Lipolysis and potassium accumulation in isolated fat cells effect of insulin and lipolytic agents

BIOCHIMICA

486 BBA

ET BIOPHYSICA

ACTA

55692

LIPOLYSIS

AND POTASSIUM ACCUMULATION

EFFECT OF INSULIN AND LIPOLYTIC

M. TOUABI

FAT CELLS

AGENTS

AND B. JEANRENAUD

Institut de Biochimie Unique, {Received

IN ISOLATED

December

University of Geneva, Geneva (Switzerland)

Ioth, 1969)

SUMMARY

The net 42K+uptake by isolated mouse fat cells has been studied. 42K+was found to accumulate in fat cells by an energy-requiring process that was stimulated by Na+ and inhibited by ouabain at concentrations as low as I.

2.

I

PM.

3. Stimulation of lipolysis by adrenaline, by ACTH or by xanthine derivatives resulted in a marked decrease in 4eK+uptake by fat cells. The magnitude of this inhibition was proportional to that of lipolysis. 4. Insulin, even in the absence of added glucose, markedly decreased the hormone- or xanthine derivative-induced lipolysis. Concomitantly, insulin prevented the decrease in *zK+ uptake induced by these lipolytic agents. 5. It is suggested that a decrease in energy availability and/or production occurs duringlipolysis, and that it is partly responsible for the observed decrease in net dzK+ uptake. Furthermore it is thought that insulin, in the absence of added glucose, prevents such a decrease in energy availability and/or production via its antilipolytic action, thereby counteracting the drop in 42K+uptake induced by lipolytic hormones and agents.

INTRODUCTION

It is now well established that K+ plays an important role in regulating adipose tissue metabolism. Thus, lipolysis induced by adrenalinely2 or by ACTH (ref. I) has been shown to decrease upon removal of K+ from the incubation medium or upon treatment of fat cells by ouabainl. These observations have been explained in part by the fact that K+ was necessary for optimal activity of the hormone-stimulated adenyl cyclasel. In addition, it has been reported that K+ lack markedly increased glucose uptake and further metabolism3-B. Whether this stimulatory effect was related to alterations of the intracellular machinery or solely to changes in the membrane or in the transport system of glucose $er se is, as yet, unknown. B&him.

Biophys. Acta, 202 (1970) 486-495

LIPOLYSIS

AND POTASSIUM

ACCUMULATION

IN FAT CELLS

487

More recently, the handling of K+ by isolated fat cell “ghosts” has been studied. It has been observed that 4aK+ accumulated in the “ghosts” by a Na+- and energydependent process similar to that described in other cells’. The present experiments have been initiated to investigate the basic requirements of K+ uptake by the isolated fat cells. It was thought that a better understanding of this process was needed if one were to eventually understand metabolic changes such as those cited above. Furthermore, as cr-aminoisobutyric acid uptake, an accumulative and energy-requiring process, was shown to be regulated by several hormones in isolated fat cells8*8, it was of interest to see whether such a regulation would apply to Kf uptake as well. A preliminary abstract of these data has been published”‘. MATERIALS

AND METHODS

5-week-old male Swiss mice from S. Ivanovas (Kisslegg im Allgau, Germany) fed ad libitum with Altromin “R” laboratory chow (Kunath Company, Aarau, Switzerland) were used throughout these studies. All experiments involved three incubation periods. During the first one, intact epididymal adipose tissues were incubated in the presence of collagenase to isolate fat cells according to RODBELL”. Fat cells thus obtained were suspended in 1.5 ml Krebs3.5 g/roe ml dialyzed human serum Ringer bicarbonate buffer (pH 7.4) containing albumin and placed in 3o-ml stoppered plastic vials. After being gassed with a CO,-0, mixture (5 :95, by vol.), cells were preincubated for 40-60 min at 37.5” in a metabolic shaker (Gallenkamp and Co., London) in the presence of the substrates, agents or hormones under study but in the absence of 4zK+. The end of the preincubation and the beginning of the incubation periods (5-40 min) coincided with the injection of 42KC1 (0.1 ml) into each vial. Net 42K+ uptake, net free fatty acid and glycerol release into the medium were measured at the end of the incubation periods. In experiments in which lipolysis was not measured, net 42K+ uptake was studied in cells which had been previously depleted of this cation by using K+-free medium during both the incubation of intact tissue with collagenase and the preincubation periods. 4BK+ was then added together with an amount of KC1 adequate to give a final concentration of 6 mM (K++dZK+) in the incubation medium. When studying lipolysis in K+-depleted cells, it was observed that time or dose curves in the presence of lipolytic hormones, particularly when used at low concentrations, gave erratic responses. This was in keeping with the need for K+ in lipolysis, as previously reported’. For that reason, when the relationship between lipolysis and net 42K+ uptake was investigated, incubation of intact tissue with collagenase and preincubation and incubation of isolated fat cells were all carried out in normal Krebs-Ringer buffer containing 6 mM K+. Injection of 42K+ into incubation vials was then performed using trace amounts of the cation in order not to alter the final 6 mM concentration. At the time of 42K+ addition, fat cells contained thus unlabeled Kf that could exchange with 42K+ during the incubation and decrease the extracellular specific activity. As the concentration of fat cells per incubation vial ranged between 25-30 mg of dried fat cells, it was calculated, attributing the cells a concentration of 150 mM K+ and an intracellular volume of about 6 ,~l/xoo mg dried fat cellss~r2, that the total amount of unlabeled K+ that could be released (assuming an unlikely comBiochim.

Biopkys.

Acta, zzw (1970) 486-495

M. TOUABI, B. JEANRENAUD

488

plete depletion) would never exceed 0.3 ,umole/I.5 ml of incubation medium. Since this theoretical amount would only change the specific activity by 3.30/o at most, it was estimated

that the measurement

of 42K+ uptake provided a reasonable

amount of K+ taken up by the cells during the incubation ft.OYzori f or fta ce 11fit s rs preincubated in K+ free medium. Incubations

were stopped

by chilling,

periods.

the incubation

estimate

of the

This was true a

vials being transferred

from the metabolic shaker to a rack placed in iced water. All further steps were performed at o-z”. As the shaking of the cell suspension ceased, fat cells quickly floated to the surface.

This enabled the rapid removal,

when lipolytic

activity

was measured

concomitantly with net 42K+ uptake, of I ml cell-free infranatant medium from each incubation vial, using a plastic automatic pipette (Eppendorf, Hamburg, Germany). This aliquot was used both for glycerol determination previously

describeds.

fat cell suspensions transferred uptake.

Fat cells present

(when aliquots

from incubation

for lipolytic

activity

incubation

exactly

acid uptakea.

the method

were not removed) previously

Suffice it to mention

as

buffer or whole were then

vials to the washing tubes in order to measure

This was done following

n-amino/r-14Clisobutyric

and free fatty acid titration,

in the remaining

described

net 42K+ for net

that this measurement

required two successive washes of the cells with cold Krebs-Ringer albumin buffer, collection of washed cells on Millipore filters, and drying and weighing of the collected cells. Dried cells on filters were then transferred water and dispersed in the water with a Vortex

to counting vials containing IO ml shaker. The cells were counted for

their radioactive content in a Packard liquid scintillation spectrometer, model 3002. The fi particles emitted by *2K+ were fast enough to allow detection by the Cerenkov radiation produced in water*“. The concentrations of substrates,

agents or hormones

medium,

and the duration

the timing

of these additions,

with each experiment. Free fatty acids were washed and titrated

according

added to the incubation

of incubation

are specified

to DOLE AND MEINERTZ~~.

Glycerol was measured enzymatically according to WIELAND’~. Organic and inorganic compounds, hormones and agents were the same as those used in previous the Isotope

experiments8pg.

Division,

Research

42K+ was obtained

as 42KC1 aqueous

solution

from

Establishment

Riser, Roskilde, Denmark. Results have been expressed as ,umoles net 42K+ uptake and as ,umoles glycerol or free fatty acid released in the medium, per g of dried fat cells.

RESULTS As may be seen in Fig. I, 42K+ uptake

by fat cells increased

rapidly with time,

reaching a maximum in 20-40 min, a maximum that is likely to represent the steady state of the concentrative capacity for 42K+. Contrary to what has been reported for fat cell “ghosts”7, the addition of pyruvate did not influence the rate of 42K+ uptake nor its steady-state levels. This suggested that, under the experimental conditions used, fat cells contained energy-yielding stores in amounts adequate to insure optimal in uptake of the cation. The energy requirement for 42K+ uptake was demonstrated further experiments summarized by Table I. The presence of oligomycin resulted in a marked drop in 42K+ content, indicating that the maintenance of a steady-state level of 4zK+ by fat cells was dependent upon a normally operating oxidative metabolism. Biochim.

Biophys.

Acta,

202 (1970)

486-495

LIPOLYSIS AND POTASSIUM ACCUMULATION IN FAT CELLS

489

Fig. I. Time-course of azK+ uptake by isolated fat cells. Incubations were carried out in 1.5 ml Krebs-Ringer bicarbonate buffer containing 3.5 g/loo ml albumin. Cells first preincubated for 40 min in a K+-free buffer, in the presence (O---O) or in the absence (0 ---- 0) of IO mM sodium pyruvate, then further incubated under the same conditions for o-40 min following addition of azK+ (6 mM final concentration). Each point is the mean of six values f S.E. Fig. a. Effect of ouabain on 42K+ uptake by isolated fat cells. Incubations were carried out in 1.5 ml Krebs-Ringer bicarbonate buffer containing 3.5 g/roe ml albumin and IO mM unlabeled sodium pyruvate. Cells first preincubated for 40 min in a K + -free buffer, in the presence (0 --- 0) or in the absence (O---O) of ouabain (0.1 mM), then further incubated under the same conditions for 0-60 min following addition of 4aK+ (6 mM final concentration). Each point is the mean of six values + S.E. TABLE

I

ENERGY REQUIREMENT

OFNET

aaK+

UPTAKE

BY

ISOLATED

FAT

CELLS

Cells first preincubated for 40 min in 1.5 ml K+-free Krebs-Ringer bicarbonate buffer containing 3.5 g/roe ml albumin, unlabeled substrate(s) or oligomycin as indicated; then further incubated with unlabeled substrate(s) or oligomycin for 40 min following addition of azK+ (6 mM final concentration). Oligomycin, I pg/ml. Results are expressed as pmoles/g dried fat cells. Each figure is the mean of six values f SE. Oligomycin

Glucose

2-Deozyglucose

CmM)

(mMI

0

0

0

:

IO0

0

;

20 IO

0 5

+

20

IO

Net 42K+ uptake

I.93

f

0.01

0.06 f& 0.06 0.02 0.74 0.82 0.22 & 5 0.01 0.04 0.18

&

0.01

The addition of glucose to oligomycin-treated cells increased net 42K+ uptake, while the simultaneous addition of z-deoxyglucose prevented this stimulatory effect of glucose. Ouabain markedly decreased net 42K+ uptake by fat cells, an effect that bore on the initial rapid phase of 42K+uptake, as well as on the steady-state levels (Fig. 2). A very significant decrease in 42K+ content could be elicited by concentrations of ouabain as low as I ,uM. (Incubations carried out as indicated in Fig. 2., 4o-min preincubation with or without I ,uM ouabain, then further incubation for 40 min with or without ouabain following the addition of 6 mM 42K+. Control cells: 9.19 & 0.64; ouabain-treated cells: 6.16 f 0.07 pmoles/g dried fat cells; mean of six values f S.E.) In addition, the effect of the glycoside could be mimicked by decreasing the concentrations of Na+ in the incubation medium (Table II). In the next series of experiments, the influence of insulin and of lipolytic agents, singly or combined, upon the net 42K+ uptake by fat cells was investigated. Table III shows that insulin failed to modify net 42K+ uptake when added to Biochim. Biophys.

Acta,

202

(1970)

486-495

M. TOUABI, B. JEANRENAUD

490 TABLE

II

EFFECT OF Na+ ON NET 42K+ UPTAKE BY ISOLATEDFAT CELLS Cells first preincubated for 40 min in 1.5 ml K+-free Krebs-Ringer bicarbonate buffer containing 3.5 g/100 ml albumin, I mM unlabeled sodium pyruvate and various Naf concentrations as indicated; then further incubated under the same conditions for 40 min following addition of 42K+ (6 mM final concentration). Nat replaced by choline. Results are expressed as pmoles/g dried fat cells. Each figure is the mean of six values * S.E.

of

Na’ concn. the medium

Net azKf

0.32 & 0.06 0.68 & 0.03

10

36.6 84.7 ‘44.4

1.52

&

0.09

3.67 & o.rr

medium table,

uptake

(mM)

containing

insulin

unlabeled

glucose

did not alter either

added substrate.

or pyruvate.

42K+ content

This may indicate,

Although

not recorded

of cells incubated

as already

suggested

in this

in the absence

of

by Fig. I, that under the

conditions used, endogenous energy-yielding substrates were sufficient to ensure optimal 42K+ accumulation, and that insulin, with or without added substrate, was unable to alter the steady-state

levels of this cation.

When

the energy-yielding

reactions

necessary for 42K+ uptake were presumaby inhibited by oligomycin, the 42K+ content of fat cells incubated in the presence of glucose or pyruvate was dramatically curtailed. Moreover, 42K+ uptake

the further

addition

of insulin

to oligomycin-treated

towards normal when glucose, but not pyruvate,

cubation medium (Table III). The effect of a constant

concentration

of adrenaline

cells restored

was present

in the in-

(r,ug/ml) upon 42K+ uptake

by fat cells is illustrated in Fig. 3. As may be seen, adrenaline-treated cells took up markedly less 42K+ than control cells; the inhibitory effect tended to increase with increasing

duration

of incubation.

line, were qualitatively by the adipocytes.

Two xanthine

similar to adrenaline

derivatives,

in their ability

caffeine to reduce

and theophyl42K+ uptake

Furthermore,

in the absence of added glucose, insulin prevented almost completely the drop in the 42K uptake brought about by adrenaline, as well as by caffeine and theophylline (Table IV). In the next series of experiments, the relationship between lipolysis and 42K+ uptake was investigated, using experimental designs previously described for studies on a-aminoisobutyric acidg. It is apparent bation of fat cells with a fixed concentration TABLE

that increasing the duration of preincuof adrenaline (Fig.4) resulted in a progres-

III

EFFECT OF INSULIN AND ~LIGOMYCIN, SINGLY OR COMBINED,ON NET 42Kf UPTAKE BY ISOLATED FAT CELLS Cells first preincubated for 40 min in I ,5 ml K&-free Krebs-Ringer bicarbonate buffer containing 3.5 g/loo ml albumin, unlabeled substrate and agent(s) as indicated; then further incubated under the same conditions for 40 min following addition of azK+ (6 mM final concentration). Oligomycin, I pg/ml; insulin, I munit/ml. Results expressed as pmoles/g dried fat cells. Each figure is the mean of six values f SE. Net 42K+ uptake

Unlabeled substrate in the medium 10 mM glucose

IO mM sodium pyruvate B&him.

Biophys.

Acta,

contvo1s

Olzg0my&

I?WGVZ

Oligomycin

4.82 & o.r7 4.59 * 0.17

I.44 * 0.05 0.38 & 0.02

4.48 + 0.07 4.58 i 0.17

4.0’ & 0.26 0.19 + 0.02

202 (1970)

486-495

+ insulin

LIPOLYSIS AND POTASSIUM ACCUMULATION IN FAT CELLS

0

15

30 Time (min)

45

$0

491

0

15

30 Time(min)

60

Fig. 3. Effect of adrenaline on a*K+ uptake by isolated fat cells. Incubations were carried out in 1.5 ml Krebs-Ringer bicarbonate buffer containing 3.5 g/1oo ml albumin and IO mM unlabeled sodium pyruvate. Cells first preincubated for 30 min in a K+-free buffer, in the presence (0 ---- 0) or in the absence (O-O) of adrenaline (I pg/ml), then further incubated under the same conditions for o-60 min following addition of raK+ (6 mM final concentration). Each point is the mean of six values * S.E. Fig. 4. Relationship between adrenaline-induced lipolysis and net rzK+ uptake by isolated fat cells, as a function of time. Incubations were carried out in I .5ml Krebs-Ringer bicarbonate buffer containing 3.5 g/100 ml albumin and 1 mM unlabeled sodium pyruvate. Cells first preincubated for 0-60 min in the presence of adrenaline (I pg/ml), then further incubated under the same conditions for 5 min following addition of trace amounts of aeKf. Cumulative glycerol or free fatty acid release during preincubation plus incubation. @K+ uptake measured during the g-min incu4aK; o----o,glycerol; o-‘-*-o, bation. Each point is the mean of twelve values & S.E. o-o, free fatty acids. TABLE EFFECT FAT

IV OFADRENALINE,XANTHINEDERIVATIVESANDINSULIN

ON

NET

azK+

UPTAKE

BYISOLATED

CELLS

Cells first preincubated for 40 min in 1.5 ml K+-free Krebs-Ringer bicarbonate buffer containing 3.5 g/1oo ml albumin, 10 mM unlabeled sodium pyruvate and agent(s) as indicated; then further incubated with pyruvate and agent(s) for 40 min following addition of 4aK+ (6 mM final concentration). Insulin, 1 munit /ml; adrenaline, 1 pg/ml; caffeine IO mM; theophylline, 1 mM.Results are expressed as pmoles/g dried fat cells. Each figure is the mean of 6 values f SE. Agent Insulin Adrenaline Adrenaline + insulin Insulin Caffeine Caffeine+insulin Theophylline Theophylline + insulin

Net 4=Kf uptake 4.59 4.58 1.53 3.51 4.21 4.47 0.80 2.86 0.80 4.28

+c 0.17 & 0.17 + 0.07 * 0.12 * 0.11 zt 0.12 * 0.05 & 0.23 * 0.07 * 0.05

sive increase in glycerol and free fatty acid release. Moreover, as lipolysis increased with time, net 4zK+ uptake progressively decreased. A similar inverse relationship between lipolytic activity and net 42K+ uptake was observed when the concentration of adrenaline in the medium was increased, the incubation time being kept constant Biochim. Biophys. Acta,

202

(1970)

486-495

M. TOUABI, B. JEANRENAUD

492

-l

Fig. 5. Effect of increasing concentrations of adrenaline on lipolysis and net 42K+ uptake by isolated fat cells. Incubations were carried out in 1.5 ml Krebs-Ringer bicarbonate buffer containing 3.5 g/roe ml albumin and I mM sodium pyruvate. Cells first preincubated for 45 min in the presence of o-1.0 pg/ml adrenaline, then further incubated under the same conditions for 40 min following addition of trace amounts of azK+. Cumulative glycerol or free fatty acid release during preincubation plus incubation. 42K+ uptake measured during the 4o-min incubation. Each point is the mean of twelve values + SE. o--o, d*K; O----O, glycerol: O-‘-.-O, free fatty acids.

(Fig. 5). Results identical to those described in Figs. 4 and 5 were obtained when ACTH (0.2-1.0 pg/ml) or caffeine (0.75-6.0 mM) were used instead of adrenaline. To see whether intracellular levels of free fatty acids and/or their derivatives might be partly responsible, as previously suggestedayg, for the inhibitory effect of lipolytic agents, experiments were designed in which intracellular free fatty acid levels were presumably increased while lipolysis was not further stimulated (Table V). It was assumed that, when using a fixed concentration of adrenaline (0.~5 pg/ml), i.e. when dealing with a constant lipolytic activity, a decrease in the extracellular concentration of albumin might result, by producing less binding sites for free fatty acids, in a higher concentration of free fatty acids within the cells. Although intracellular free fatty acids were not measured during these experiments, it is evident (Table V) that a concentration of adrenaline that was ineffective in decreasing 42K+ uptake when added to the medium containing 3.5 g/roe ml albumin did become inhibitory when added to the medium containing a low concentration of albumin. The next series of experiments was carried out to study the influence of insulin upon both lipolysis and 42K+ uptake in fat cells treated with lipolytic hormones or agents. The data summarized in Table VI shows that insulin, in the presence of either TABLE EFFECT

\’ OF

INHIBITORY

LOWERING ACTION

THE OF

ALBUMIN

ADRENALINE

CONCENTRATION OK

NET

IN

THE

42K+ UPTAKE BY

INCUBATION ISOLATED

MEDIUM FAT

UPON

THE

CELLS

Cells iirst preincubated for 40 min in I .5 ml K+-free Krebs-Ringer bicarbonate buffer containing .albumin as indicated, IO mM unlabeled sodium pyruvate and adrenaline (0.25 pg/ml) ; then further incubated under the same conditions for 40 min following addition of 42K+ (6 mM final concentration). Results are expressed as percent of controls. Each figure is the mean of six values & S.E. HlWmCWle

__. Adrenaline Adrenaline

Albumin concn. of the medium (g/100 ml)

3.6

Biophys.

Acta,

.. 100 + ‘3.4

96 & 8.7* IOO f 7.6

3.6 0.45 0.45

74.2

* Difference from control: ** Difference from control: Biochim.

aaK+ uptake (% of control)

202

not significant. P < 0.01. (1970) 486-495

&

4.0**

LIPOLYSIS AND POTASSIUM ACCUMULATION IN FAT CELLS TABLE EFFECT ISOLATED

493

VI OF ADRENALINE, FAT CELLS:

ACTH

AND

ITS PREVENTION

XANTHINE

DERIVATIVES

ON LIPOLYSIS

AND

raK+

UPTAKE

BY

BY INSULIN

Cells first preincubated for 50 min in 1.5 ml normal Krebs-Ringer bicarbonate buffer containing 3.5 g/100 ml albumin, unlabeled substrate and hormone(s) as indicated; then further incubated under the same conditions for 30 min following addition of trace amount of azKf. Adrenaline, 0.5 ,ug/ml; ACTH, I ,wg/ml; caffeine, 1.5 mM; theophylline, I mM; insulin, I munit/ml. Results are expressed as pmoles/g dried fat cells. Each figure is the mean of twelve values 5 S.E. Agent

Unlabeled substrate in the medium (I mM)

Net 42K+ @take

Net glycerol release

Net free fatty acid release

Pyruvate

1.21 & 0.06 0.4’ * 0.02 * 0.04 0.93 I.33 * 0.01 0.69 & 0.04 1.21 & 0.03 I.55 xt 0.03 0.62 & 0.06 1.26 k 0.03 1.26 & 0.05 0.65 & 0.05 3.3r * 0.18 I.45 i 0.09 3.41 & 0.06 & 0.05 1.60 0.90 k 0.06 I.55 * 0.10

8.04 52.40 32.99 5.74 49.51 39.05 7.08 52.42 39.40 8.36 58.41 39.38 1.64 63.43 37.64 1.66 57.02 37.76

2.38 62.59 37.23 13.73 129.17 62.76 5.42 65.06 44.24 5.11 74.73 26.84 4.48 179.4

agents-treated

cellstinsulin:

Adrenaline Adrenaline + insulin Glucose Adrenaline Adrenaline+insulin Pyruvate ACTH ACTH + insulin Glucose ACTH ACTH + insulin

1.16

Pyruvate Caffeine Caffeine + insulin Pyruvate Theophylline * Theophylline+insulm * Lipolytic agents-treated measurements.

cells ZJS.lipolytic

&

0.09

* zt i f zk + 5 & * * 5 i * zt 5 & * *

0.31 0.80 1.64 2.16 2.03 1.86 0.81 3.73 I.96 0.92 I.29 4.41 0.52 5.19 115.97 f 6.61 1.82 * 0.11 133.35 zt 3.11 86.01 * 6.93

0.41 0.54 2.45 1.13 0.87 0.57 1.66 0.41 0.74 0.86 2.68 2.84 0.16 6.72 2.37 0.13 3.29 2.44 P

<

0.001

* f * & * f & & zt * * f * &

for all

pyruvate or glucose, did significantly prevent the drop in 42K+ uptake brought by adrenaline, ACTH, caffeine or theophylline. Of further importance was the finding that insulin concomitantly decreased the release of both glycerol and free fatty acids induced by all lipolytic hormones or agents tested. DISCUSSION

These experiments demonstrate that isolated fat cells take up 42K+, as previously shown for fat cell “ghostsJJ7. As reported for cr-amino[r-‘*C]isobutyric acid uptake by fat cells *pa,significant daily variations in the absolute values of 42K+ content were observed. The reason for these variations is not known, but comparisons were therefore always made within one experiment. The finding that ouabain decreased 42K+ content of the aclipocytes at concentrations known to specifically inhibit the active coupled transport of Naf and K+ in other cells+ and the observations that 42K+ uptake was stimulated by Na+ and was energy-dependent provide good, although indirect, evidence that 42K+ content at steady state does represent an accumulation of the cation against a concentration gradient. In these studies, actual concentrations of intracellular K+ were not measured because of the technical difficulties involved in accurately determining intracellular water space of isolated fat cells 8,12. However, considering a maximal 42K+ uptake of 5 pmoles/g dried fat cells, it could be calculated that the concentrative capacity of the adipocytes was between 50 mM (assuming an intracellular water space of IOO ,ul/gdried fat cells)* and 125 mM (water space of 40 ,d/g dried fat cells)12. This Biochim. Biophys. Acta,

202

(1970)

486-495

M. TOUABI,

494

B. JEANRENAUD

clearly indicates that intracellular K+ is considerably higher than extracellular Kf and that accumulation against a concentration gradient does occur. Net 42K+ uptake by isolated fat cells appears to be modulated by various hormonal stimuli. Adrenaline, ACTH, and xanthine derivatives were markedly inhibitory (Table VI). Insulin, when present alone in the incubation medium, influenced 42K+ uptake only when the energy supply was limited, e.g. in the presence of oligomycin. Under these conditions, insulin restored 42K+ uptake towards normal when glucose, but not pyruvate, was present (Table III). This is consistent with an effect of the hormone upon glucose entry and further metabolism, the needed energy being presumably supplied via the glycolytic pathway. This concept is strengthened by the observation that the addition of glucose to oligomycin-treated cells resulted in an effect that was qualitatively similar to that of insulin, and that it could be prevented by simultaneous addition of 2-deoxyglucose (Table I). However, experiments in which insulin markedly counteracted, in the absence of glucose, the inhibitory effect of lipolytic hormones or agents upon 42K+ uptake (Tables IV and VI) do indicate that the effect of the hormone is not solely related to glucose metabolism but also to its well-substantiated though ill-understood antilipolytic actionr7. These studies also demonstrate that there exists a striking inverse relationship between lipolysis and 42K+ uptake by fat cells. Thus, when free fatty acid and glycerol release was stimulated by adrenaline, caffeine or theophylline, net 42K+ uptake was reduced (Figs. 4 and 5, Table VI). On the contrary, when adrenaline- or xanthine derivative-induced lipolysis was inhibited by insulin, 42K+ uptake was within normal control values (Table VI). It is possible that the energy necessary for 42K+ accumulation may be the clue as to the nature of the opposing effects of insulin and lipolytic agents. Indeed, it should be mentioned that most changes in 42K+ uptake observed during this study can be rather satisfactorily related to changes in ATP levels measured in isolated fat cells incubated under similar conditions I*. As discussed in the preceding paper9 ATP depletion during lipolysis, although unknown in its origin, might be related in part to one or a combination of the following factors: (a) cyclic AMP formation; (b) interference with ATP production by free fatty acids or some other product(s) of lipolysis; (c) enzyme protein release with secondary alteration in the intracellular machinery. This latter possibility has been considered to be an unlikely one under the experimental conditions used in these studiess. Furthermore, nonspecific or structural damage of the fat cells during lipolysis ilz vitro has been ruled outa. Finally, it is possible that the regulation of K+ uptake in fat cells may have some functional link with that of glucose. It has been shown that K+ lack resulted in a stimulation of glucose metabolism in isolated fat cells 3-6. On the other hand, lipolytic hormones as well as fatty acids have also been reported to increase glucose metabolism when added to adipose tissue or isolated fat cells incubated in a high (3-4 g/roe ml) albumin medium311+21. As lipolytic activity appears, from these data, to result in a lowering of the K+ content of the adipocytes, it is conceivable that such a decrease in K+ might be partly responsible for the observed stimulation of glucose metabolism brought about by the lipolytic hormones. Also, because of the known influence of Kf on the lipolytic processryz, the observed changes in 42K+ content within fat cells may possibly be involved in the fine regulation of lipolysis, of which, despite extensive investigations (reviewed in refs. 17 and 22), rather little is known as yet. Biochim.

Biophys.

Ada,

xn

(1970) 486-495

LIPOLYSIS

AND POTASSIUM

ACCUMULATION

IN FAT CELLS

495

ACKNOWLEDGMENTS

The authors are greatly indebted to Misses E. Hiimmerich and T. Brsndle for their excellent technical assistance. This study was supported by Grant No. 4848.3 from the Fonds National Suisse de la Recherche Scientifique, Berne, Switzerland. REFERENCES

I R. J. Ho, B. JEANRENAUD, TH. POSTERNAK AND A. E. RENOLD, Biochim. Biophys.

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