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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 REFERENCES Aizawa T, Sato Y, Ishihara F, Taguchi N, Komatsu M, Suzuki N, Hashizume K and Yamada T. ATP-sensitive K+ channel-independent glucose action in rat pancreatic beta-cell. Am J Physiol 1994; 266(3 Pt 1):C622-7 Aizawa T, Sato Y, Komatsu M and Hashizume K. ATP-sensitive K+ channelindependent, insulinotropic action of glucose in the B-cells. Endocr Regul 1992; 26(4):159-62 Aleyassine H. 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