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Bioelectrochemistry and Bioenergetics, 21 (1989) 333-342

A section of J. Electroanal. Chem., and constituting Vol. 275 (1989) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

Changes in membrane potential of intact adipocytes measured with fluorescent dyes

Frederick Bailey, Nathalie Hill and Tadeusz Malinski *

Department of Chemistry, Oakland Uniuersity, Rochester, MI 48309-4401 (U.S.A.)

Frederick Kiechle

Department of Clinical Pathologv, William Beaumount Hospital, Royal Oak, MI 48072 (U.S.A.)

(Received 12 May 1988; in revised form 8 February 1989)

ABSTRACT

The change in transmembrane potential of rat adipocytes was measured using the fluorescent probes 3,3’-diethylthiadicarbocyanine iodide, [di-SC,(S)], and 3,3’-dipropylthiadicarbocyanine iodide, [di- SC,(S)]. The fluorimetric technique was calibrated by altering the potassium ion concentration while keeping the sum of potassium and sodium ions at a constant concentration of 153 mM. A 3% change in relative fluorescence was equivalent to a membrane potential change of 1 mV. This method was used to measure changes in transmembrane potential by insulin and two insulin-mimetic agents: wheat germ agglutinin and ethanolamine.

INTRODUCTION

The regulation of biological processes by insulin and insulin-mimetic agents is associated with changes in ion transport which are followed by transmembrane potential changes [l-3]. Therefore, measurements of the membrane potential changes are necessary to elucidate the mechanism of insulin action. Direct electrophysiologi- cal techniques (microelectrodes) [4] as well as fluorimetric and radiochemical methods [5,6] have been used to estimate a transmembrane potential &,., [1,4]. Relatively small cells, such as adipocytes, are difficult to impale with microelec- trodes and in addition have large water-insoluble inclusions. Due to small water space (about 4% of the total volume in mature adipocytes), the microelectrode tip may be clogged with insoluble fatty globules.

l To whom correspondence should be addressed.

0302-4598/89/$03.50 Q 1989 Elsevier Sequoia S.A.

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The use of fluorescent dyes for estimation of the potential of adipocytes has been difficult apparently due to problems encountered during calibration [7]. Calibration

of the potential in the fluorimetric method is usually done by altering the external concentration of potassium in the presence of valinomycin [5]. The resultant changes in the potential are followed by changes in fluorescence of the dyes. We have confirmed the results of other investigators [7] which demonstrate that this method of calibration cannot be applied to adipocytes since valinomycin induces only small changes in fluorescence which are comparable with the experimental

error, i.e. *0.6%. The reason for a lack of change in observed fluorescence after addition of valinomycin is not clear. However, it may be attributed to the slow rate of change in the equilibrium of potassium ions in the presence of valinomycin.

We report here an alternative method of calibration of relative potential changes in intact adipocytes which does not require the use of valinomycin. This fluorimetric method appears to be especially useful for studies of alterations of the transmem- brane potential of adipocytes by insulin or insulin-mimetic agents.

EXPERIMENTAL

Materials Male Sprague-Dawley rats (loo-140 g) were acquired from Harlan Sprague

Dawley. 3,3’-diethylthiadicarbocyanine iodide [di-S&(5)] was obtained from Kodak and 3,3’-dipropylthiadicarbocyanine iodide [di-SC,(S)] from Molecular Probes, Inc. Collagenase, Tris(hydroxymethy1) aminomethane hydrochloride (Tris), wheat germ agglutinin (Triticum vulgaris lectin), ethanolamine, KCl, NaCl, CaCl,, and MgSO, were purchased from Sigma. KH2P04 was obtained from Mallinckrodt Chemical Works. Bovine serum albumin (BSA), Fraction V, was purchased from Boehringer Marmheim Biochemicals. Porcine insulin was donated by S.R. Fields of Eli Lilly. [3H]-Methyltriphenylphosphonium iodide was purchased from DuPont.

Methods Adipocytes were prepared from rat epididymal fat pads digested for 45 min in a

metabolic shaker (70 RPM, 37 o C) in a solution containing 0.42 mg/ml collagenase, and 12.0 ml KRP solution (pH 7.4, 37’ C). The KRP solution contained: 3% BSA, 0.2% D-glucose, 1.28 mM NaCl, 5.2 mM KCl, 1.4 mM MgSO,, 10 mM Na,HPO, and 1.4 mM CaCl, .2 H,O. The cells were then filtered through a silk screen. The cells were washed twice in KRP buffer (pH 7.4, 37°C) and twice with 24.6 mM Tris buffer (pH 7.4, 37OC). The Tris buffer contained; 25 mM NaCl, 128 mM KCl, 1.0 mM CaCl,, 1.0 mM MgSO,, 1.0 mM KH,PO,, 24.6 mM Tris HCl, 1.0 mM D-glucose, and 3% BSA stored at pH 7.4, 37 o C. After each washing the cells were centrifuged at 1700-1800 RPM (6.2 X lo4 g) for 30 s and the liquid was pipetted out after each centrifugation. The adipocytes were diluted 1 : 50 compared to their packed cell volume with Tris buffer. The cells were then separated into two portions of approximately equal concentration and stored in a shaker bath at 37 o C.

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Ethanolamine and wheat germ agglutinin stock solutions (prepared fresh daily) were made in Tris buffer.

The fluorimetric measurements were performed using a RF 5000 U Shimadzu Scientific Instrument spectrophotofluorometer. A standard 1 cm pathlength quartz fluorescent cuvette was equipped with a magnetic stirrer to keep the cell suspension homogeneous. The temperature was kept constant at 37°C by water circulating through the cuvette holder. The excitation wavelength was set at 647 and 620 nm and the emission wavelength at 651 and 669 nm for di-SC,(S) and di-SC,(S), respectively. Three ml of Tris buffer (pH 7.4, 37 o C) (without the 1.0 mM glucose or 3% BSA) and 10.0 ~1 of 3 mM dye was equilibrated to 37” C in the cuvette. Cell + Tris buffer suspension was added to achieve a final concentration of adipo- cytes in the cuvette of approximately 2.5% by volume. The concentration of sodium ions in the cuvette was the sum of the concentration of Na+ in the ethanolamine or wheat germ agglutin + Tris buffer solution, [Na+lX, and in adipocyte + Tris buffer solution [Na+],. The total concentration of [Na+], + [Na+], was kept constant at 25 m&f. The concentration of potassium was also kept constant at 128 mM (except during the calibration procedure). Compared to the fluorescence, F,, of the control adipocyte cell solution, an increase or decrease of fluorescence, F, was measured after addition of insulin, wheat germ agglutinin or ethanolamine. Hyperpolarization or depolarization of the membranes was designated by a negative or positive value of AF/Fo, respectively, where AF = F - F,. A radiochemical method of measure- ment of membrane potential, using (3H)TPMP+ as a tracer, was used according to a procedure published previously [6].

RESULTS AND DISCUSSION

In excitable cells, like nerve or muscle cells, a membrane potential can be altered following a change of the potential between the outer and inner aqueous phase of the cell. This kind of potential, which can be changed rapidly from one state to another, is designated the action potential. In non-excitable cells, such as adipo- cytes, there is no action potential, and it is gratuitous to refer to adipocyte potentials as resting. Therefore, we will refer to this adipocyte potential as a transmembrane potential &,. The transmembrane potential is influenced by non-equal distributions of ions (mainly potassium and sodium ions) on the outer (0) and inner (i) face of the membrane/aqueous interface. This potential can be described by the Nemst equation as:

RT PKIK+lo + PNa[Naflo + a kc 7 In Px[K+]i + P,,[Na+]i + b (1)

where P, and P,, are the permeability coefficients for potassium and sodium ions, respectively; a and b are constants [l]. The fast fluorescent dyes can be adsorbed on the outer surface of the membrane and the fluorescence of these dyes will be affected by the membrane potential. The slow fluorescent dyes will diffuse through the membrane and the distribution of these dyes between the inner and outer face of

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the membrane will be affected by the transmembrane potential. The change of the membrane potential results in linear changes of the intensity of the fluorescence, F,

according to the equation:

F=K+,+c (2)

where K and c are constants [5]. An addition of valinomycin will change the permeability and distribution of

potassium ions but not of sodium ions. Therefore, c$+,, will become a function of the change of the distribution of K+ only, and eqn. (1) will be simplified:

P;[K+]: + d

P,*[K+]* +e (3)

where Pz and [K+]* are the permeability coefficient and concentration of potas- sium in the presence of valinomycin respectively and d and e are constants. (P, has been determined routinely by changing the external concentration of K+ and measuring the internal concentration of K+ in the presence of valinomycin and cyanine dyes, and employing eqns. (2) and (3) [Xl. There are, however, a few major drawbacks inherent in ion distribution determinations using vahnomycin. The rate of change of the equilibration of potassium ions may be relatively slow in the

presence of valinomycin so that it may not be suitable for measurements [9]. In cells such as white adipocytes, where it is difficult to estimate accurately the intracellular water space, determination of the intracellular ion concentrations of potassium is very difficult. In addition, only minimal changes (within experimental error) of fluorescence were observed in the presence of valinomycin in the range of [K+] from 10 to 500 mM (data not shown). Therefore, valinomycin could not be used for a “standard” calibration procedure. This is a confirmation of previous observations reported by Petrozzo and Zierler [7] concerning calibration of membrane potential in intact adipocytes.

However, the fluorescent dyes [di-SC,(S)] and [di-X,(5)] did demonstrate a stable fluorescent signal in the presence of intact rat epididymal adipocytes. The intensity of the fluorescent signal increased with the increase of potassium ion concentration when the ionic strength of the solution is kept constant, by addition of sodium ions (the total concentration of [Na+] + [K+] was always at a constant level of 153 mM). It has been shown previously that the membrane potential of rat white adipocytes is determined by transmembrane Kt and Nat gradients [6]. The effects of other ions are negligible. Both K+ and Nat ions influence the membrane potential only when the external concentration of potassium ions does not exceed 100 mM. However, at a K+ concentration of about 100 mM, the potassium ion gradient becomes the dominant controller in transmembrane potential and the distribution of potassium reflects the transmembrane potential +, in a Nemstian fashion (6) i.e.:

+,,, = 59 log[K+], + h (4)

where h is constant. Analysis of the relationship between the fluorescence intensity and the logarithm of the potassium concentration plot (Fig. 1) demonstrates that

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'c-1 2.bO 2b5 2,;0 2.!5 2.bO

1% ( [ K+l, 1 mW

Fig. 1. Variation of fluorescent intensity (arbitrary increments) of di-SC,(S) in medium with different [K+], when [Na+],+[K+],=constant; concentration of dye was 3 PM in 2.5% suspension of adipocytes.

this plot is a sigmoidal shape. A small increase of fluorescence is observed in the region of K+ concentration from 30 to 80 mM. However, starting from a concentra- tion of K+ of 85 mM, a significant increase of fluorescent signal is observed. At concentrations of K+ higher than 140 mM, an inflection point is observed and an increase of [K+] beyond this point causes only a small increase in signal.

A plot of F vs. log[K+] in the range of concentrations between can be represented by the equation:

F=f log[K+],+g

the fluorescence

85 and 140 mM

(5) where f and g are constants. The slope (f) is independent of the cyanine dye used, within experimental error (f = 76 k 2 and 74 + 2 for di-SC,(S) and di-SC,(S), respectively). Therefore, based on the linearity between the fluorescence and mem- brane potential from eqns. (4) and (5), the percent change (AF/F,) of 3% is equivalent to the potential change A+, = 1 mV. AF is the difference between fluorescent intensity (F), measured for dye added to incubated cells with insulin, and fluorescent intensity (F,), measured in the absence of insulin. An increase (AF > 0) and decrease (AF < 0) of fluorescent signal, after addition of insulin or insulin-mimetic agent, will be due to depolarization and hyperpolarization of cell membranes, respectively. The measurement of relative values of fluorescence eliminates the contribution of scattered light to the measured signal.

The method was tested using several ligands including insulin and two insulin- mimetic agents: wheat germ agglutinin and ethanolamine.

Insulin-induced hyperpolarization of membrane potential Insulin alters the distribution of ions by altering the net ionic flux between the

cell and its environment [lo]. The hypothetical mechanism of insulin action is that

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time / min

Fig. 2. Change of fluorescent intensity (arbitrary increments) with time of di-SC,(S) at a final

concentration of 3 pM in 2.5% suspension of adipocytes in 50 mM Tris buffer, pH 7.4 containing 25

mM NaCl and 128 mM KC1 without insulin (upper trace) and with 100 $J/ml insulin added (lower

trace). AF/F, = - 5.4%, which is equivalent to - 1.8 mV. AF measurements were made 7.2 mm (t = 0)

after the addition of insulin.

insulin alters the electrochemical potential gradient or the permeability of the barrier to the diffusion of ions [11,12]. Insulin can also alter directly or indirectly either the coupling or metabolic processes to which the ion flux is coupled. As a result, an increase of the absolute value of the electrical potential difference, called hyperpolarization, has been observed. However, several previous attempts to use a fluorimetric method to measure this insulin-induced hyperpolarization in adipocytes were not successful. In a preliminary report, Petrozzo and Zierler [7] used the fluorescent probe 3-ethyl-2-[5-(3-ethyl-2-benzothiazolinyl-l,3-pentadienyl] benzothiazolium iodide to measure the membrane potential in adipocytes but did not convert changes in fluorescent intensity to membrane potential, apparently due to problems encountered in calibration. Davis et al. [13] failed to achieve a stable fluorescent signal from adipocytes incubated with the anionic dye bis-oxonal. However, we were able to measure potential changes in intact adipocytes using either di-SC,(S) or di-SC,(S). Figure 2 shows a decrease of fluorescence in the presence of insulin and Fig. 3 demonstrates that insulin concentrations from 1 to 1000 $J/ml hyperpolarize adipocyte membranes. Hyperpolarization increases very rapidly at low concentrations of insulin and reaches a level of about 2.1 mV at a 2 pU/ml concentration of insulin. A further increase of the concentration of insulin causes a slight decrease of hyperpolarization, reaching a minimum at 100 $J/ml, the physiological concentration of insulin. After this point, a significant increase of hyperpolarization is observed up to 2.9 mV at 1000 /.JJ/ml of insulin. In order to verify the measurement obtained by the fluorimetric method, we used a radiochem- ical method under similar experimental conditions. A lipophilic cation, (3H)TPMP+, was used as a probe for the measurement of insulin-induced hyperpolarization at the physiological concentration of insulin (100 $J/rnl). (3H)TPMP+ will be distrib-

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Fig. 3. Effect of concentration on insulin-induced hyperpolarization of intact rat adipocytes. Six

measurements of membrane potential were made with each concentration of insulin. The error bar

represents the standard deviation of the mean.

uted between the two sides of the membrane according to the potential difference:

A&,=% In [ (3~)~~~~+]i

[ (3~)~~~~+] o (6)

When the potential is perturbed by the addition of insulin, the membrane potential change will be described by the equation:

AGm=F In [ (3~)~~~~+]0/[ (3~)~~~~+] i

[ (3~)~~~~+] ,*/[ (3~)~~~~+] ; (7)

where (3H)[TPMP+] and t3H)[TPMP+]* are the cell concentrations before and after the addition of insulin, respectively. The hyperpolarization of the membrane potential of adipocyte cells in the presence of 100 pU/ml of insulin was found to be 2.8 * 0.4 mV by the above radiochemical method.

The magnitude of insulin-induced hyperpolarization is similar to that reported by Cheng et al. [6], using (3H)TPMPf: - 3 mV. They observed a small response at low insulin concentrations (0.1 to 1 &t/ml) which rapidly progressed to a level of maximal respone ( - 3 mV) at 100 and 1000 @J/ml. Insulin-induced hyperpolariza- tion measured by using (3H)TPMP+ is about 1 mV higher compared to the fluorescence method. This difference may be attributed to the observation that the hyerpolarization measured with (3H)TPMP+ in intact adipocytes may represent both mitochondrial and plasma membrane potential changes [13]. The precise mechanism by which insulin hyperpolarizes membranes is not settled. Some experi- ments have been interpreted to mean that insulin stimulates an ouabain-inhibitable

340

electrogenic Na+-K+ exchange pump. However, other studies come to an opposite conclusion [l]. Other experiments are interpreted to mean that insulin-induced

hyperpolarization is caused by increased K+ conductance, while still others are interpreted to mean that insulin-induced hyperpolarization is caused by decreased K+ conductance. To explore the mechanism responsible for this hyperpolarization further, we developed a new method for examining the presence of insulin [14]. By using cell culture (BC3H-1 myocytes)-indium oxide semiconductor electrodes and ac impedance measurements, we found that the pattern of change of conductivity of cells in the presence of insulin follows almost exactly the pattern of change of the potential presented in this work (Fig. 3). Insulin at concentrations below 1 $_J/ml failed to alter the conductivity significantly, but 10 pU/ml insulin increased the conductivity 2.8% compared to control. Higher concentrations of insulin rapidly increased the conductivity to a plateau at the physiological concentration of insulin (100 $J/rnl) of a 67% increase.

Insulin-mimetic agents - wheat germ agglutinin and ethanolamine Wheat germ agglutinin and ethanolamine mimic the actions of insulin in fat cells

[15-171. The effect of wheat germ agglutinin is probably mediated by direct interaction with the insulin receptor [15]. The mechanism by which ethanolamine increases glucose transport and increases pyruvate dehydrogenase activity synergisti- cally in the presence of insulin is unknown [17].

A potential dose response curve for wheat germ agglutinin is shown in Fig. 4. The shape of this curve is analogous to that observed for insulin. A rapid hyperpolari- zation of adipocyte membranes is observed at concentrations of wheat germ

,/ 1 1

0.0 1.0 20 log(c/ mM)

Fig. 4. Effect of concentration on wheat germ agglutinin-induced hyperpolarization of intact rat adipocytes. Five measurements of membrane potential were made with each concentration of wheat germ agglutinin. The error bar represents the standard deviation of the mean.

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6.0 I 1.0 20

log(c/mM)

Fig. 5. Effect of concentration on ethanolamine-induced depolarization of intact rat adipocytes. Five

measurements of membrane potential were made with each concentration of ethanolamine. The error bar

represents the standard deviation of the mean.

agglutinin as low as 2 mM. An increase in concentration causes an increase of hyperpolarization and this increase is linear vs. the logarithm of the concentration, up to 5 mM, when the hyperpolarization is - 1.4 mV. A further increase of the concentration results in a slight decrease of the hyperpolarization, reaching a minimum at a concentration of about 10 mM. After this point, an increase of hyperpolarization is observed up to - 2.5 mV at 100 m M of wheat germ agglutinin.

Ethanolamine has shown a remarkably different potential-concentration re- sponse from that observed for insulin and wheat germ agglutinin. At a low concentration of ethanolamine, a depolarization of membrane potential of the adipocytes was observed (Fig. 5) with a maximum depolarization at a concentration of 10 mM. A further increase of the concentration of ethanolamine gives a decrease of the depolarization down to 0.5 mV at 100 mM.

The method described here using two fluorimetric dyes can be used to study the effect of hormones or drugs on the plasma potential of intact adipocytes. However, these reactions may be altered by the presence of higher than physiological con- centrations of potassium ions. In order to investigate the possible potassium dependence, the polarization measurements should be made for several potassium concentrations in the range between 30 and 140 mM ([K+] + [Na+] = 153 mM). The accuracy and detection limit will be better at higher concentrations of potas- sium due to a higher fluorescence intensity which implicates a higher signal to noise ratio.

ACKNOWLEDGEMENT

This research was Research Institute.

supported by a grant from William Beaumont Hospital

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REFERENCES

1 K. Zierler in M.D. Hollenberg (Ed.), Insulin. It’s Receptors and Diabetise, Marcel Dekker, New

York, 1985, p. 141.

2 K. Zierler, E.M. Rogers, R.W. Scherer and F. Wu, Am. J. Physiol., 249 (1985) E17.

3 F. Wu and K. Zierler, Am. J. Physiol., 249 (1985) E12.

4 U.V. Lassen, A.M.T. Nielsen, L. Pape and L.O. Simonsen, J. Membr. Biol., 6 (1971) 269.

5 A.S. Waggoner, AMU. Rev. Biophys. Bioeng., 8 (1979) 47.

6 K. Cheng, H.C. Haspel, M.L. Vallano, B. Osotimehin and M. Sonenberg, J. Membr. Biol., 56 (1980)

191.

7 P. Petrozzo and K. Zierler, Fed. Proc. Fed. Am. Sot. Exp. Biol., 25 (1976) 602.

8 L.B. Cohen and B.M. Salzberg, Rev. Physiol. Pharmacol., 83 (1978) 35.

9 J.P. Leader in A.D.C. Ma&night and J.P. Leader (Eds.), Epithelial Ion and Water Transport, Raven

Press, New York, NY, 1981.

10 K.L. Zierler in R.O. Greep, E.P. Astwood, D.F. Steiner, N. Freinkel and S.R. Geiger (Eds.),

Handbook of Physiology, Sect. 7: Endocrinology, Vol. 1, Washington, DC, Am. Physiol. Sot., 1972,

Ch. 22, p. 347.

11 R.D. Moore, Biochim. Biophys. Acta, 737 (1983) 1.

12 K. Zierler and F.-S. Wu, Chem. Res., 34 (1986) 727A.

13 R.J. Davis, M.D. Brand and B.R. Martin, B&hem. J., 196 (1981) 133.

14 T. Mahnski, A. Ciszewski, N. Hill, D. Dandurand and F. Kiechle, FASEB J., 3 (1989) A1292.

15 P. Cuatrecasas and G.P.E. Tell, Proc. NatJ. Acad. Sci. USA, 70 (1973) 485.

16 J. Shechter and B.A. Sela, Biochem. Biophys. Res. Commun., 98 (1981) 367.

17 G. Barseghian, T. Papoian, D.L. Hwang, A. Roitman and A. Lev-Ran, B&hem. Biophys. Res.

Commun., 121 (1984) 413.