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Electrolyte-activated lipolysis in vitro: Modifying effect of calcium
Electrolyte-Activated Lipolysis In Vitro: Modifying Effect of Calcium By SHELDONJ. BLEICHER, LEONARDFARBER, ARTHUR LEWIS AND MARTIN G. GOLDNER In vitro experiments, employing rat epididymal fat tissue, have shown spontaneous lipolysis to be induced by Cafree media containing high K concentrations. Electrolyte-activated lipolysis proceeds linearly over a 2 hour incubation period and is not affected by omission of albumin from incubation media. Inclusion of 2.5 mEq./L. Ca in the medium sensitizes adipose tissue to low concentrations of K but results in a biphasie response, wherein lipolysis in all-K media is less than in mixed N a / K media. Neither Mg nor buffer anions were found to play any role in the response, except that in all-PO4 buffers lipolysis developed at lower K eoneen-
tratlons than in Ca-free C1-HCO3 buffers; a similar effect was achieved by adding EDTA to CI-HCO 3 buffer. Cysteine did not modify the response to Ca. Although the response to Na/K buffers in the presence of Ca is not specific, in that similar effects were achieved in mixed Na/choline buffers, extrapolation to the in vivo state suggests that Na/K flux may play a role in regulating the basal state of adipose tissue, in modifying the capacity to respond to lipalytie stimuli, and serve in orienting the tissue in generally anabolie or catabolic directions. (Metabolism 15: No. 8, August, 742-748, 1966)
D I P O S E T I S S U E M E T A B O L I S M , in normal or diabetic animals, is generally viewed in terms of the direct interplay of hormones. The possibility exists, however, that additional factors affect the "basal" activity of this tissue and modify its responsiveness to hormones. For this reason, investigations of the effects of electrolytes on in vitro lipolysis were initiated. It was found that certain rnono- and divalent cations exhibited considerably different effects on spontaneous lipolysis, w h e n each constituted the sole cation present in the incubation medium. 1 K, of all the cations studied, h a d the greatest lipolytic potency; Cs, NH4 and choline were equal to each other in lipolytic effect but less active than K; Ca and Na, themselves equal in lipolytic efficacy, were appreciably less active than any of the preceding ions; while Li and Mg From the Metabolic Research Unit, Department of Medicine, Jewish Hospital of Brooklyn, and the Department of Medicine, Downstate Medical Center, State University of New York, Brooklyn, New York. Supported by National Institutes of Health Grant AM-08102, T1 AM-5338, FR-5503; and a Grant-in~Aid from the Ciba Pharmaceutical Co., Summitt, New Jersey. Received Mar. 18, 1966. SHELDONJ. BLEICUER, M.D.: Physician-in-Charge, Metabolic Research Unit, The Jewish Hospital of Brooklyn, Assistant Professor of Medicine, State University of New York, Downstate Medical Center. ARTHURLEWIS, M.S.: Laboratory Technician, Metabolic Research Unit, The Jewish Hospital of Brooklyn. LEONARDFARBE~, B.A.: Fourth year medical student, Downstate Medical Center, State University of New York (presently, intern at Boston University Medical Service, Boston City Hospital). MARTIN G. GOLDNER,M.D.: Director, Department of Medicine, The Jewish Hospital of Brooklyn, Professor of Medicine, State University of New York, Downstate Medical Center. 742
743
ELECTROLYTE-ACTIVATED LIPOLYSIS
were least active. The marked differences among the physiologically inportant cations, Na, K, Ca and Mg, suggested possible interrelated activities and led to the studies comprising this report. The data presented here demonstrate that spontaneous lipolysis in surviving fragments of rat epididymal fat tissue m a y be markedly affected by the ratios of Na and K in incubation media, and that this response is modified b y the presence of Ca. METHODS Standard buffer mixtures were prepared in which Na and K content of each cation (expressed in mEq./L.) was reciprocally varied as follows: Na/K, 154/0; 103/51; 51/103; and 0/154. Anionic composition of the buffers, in the absence of albumin, was 154 mEq./L. PO4, or a mixture of "25 mEq./L. HCO 3 and 129 mEq./L. CI. Glucose was omitted from all media. When albumin was present in the media it had been defatted by extraction with Dole's extraction mixture, 2 washed with ethanol and ether, and redissolved in water distilled from glass to a final concentratiin of 2 Gin/100 ml. Solution of the albmnin was accomplished by the cautious addition of 4N NaOH or KOH, to provide a Na or K medium, respectively. After the albumin had been dissolved and pH adjusted to 7.6, 25 mEq./L. NaHCO3 or KHCOz was added. Na or K content of the albumin solution was determined by flame photometry, and sufficient NaC1 or KC1 added to bring the cation content to 154 mEq./L. When added, Ca was present at 2.5 mEq./L. (chloride salt) unless otherwise stated; in one experiment with Na/K 103/51, lO-2M cysteine or 2.5 mEq./L. Ca were added, individually or together, while in another experiment the effect of 1 mg./ml. EDTA* in this medium was observed. Albumin-free TRIS{ buffer was employed when studying the effect of 2.5 mEq./L. Ca in Na/K or Na/Choline* 103/51 media, and also in the preparation of 300 mM sucrose and Na buffers. Epididymal fat tissue was obtained from fed Wistar rats, killed by stunning and decapitation. Each fat pad was longitudinally bisected, one fragment serving as control for the other 3 fragments from the same rat. Incubation and processing of tissues and media, and statistical analysis of results, were as previously reported.~ RESULTS
Typical experiments with varied proportions cf Na and K in C]-HCOs buffer, in the absence of Ca and the presence or absence of albumin, are presented in Table 1 (lines A and B): significant lipolysis developed at those K eoncentrations greater than N a / K 103/51, and was maximal in an all-K medium. Lipolyric response to K was the same in the absence of albumin (thus preventing release of free fatty acids from the tissue) z or in its presence. Replaeement of C1-HCO3 and PO4 buffer, free of Ca and albumin ( T a b l e 1, line C), produced a pattern of inereasing lipolysis with increasing K concentrations similar to that seen in C1-HCO~ buffer, differing only in as much as significant lipolysis was now induced at a lower K eoneentration. The presence of 1 mg./ml. E D T A in a Ca-free CI-HCO,~ m e d i u m of N a / K 103/51 was associated with a signifieant increase in lipolysis above control of 197 per eent (control -- N a / K 103/51, 2.33 IxM/Gm/2 hours; E D T A present, 4.62 / x M / G m . / 2 hours; N = 6, P < 0.01). This should be c o m p a r e d with the response to the same N a / K mixture in an all PO4 buffer (Table 1, line C, increase of 206 per eent, P < 0.01 ). *EDTA, Ethylenediamine tetracetate, sodium salt, purchased from Eastman Chemical Company. CTBIS, 0.025 M, pH 7.40, THIS(Hydroxy)Aminomethane, Sigma Chemical Company. ~Choline chloride, Sigma Chemical Company.
744
B L E I C H E t l , FARBElt, L E W I S AND GOLDNER
Table 1.--Effect of Ca or Albumin on Lipolytic Response to Na and K Experimental Group
A
Buffer Cation Mixtures (mEq./L.) No. of Rats N a / K
154/0
103/51
51/103
2.178 2.10 3.01 --+0.05 • • B 7 2.59 3.11 3.98 • • • C 6 2.15 4.36 6.26 • • • D 6 2.17 7.06 9.94 • • • E 6 2.12 8.57 8.20 • • • ~Values are total free fatty acids generated, tion • 1 S.E.
0/154
5
Albumin Calcium
Anions
8.07
--
--
C1-HCO s
8.82
q-
--
C1-HCOz
7.62
--
--
PO4
6.66
--
+
CI-HCO3
• • • • 5.61 + + C1-HCO3 • expressed as ~M/Gm. fat/2 hour incuba-
Incubation of fat tissue in Na-K media containing 2.5 m E q . / L . Ca changed the pattern of lipolytie response to a biphasic one (Table 1, lines D and E). Free fatty acid production in Ca-supplemented N a / K 103/51 medium was significantly increased above that in all-Na medium, but lipolysis in an all-K medium was substantially lower than that in N a / K 51/103. Again, the presence or absence of albumin did not modify this response to added Ca (Table 1, lines D and E ). CI-HCO3 buffers with 2.5 mEq./L. Ca present, of the following N a / K composition, were tested: 154/0; 149/5; 77/77; 23/131; 0/154. The results (Fig. 1A) show a broad plateau of lipolytic response extending over the region N a / K 103/51 to N a / K 23/131, with significant decline from the plateau at N a / K 0/154. The presence of so little as 5.0 m E q . / L . K in a medium containing 149 mEq./L. Na and 2.5 mEq./L. Ca was associated with a small but significant increase in free fatty acid release over that seen in 154 m E q . / L . Na and 2.5 mEq./L. Ca ( N a / K 154/0, 1.27 -+- 0.17 ttM free fatty acid/Gm, fat/2 hours, N = 12; N a / K 149/5, 1.77 -----0.13/xM/Gm./2 hours, N = 33; 0.05 < P < 0.02). Because the effect of Ca on lipolysis appeared to differ markedly at N a / K 0/154 and N a / K 103/5i, these 2 points were subjected to careful re-evaluation. The addition of 2.5 mEq./L. Ca to a medium containing N a / K 103/51 increased free fatty acid release by 240 per cent (N = 7, P < 0.02), while the addition of 2.5 mEq./L. Ca to a medium containing N a / K 0/154 diminished lipolysis by 18 per cent (N = 7, P < 0.05). In other experiments, the depressant effect of Ca on lipolysis in all-K media, while somewhat variable, has ranged up to 40 per cent. While inclusion of 2.5 mEq./L. Ca modified K-induced lipolysis, addition of this or higher concentrations of Ca failed to show any such effect in an all-Na medium, and showed no greater effect in N a / K 103/51: N a / K 154/0, no Ca, 2.51 ixM/Gm./2 hours; 2.5 m E q . / L . Ca, 2.35 /xM/Gm./2 hours; 15 m E q . / L . Ca, 2.39 tzM/Om./2 hours; N a / K 103/51, no Ca, 2.88 IzM/Gm./2 hours; 2.5 mEq./L. Ca, 6.70 /zM/Gm./2 hours; 15 m E q . / L . Ca, 6.92 IxM/Gm./2 hours. When Ca concentrations of 15 mEq./L, were employed, turbidity developed
ELECTROLYTE-ACTIVATED
745
LIPOLYSIS,
CALCIUM
CALCIUM NOT PRESENT
PRESENT
O.
170-
(s)
160 -
.J 150-] r
T t~ S.E.
14.0 -
i 130-' o
"
120
(2S)
2
t-Z
"'
ItO-
n
IO0"q 125)
= W
/
o')
,,,
90-
"' J
80-
u') ,,:Z
tu nO
70-
(D
< >b-
$)
60-
50uJ 4 0 -
w
t
(12:)
t/.. .[
~{(12)
I
I
51
0
20 I0
ol-9- , 154
131
I
103
|
I
77
51
I I
$
I
0
Na
154
6
103
mEq/L
Fig. 1.--Lipolytic response to varying ratios of Na/K in the presence (a) o r absence (b) of 2.5 mEq./L. Ca, All experiments were conducted in albumin-free CI-HCO 3 media, except for those represented by the dashed line (b) in which Cafree a]l-PO4 buffer was employed. Nmnbers in parentheses refer to the number of animals contributing to each point. All experiments contained one flask of Na/K 0/154, with or without Ca as appropriate, to serve as control (Na/K 0/154 = 100 per cent). Absolute free fatty acid release at Na/K 0,,/154 in the presence of Ca, however, was lower than in its absence (see text). Free fatty acid release in Na/K 154/0 was equivalent in both media (see text). during incubation indicating the precipitation of CaCO3 and, therefore, the value of 15 m E q . / L , refers to the initial C a concentration. Inclusion of 10- 2 M eysteine failed to modify either basal lipolysis in N a / K 103/51, or the augmenting effect noted on the addition of 2.5 m E q . / L . Ca: control, 2.06 /xM/Gm./2 hours; cysteine added, 2.10 /.~M/Gm./2 hours; Ca added, 4.69
746
BLEICHER, FARBER, LE~VIS AND GOLDNER
/xM/Gm./2 hours; cysteine and Ca added, 4.47 #M/Gm./2 hours; N = 6; P for effect of Ca above control, alone or with cysteine, < 0.02. Addition of 2.5 mEq./L. Ca to media containing either Na/K 103/51 or Na/choline 103/51 (TR1S buffer) resulted in greater than a twofold increase in lipolysis: Na/K 103/51 control, 1.42 /xM/Gm./2 hours; with Ca, 3.84 /zM/Gm./2 hours; Na/choline 103/51 control, 1.41/xM/Gm./2 hours; with Ca, 3.38 tzM/Gm./2 hours; N = 6; P for Ca in either Na/K or Na/choline medium < 0.02. Mg at a concentration of 2.0 mEq./L., alone, or in combination with 2.5 mEq./L. Ca, had no effect on lipolysis in Na/K 103/51: no additions, 3.15 /zM/Gm./2 hours; Ca, 9.75 tzM/Gm./2 hours; Mg, 2.95 txM/Gln./2 hours; Ca + Mg, 11.3/xM/Gm./2 hours; P value for Ca xersus Ca + Mg > 0.10, N = 6. Spontaneous lipolysis in isosmotic NaC1 or sucrose, in TRIS buffer, was found equivalent: NaC1, 1.41/xM/Gm. fat/2 hours; sucrose, 1.43/zM/Gm. fat/2 hcurs; N = 12; P > 0.80. Linearity of lipolysis over a 2 hour period, in either Na/K 154/0 or Na/K (devoid of Ca and Mg), was established by incubating epididymal fat fragments in appropriate media for 1 hour and then transferring them to fresh media for a second hour of incubation. In 5 such experiments, each performed in duplicate, free fatty acid release into the medium at the end of the first hour of incubation was 52.9 per cent of total release in Na and 50.2 per cent of the total release in K. These did not differ significantly from each other (P > 0.50), nor was the release in either medium significantly different from 50 per cent release in the first hour (P for Na > 0..80, P for K > 0.20). Although there was no appreciable difference in the fractional release of free fatty acids (i.e., medium free fatty acids/medium + tissue free fatty acids) into Na medium from that into K medium (P > 0.50), lipolysis was greater in the K medium: Na, 2.67/xM/Gm. fat/2 hours; K, 6.85/zM/Gm. fat/2 hours; N = 5; P < 0.01. DISCUSSION These experiments demonstrate a reciprocal effect of K and Na on spontaneous lipolysis in rat epididymal fat tissue in vitro. Comparable effects are observed whether or not albumin is present in the incubation medium to serve as free fatty acid acceptor. The latter observations, as well as those dealing with fractional release of free fatty acids in Na or K media, indicate that the phenomena observed are related to the lipolytic process per se and not to the transfer of generated free fatty acids from tissue to medium. Lipolysis induced in either all-Na or all-K media proceeds linearly over a 2 hour incubation period, suggesting that the lipolytic effects observed are not due to the sudden disruption of tissue when introduced into unphysiologic media. Lipolysis is maximal in all-K media and decreases with decreasing K concentrations, until baseline levels are reached at Na/K 103/51 and below. A biphasic response to K is induced if 2.5 mEq./L. Ca are added to the media. While Ca does not affect lipolysis in an all-Na medium, it significantly potentiates the lipolytie response to so little as 5 mEq./L. K. Over the region Na/K 77/77 to 23/131 lipolysis is maximal when Ca is present, but declines at higher K concentrations.
ELECTROLYTE-ACTIVATED LIPOLYSIS
747
Anions play little or no role in electrolyte-induced lip0iysis, since the response to K is seen with PO4, C1-HCOa, or TRIS-C1 buffers) In PO4 buffers, however, increasing K concentrations evoke a virtually linear lipolytic response, contrasting with the lag in response noted in Ca-free C1-HCO3 buffer (Fig. 1B). Conceivably, removal of membrane Ca by complex formation with PO4 might alter (i.e., increase) membrane permeability to K. Such an increase in permeability, in the absence of Ca, has been noted with erythrocytes4 and Necturus kidney slices. ~ Further support for this view is found in the virtually identical increase in lipolysis in the same medium, Ca-free Na/K 103/51, in an all PO4 buffer or in a C1-HCO~ buffer containing i mg./ml. EDTA. It is paradoxical, then, that inclusion of Ca in C1-HCO3 media permits prompt lipolysis in response to low concentrations of K. This paradox may be apparent rather than real, in that incubation in Ca-flee C1-HCO3 media could wash Ca from a critical intracellular site and make the lipolytic mechanism afl'ected by K less sensitive but not wholly unresponsive. This interpretation is supported by the markedly different slopes of response at lower K concentrations in PO4 as opposed to Ca-supplemented C1-HCO.~ buffers (Fig. 1, and Table 1, lines C and D). Although the site of action of Ca is not presently known, failure of cysteine to modify the augmentation in lipolysis seen on the addition of Ca suggests that this effect is not on membrane sulfhydryl groups. Further, Rizack has shown that Ca may substitute for adenosine 3', 5'-phosphate (cyclic AMP) in activating a cell-free epinephrine-sensitive lipolytie system when ATP and Mg are also present. 6 Failure of Mg to activate or augment lipolysis in the experiments reported here does not exclude a role for this ion in the lipolytic process activated by K, since Mg may be firmly attached at its site of activity, not depleted by the methods applied here, and consequently no effect noted upon its addition. The lipase system or systems activated by K are presently unknown. Rizack 7 and Vaughan, Berger and Steinberg s have extracted lipolytic systems from rat epididymal fat. While both preparations presumably contain monoglyceridase activity, that of Vaughan and co-workers also contains an hormonally-responsive triglyceridase. As the latter preparation was obtained by homogenizing fat in 0.15 M KC1, it would seem plausible that the lipolytie activation demonstrated with K in surviving tissue fragments is similarly, at least in part, the hormonally-responsive triglyceridase. It may be asked whether these results demonstrate lipolytic activation in response to decreasing concentrations of Na or to increasing concentrations of K, since in these experiments the ions were varied reciprocally. The absence of Na from incubation media does not simultaneously induce lipolysis since, as has been previously demonstrated, 1 the cation replacing Na determines the direction of response: less lipolysis than that in Na buffers is seen with Mg or Li solutions, while lipolysis greater than that in Na is seen with NH4, choline or Cs solutions. Further, equal spontaneous lipolysis was found in isosmotic sucrose or Na solutions. Electrolyte-influenced lipolysis should not, then, be regarded in the general sense as limited to Na and K. Since Ca augments lipolysis in Na/choline 103/51 as well as Na/K 103/51, reservations regarding Na-K specificity also apply to this phenomenon. Nevertheless, in the restricted
748
BLEICIIER, FARBEB,~ LEWIS AND GOLDNEIR
setting pertinent to most functioning biological systems, the strikingly divergent and sensitive response to 2 physiologically important ions, Na and K, in the presence of a third, Ca, would suggest some reciprocal role for the former in affecting lipolysis directly (perhaps mediated by hormonal effects on Na-K flux), or in modifying hormonally-activated lipolysis. Support for the latter is found in the observation of Lopez, White and Engel, 9 that the in vitro lipolytic response to A C T H and epinephrine was augmented by the inclusion of K in the incubation medium. While the experiments reported here have been concerned with lipolysis, certain observations in the literature regarding the effect of electrolytes on other aspects of adipose tissue metabolism should be noted. Leonards, Landau and Bartsch 1~ have shown that glucose uptake is increased by incubation of epididymal fat in K-free media (confirmed in this laboratory, unpublished observations), and that lipid synthesis is promoted under these conditions. The lipolytic response to Na-K mixtures interestingly parallels the Na-K activation of ATPase, 11 the latter thought to play a role in active cation transport. These presently unconnected observations suggest that cation flux may affect adipose tissue energy stores, and serve to link and correlate metabolic processes in this tissue. ADDENDUM
With regard to the suggestion, above, that lipolysis may be "mediated by hormonal effects on Na-K flux," we have determined that K plays a critical role in lipolysis induced by growth hormone (Potassium Dependence of Growth Hormone Activated Lipolysis In Vitro, J. Clin. Invest. 45: In press, 1966 (Abstract)) and by TSH, ACTH, and epinephrine in vitro. REFERENCES
1. Bleiclaer, S. J,, Lewis, A., Farber, L., and Goldner, M. G.: Effect of monoand divalent cations on in vitro lipolysis. Proe. Soc. Exp. Biol. Med. 121: 980, 1966. 2. Dole, V. P.: A relation between nonesterified fatty acids in plasma and the metabolism of glucose. J. Clin. Invest. 35:150, 1956. 3. Reshef, L., Shafrir, E., and Shapiro, B.: In vitro release of unesterified fatty acids by adipose tissue. Metabolism 7:723, 1958. 4. Maize/s, M.: In A. Kleinzeller and A. Kotyk (Eds.): Membrane Transport and Metabolism. London, Academic Press, 1961, p. 257. 5. Solomon, A. K.: Ibid, p. 98. 6. Rizack, M. A.: Aetivat'on of an epinephrine-sensitive lipolytic activity from adipose tissue bg adenosine-3'5'-phosphate. J. Biol. Chem. 239:392, 1964.
7 . - - : An epinephrine-sensitive lipolytie activity in adipose tissue. J. Biol. Chem. 236:657, 1961. 8. Vaughan, M., Berger, J. E., and Steinberg, D.: Hormone-sensitive lipase and monoglyceride lipase activities in adipose tissue. J. Biol. Chem. 239:401, 1964. 9. Lopez, E., White, J. E., and Engel, F. L.: Contrasting requirements for the lipolytic action of eorticotropin and epinephrine on adipose tissue in vitro. J. Biol. Chem. 234:2254, 1959. 10. Leonards, j, R., Landau, B. R., and Bartsch, G.: Assay of insulin-like activity with rat epididymal fat pad. J. Lab. Clin. Med. 60:552, 1962. 11. Skou, j. c.: Enzymatic basis for active transport of Na and K across cell membrane. Physiol. R e v . 45:596, 1965.
tratlons than in Ca-free C1-HCO3 buffers; a similar effect was achieved by adding EDTA to CI-HCO 3 buffer. Cysteine did not modify the response to Ca. Although the response to Na/K buffers in the presence of Ca is not specific, in that similar effects were achieved in mixed Na/choline buffers, extrapolation to the in vivo state suggests that Na/K flux may play a role in regulating the basal state of adipose tissue, in modifying the capacity to respond to lipalytie stimuli, and serve in orienting the tissue in generally anabolie or catabolic directions. (Metabolism 15: No. 8, August, 742-748, 1966)
D I P O S E T I S S U E M E T A B O L I S M , in normal or diabetic animals, is generally viewed in terms of the direct interplay of hormones. The possibility exists, however, that additional factors affect the "basal" activity of this tissue and modify its responsiveness to hormones. For this reason, investigations of the effects of electrolytes on in vitro lipolysis were initiated. It was found that certain rnono- and divalent cations exhibited considerably different effects on spontaneous lipolysis, w h e n each constituted the sole cation present in the incubation medium. 1 K, of all the cations studied, h a d the greatest lipolytic potency; Cs, NH4 and choline were equal to each other in lipolytic effect but less active than K; Ca and Na, themselves equal in lipolytic efficacy, were appreciably less active than any of the preceding ions; while Li and Mg From the Metabolic Research Unit, Department of Medicine, Jewish Hospital of Brooklyn, and the Department of Medicine, Downstate Medical Center, State University of New York, Brooklyn, New York. Supported by National Institutes of Health Grant AM-08102, T1 AM-5338, FR-5503; and a Grant-in~Aid from the Ciba Pharmaceutical Co., Summitt, New Jersey. Received Mar. 18, 1966. SHELDONJ. BLEICUER, M.D.: Physician-in-Charge, Metabolic Research Unit, The Jewish Hospital of Brooklyn, Assistant Professor of Medicine, State University of New York, Downstate Medical Center. ARTHURLEWIS, M.S.: Laboratory Technician, Metabolic Research Unit, The Jewish Hospital of Brooklyn. LEONARDFARBE~, B.A.: Fourth year medical student, Downstate Medical Center, State University of New York (presently, intern at Boston University Medical Service, Boston City Hospital). MARTIN G. GOLDNER,M.D.: Director, Department of Medicine, The Jewish Hospital of Brooklyn, Professor of Medicine, State University of New York, Downstate Medical Center. 742
743
ELECTROLYTE-ACTIVATED LIPOLYSIS
were least active. The marked differences among the physiologically inportant cations, Na, K, Ca and Mg, suggested possible interrelated activities and led to the studies comprising this report. The data presented here demonstrate that spontaneous lipolysis in surviving fragments of rat epididymal fat tissue m a y be markedly affected by the ratios of Na and K in incubation media, and that this response is modified b y the presence of Ca. METHODS Standard buffer mixtures were prepared in which Na and K content of each cation (expressed in mEq./L.) was reciprocally varied as follows: Na/K, 154/0; 103/51; 51/103; and 0/154. Anionic composition of the buffers, in the absence of albumin, was 154 mEq./L. PO4, or a mixture of "25 mEq./L. HCO 3 and 129 mEq./L. CI. Glucose was omitted from all media. When albumin was present in the media it had been defatted by extraction with Dole's extraction mixture, 2 washed with ethanol and ether, and redissolved in water distilled from glass to a final concentratiin of 2 Gin/100 ml. Solution of the albmnin was accomplished by the cautious addition of 4N NaOH or KOH, to provide a Na or K medium, respectively. After the albumin had been dissolved and pH adjusted to 7.6, 25 mEq./L. NaHCO3 or KHCOz was added. Na or K content of the albumin solution was determined by flame photometry, and sufficient NaC1 or KC1 added to bring the cation content to 154 mEq./L. When added, Ca was present at 2.5 mEq./L. (chloride salt) unless otherwise stated; in one experiment with Na/K 103/51, lO-2M cysteine or 2.5 mEq./L. Ca were added, individually or together, while in another experiment the effect of 1 mg./ml. EDTA* in this medium was observed. Albumin-free TRIS{ buffer was employed when studying the effect of 2.5 mEq./L. Ca in Na/K or Na/Choline* 103/51 media, and also in the preparation of 300 mM sucrose and Na buffers. Epididymal fat tissue was obtained from fed Wistar rats, killed by stunning and decapitation. Each fat pad was longitudinally bisected, one fragment serving as control for the other 3 fragments from the same rat. Incubation and processing of tissues and media, and statistical analysis of results, were as previously reported.~ RESULTS
Typical experiments with varied proportions cf Na and K in C]-HCOs buffer, in the absence of Ca and the presence or absence of albumin, are presented in Table 1 (lines A and B): significant lipolysis developed at those K eoncentrations greater than N a / K 103/51, and was maximal in an all-K medium. Lipolyric response to K was the same in the absence of albumin (thus preventing release of free fatty acids from the tissue) z or in its presence. Replaeement of C1-HCO3 and PO4 buffer, free of Ca and albumin ( T a b l e 1, line C), produced a pattern of inereasing lipolysis with increasing K concentrations similar to that seen in C1-HCO~ buffer, differing only in as much as significant lipolysis was now induced at a lower K eoneentration. The presence of 1 mg./ml. E D T A in a Ca-free CI-HCO,~ m e d i u m of N a / K 103/51 was associated with a signifieant increase in lipolysis above control of 197 per eent (control -- N a / K 103/51, 2.33 IxM/Gm/2 hours; E D T A present, 4.62 / x M / G m . / 2 hours; N = 6, P < 0.01). This should be c o m p a r e d with the response to the same N a / K mixture in an all PO4 buffer (Table 1, line C, increase of 206 per eent, P < 0.01 ). *EDTA, Ethylenediamine tetracetate, sodium salt, purchased from Eastman Chemical Company. CTBIS, 0.025 M, pH 7.40, THIS(Hydroxy)Aminomethane, Sigma Chemical Company. ~Choline chloride, Sigma Chemical Company.
744
B L E I C H E t l , FARBElt, L E W I S AND GOLDNER
Table 1.--Effect of Ca or Albumin on Lipolytic Response to Na and K Experimental Group
A
Buffer Cation Mixtures (mEq./L.) No. of Rats N a / K
154/0
103/51
51/103
2.178 2.10 3.01 --+0.05 • • B 7 2.59 3.11 3.98 • • • C 6 2.15 4.36 6.26 • • • D 6 2.17 7.06 9.94 • • • E 6 2.12 8.57 8.20 • • • ~Values are total free fatty acids generated, tion • 1 S.E.
0/154
5
Albumin Calcium
Anions
8.07
--
--
C1-HCO s
8.82
q-
--
C1-HCOz
7.62
--
--
PO4
6.66
--
+
CI-HCO3
• • • • 5.61 + + C1-HCO3 • expressed as ~M/Gm. fat/2 hour incuba-
Incubation of fat tissue in Na-K media containing 2.5 m E q . / L . Ca changed the pattern of lipolytie response to a biphasic one (Table 1, lines D and E). Free fatty acid production in Ca-supplemented N a / K 103/51 medium was significantly increased above that in all-Na medium, but lipolysis in an all-K medium was substantially lower than that in N a / K 51/103. Again, the presence or absence of albumin did not modify this response to added Ca (Table 1, lines D and E ). CI-HCO3 buffers with 2.5 mEq./L. Ca present, of the following N a / K composition, were tested: 154/0; 149/5; 77/77; 23/131; 0/154. The results (Fig. 1A) show a broad plateau of lipolytic response extending over the region N a / K 103/51 to N a / K 23/131, with significant decline from the plateau at N a / K 0/154. The presence of so little as 5.0 m E q . / L . K in a medium containing 149 mEq./L. Na and 2.5 mEq./L. Ca was associated with a small but significant increase in free fatty acid release over that seen in 154 m E q . / L . Na and 2.5 mEq./L. Ca ( N a / K 154/0, 1.27 -+- 0.17 ttM free fatty acid/Gm, fat/2 hours, N = 12; N a / K 149/5, 1.77 -----0.13/xM/Gm./2 hours, N = 33; 0.05 < P < 0.02). Because the effect of Ca on lipolysis appeared to differ markedly at N a / K 0/154 and N a / K 103/5i, these 2 points were subjected to careful re-evaluation. The addition of 2.5 mEq./L. Ca to a medium containing N a / K 103/51 increased free fatty acid release by 240 per cent (N = 7, P < 0.02), while the addition of 2.5 mEq./L. Ca to a medium containing N a / K 0/154 diminished lipolysis by 18 per cent (N = 7, P < 0.05). In other experiments, the depressant effect of Ca on lipolysis in all-K media, while somewhat variable, has ranged up to 40 per cent. While inclusion of 2.5 mEq./L. Ca modified K-induced lipolysis, addition of this or higher concentrations of Ca failed to show any such effect in an all-Na medium, and showed no greater effect in N a / K 103/51: N a / K 154/0, no Ca, 2.51 ixM/Gm./2 hours; 2.5 m E q . / L . Ca, 2.35 /xM/Gm./2 hours; 15 m E q . / L . Ca, 2.39 tzM/Om./2 hours; N a / K 103/51, no Ca, 2.88 IzM/Gm./2 hours; 2.5 mEq./L. Ca, 6.70 /zM/Gm./2 hours; 15 m E q . / L . Ca, 6.92 IxM/Gm./2 hours. When Ca concentrations of 15 mEq./L, were employed, turbidity developed
ELECTROLYTE-ACTIVATED
745
LIPOLYSIS,
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CALCIUM NOT PRESENT
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Fig. 1.--Lipolytic response to varying ratios of Na/K in the presence (a) o r absence (b) of 2.5 mEq./L. Ca, All experiments were conducted in albumin-free CI-HCO 3 media, except for those represented by the dashed line (b) in which Cafree a]l-PO4 buffer was employed. Nmnbers in parentheses refer to the number of animals contributing to each point. All experiments contained one flask of Na/K 0/154, with or without Ca as appropriate, to serve as control (Na/K 0/154 = 100 per cent). Absolute free fatty acid release at Na/K 0,,/154 in the presence of Ca, however, was lower than in its absence (see text). Free fatty acid release in Na/K 154/0 was equivalent in both media (see text). during incubation indicating the precipitation of CaCO3 and, therefore, the value of 15 m E q . / L , refers to the initial C a concentration. Inclusion of 10- 2 M eysteine failed to modify either basal lipolysis in N a / K 103/51, or the augmenting effect noted on the addition of 2.5 m E q . / L . Ca: control, 2.06 /xM/Gm./2 hours; cysteine added, 2.10 /.~M/Gm./2 hours; Ca added, 4.69
746
BLEICHER, FARBER, LE~VIS AND GOLDNER
/xM/Gm./2 hours; cysteine and Ca added, 4.47 #M/Gm./2 hours; N = 6; P for effect of Ca above control, alone or with cysteine, < 0.02. Addition of 2.5 mEq./L. Ca to media containing either Na/K 103/51 or Na/choline 103/51 (TR1S buffer) resulted in greater than a twofold increase in lipolysis: Na/K 103/51 control, 1.42 /xM/Gm./2 hours; with Ca, 3.84 /zM/Gm./2 hours; Na/choline 103/51 control, 1.41/xM/Gm./2 hours; with Ca, 3.38 tzM/Gm./2 hours; N = 6; P for Ca in either Na/K or Na/choline medium < 0.02. Mg at a concentration of 2.0 mEq./L., alone, or in combination with 2.5 mEq./L. Ca, had no effect on lipolysis in Na/K 103/51: no additions, 3.15 /zM/Gm./2 hours; Ca, 9.75 tzM/Gm./2 hours; Mg, 2.95 txM/Gln./2 hours; Ca + Mg, 11.3/xM/Gm./2 hours; P value for Ca xersus Ca + Mg > 0.10, N = 6. Spontaneous lipolysis in isosmotic NaC1 or sucrose, in TRIS buffer, was found equivalent: NaC1, 1.41/xM/Gm. fat/2 hours; sucrose, 1.43/zM/Gm. fat/2 hcurs; N = 12; P > 0.80. Linearity of lipolysis over a 2 hour period, in either Na/K 154/0 or Na/K (devoid of Ca and Mg), was established by incubating epididymal fat fragments in appropriate media for 1 hour and then transferring them to fresh media for a second hour of incubation. In 5 such experiments, each performed in duplicate, free fatty acid release into the medium at the end of the first hour of incubation was 52.9 per cent of total release in Na and 50.2 per cent of the total release in K. These did not differ significantly from each other (P > 0.50), nor was the release in either medium significantly different from 50 per cent release in the first hour (P for Na > 0..80, P for K > 0.20). Although there was no appreciable difference in the fractional release of free fatty acids (i.e., medium free fatty acids/medium + tissue free fatty acids) into Na medium from that into K medium (P > 0.50), lipolysis was greater in the K medium: Na, 2.67/xM/Gm. fat/2 hours; K, 6.85/zM/Gm. fat/2 hours; N = 5; P < 0.01. DISCUSSION These experiments demonstrate a reciprocal effect of K and Na on spontaneous lipolysis in rat epididymal fat tissue in vitro. Comparable effects are observed whether or not albumin is present in the incubation medium to serve as free fatty acid acceptor. The latter observations, as well as those dealing with fractional release of free fatty acids in Na or K media, indicate that the phenomena observed are related to the lipolytic process per se and not to the transfer of generated free fatty acids from tissue to medium. Lipolysis induced in either all-Na or all-K media proceeds linearly over a 2 hour incubation period, suggesting that the lipolytic effects observed are not due to the sudden disruption of tissue when introduced into unphysiologic media. Lipolysis is maximal in all-K media and decreases with decreasing K concentrations, until baseline levels are reached at Na/K 103/51 and below. A biphasic response to K is induced if 2.5 mEq./L. Ca are added to the media. While Ca does not affect lipolysis in an all-Na medium, it significantly potentiates the lipolytie response to so little as 5 mEq./L. K. Over the region Na/K 77/77 to 23/131 lipolysis is maximal when Ca is present, but declines at higher K concentrations.
ELECTROLYTE-ACTIVATED LIPOLYSIS
747
Anions play little or no role in electrolyte-induced lip0iysis, since the response to K is seen with PO4, C1-HCOa, or TRIS-C1 buffers) In PO4 buffers, however, increasing K concentrations evoke a virtually linear lipolytic response, contrasting with the lag in response noted in Ca-free C1-HCO3 buffer (Fig. 1B). Conceivably, removal of membrane Ca by complex formation with PO4 might alter (i.e., increase) membrane permeability to K. Such an increase in permeability, in the absence of Ca, has been noted with erythrocytes4 and Necturus kidney slices. ~ Further support for this view is found in the virtually identical increase in lipolysis in the same medium, Ca-free Na/K 103/51, in an all PO4 buffer or in a C1-HCO~ buffer containing i mg./ml. EDTA. It is paradoxical, then, that inclusion of Ca in C1-HCO3 media permits prompt lipolysis in response to low concentrations of K. This paradox may be apparent rather than real, in that incubation in Ca-flee C1-HCO3 media could wash Ca from a critical intracellular site and make the lipolytic mechanism afl'ected by K less sensitive but not wholly unresponsive. This interpretation is supported by the markedly different slopes of response at lower K concentrations in PO4 as opposed to Ca-supplemented C1-HCO.~ buffers (Fig. 1, and Table 1, lines C and D). Although the site of action of Ca is not presently known, failure of cysteine to modify the augmentation in lipolysis seen on the addition of Ca suggests that this effect is not on membrane sulfhydryl groups. Further, Rizack has shown that Ca may substitute for adenosine 3', 5'-phosphate (cyclic AMP) in activating a cell-free epinephrine-sensitive lipolytie system when ATP and Mg are also present. 6 Failure of Mg to activate or augment lipolysis in the experiments reported here does not exclude a role for this ion in the lipolytic process activated by K, since Mg may be firmly attached at its site of activity, not depleted by the methods applied here, and consequently no effect noted upon its addition. The lipase system or systems activated by K are presently unknown. Rizack 7 and Vaughan, Berger and Steinberg s have extracted lipolytic systems from rat epididymal fat. While both preparations presumably contain monoglyceridase activity, that of Vaughan and co-workers also contains an hormonally-responsive triglyceridase. As the latter preparation was obtained by homogenizing fat in 0.15 M KC1, it would seem plausible that the lipolytie activation demonstrated with K in surviving tissue fragments is similarly, at least in part, the hormonally-responsive triglyceridase. It may be asked whether these results demonstrate lipolytic activation in response to decreasing concentrations of Na or to increasing concentrations of K, since in these experiments the ions were varied reciprocally. The absence of Na from incubation media does not simultaneously induce lipolysis since, as has been previously demonstrated, 1 the cation replacing Na determines the direction of response: less lipolysis than that in Na buffers is seen with Mg or Li solutions, while lipolysis greater than that in Na is seen with NH4, choline or Cs solutions. Further, equal spontaneous lipolysis was found in isosmotic sucrose or Na solutions. Electrolyte-influenced lipolysis should not, then, be regarded in the general sense as limited to Na and K. Since Ca augments lipolysis in Na/choline 103/51 as well as Na/K 103/51, reservations regarding Na-K specificity also apply to this phenomenon. Nevertheless, in the restricted
748
BLEICIIER, FARBEB,~ LEWIS AND GOLDNEIR
setting pertinent to most functioning biological systems, the strikingly divergent and sensitive response to 2 physiologically important ions, Na and K, in the presence of a third, Ca, would suggest some reciprocal role for the former in affecting lipolysis directly (perhaps mediated by hormonal effects on Na-K flux), or in modifying hormonally-activated lipolysis. Support for the latter is found in the observation of Lopez, White and Engel, 9 that the in vitro lipolytic response to A C T H and epinephrine was augmented by the inclusion of K in the incubation medium. While the experiments reported here have been concerned with lipolysis, certain observations in the literature regarding the effect of electrolytes on other aspects of adipose tissue metabolism should be noted. Leonards, Landau and Bartsch 1~ have shown that glucose uptake is increased by incubation of epididymal fat in K-free media (confirmed in this laboratory, unpublished observations), and that lipid synthesis is promoted under these conditions. The lipolytic response to Na-K mixtures interestingly parallels the Na-K activation of ATPase, 11 the latter thought to play a role in active cation transport. These presently unconnected observations suggest that cation flux may affect adipose tissue energy stores, and serve to link and correlate metabolic processes in this tissue. ADDENDUM
With regard to the suggestion, above, that lipolysis may be "mediated by hormonal effects on Na-K flux," we have determined that K plays a critical role in lipolysis induced by growth hormone (Potassium Dependence of Growth Hormone Activated Lipolysis In Vitro, J. Clin. Invest. 45: In press, 1966 (Abstract)) and by TSH, ACTH, and epinephrine in vitro. REFERENCES
1. Bleiclaer, S. J,, Lewis, A., Farber, L., and Goldner, M. G.: Effect of monoand divalent cations on in vitro lipolysis. Proe. Soc. Exp. Biol. Med. 121: 980, 1966. 2. Dole, V. P.: A relation between nonesterified fatty acids in plasma and the metabolism of glucose. J. Clin. Invest. 35:150, 1956. 3. Reshef, L., Shafrir, E., and Shapiro, B.: In vitro release of unesterified fatty acids by adipose tissue. Metabolism 7:723, 1958. 4. Maize/s, M.: In A. Kleinzeller and A. Kotyk (Eds.): Membrane Transport and Metabolism. London, Academic Press, 1961, p. 257. 5. Solomon, A. K.: Ibid, p. 98. 6. Rizack, M. A.: Aetivat'on of an epinephrine-sensitive lipolytic activity from adipose tissue bg adenosine-3'5'-phosphate. J. Biol. Chem. 239:392, 1964.
7 . - - : An epinephrine-sensitive lipolytie activity in adipose tissue. J. Biol. Chem. 236:657, 1961. 8. Vaughan, M., Berger, J. E., and Steinberg, D.: Hormone-sensitive lipase and monoglyceride lipase activities in adipose tissue. J. Biol. Chem. 239:401, 1964. 9. Lopez, E., White, J. E., and Engel, F. L.: Contrasting requirements for the lipolytic action of eorticotropin and epinephrine on adipose tissue in vitro. J. Biol. Chem. 234:2254, 1959. 10. Leonards, j, R., Landau, B. R., and Bartsch, G.: Assay of insulin-like activity with rat epididymal fat pad. J. Lab. Clin. Med. 60:552, 1962. 11. Skou, j. c.: Enzymatic basis for active transport of Na and K across cell membrane. Physiol. R e v . 45:596, 1965.