Molecular and Cellular Endocrinology, 86 (1992) 49-56 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303.7207/92/$05.00

MOLCEL

02775

Lactate transport in insulin-secreting p-cells: contrast between rat islets and HIT-T15 insulinoma cells L. Best, R. Trebilcock

and S. Tomlinson

Department of Physiological Sciences, Unirlersity of Manchester, Manchester Royal Infirmary, Oxford Road, Manchester Ml3 9WL, UK (Received

Key words: Pancreatic

islet; Insulin

release;

10 December

Lactate

transport;

1991; accepted

Intracelluktr

16 March

1992)

pH

Summary The transport of L- and o-lactate into rat pancreatic islets and HIT-T15 insulinoma cells was studied substrate at room temperature and by following changes in by measuring uptake of 14C-labelled intracellular pH (pHi) in islets and HIT-T15 cells loaded with 2’,7’-bis(carboxyethyl)-5’(6’)-carboxyfluorescein (BCECF). Uptake of L-lactate into HIT-T15 cells was rapid, reaching equilibrium after 5 min was markedly inhibited by a-cyano-4-hydroxycinnawith an apparent K, value of 4.8 mM. Transport mate, a-fluorocinnamate, quercetin and p-chloromercuribenzenesulphonate (pCMBS), and was enhanced in citrate medium. Uptake of o-lactate was less rapid, apparent eqilibrium not being reached within 10 min. In contrast to HIT-T15 cells, rat pancreatic islets showed greatly reduced rates of transport of L- and D-lactate together with a correspondingly lower degree of inhibition by cY-cyano-4-hydroxycinnamate. The addition of L- or D-lactate to HIT-T15 cells, but not dispersed islet cells, resulted in a marked and rapid intracellular acidification followed by a gradual recovery. In both HIT-T15 cells and isolated islets, the rates of transport of both L- and o-lactate in the presence of Lu-cyano-4-hydroxycinnamate were significantly greater in a depolarising K+ medium compared to the normal Na+ medium. These observations suggest that native rat islet cells have considerably reduced activity of the lactate-/H’ transport system compared to HIT-T15 insulinoma cells. There is evidence in both cell types of an additional electrogenic pathway for lactate which might play a role in coupling lactate efflux to p-cell depolarisation.

Introduction Correspondence to: Dr. L. Best, Multipurpose Building, Manchester Royal Infirmary, Oxford Road, Manchester Ml3 9WL, UK. Abbreviations: BCECF, 2’,7’-bistcarboxyethylk5’(6’)carboxyfluorescein; pCMBS, p-chloromercuribenzenesulphonate; DMO, 5,5’-dimethyloxazolidine-2,4-dione.

The stimulation of insulin, secretion by glucose is dependent upon metabolism of the hexose within the p-cell (Malaisse et al., 1979; Ashcroft, 1980). Whilst a significant amount of glucose is completely oxidised to carbon dioxide, up to 50%

of glucose utilised by the P-cell can be accounted for by the formation of lactate (Malaisse et al., 1976; Sener and Malaisse, 1976; Best et al., 1989) which is lost from the cell into the extracellular medium (Best et al., 1989). Little is at present known regarding the mechanism(s) by which this extrusion of lactate from the /3-cell occurs or indeed the physiological importance of lactate formation and efflux in the P-cell. The process of lactate transport across the plasma membrane has been shown to be mediated largely via a specific H+-linked carrier mechanism in a wide variety of cell types, including erythrocytes (Halestrap, 19761, hepatocytes (Edlund and Halestrap, 1988) skeletal and cardiac muscle (Koch et al., 1981; Poole et al., 1989) and Ehrlich ascites cells (Spencer and Lehninger, 1976). This carrier has been characterised largely by its susceptibility to certain inhibitors, particularly derivatives of cinnamic acid (Halestrap and Poole, 1989). There is evidence, based upon selectivity for L-lactate over the o-enantiomer, that at least two such carriers may exist. Thus, Dlactate is a very poor substrate for the erythrocyte and hepatocyte carrier (Halestrap, 1976; Edlund and Halestrap, 1988), whilst that in cardiac myocytes has a low stereoselectivity for L- over Dlactate (Poole et al., 1989). In additional to the specific, carrier-mediated lactate transport systcm, a non-specific, non-saturable pathway of passive diffusion of the undissociated acid is also thought to occur, particularly at high concentrations of lactate (Halestrap and Poole, 1989). The present study demonstrates the existence of a lactate transporter with low selectivity for Lover o-lactate in a cloned hamster insulinoma cell line (HIT-T15). In contrast, rat pancreatic islets exhibited greatly reduced activity of this transport system. We have also obtained evidence in both HIT-TX cells and islets of an electrogenic pathway for lactate across the plasma membrane. The possible physiological implications of these findings are discussed. Materials

and methods

Pancreatic islets were isolated from fed adult rats by collagenase digestion (Lacy and Kostianovsky, 1967). Dispersed islet cells were ob-

tained by mechanical agitation of islets in a nominally Ca’+- and Mg’+-free medium. HIT-T15 cells were grown in culture in RPM1 supplemented with 10% foetal calf serum, 50 U/ml penicillin, 50 pg/ml streptomycin and 2.5 pg/ml fungizone. The medium was replaced every 3 days and the cells passaged once per week. The expcriments described here used cells between passage Nos. 70 and 80. Measurement of lactate transport Islets and cells were incubated in a medium with the following ionic composition (mmol/l): Na+ 120, Kt 5, Mg2+ 1, Ca*+, Cl- 129, buffered with Hepes 20 to pH 7.35. In certain experiments, a depolarising medium was used containing K’ 120 and Na.+ 5. Albumin was omitted from the media since it is known to bind cinnamate derivatives. For transport assays, an oil-filtration technique was employed (Best et al., 1991). For each experimental condition, a time zero measurement was performed at 0°C in the presence of 10 mM cu-cyano-4-hydroxycinnamate. This value, which represented a rapid lactate binding component (Edlund and Halestrap, 1988), was subtracted from all other measurements. Lactate transport measurements were expressed as nmol/pl intracellular water space. Measurement of intracellular pH (pHiI in HIT- T15 cells and isolated islets pHi was estimated by incubating HIT-T15 cells or rat islets in the presence of ‘H,O (20 pCi/ml), 14C-DM0 (50 mCi/mmol; 2 pCi/ml) and 1 mM unlabelled L-lactate essentially as described above. pHi was calculated as: 6.13 + log([DMO,]

x (1 + 10P”“~h.‘.i) -

l/[DMO,,I).

Changes in pHi were monitored in stirred suspensions of HIT-T15 cells or dispersed islet cells loaded with BCECF as described previously (Best et al., 1988; Lynch et al., 1989). L- and o-lactate, a-cyano-4_hydroxycinnamate, cu-fluorocinnamate, pCMBS, quercetin, silicone oil MS550 and dinonylphthalate were obtained from the Sigma Chemical Company, Poole, Dorset,

51

UK. L-[U-‘4C]lactate (169 mCi/mmol), D-W“C]lactate (40 mCi/mmol), “H-inulin (l-5 Ci/mmol), 14C-DM0 (50 mCi/mmolI and “H,O (5 mCi/ml) were purchased from Amersham International, Amersham, Bucks., UK. Results Time courses of L- and o-lactate uptake into HIT-T15 cells are shown in Fig. 1. Transport of 1 mM L-lactate (Fig. 1 A) was rapid, reaching equilibrium within 5 min and was markedly inhibited by 10 mM a-cyano-4-hydroxycinnamate and also by cu-fluorocinnamate, quercetin, pCMBS and by amiloride (Table 1I. Uptake of L-lactate was enhanced in a medium containing 84 mM sodium citrate which results in (A)

1.2

1.0

0.6

gz

c: 1

0.6

CUO

?E Jc

an alkaline cytosolic compartment. The values for pHi in HIT-T15 cells calculated from 14C-DM0 distribution were 7.42 + 0.01 (n = 4) and 7.49 f 0.01 (n = 4) in normal and citrate media respectively. From these figures, it can be calculated that the intracellular lactate concentration in cells incubated in 1 mM lactate at equilibrium should be approximately 1.05 mM and 1.23 mM in normal and citrate media respectively. These values correspond closely to the experimental data. In the case of 10 mM L-lactate (Fig. lBI, the extent of inhibition by cu-cyano-4-hydroxycinnamate was reduced, probably reflecting increased passive diffusion of the undissociated acid at this higher concentration. HIT-T15 cells were also found to transport o-lactate (Fig. 1C and D) although at a comparatively reduced rate.

0.4 0.2 0.0 0

2

4

6

0

010

I

2

4

6

Time (min)

4

6

610

Time (min)

Time (min)

0

2

6

10

b 0

1

1

I

I

,

2

4

6

6

10

Time (min)

Fig. 1. Time course of uptake of lactate by HIT-T15 cells. Suspensions of approximately 10’ cells were incubated with either 1 mM (A) or 10 mM (B) L-[ ‘JC]lactate or with 1 mM (C) or 10 mM (D) D-[ “Cllactate either in standard NaCl buffer t+, 0) or 84 mM Each point represents the mean of citrate buffer t n ) in the absence ( +) or presence (0) of 10 mM a-cyano-4-hydroxycinnamate. 2-6 determinations using separate batches of cells.

52

The time courses for lactate transport by isolated pancreatic islets are shown in Fig. 2. Uptake of 1 mM and 10 mM L-lactate (A and B) and 1 mM D-lactate (C) occurred at a considerably reduced rate compared to HIT-T15 cells, whilst a-cyano-4-hydroxycinnamate had a correspondingly smaller inhibitory effect on uptake. The apparent pHi values for islets, as judged from 14C-DMO distribution at equilibrium, were 7.15 f 0.015 (n = 3) and 7.23 f 0.012 (n = 3) in normal and citrate medium respectively. From these figures, one would predict intracellular lactate concentrations at equilibrium (in 1 mM external lactate) of 0.56 and 0.68 mM respectively. From the experimental data (Fig. 2A) it is clear

that these values were not attained within a 10 min incubation period, suggesting that the distribution of 1 mM lactate in islet cells does not reach equilibrium within this period. Uptake of lactate in HIT-T15 cells and dispersed rat islet cells was also monitored by measuring changes in intracellular pH in stirred suspensions of cells loaded with the pH-sensitive fluorescent dye BCECF. As depicted in Fig. 3, the addition of 10 mM L-lactate elicited a rapid, marked decrease in fluorescence, corresponding to a fall in pHi of approximately 0.3 units, followed by recovery and subsequent ‘overshoot’ alkalinisation. p-Lactate had a similar effect (not shown). Preincubation of the cells with cu-fluoro-

,

I

I

I

I

I

0

2

4

6

6

10

Time (min) (c)

,

0

0.3

I

I

1

I

1

I

I

I

I

2

4

6

6

10

0

2

4

6

Time (min) Fig. 2. Time course of uptake of lactate by isolated rat islets. Groups (B) I_-[‘“Cjlactate or 1 mM D-[ “C]lactate (C) in either standard NaCl (+, W) or presence f 0. 0) of 10 mM cY-cyano-4-hydroxycinnamate. separate batches

1

6

10

Time (min) of 100 islets were incubated with either 1 mM (A) or 10 mM buffer ( +. 0) or 84 mM citrate buffer (H, 0) in the absence Each point represents the mean of 2-4 determinations using of islets.

53

A HIT

TABLE

T15cells

10 mM L

lactate

10mM

1

UPTAKE OF L-LACTATE VARIOUS CONDITIONS

Na acetate DHI

INTO HIT-T15

CELLS

UNDER

Suspensions of 10h cells were incubated for 5 min with 1 mM t_-[‘4C]lactate under the conditions indicated. The figures in parentheses indicate the number of replicate determinations.

6 Diswrsed 1OmM

L

islet cells 10mM

lactate

Na acetate - 7.2

.

7.1

I.-Lactate (nmol/~l

Control 1 mM amiloride 84 mM Na citrate 10 mM cu-cyano-4hydroxycinnamate 5 mM cy-fluorocinnamate 0.25 mM quercetin 0.25 mM pCMBS

1.03 + 0.08 (61 0.76 f 0.05 (31 1.60 * 0.04 (3)

< 0.05 < 0.001

0.48 f 0.05 (51

< 0.001

0.50 + 0.06 (31 049 * 0.05 (3) 0.20+ 0.01 (3)

< 0.001 < 0.001 < 0.001

i/c,

Fig. 3. Effect of L-lactate and acetate on intracellular pH (pHi) in stirred suspensions of HIT-T15 cells and dispersed islet cells loaded with BCECF. Each trace is representative of 3-5 recordings showing similar results.

cinnamate, a non-fluorescent inhibitor of the lactate transporter, almost completely prevented lactate-induced acidification, but did not affect the acidification elicited by the weak acid acetate, which enters the cell predominantly by diffusion of the undissociated acid. Neither cy-fluorocinnamate nor pCMBS (trace not shown) affected

0

0.2

0.4

l/[L-lactate]

0.6 CmM-‘)

0.8

intracellular

resting levels of pHi. In the case of islet cells, addition of L-lactate failed to cause any detectable acidification, although acetate resulted in an acidification comparable to that observed with HIT-T15 cells. In order to estimate K, values for lactate transport in HIT-T15 cells, the initial rates of 14C-lactate uptake were measured at concentrations of L-lactate between 0.1 and 10 mM, subtracting the non-carrier-mediated transport in the presence of a-cyano-4-hydroxycinnamate (Fig. 4). These data indicate an approximate K, for Llactate of 4.8 mM. In comparison, the corre-

w 1 min.

-0,2

uptake i/c water)

P

Conditions

I.0

-0.12

-0.04

0.04

0.12

0.20

l/[L-lactate](mM-‘)

Fig. 4. Kinetics of L-lactate uptake into HIT-T15 cells. Initial rates of uptake were calculated either from uptake of I.-[‘“Cllactate at 1 min having subtracted non-carrier-mediated transport in the presence of cu-cyano-4-hydroxycinnamate (left panel) or from the change in intracellular pH (pHi1 30 s after the addition of L-lactate to BCECF-loaded cells having subtracted non-carrier-mediated transport in the presence of cu-fluorocinnamate (right panel). Each point represents the mean of 3-4 determinations using cells from separate batches. The linear regression coefficients are 0.972 (left panel) and 0.986 (right panel).

(A)

(8)

0.4-I

0.5+

0.0-

k 0

I 5

I 10

I 15

1 20

o.o-

r 0

I 5

I 15

r 10

I 20

Time (min)

Time (min)

IO” cells were incubated Fig. 5. Uptake of L.- and u-lactate by HIT-T15 cells in Na + and K’ media. Suspensions of approximately in the presence of 10 mM ru-cyano-4-hydroxycinnamate with 1 mM t.-[‘“C]lactate (A) or 1 mM o-[‘4C]lactate (B) in either Na‘ ( q ) or K+ ( + ) medium (see Materials and methods for details). Each point represents the mean f SEM of 3-6 determinations using separate batches of cells. * p < 0.05.

sponding value obtained by measuring the initial rate of acidification following the addition of lactate G-30 mM) was approximately 7.1 mM. It should be emphasised that measurement of changes in pHi only permits a quantitative assessment of lactate transport rates at relatively high concentrations of lactate (10 mM and above). The obvious differences in apparent V,,, of the car-

;

;

lb

Time (min)

1'5

2b

rier using the two methods probably result from the intracellular H+-buffering capacity of the cells, the pHi method thereby giving a large underestimate. In an attempt to explore the possibility of an electrogenic pathway for lactate in insulin-secreting cells, 14C-lactate uptake was measured in cells incubated in a depolarising K+ medium com-

Ii

;

lb

1'5

2b

Time (min)

Fig. 6. Uptake of L- and o-lactate by isolated rat islets in Na+ and K+ media. Groups of 100 islets were incubated of 10 mM cu-cyano-4-hydroxycinnamate with 1 mM L-[‘4C]lactate (A) or I mM D-l’4C]lactate (B) in either Nat medium (see Materials and methods for details). Each point represents the mean*SEM of 3-4 determinations batches of islets. * p < 0.05.

in the presence (0) or Kt (+l using separate

55

pared with cells incubated in normal Na+ medium. In these experiments, cells were incubated in the presence of a-cyano-4-hydroxycinnamate in order to minimise contribution from the carrier-mediated pathway. Similarly, a low concentration of substrate (1 mM) was used in order to minimise the passive diffusion component. As shown in Fig. 5, enhanced rates of uptake of both L- and o-lactate were consistently observed in depolarised compared with non-depolarised HIT-T15 cells. Fig. 6 shows the results of similar experiments with isolated islets. Again, a small, though significant increase in the rate and extent of L- and p-lactate uptake was apparent in islets incubated in a depolarising KC1 medium compared with normal NaCl medium. Discussion The results of this study provide evidence for a lactate transport system in HIT-T15 insulinoma cells. The finding that lactate uptake was impaired by amiloride and enhanced in citrate medium, conditions which would be expected to lower and raise, respectively, intracellular pH, suggests that lactate transport is H+-linked. In common with a wide variety of other tissues (Halestrap and Poole, 1989), this carrier was inhibited by cinnamate derivatives, quercetin and by pCMBS. Inhibition by these agents is the result of a direct interaction with the lactate carrier (Halestrap and Poole, 1989) and not, as shown in the present study for the cinnamate derivative and pCMBs, secondary to an effect upon pHi. A somewhat unexpected finding in the present study was the markedly reduced activity of the lactate-/H+ transport system in freshly-isolated rat pancreatic islets. This was reflected not only by the low rate of lactate uptake but also by the low sensitivity of uptake to a-cyano-5-hydroxycinnamate and the failure of lactate to acidify islet cells. Since islets are comprised of at least three cell types, it is impossible at present to ascribe the existing transport activity to any particular cell type. However, it seems an inescapable conclusion that native rat p-cells have a greatly reduced activity of the lactate carrier compared to

the cultured hamster P-cell line. We have recently found similar lactate transport characteristics to the HIT-T15 cells in the RINm5F cell line (Best et al., 1991), suggesting that the difference between cultured insulinoma cells and native islets is not due to species differences. Whether, on the other hand, the lactate carrier becomes expressed in tumoural/cultured insulin-secreting cells remains to be established. The possible existence of an additional, electrogenic pathway for lactate has been suggested by others (Edlund and Halestrap, 1988; Poole et al., 1989) on the basis of a lower-than-predicted intracellular distribution of lactate at equilibrium. Such a pathway might be of particular importance in the &cell, given the depolarising effect of lactate and related compounds on HIT-T15 cells (Meats et al., 1989; Lynch et al., 1991). The observation that HIT-T15 cells and islets show an enhanced rate and extent of lactate uptake when incubated in a depolarising KC1 medium, compared to the normal NaCl medium, suggests that an electrogenic pathway for lactate does indeed exist in p-cells. The apparent low activity of this pathway compared to the H+-linked carrier in HIT-T15 cells makes it unlikely that the former contributes greatly to the extrusion of endogenous lactate from the cell under normal circumstances. However, in the case of the native pancreatic P-cell, the reduced activity of the lactate-/H+ carrier would tend to favour lactate efflux via the electrogenic route. This process might be of particular importance in view of the large quantities of lactate produced during glucose stimulation of the P-cell (Malaisse et al., 1976; Sener and Malaisse, 1976; Best et al., 1989). Such a mechanism would be consistent with earlier findings that rat islets can be activated not only by the addition of lactate (and related anions), but more strikingly by the subsequent rapid withdrawal of the anion (Best et al., 1988, 1989; Yates et al., 1990). An electrogenic route of endogenous lactate efflux could also be of importance in coupling glucose metabolism in the p-cell to plasma membrane depolarisation. In this respect, it is of interest that HIT-T15 cells, which are, in common with several cloned insulinoma cell lines, poorly responsive to glucose compared to native islet cells, also express a highly active

elcctroneutral lactate-/H+ carrier. Whether depolarisation of the P-cell membrane in response to glucose is in any way dependent upon the pathways by which endogenously formed lactate is extruded from the cell remains to be established.

Best, L., Trebilcock, Pharmacol. Edlund,

R. and Tomlinson,

S. (IYYI)

Biochem.

41, 405-40Y.

G.L.

and Halestrap,

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117~126. Halestrap,

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Biochem.

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J. 156, 19.1~207.

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(IYXY)

in Anion

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(Hamasaki,

Elsevier.

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Amster-

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Acknowledgements

Koch, A.. Webster,

B. and Lowell.

S. (1081)

Biophys. J. 36,

775-796.

This work was supported by the North-West Regional Health Authority, the William Hewitt Trust and the Nuffield Foundation. We should like to thank Drs. R.C. Poole, A.P. Halestrap and J.D. McGivan, University of Bristol, for helpful discussion.

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