Rapid insulin-induced exocytosis in white rat adipocytes

Helena Chowdhury; Marko Kreft; Robert Zorec

Rapid insulin-induced exocytosis in white rat adipocytes

Helena Chowdhury

2002, Pflügers Archiv: European Journal of Physiology

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Insulin is believed to increase glucose permeability of adipocytes by regulating the incorporation of glucose transporters into the plasma membrane by exocytosis. This process involves fusion of membranebound cellular compartments with the plasma membrane, thus influencing the plasma membrane area. However, insulin-induced changes in plasma membrane area have not yet been demonstrated. In the present study we monitored fluorescence intensity with a confocal microscope to study the effect of insulin on adipocyte plasma membrane area. After cell isolation and adhesion to a glass cover-slip, adipocytes were stained with the dye FM1-43, a membrane area reporter. At rest, the rate of fluorescence intensity increase was initially high, but gradually stabilized at 2%/min. This steady increase in fluorescence is due to a slow rate of exocytosis coupled to endocytosis, since the removal of FM1-43 from the bath did not abolish FM1-43 fluorescence. Insulin addition caused an abrupt increase of fluorescence intensity of 4%/ min, which was significantly higher than in controls. These results suggest rapid, insulin-induced incorporation of new membrane into the plasma membrane by exocytosis.

Pflugers Arch - Eur J Physiol (2002) 445:352–356 DOI 10.1007/s00424-002-0938-2 CELL AND MOLECULAR PHYSIOLOGY Helena H. Chowdhury · Marko Kreft · Robert Zorec Rapid insulin-induced exocytosis in white rat adipocytes Received: 9 May 2002 / Revised: 29 August 2002 / Accepted: 16 August 2002 / Published online: 11 October 2002 Springer-Verlag 2002 Abstract Insulin is believed to increase glucose permeability of adipocytes by regulating the incorporation of glucose transporters into the plasma membrane by exocytosis. This process involves fusion of membranebound cellular compartments with the plasma membrane, thus influencing the plasma membrane area. However, insulin-induced changes in plasma membrane area have not yet been demonstrated. In the present study we monitored fluorescence intensity with a confocal microscope to study the effect of insulin on adipocyte plasma membrane area. After cell isolation and adhesion to a glass cover-slip, adipocytes were stained with the dye FM1-43, a membrane area reporter. At rest, the rate of fluorescence intensity increase was initially high, but gradually stabilized at 2%/min. This steady increase in fluorescence is due to a slow rate of exocytosis coupled to endocytosis, since the removal of FM1-43 from the bath did not abolish FM1-43 fluorescence. Insulin addition caused an abrupt increase of fluorescence intensity of 4%/ min, which was significantly higher than in controls. These results suggest rapid, insulin-induced incorporation of new membrane into the plasma membrane by exocytosis. Keywords Confocal microscopy · FM1-43 · Insulin · Exocytosis · White adipocytes H.H. Chowdhury · M. Kreft · R. Zorec ()) Laboratory of Neuroendocrinology – Molecular Cell Physiology, Institute of Pathophysiology, Medical School, University of Ljubljana, ZaloÐka cesta 4, 1000 Ljubljana, Slovenia e-mail: [email protected] Tel.: +386-1-5437020 Fax: +386-1-5437021 H.H. Chowdhury · M. Kreft · R. Zorec Celica Biomedical Sciences Centre, Stegne 21, 1000 Ljubljana, Slovenia Introduction White adipocytes constitute one of the most important target cells for insulin action in the body. Upon a rise of glucose concentration in the plasma, b-cells in pancreas begin to secrete insulin, which is blood-borne and binds to receptors on target cells, mainly adipocytes and skeletal muscle cells. Insulin binding to receptors triggers an increase in the permeability of the plasma membrane to glucose [27]. The increased glucose transport is thought to be due to an increased number of glucose transporter isoform 4 (GLUT4) molecules in the plasma membrane [20]. At rest, GLUT4 is localized mainly in the intracellular microsomal compartment of adipocytes [14, 23]. After stimulation with insulin these transporters are thought to translocate to the plasma membrane [11, 19, 23, 24]. Although indirect, several lines of evidence support the existence of insulin-induced exocytosis of GLUT4-bearing vesicles. Firstly, studies using cellsurface labelling of glucose transporters have demonstrated GLUT4 translocation to the plasma membrane after insulin stimulation [10]. Secondly, exocytosis appears to require specialized proteins, termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) [12] and several studies have reported the co-localization of GLUT4 with various SNARE proteins, indicating that the GLUT4 membrane compartment is equipped with essential proteins that are required in regulated exocytosis [4, 8, 9, 21]. Thirdly, exocytosis of vesicles is associated with an increase in the plasma membrane surface area that can be monitored by electrophysiological measurements of membrane capacitance [17]. Using this technique, exocytotic activity has been recorded in rat adipocytes [15], however the effects of insulin on surface area changes have not yet been studied. If insulin were to induce the fusion of translocated GLUT4-bearing vesicles with the plasma membrane, an insulin-induced increase in the surface area of the plasma membrane should be detectable in a single adipocyte. In this study we used a fluorimetric method to study membrane dynamics of cultured single white adipocytes. 353 We employed confocal microscopy to observe the changes of plasma membrane area using the styryl fluorescent dye FM1-43 [5]. The fluorescence intensity of stained membrane increases in proportion to the rate of membrane addition [2]. We show here that the addition of insulin increases the fluorescence intensity of FM1-43stained adipocyte plasma membrane. These results show directly that in, a single adipocyte, insulin regulates membrane dynamics, probably by regulating exocytosis associated with secretion [1] and/or transporter density regulation [6]. Upon vesicle fusion membrane area increases, hence the FM143 fluorescence intensity increases due to staining of freshly fused membrane [18]. The time-dependent increase in fluorescence intensity was analysed by differentiating the signal and fitting a function to the data consisting of a sum of an exponential term and a constant (Sigmaplot, SPSS, Chicago, Ill., USA). The effect of insulin, ATP and vehicle addition onto cells was quantified by measuring changes in FM1-43 fluorescence intensity relative to the resting levels (fluorescence intensity at the time of drug addition) 20 and 60 s after stimulation. In the present study, the timedependent changes of FM1-43 fluorescence intensity of a 2-mthick rim of the plasma membrane area were determined. All experiments were performed at room temperature. Data are given as means€SEM. Materials and methods Results Adipocyte isolation and incubation As already reported [15] cell preparation and adherence is a major methodological challenge in experiments with isolated adipocytes. Once white fat cells were bathed in the recording medium, we began to scan cells with the argon laser to determine the background fluorescence. Upon the addition of 5 M FM1-43, fluorescence intensity of the cell increased, eventually attaining a slow, steady increase (Fig. 1). The persistent increase in fluorescence may indicate cumulative exocytosis if the dye is internalised by vesicle endocytosis [18]. Indeed, after removal of FM1-43, cells retained a residual fluorescence (R, 32€3%, n=18), indicating dye internalisation by endocytosis. We evaluated the rate of steadystate fluorescence increase. Figure 1 shows a representative recording of the time-dependent increase in fluorescence intensity. After exposure to FM1-43, membrane fluorescence intensity increased initially rapidly due to the dye loading into the plasma membrane and subsequently more slowly with a stable rate of around 2.0€0.4%/min, determined by fitting an exponential function with a constant to the averaged data of time derivatives of the fluorescence intensity signal (Fig. 1B). The equation to the line of best fit was: Wistar rats (200–250 g) were killed by exposure to an atmosphere of 100% CO2 followed by decapitation. Epididymal fat pads were harvested and adipocytes prepared as described [22]. Briefly, the fat pads were immersed in Earl’s balanced salt solution (EBSS) containing 4% bovine albumin, streptomycin (1 g/ml) and penicillin (1 unit/ml). Adipose tissue was minced and subjected to collagenase (GIBCO, UK) digestion (2 mg/ml) for 1 h at 37 C. The suspension was centrifuged and the floating adipocytes collected and filtered through 400-m nylon mesh to remove tissue pieces. The cell suspension was then centrifuged at 400 g for 1 min. Adipocytes floated to the surface and the stromal-vascular cells (capillary, endothelial, mast, macrophage and epithelial cells) sedimented. Adipocytes were collected from the supernatant of the suspension and resuspended in EBSS. The centrifugation was repeated 3 times. The floating fat cells were resuspended in DMEM supplemented with 10% fetal bovine serum (FBS), streptomycin (1 g/ml, Sigma) and penicillin (1 unit/ml, Sigma). After isolation, cells were allowed to adhere to glass cover-slips coated with the biological adhesive Cell-Tak according to the manufacturer’s instructions (Becton Dickinson Collaborative Biochemical, Bedford, Mass., USA) and published reports [15]. Culture dishes with adipocyte-covered glass cover-slips were kept in an incubator at 37 C, 95% humidity and 5% CO2 in DMEM/FBS/antibiotic solution. Cells were used for experiments 0–5 days after isolation. Confocal microscopy and fluorimetry Isolated adipocytes were placed in a recording chamber on the confocal microscope (LSM 510, Zeiss, Oberkochen, Germany). The recording medium consisted of (in mM): NaCl 131.8, CaCl2 1.8, KCl 5, MgCl2 2, HEPES/NaOH 10, d-glucose 10, NaH2PO4 0.5, NaHCO3 5; pH 7.2. The recording medium also contained 5 M FM1-43 (Molecular Probes, Eugene, Ore., USA), diluted previously in DMSO to obtain a 1 mM stock. The experiments were performed by exposing cells to FM1-43 for 3–5 min before the addition of the drug. After addition of FM1-43 to the recording medium the final concentration of DMSO was less than 0.5%, which did not affect the time course of FM1-43 fluorescence intensity of adipocytes (not shown). During experiments we added insulin (final concentration 870 nM) or ATP (100 M) or insulin vehicle, respectively. Insulin was prepared as an 87 M stock solution in acidified H2O (pH 2, using glacial acetic acid, approx. 1%). To prepare a 20 mM stock solution of ATP, Na2ATP was diluted in water. The fluorescence images of FM1-43 stained cells were acquired through a planapochromatic oil-immersion objective (40, NA=1.3), excited by the 488 nM argon laser line and filtered with the 505 nM low-pass emission filter. Images were scanned for 5 min with a 5-s scan interval and analysed quantitatively using the LSM 510 Time series software (Zeiss). RFII (%/min)=(2.0€0.4)+(4981€1340)*e((0.041€0.002)*t(s)) where RFII is the rate of fluorescence intensity increase at time t. A similar rate of increase of FM1-43 fluorescence intensity (0.8€0.2%/min, n=138) was determined prior to the addition of drugs by fitting a line through data points (more than ten) just prior to the drug application. The persistent slow increase in FM1-43 fluorescence intensity suggests slow exo-, endocytic membrane recycling in resting adipocytes, as reported previously [13]. To establish whether insulin stimulates membrane dynamics, we added a bolus (see Materials and methods) of insulin to a final concentration of 870 nM. In controls we applied the vehicle solution only or ATP (100 M). The application of ATP results in an increase of electrophysiologically determined membrane surface area in white adipocytes [15]. Figure 2 shows two images of the same live adipocyte, before (Fig. 2, left) and after (Fig. 2, right) insulin addition. In the presence of insulin the fluorescence intensity of the cell perimeter increased

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Rapid insulin-induced exocytosis in white rat adipocytes

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