding for tRNA(Leu, UUR) has been found (Maassen et al., 1996; Maassen &
Kadowaki, 1996; Velho et al., 1996; van den Ouweland et al., 1999). Since this
mutation affects the translational efficiency of the mitochondrial DNA, it can be
expected to have effects also on the oxidative phosphorylation and the concomitant
ATP-production in the mitochondria.
Other sub-types of type 2 diabetes can also be related to defects in the cell
metabolism, although the effect on the insulin release pattern has not been thoroughly
investigated. The transcription factor hepatocyte nuclear factor 4α (HNF-4α) is
critical for regulating expression of numerous genes in vivo (Duncan et al., 1997).
The diabetic phenotype (MODY1) (Yamagata et al., 1996a; Herman et al., 1997; Hani
et al., 1998) results from a mutation of HNF-4α which decreases the expression of
9
essential genes involved in glucose metabolism (Stoffel & Duncan, 1997). MODY3 is
caused by mutations in HNF-1α (Vaxillaire et al., 1995; Yamagata et al., 1996b;
Vaxillaire et al., 1997), which regulates the expression of pyruvate kinase in liver
cells (Liu & Towle, 1995; Yamada et al., 1997) and may have similar functions in ßcells. From in vitro experiments it was found that mutations of HNF-1α are associated
with impaired glucose signaling, resulting in impaired insulin secretion and [Ca2+]i
response (Dukes et al., 1998). A common trait for the genetic mutations in MODY,
MIDD and patients with Tarui’s syndrome is that the affected genes code for
enzymes, which play crucial roles in cell metabolism.
Stimulus-secretion coupling of the ß-cell
An important step towards resolving the nature of the insulin oscillations was
taken when the isolated perfused pancreas was shown to produce pulsatile release of
insulin (Stagner et al., 1980). The pulses were amplitude-regulated and had
approximately 6 minutes duration. Oscillations with similar properties were also
observed in the blood of patients with a pancreas transplant (O'Meara et al., 1993).
Even individual islets of Langerhans show amplitude-regulated oscillations of insulin
release (Bergsten & Hellman, 1993a) with similar properties as in the perfused
pancreas (Stagner et al., 1980).
Glucose metabolism of the ß-cell
The ß-cell is a fuel-sensing cell, responding to increases in the concentration
of nutrients with stimulation of its metabolism, leading to an increase in ATP. This
nucleotide is probably the most important metabolic factor in stimulus-secretion
coupling (see below). In rodents, the major stimulus, glucose, enters the ß-cell by
10
facilitated diffusion via the high capacity glucose transporter, type 2 (GLUT-2)
(Thorens et al., 1988; Johnson et al., 1990). Human ß-cells express predominantly
GLUT-1, which has a lower Km and Vmax than GLUT-2 (De Vos et al., 1995).
However, the capacity is sufficient to equilibrate the glucose concentration across the
plasma membrane and the transport is not rate-limiting for glucose metabolism.
Conversion of glucose to glucose-6-phosphate by glucokinase is the ratelimiting step in the breakdown of glucose in the ß-cell and glucokinase has been
proposed to be the “glucose sensor” of the cell (Matschinsky, 1996). The clinical
importance of this enzyme is evident from studies on MODY2-patients, with
mutations in the glucokinase gene (Froguel et al., 1992). Also, in the glucokinase
knockout mouse, glucose fails to stimulate insulin secretion (Sakura et al., 1998).
Another glycolytic enzyme with regulatory properties in the ß-cell is PFK, which
catalyzes the conversion of fructose-6 phosphate to fructose-1, 6-bisphosphate. Three
different isoenzymes have been described; the liver-, muscle- and platelet-subtypes
(Dunaway et al., 1988). Although the islet contains all isoforms of the enzyme, only
the muscle-type is activated in the islet under physiological conditions (Yaney et al.,
1995). PFK-M has been proposed to be responsible for spontaneous glycolytic
oscillations in muscle extracts (Tornheim & Lowenstein, 1976; Andres et al., 1990),
but so far such oscillations have not been demonstrated in ß-cells.
In the ß-cell, glycolysis dominates over glycogen and fatty acid synthesis
(Sener & Malaisse, 1984). The three-carbon compounds produced by glycolysis are
preferentially shuttled to the mitochondrial metabolism due to the low activity of
lactate dehydrogenase (Sekine et al., 1994). Moreover, cytosolic NADH is efficiently
removed from the cytosol to the mitochondria by transfer of hydrogen via shuttle
systems (MacDonald, 1990; Eto et al., 1999). Apart from supplying three-carbon
11
compounds to the citric acid cycle, NADH produced in glycolysis appears to be
important for signal transduction (Dukes et al., 1994).
By producing most of the ATP, the mitochondrial metabolism plays a central
role in the stimulus-secretion coupling of the ß-cell (Maechler & Wollheim, 1998).
Depletion of mitochondrial DNA from ß-cell lines results in defective ATP
production as well as impaired insulin secretion (Soejima et al., 1996; Kennedy et al.,
1998; Tsuruzoe et al., 1998). The recent report that mitochondrially derived glutamate
is a messenger coupling glucose metabolism to exocytosis by acting on the secretory
granules further stresses the key role of the mitochondria in the stimulus-secretion
coupling (Maechler & Wollheim, 1999).
Insulin release and ionic fluxes
The ß-cell is equipped with KATP channels whose activity keeps the ß-cell
hyperpolarized under resting conditions (Ashcroft et al., 1984). The increase in the
ATP/ADP-ratio obtained with glucose metabolism closes these channels, resulting in
depolarization (Ashcroft et al., 1984; Cook & Hales, 1984). The subsequent opening
of voltage-dependent Ca2+-channels leads to influx of Ca2+ and rise of [Ca2+]i
(Rorsman et al., 1988), which stimulates exocytosis of insulin granules (Wollheim et
al., 1996).
With the discovery that hypoglycemic sulphonylureas, which are extensively
used in the treatment of type 2 diabetes, reduce the permeability of the KATP channel
(Sturgess et al., 1985), the mechanism of action of these drugs became apparent. The
related sulfonamide diazoxide has the opposite effect in opening the channel. Under
physiological conditions this channel opener causes hyperpolarization and inhibition
of insulin secretion (Wong et al., 1967; Basabe et al., 1970; Henquin et al., 1982). In
the presence of diazoxide, the membrane potential is essentially controlled by the K+
12
equilibrium potential. Consequently it is possible to effectively control the membrane
potential independent of glucose by elevating K+. Using this approach it has been
shown that glucose can stimulate insulin secretion not only by depolarization, but also
by other mechanisms (Aizawa et al., 1992; Gembal et al., 1992; Gembal et al., 1993;
Aizawa et al., 1994). This KATP channel-independent mechanism has also been
demonstrated in human islets (Straub et al., 1998).
Measurements of [Ca2+]i in single ß-cells (Grapengiesser et al., 1988) and
intact islets (Valdeolmillos et al., 1989) have shown that [Ca2+]i is regulated in an
oscillatory manner. These oscillations have a frequency of 0.2 – 0.5 min-1 and are
tightly coupled to the pulsatile release of insulin (Gilon et al., 1993; Bergsten et al.,
1994). These slow insulin oscillations are composed of rapid secretory phenomena
with a frequency of 3 – 5 min-1 (Bergsten & Hellman, 1993b), corresponding to the
regular variations in membrane potential (Rosario et al., 1986) and fast
[Ca2+]i-oscillations (Bergsten, 1995).
Apart from triggering exocytosis, the intermittent high [Ca2+]i may also
decrease the risks for an intracellular overload of Ca2+ in the ß-cell. Prolonged
elevation of [Ca2+]i is coupled to the initiation of apoptotic signals (Berridge, 1994;
Trump & Berezesky, 1995). In support of such a notion increased cell death has been
observed at elevated glucose concentrations when oscillations of [Ca2+]i are absent
(Efanova et al., 1998).
Origin of insulin pulses
The oscillations in circulating insulin are due to the pulsatile release of insulin
from the pancreas and the islets of Langerhans. It has not been possible to directly
measure kinetics of insulin release from individual ß-cells due to limited sensitivity of
the assays. Different indirect techniques have been used to measure insulin from
13
single ß-cells such as detection of 5-hydroxytryptamine (5-HT) and zinc. The 5-HT is
concentrated in the secretory granules and released together with insulin (Gylfe,
1978). Using amperometric detection of 5-HT with microelectrodes, secretion has
been studied in individual ß-cells pre-loaded with this amine (Smith et al., 1995). A
limitation with amperometry is that the microelectrode measures the concentration in
a limited volume close to the cell, rather than the total secretion. In a recently
developed technique extracellular zinc is measured around single ß-cells using the
fluorogenic zinc-indicator Zinquin and confocal microscopy (Qian et al., 2000).
Insulin is crystallized with zinc in the secretory granules and release can thus be
monitored.
The aberrant insulin pattern in patients with type 2 diabetes makes it important
to explore how pulsatile insulin release is generated. Pulsatile insulin secretion
follows from changes in the membrane potential (Ashcroft & Rorsman, 1989),
oscillations of [Ca2+]i (Pralong et al., 1994; Kennedy & Wollheim, 1998) and an
oscillatory metabolism (Tornheim, 1997), but the temporal contribution of the
different phenomena is not clear. A quantitative and qualitative study on the
contribution of metabolic and ionic oscillations to the insulin pulses indicate that
[Ca2+]i oscillations are more effective in inducing insulin pulses than metabolic ones
(Ravier et al., 1999). However, observations that increased metabolism precedes
changes in ionic fluxes (Civelek et al., 1996; Nilsson et al., 1996) indicate a primary
role of oscillations in metabolism as generator of pulsatile insulin release.
14
AIMS
The aims of the thesis were to investigate the insulin release pattern from
single islets of Langerhans when:
c [Ca2+]i is low and stable
c [Ca2+]i is elevated and non-oscillating
c the KATP channel-independent pathway is activated
c glucose metabolism is inhibited
15
METHODS
Islets of Langerhans
The ob/ob-mouse
Islets of Langerhans were obtained from ob/ob-mice taken from a local colony
(Hellman, 1965). This choice was made as previous studies have indicated that the
vast majority of the islet cells from these animals are insulin producing ß-cells and
that the islets respond normally to glucose and other stimulators of insulin release
(Hahn et al., 1974). In addition to the high proportion of ß-cells, the ob/ob-islet has
the advantage of being large, which results in a higher insulin release per islet. This is
particularly important when studying variations of basal insulin secretion.
The ob/ob-mouse was raised from selective breeding of normal house mice
(Ingalls et al., 1950). It carries a mutation in the ob-gene on chromosome number 6
(Zhang et al., 1994), which results in the formation of dysfunctional leptin from the
fat cells. Plasma leptin concentrations are correlated to the amount of body fat, acting
as a lipid sensor in a negative feedback control between the adipose tissue and the
satiety center in the hypothalamus. Since the ob/ob-mouse is unable to produce
functional leptin, increase in food intake, lipid uptake and obesity follows.
Paracrinology of the islet of Langerhans
The islet of Langerhans is composed of insulin producing ß-cells, glucagon
producing α-cells, somatostatin producing ∂-cells and PP-cells producing pancreatic
polypeptide. Both glucagon (Lang et al., 1982; Jaspan et al., 1986) and somatostatin
(Matthews et al., 1987) display oscillations in the blood synchronous to those of
insulin. Also, determination of the [Ca2+]i patterns from single α- ,∂- and PP-cells
16
have revealed oscillations under different conditions (Berts et al., 1995; Berts et al.,
1996; Berts et al., 1997; Liu et al., 1999).
Most ß-cells are juxtaposed to other ß-cells (Grube et al., 1983; Jörns et al.,
1988) but they also have contact with neighboring α- and ∂-cells. The cells can
connect via gap junctions (Orci et al., 1975; Meda et al., 1980; Michaels & Sheridan,
1981; Meda et al., 1982) allowing propagation of depolarization and passage of ions
and small molecules between the cells. Interactions between the cells can also take
place via secreted hormones and other paracrine factors (Lernmark & Hellman, 1970;
Orci & Unger, 1975). The ß-cell expresses high-affinity glucagon receptors and
glucagon is a powerful stimulus of insulin secretion (Schuit & Pipeleers, 1985;
Pipeleers, 1987; Van Schravendijk et al., 1990). Somatostatin inhibits insulin
secretion, but due to the high concentrations necessary to evoke this effect it is
unlikely to occur in vivo (Schuit et al., 1989; Van Schravendijk et al., 1990). The islet
blood flow is from the centrally located ß-cells via the α-cells to the ∂-cells (Stagner
& Samols, 1992), indicating an influence of the ß-cells on the α- and ∂-cells rather
than the opposite. However, a paracrine release of glucagon from the α-cells is
required for the maintenance of normal cAMP-levels in the ß-cell (Schuit & Pipeleers,
1985).
Preparation of islets of Langerhans
The ob/ob-mouse was anesthetized using ether or CO2 and then decapitated.
The pancreas was removed and transferred to ice-cold medium containing (in mM)
Na+ 125, K+ 5.9, Mg2+ 1.2, Ca2+ 1.3, Cl- 136 and HEPES 25, pH 7.4. Fat and large
vessels were excised before the pancreas was cut into small pieces and collagenase
digested (6.67 mg/ml) at 37° C for 15 – 20 min. The islets were washed and
handpicked. Freshly isolated islets were used for all insulin release studies. For the
17
experiments involving [Ca2+]i–measurements, the islets were cultured over-night in an
atmosphere of 5 % CO2 in humidified air in a RPMI 1640 medium containing 5.5 mM
glucose supplemented with 10% fetal calf serum.
Experimental design of the insulin measurements
Perifusion system
A single islet was placed in a 10 µl-chamber at 37° C and perifused at a
constant flow rate with a pre-heated (37° C) buffer containing 3 mM glucose. The
perifusion buffer contained (in mM) Na+ 125, K+ 5.9, Mg2+ 1.2, Ca2+ 1.3, Cl- 136, and
HEPES 25, pH 7.4, supplemented with 0.1% BSA (w/v). When K+ was increased to
30.9 mM, Na+ was isoosmotically reduced. The flow rate was kept constant
throughout each experiment with the aid of a peristaltic pump placed before the
chamber. To avoid pressure fluctuations, a pump with multiple rollers was chosen
(Ismatec Reglo 4/12). Elastic tubing was used to dampen remaining fluctuations. The
possible influence of the pump on pulsatile insulin release was evaluated by using
hydrostatic pressure for delivery of medium. These procedures clarified that the pump
did not affect the secretory pattern. Variation in the flow rate of 150-200 µl/min was
allowed between experiments. These variations did not affect the insulin release
pattern. After 60-75 min of introductory perifusion the perifusate was collected in 20sec fractions directly into microtiter plates. Insulin was assayed by a competitive
ELISA (see below). The rate of insulin release was normalized to islet dry weight
after freeze-drying and weighing on a quartz fiber balance. Control experiments were
performed by perfusing empty chambers with medium containing 100 pM insulin
(Fig. 1A). The recovery in such experiments was 58 ± 4% and the variation
18
considerably greater than when pipetting the same solution directly into microtiter
plates (95 ± 1 % recovery).
A
B
200
INSULIN RELEASE (pM)
INSULIN (pM)
200
100
100
5 MIN
5 MIN
30
% power
% power
30
20
10
0
20
10
0
5
10
15
20 25
mHz
5
10
15
20 25
mHz
Figure 1. Frequency analysis of insulin from an empty chamber (A) perfused with buffer containing
100 pM insulin and an islet-containing chamber (B) perfused with buffer containing 11 mM glucose.
The lower panels show how different frequencies contribute to the overall pattern. The dotted line (A)
shows repetitive samples of 100 pM insulin pipetted directly into the microtiter plate.
The loss of insulin in the control perfusion studies is probably due to insulin binding
to the plastic tubing, and the variation may result from uneven release of such bound
insulin from the mechanically stressed Tygon™ tubing in the peristaltic pump. It is
therefore pertinent to note that the pump is placed before the chamber in experiments
with islets and that the total length of the tubing exposed to insulin is much shorter.
The time required for medium to reach the chamber is consequently 90 sec but it takes
only 20 sec for the released insulin to reach the microtiter plate via a narrow Teflon™
tube. Nevertheless, comparing the insulin variations in these unfavourable control
19
experiments with those obtained from an islet exposed to 11 mM glucose it was clear
that the pulsatile secretion resulted in variations with higher amplitude and a
characteristic dominating frequency component, which was lacking in the control
experiments (Fig. 1).
Enzyme-linked immuno-sorbent assay (ELISA)
Freeze-dried guinea-pig anti-bovine insulin serum, produced in our laboratory
according to Wright (Wright et al., 1968), was diluted 1:10.000 in a phosphate buffer,
pH 7.4, containing (in mM) Na2HPO4 37 and NaH2PO4 6. IgG-certified microtiter
plates (Nunc, Roskilde, Denmark) were coated with antibodies by incubation
overnight at 4° C with 100 µl of the diluted anti-serum. On the following day, the
plates were washed with a medium containing (in mM) NaCl 137, KCl 2.7, Na2HPO4
7.4, and KH2PO4 1.5 together with 0.05% Tween20 (v/v), pH 7.2. A blocking solution
(100 µl) with 1 % casein (Sigma Chemical Co., St. Louis, MO) dissolved in the same
buffer as used in the perifusion was then added and the plates were incubated at 37° C
for 2 hours. The blocking procedure was performed to minimize unspecific binding of
insulin to the plastic. Before the experiments the plates were washed and rat insulin
standards (Novo Nordisk, Bagsvaerd, Denmark) were added to each plate. Hundred
microliter buffer was added to the remaining wells. The perifusate was collected
directly into the buffer-containing wells of the microtiter plates. To keep the volume
constant, a volume corresponding to the perifusate was removed from each well
immediately before the delivery of perifusate. The plates were thereafter incubated
over-night at 4° C. On the following day, insulin peroxidase (25 mU/ml ; Sigma
Chemical Co., St. Louis, MO) dissolved in phosphate buffer supplemented with 60
mg/ml BSA and 10 mM NaCl was added to the plates after washing. The plates were
incubated for 4 hours at 4° C, before the next wash. Twenty mM tetramethylbenzidine
20
(Sigma Chemical Co., St. Louis, MO) and 0.1 M K+-citrate solution were mixed 1:25
(v/v, pH 4.25) and pipetted (100 µl) into the plates, which were incubated in the dark
at room temperature for one hour before the color-reaction was measured with a
spectrophotometer (iEMS, Labsystems, Helsinki, Finland). Amounts of insulin down
to 100 amol were obtained from linear standard curves in semilogarithmic plots.
Using 96-well microtiter plates for ELISA, unexpectedly high or low spectral
densities are sometimes observed in peripheral wells. This so-called edge-effect has
been attributed to temperature differences between central and peripheral wells and
illumination of light sensitive substrates (Esser, 1997). Extensive precautions were
taken to reduce this phenomenon. All plates were tested to avoid batches with
irregularities in the plastic. The plates were stored at 4° C before being used. The
plates were temperature equilibrated for 20 min before every washing and application
of medium. The outer row of wells on each microtiter plate was not used. Using these
procedures, the inter- and intra-assay variations of the ELISA were less than 10%
(Bergsten & Hellman, 1993a).
Experimental design of the [Ca2+]i measurements
Perifusion system
When measuring insulin release simultaneously with [Ca2+]i, it is important to
keep the chamber volume as small as possible to avoid dilution of insulin in the
perifusate. This was achieved by using a closed 15 µl chamber as described elsewhere
(Bergsten, 1995). Briefly, the islet was attached to a poly-L-lysine-coated coverslip
serving as the bottom of the chamber. The top of the chamber consisted of a
cylindrical perspex block, which had a central 15 µl cavity provided with channels for
21
the inflow and outflow perifusion medium. When [Ca2+]i was measured alone, the
experiments were performed with a 150 µl open superfusion chamber (Bergsten et al.,
1994).
Recordings of [Ca2+]i
Individual islets were loaded with 2 µM fura 2-acetoxymethyl ester in the
presence of 3 mM glucose for 50 min. The chamber with the islet (see above) was
placed on the stage of an inverted microscope (Diaphot; Nikon Inc.) within a climate
box maintained at 37° C. The microscope was equipped for epifluorescence
illumination with a 75 W xenon lamp and a 100x oil immersion fluorescence
objective. A filter changer of a time-sharing multichannel spectrophotofluorometer
(Chance et al., 1975) provided excitation light flashes of 1 ms duration at 340 and 380
nm every 10 ms and the emission was measured at 510 nm with a photomultiplier.
Calculation of [Ca2+]i was performed using the equation
[Ca2+]i = KD * F0/FS * (R – Rmin) / (Rmax – R)
(Grynkiewicz et al., 1985)
where KD is the dissociation constant of the indicator fura-2. The KD employed was
224 nM (Grynkiewicz et al., 1985). R is the ratio of the fluorescence at the excitation
wavelengths 340 and 380 nm. Rmax and FS are the 340/380 nm fluorescence excitation
ratio and 380 nm fluorescence at saturating Ca2+ concentrations (10 mM),
respectively. Rmin and F0 are the corresponding values in a medium lacking Ca2+.
Data analysis
Frequency determination of insulin pulses and [Ca2+]i oscillations was done by
Fourier transformation using the Igor software (WaveMetrics Inc., Lake Oswego,
OR). Determination of significant oscillations was based on the signal-to-noise ratio
22
described previously (Bergsten, 1995). Results were presented as means ± SEM.
Differences in secretory rates were evaluated with Students’ t-test for paired and
unpaired observations.
RESULTS AND DISCUSSION
Pulsatile insulin release at stable [Ca2+]i (papers I and II)
Insulin release at low [Ca2+]i
Under basal conditions, when the ß-cell remains hyperpolarized due to
openings of the KATP channels, [Ca2+]i is low and stable (Grapengiesser et al., 1988;
Gilon et al., 1993; Bergsten et al., 1994). [Ca2+]i therefor differs from the insulin
pattern in the plasma under the fasting situation, where insulin exhibits prominent
oscillations despite a prevailing non-stimulatory glucose concentration (Goodner et
al., 1977; Jaspan et al., 1986). Using the activity of the KATP channels as an indicator
of the ATP/ADP ratio, it has also been possible to demonstrate primary oscillations of
the basal metabolism in ß-cells with a stable and low [Ca2+]i (Dryselius et al., 1994).
Against this background, we determined whether insulin release is also pulsatile in the
absence of stimulated entry of Ca2+. At 3 mM glucose, insulin release was pulsatile
with a frequency of about 0.4 min-1. Even if glucose was removed from the perifusing
medium, the pulses persisted with unaffected frequency. Under these conditions
glucagon-producing α-cells demonstrate [Ca2+]i oscillations (Berts et al., 1995), which
may result in pulsatile release of the hormone (Stagner et al., 1980). Glucagon
stimulates cAMP formation in the ß-cell and there is evidence that paracrine release of
glucagon is a prerequisite for the maintenance of normal cAMP-levels in the ß-cell
(Schuit & Pipeleers, 1985). To investigate if pulsatile release of glucagon could give
23
rise to cAMP-dependent pulses of insulin from the ß-cell, the effect of clonidine,
which blocks the formation of cAMP in the ß-cell (Garcia-Morales et al., 1984), was
tested in the absence of glucose. Clonidine was unable to affect the frequency of the
insulin pulses, but suppressed the average secretory rate. We therefor concluded that
pulsatile insulin secretion under basal conditions is independent of oscillations in
[Ca2+]i and that the pulsatility is an intrinsic property of the ß-cell. Although usually
not observed in ß-cells or islets (Grapengiesser et al., 1988; Gilon et al., 1993;
Bergsten et al., 1994), oscillations of [Ca2+]i at basal glucose concentrations have been
reported in one study (Longo et al., 1991). This observation may be explained by
recordings from superficially located α-cells, which are oscillating at basal glucose
concentrations (Berts et al., 1995).
Insulin release at high [Ca2+]i
The importance of Ca2+ in insulin secretion can be demonstrated by removing
the ion from the medium (Grodsky & Bennett, 1966; Milner & Hales, 1967) or
blocking voltage-dependent Ca2+ channels (Devis et al., 1975), which abolishes
glucose-stimulated insulin secretion. In permeabilized ß-cells an increase in the
external Ca2+ concentration stimulates insulin secretion (Yaseen et al., 1982). The
discovery of a tight correlation between oscillations in [Ca2+]i and insulin release
(Gilon et al., 1993; Bergsten et al., 1994) was interpreted as oscillations in [Ca2+]i
pacing the secretory pulses. When it was found that basal insulin release is pulsatile
also when [Ca2+]i is low and stable, it became important to study the kinetics of
secretion under conditions with an elevated and stable [Ca2+]i. During exposure to
3 mM glucose both insulin secretion and [Ca2+]i increased immediately upon
depolarization with K+. However, whereas no oscillations were observed in [Ca2+]i,
insulin release was pulsatile with a pronounced initial peak followed by others with
24
decreasing amplitudes. Sustained elevation of [Ca2+]i and pulsatile insulin release
were also observed during tolbutamide depolarization in the presence of 3 mM
glucose. Similar to the situation with K+-depolarization, the amplitude of the insulin
pulses decreased with time. Also in the presence of tolbutamide and 11 mM glucose a
sustained elevation of [Ca2+]i was observed. Corresponding measurements of insulin
release showed pulses with constant amplitude. These results indicate that elevation of
[Ca2+]i is not sufficient for maintaining secretion if energy is not supplied for energyrequiring processes like the recruitment of insulin granules (Eliasson et al., 1997). The
inability to maintain insulin secretion in response to K+ or tolbutamide depolarization
at basal glucose concentrations may consequently result from insufficient metabolism
and energy production in the ß-cells.
The observation of pulsatile insulin release when exposing the islets to
tolbutamide is important since sulfonylureas such as tolbutamide are widely used to
enhance insulin secretion in type 2 diabetes (Turner et al., 1999). The findings are
consistent with increased amplitude of plasma insulin concentrations in humans
infused with tolbutamide (Matthews et al., 1983a). Even with the high tolbutamide
concentrations (1 mM) used here to close all KATP channels (Trube et al., 1986),
insulin release remained pulsatile both at basal and stimulatory glucose
concentrations, despite high and stable [Ca2+]i levels. It should be noted that [Ca2+]i in
single ß-cells oscillates at lower concentrations of the drug (Grapengiesser et al.,
1990), although this could not be shown in isolated islets (Mariot et al., 1998).
25
Pulsatile insulin release independent of KATP channel activity
(papers I, II and III)
Under normal conditions, the KATP channel couples the generation of ATP to
pulsatile release of insulin via depolarization and influx of Ca2+ (Ashcroft et al., 1984;
Cook & Hales, 1984; Ashcroft & Rorsman, 1990). However, insulin secretion also
occurs independently of the KATP channel. This KATP channel-independent secretion
can be modulated by glucose (Aizawa et al., 1992; Gembal et al., 1992; Gembal et al.,
1993; Aizawa et al., 1994) and is also present in humans (Straub et al., 1998). To
evaluate the role of the KATP channel-independent pathway in pulsatile insulin release
we exposed the islets to diazoxide alone, or in combination with K+ depolarization to
elevate [Ca2+]i.
Diazoxide opens KATP channels and thereby hyperpolarizes the ß-cells. (Trube
et al., 1986). In the absence of glucose, 400 µM diazoxide had no effects on either
frequency or amplitude of the insulin pulses. An increase of the glucose concentration
to 11 or 20 mM under the continued presence of diazoxide did not affect the secretory
pattern. When insulin release was stimulated with 11 mM glucose, application of
diazoxide inhibited secretion to basal levels, but with maintained pulsatility. However,
the [Ca2+]i oscillations in the presence of 11 mM glucose disappeared and became low
and stable. When islets perifused with 11 mM glucose were depolarized with K+ in
the presence of diazoxide, the insulin pulse amplitude was restored. [Ca2+]i also
increased, but without oscillations. Exposing islets to diazoxide and an elevated K+
concentration increased insulin release already at 3 mM glucose, due to elevation of
[Ca2+]i. It was concluded that glucose can modulate pulsatile insulin secretion also via
the KATP channel-independent pathway and that this is done by changing the
amplitude of the pulses without affecting the frequency.
26
The observation of pulsatile insulin secretion also by the KATP channelindependent pathway is in conflict with another study (Jonas et al., 1998), where
[Ca2+]i oscillations seem to be a prerequisite for pulsatile secretion. The latter results
are difficult to evaluate since glucose-induced pulsatile insulin release by this group
has only been studied in the presence of very high extracellular Ca2+ (Gilon et al.,
1993). This manipulation amplifies secretion (Atwater et al., 1983; Gilon & Henquin,
1992), making it possible to measure insulin with less sensitive techniques.
Furthermore, a high extracellular Ca2+ concentration increases the time between bursts
of action potentials (Atwater et al., 1983). The reports of 2 min insulin pulses
correlated to the membrane potential changes may therefor correspond to the
15 – 25 s transients recorded in the presence of physiologic Ca2+ concentrations
(Bergsten & Hellman, 1993b).
In the KATP channel-independent pathway, when no [Ca2+]i oscillations are
present, metabolic oscillations may be the factor generating pulsatile insulin release.
When variations in the ATP/ADP-ratio are prevented from influencing [Ca2+]i, they
may generate pulsatile secretion by providing energy for exocytosis (Eliasson et al.,
1997). Mitochondrially derived glutamate may be another factor linking an oscillatory
metabolism to secretion via the KATP channel-independent pathway (Maechler &
Wollheim, 1999). In islets exposed to physiological concentrations of glucose, the
insulin pulses are synchronized with oscillations in [Ca2+]i (Bergsten et al., 1994). In
this situation fluctuations of metabolism and [Ca2+]i oscillations act synergistically
and produce pulsatile insulin secretion (Longo et al., 1991; Civelek et al., 1996; Jung
et al., 1999; Jung et al., 2000; Ortsäter et al., 2000). Regarding both metabolic factors
and [Ca2+]i as messengers for insulin release, it is possible to understand that insulin
27
release remains pulsatile also if one of the factors is not oscillating (Ravier et al.,
1999).
Pulsatile insulin release and glucose metabolism (paper IV)
Impaired glucose metabolism in pancreatic ß-cells has been implicated in the
development of the disturbed plasma insulin pattern seen in type 2 diabetes
(Matschinsky, 1996). Support for this idea has been obtained from the altered plasma
insulin patterns in patients with mutations in genes that play crucial roles in the cell’s
metabolism (Froguel et al., 1992; Byrne et al., 1994; Ristow et al., 1999).
When islets are exposed to inhibitors of metabolism, insulin release decreases
(Aleyassine, 1970; Georg et al., 1971; Hellman et al., 1973; Pagliari et al., 1975;
Zawalich et al., 1977; Mertz et al., 1996). To evaluate the effect of metabolic
inhibition on the insulin pulses, isolated islets were subjected to iodoacetamide (IAA),
antimycin A or 2, 4-dinitrophenol (DNP) both at stimulatory and basal glucose
concentrations. IAA inhibits glycolysis, thereby reducing the formation of metabolites
for the citric acid cycle. Such a reduction also produces a block of ATP-production in
the mitochondria. Antimycin A and DNP impair mitochondrial ATP-production
directly. Whereas antimycin A interferes with the electron transport chain and inhibits
mitochondrial flux, the uncoupler DNP dissipates the proton gradient supplying the
force for ATP-generation without inhibiting the mitochondrial flux.
At basal glucose concentrations (3 mM), metabolic inhibition with IAA tended
to induce a slight increase of pulsatile insulin release without affecting the frequency
of the pulses. Corresponding measurements of [Ca2+]i also showed a slight increase
but without oscillations. Whereas basal insulin release rose slowly with continued
pulsatility in the presence of the mitochondrial inhibitors antimycin A and DNP, a
28
prompt increase followed by a sustained elevation was observed in [Ca2+]i. In the
presence of 11 mM glucose pulsatile insulin release was decreased to basal levels
when applying the different metabolic inhibitors. The frequency of the insulin pulses
remained unaffected by metabolic inhibition during all conditions. The oscillatory
[Ca2+]i induced by 11 mM glucose was replaced by stably elevated levels when
metabolic inhibitors were added. This dissociation between insulin release and [Ca2+]i
indicates that a rise in [Ca2+]i is a weak stimulus for secretion when ATP is lacking.
The [Ca2+]i pattern obtained with metabolic inhibitors may be the consequence of an
inability to maintain ion gradients due to energy deprivation and/or influence of the
inhibitors on the intracellular Ca2+ stores.
Despite metabolic inhibition, insulin secretion from isolated islets remained
pulsatile in the presence or absence of glucose in the perifusing medium. Although it
is possible that the cell can circumvent derangement in its metabolism and continue
insulin secretion with reduced amplitude, another explanation could be incomplete
penetration of the inhibitors through the islet. In diabetic patients with mutations
causing impairments in enzymes affecting the metabolism (Lang et al., 1981; Froguel
et al., 1992; Ristow et al., 1999) insulin release persists, although severely deranged.
29
CONCLUSIONS
c Insulin release from single islets of Langerhans is pulsatile under basal
conditions when [Ca2+]i is low and stable.
c Pulsatile insulin release also persists when [Ca2+]i is steadily elevated,
as when the islet is depolarized.
c Elevated [Ca2+]i is not sufficient to maintain secretion, which also
requires a supply of energy.
c Reduction in energy production decreases the amplitude but does not
affect the frequency of the insulin pulses.
c The KATP channel-independent pathway generates amplitudemodulated pulsatile release of insulin in response to glucose.
c The pulse frequency does not seem to depend on the paracrine
secretion of glucagon from neighboring α-cells, but is intrinsic to the
ß-cell.
30
SAMMANFATTNING PÅ SVENSKA
Insulinkoncentrationen i blodet varierar regelbundet med en periodicitet om
ungefär tio minuter. Dessa variationer, som också återfinns hos den isolerade
bukspottkörteln, uppstår som ett resultat av samordning av rytmisk frisättning av
hormonet från de enskilda Langerhanska öarna. Plasmainsulinoscillationerna hos
typ 2 diabetiker har lägre amplitud och är mer oregelbundna än hos friska individer.
Detta anses vara en viktig orsak till att mängden insulinreceptorer minskar hos
diabetiker och bidrar till att deras insulinkänslighet minskar. Det är därför
betydelsefullt att finna mekanismerna bakom den rytmiska insulinfrisättningen från
den Langerhanska ön och klarlägga orsakerna till det förändrade
plasmainsulinmönstret vid typ 2 diabetes.
Glukosspjälkningen i de insulinproducerande ß-cellerna resulterar i produktion
av ATP, vilket minskar genomsläppligheten för kaliumjoner (K+) i cellmembranens
ATP-känsliga kaliumkanaler (KATP-kanaler). Den depolarisering som följer av detta
öppnar spänningsberoende kalciumkanaler i cellmembranet varvid kalciumjoner
(Ca2+) strömmar in i cellen och den cytoplasmatiska Ca2+-koncentrationen ([Ca2+]i)
stiger. Detta är den viktigaste signalen för insulinfrisättning.
Vid närmare studium av de Langerhanska öarna uppvisar en rad intracellulära
faktorer och funktioner en rytm som liknar den för insulinfrisättningen. [Ca2+]i är en
sådan faktor vars regulatoriska roll upptäcktes redan tidigt, då det visade sig att
glukosstimulerad insulinfrisättning hämmas om Ca2+ tas bort från det omgivande
mediet eller om Ca2+-kanalerna i cellmembranet blockeras. Eftersom [Ca2+]i oscillerar
med en frekvens motsvarande den rytmiska insulinfrisättningens och svängningar i
[Ca2+]i och insulin dessutom befunnits vara synkroniserade föreslogs [Ca2+]ioscillationer som initierande för pulserna i insulinfrisättningen. Mot denna hypotes
stod emellertid observationen att insulinoscillationer i blodet även kan registreras
under fasta, vilket motsvarar glukosnivåer som inte stimulerar insulinfrisättningen.
Med bakgrund av detta studerades mekanismer som kan ligga bakom den rytmiska
insulinfrisättningen från enskilda Langerhanska öar och speciellt [Ca2+]i:s roll för
uppkomsten av insulinoscillationerna.
31
Arbete I: I detta arbete studerades insulinfrisättningen och [Ca2+]i från enskilda
Langerhanska öar vid glukoskoncentrationer som inte depolariserar ß-cellerna.
Förändringar i insulinnivåerna mättes med ELISA-teknik och [Ca2+]i bestämdes med
hjälp av dubbelvåglängdsfluorometri och Ca2+-indikatorn fura-2. Eftersom mängden
insulin som frisätts under de beskrivna situationerna är små var en känslig
analysmetod för insulin, samt användandet av Langerhanska öar från ob/ob-möss,
som innehåller en stor andel ß-celler, en förutsättning för studien. Förutom låga
glukoskoncentrationer perifunderades öarna med klonidin, som blockerar cAMPbildningen i ß-cellerna, och diazoxid, som hyperpolariserar ß-cellerna genom att
öppna KATP-kanalerna i cellmembranen. Trots att [Ca2+]i var låg och utan oscillationer
varierade insulinmängderna i de olika experimentella situationerna rytmiskt med
samma frekvens som vid glukosstimulering under alla försökssituationer.
Insulinsekretionen reglerades genom förändringar av insulinpulsernas amplitud.
Arbete II: I detta arbete undersöktes hur insulinfrisättningen påverkas då [Ca2+]i är
förhöjt men inte oscillerar, som är fallet då ön perifunderas med höga
kaliumkoncentrationer eller med tolbutamid. Tolbutamid är ett sulfonylureapreparat
som används vid behandling av typ 2 diabetes. Också i dessa situationer var
insulinsekretionen rytmisk. Insulinfrisättningens rytmicitet torde sålunda bero på
andra faktorer än [Ca2+]i under dessa betingelser, troligen en oscillerande metabolism.
Arbete III: I arbete III studerades insulinfrisättningen då KATP-kanalerna hölls
permanent öppna med diazoxid samtidigt som K+-koncentrationen i det omgivande
mediet höjdes för att depolarisera cellen och därmed öka [Ca2+]i och stimulera
insulinfrisättningen. Under dessa betingelser kvarstod insulinpulserna, trots den
stabila [Ca2+]i-nivån. Insulinsekretionen kunde fortfarande stimuleras med ökande
glukoskoncentrationer, trots att KATP-kanalerna inte tilläts variera sin aktivitet. Ingen
av manipulationerna påverkade insulinpulsernas frekvens nämnvärt.
Arbete IV: För att ytterligare penetrera sambandet mellan metabolismen och den
rytmiska insulinfrisättningen användes inhibitorer av metabolismen.
Energiproduktionen i cellen, i form av ATP, blockerades dels i glykolysen med
jodoacetamid, dels i mitokondrien med antimycin A eller dinitrofenol. Metabol
32
hämning av glukos-stimulerad insulinfrisättning manifesterade sig genom en
dramatisk sänkning av insulinpulsernas amplitud till basalnivån, medan deras
frekvens förblev oförändrad. [Ca2+]i steg och de glukosinducerade oscillationerna
ersattes med en konstant nivå. När de metabola hämmarna sattes till vid låga
glukosnivåer tenderade insulinfrisättningen att öka med tiden. Motsvarande mätningar
av [Ca2+]i gav med den glykolytiska inhibitorn jodoacetamid en liknande höjning,
medan effekten av de mitokondriella inhibitorerna var en [Ca2+]i-stegring följd av en
förhöjd nivå som inte uppvisade några oscillationer.
Sammanfattningsvis kan konstateras att den rytmiska insulinfrisättningen kvarstår då
[Ca2+]i inte oscillerar. Detta gäller såväl när [Ca2+]i är lågt, som vid frånvaro av
stimulering, eller vid högt [Ca2+]i, som vid depolarisering med förhöjda
kaliumkoncentrationer eller tolbutamid. Insulinnivåerna tycks vara beroende såväl av
den rådande [Ca2+]i och energitillgången i ß-cellen. Frekvensen på insulinpulserna
beror på inneboende egenskaper hos ß-cellen och verkar inte styras av faktorer utanför
den.
33
ACKNOWLEDGEMENTS
Jag vill framföra mina varmaste TACK till följande personer:
Min handledare Peter Bergsten för din aldrig sinande
entusiasm och uppmuntran, oavsett omständigheterna och
trots min ångermanländska skepsis mot allting. ”Foten i
kläm? Jajamensan!”
Kristina Mustajärvi, min ELISA-mentor framför alla.
Alla ELISAs kämpar genom åren: Emma Andersson,
Johannes Blom, Russell Brown, Mina Eriksson, Andrea
Hinas, Jian-Man Lin, Erik Mörtberg, Henrik Ortsäter, Fredrik
Palm, E-ri Sol och Tea Sundsten, som alla vet vad
”ippa”betyder.
Doktorander, utbytesstudenter och gästforskare i gruppen,
med ett särskilt tack till Staffan Dryselius, Michael
Eberhardson och Anders Tengholm för intressanta
diskussioner om livet, universum och allting.
TA-personalen, särskilt Linnéa Brandt, Agneta Bäfwe,
Héléne Dansk, Birgitta Jönzén och Karin Öberg för er
sagolika förmåga att fixa saker.
Eva Grapengiesser, Erik Gylfe och Bo Hellman för den
forskningsfrämjande atmosfären i gruppen.
Prefekterna Arne Andersson, Birger Peterson och Godfried
Roomans.
Övriga vänner och kollegor vid Institutionen för medicinsk
cellbiologi.
Mina f.d. kursare, särskilt torsdagarnas trogna
lunchsällskap: Johanna Andræ, Cecilia Annerén, Helena
Brändström, Ulf Holmbäck och Elisabet Roman.
34
Mina bästisar Marit Jönsson, Helena Löthman-Ybo, Daniel
Markstedt och Peder Olofsson, för att ni effektivt distraherat
mig i mitt forskningsarbete.
Mamma Ann-Christine, pappa Björn, Patrik, Helena och
Gonasjonas, som trots att ni aldrig riktigt förstått vad jag
sysslat med alltid varit lika uppmuntrande och
entusiastiska.
Fredrik, världens bäste avhandlingscoach m.m.
Alla andra goda vänner och alla snälla släktingar, ingen
nämnd, ingen glömd.
This work was performed at the Department of Medical
Cellbiology, Uppsala University, Uppsala. Financial
support was received from the Swedish Medical Research
Council (grants 12X-562, 12X-6240 and 12X-11203), the
Novo Nordisk Foundation, the Swedish Diabetes
Association, the Family Ernfors Foundation, the Marcus
and Amalia Wallenberg Foundation, the Göran Gustafsson
foundation and the Swedish Society for Medical Research.
Det kommer inte att bli enklare i fortsättningen, men det kommer i alla fall att bli
mindre svårt.
Tommy Boustedt
35
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APPENDIX: PAPER I – IV
Advance in science comes by laying brick upon brick, not by sudden erection of fairy palaces.
- J. S. Huxley
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