Chase, L.R., G.D. Aurbach: The effects of parathyroid hormone on the concentration of adenosine 3',5'-monophosphate in skeletal tissue in vitra. J.BioI.Chem. 245: 15201526 (1970) Chase, L.R., G.D. Aurbach: Renal adenyl cyclase: anatomically separate sites for parathyroid hormone and vasopressin. Science (Washington) 159: 545-547 (1968) Chase, L.R., G.L. Melson, G.D. Aurbach: Pseudohypoparathyroidism: defective excretion of 3',5'-AMP in response to parathyroid hormone. J.Oin.lnvest. 48: 1832-1844 (1969) Chase, L.R., S.A. Fedak, G.D. Aurbach: Activation of skeletal adenyl cycIase by parathyroid hormone in vitra. Endocrinology 84: 761-768 (1969) Chernick, S.: Determination of glycerol in acyl glycerols. Methods in Enzymol. 14: 627-630 (1969) Dousa, T., T. Rychlik: The effect of parathyroid hormone on adenyl cycIase in rat kidney. Biochim.Biophys.Acta 158: 484-486 (1968) GilTTllln, A,.G.: A protein binding assay for adenosine 3',5'monophosphate. Proc.Nat.Acad.Sci. U.S.A. 67: 305-312 (1970) Gozariu, L., K. Forster, J.D. Faulhaber, H. Minne, R. Ziegler: Parathyroid hormone and calcitonin: influences upon lipolysis of human adipose tissue. Horm.Metab.Res. 6: 243-245 (1974) JaTTett, L., A.L. Steiner, R.M. Smith, D.M. Kipnis: The involvement of cycIic AMP in the hormonal regulation of protein synthesis in rat adipocytes. Endocrinology 90: 1277-1284 (1972) Khoo, J.c., D. Stein berg, D. Thompson, S.E. Mayer: Hormonal regulation of adipocyte enzymes. J.BioI.Chem. 248: 3823-3830 (1973)

195

Kono, T., F. W. Barham: Effects of insulin on the levels of adenosine 3',5'-monophosphate and lipolysis in isolated rat epididymal fat cells. J.BioI.Chem. 248: 7417-7426 (1973) Lech, J.1., D.N. Calvert: Protein content and osmotic behavior of isolated fat cells. J.Lipid Res. 7: 561-564 (1966) Littledike, T., C.D. Hawker: Extraction, purification and partial characterization of porcine parathyroid hormone. Endocrinology 81: 261-266 (1967) Loten, E. G., J. G. T. Sneyd: Evidence for separate sites of action for the antilipolytic effects of insulin and prostaglandin E J • Endocrinology 93: 1315-1322 (1973) Moxley, M.A., N.H. Bell, S.R. Wagle, D.O. Allen, J. Ashmore: Stimulation by parathyroid hormone of glucose and urea production in isolated liver cells. Am.J.Physiol. 227: 1058-1061 (1974) Potts, 1. T., Jr., G. W. Tregear, H. T. KeutTTlllnn, H.D. Niall, R. Sauer, L.J. Deltos, B.F. Dawson, M.L. Hagan, G.D. Aurbach: Synthesis of a biologically active N-terminal tetratriacontapeptide of parathyroid hormone. Proc.Nat. Acad.Sci. U.S.A. 68: 63-67 (1971) Sinha, T.K., P. Thajchayapong, S. Queener, D.O. Allen, N.H. Bell: On the lipolytic action of parathyroid hormone in man. Metabolism 25: 251-260 (1976) Werner, S., H. Löw: Stimulation of lipolysis and calcium accumulation by parathyroid hormone in rat adipose tissue in vitro after adrenalectomy and administration of high doses of cortisone acetate. Horm.Metab.Res. 5: 292-296 (1973)

Requests for reprints should be addressed to: N.H. Bell, VA Hospital, 1481 West Tenth Street, lndianapolis, lndiana 46202 (USA)

Horm. Metab. Res. 8 (1976) 195-201

© Georg Thieme Verlag Stuttgart

The Role of Calcium Ion in Epinephrine Activation of Lipolysis R.J. Schimmel Department of Physiology, University of Pittsburgh, Pittsburgh, PA, USA

Summary Adipocytes were prepared by collagenase digestion of rat epididymal adipose tissue and incubated for 5, 15, or 30 minutes in Krebs-Ringer bicarbonate buffer containing albumin (40 mg/mI), glucose (1 mg/mI) and epinephrine. Calcium ion was present in some incubations at concentration of 2.5 mM and omitted from others; media with no added calcium contained 1.0 mM EGTA thereby producing a final calcium concentration of< 10- 7 M. Glycerol release and accumulation of cycIic AMP were measured. Basal lipolysis and cell cycIic AMP levels were increased slightly but not significantly when adipocytes werc incubated in calcium free media. Lipolysis could be activated with epinephrine Received: 6 Sept. 1975

Accepted: 5 Jan. 1976

in the absence of calcium but the sensitivity of the lipolytic response was greatly reduced; however, the maximum lipolytic response to epinephrine was not decreased in calcium free media. Similarly, incubation of adipocytes in calcium free media resulted in decreased accumulation of cycIic AMP in response to epinephrine but only when sulrmaximum concentrations of the catecholamine were present. Varying the extracellular calcium concentration showed that a concentration of at least 1O-s M was optimal for epinephrine activation of lipolysis. These observations are considered in ac cord with the view that activation of adenylate cycIase is facilitated by calcium ion.

Key-Words: Lipolysis - Calcium - Epinephrine - cAMP

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The Role of Calcium Ion in Epinephrine Activation of Lipolysis

R.J. Schimmel

Introduction Although it is weH established that calcium ion is required for ACfH activated lipolysis (Katoes, Largis and Allen 1974) the influences of extracellular calcium on catecholamine accelerated lipolysis are unresolved. Lopez, White and Engel (1959) first reported that lipolysis activated with epinephrine was undiminished in calcium free media. More recently, however, several groups of investigators (Mosinger and Vaughan 1967, Fassina and Contessa 1967, Elendie, Alm and Löw 1970) have shown slight, but apparently statistically significant reductions in lipolysis in response to epinephrine or norepinephrine present at concentrations much less than employed in the study of Lopez et al. (l959). A previous report from this laboratory (Schimmel 1973) appeared to reconcile these observations by showing that the sensitivity of the lipolytic response to catecholamines is reduced in calcium free media, whereas, the maximum lipolytic response is unaffected under the same conditions. Because the lipolytic response to theophylline, cyclic AMP or dibutyryl cyclic AMP is not reduced in the absence of calcium, it has been proposed that calcium was required for optimal formation of cyclic AMP, not for the action of cyclic AMP (Mosinger and Vaughan 1967, Schimmel 1973). This proposal has not as yet been tested by direct measurement of cyclic AMP in isolated adipocytes incubated with catecholamines. The present experiments were undertaken to provide such measurements. In addition, this communication presents dose response relations for calcium ion and lipolysis activated with epinephrine. Materials aod Methods

cium concentrations of .2 to .5 mM; with 1.0 mM EGTA present the free calcium concentration was calculated to be less than lO-'M (Portzehl, ColdweIl and Ruegg 1964). The incubation was terminated by immersing the cells and medium in boiling water for 30-40 seconds. The boiled extraet was centrifuged for 2 minutes at 3000 RPM and total (medium + cells) cyclic AMP was determined on duplicate 25 pi and 50 pi aliquots of the supernatant of the unmodüied boiled extract by a modüication (Mashiter, Mashiter, Hauger and Field 1973) of the protein binding assay described by Gilmtzn (1970). This procedure for extraction and assay of cyclic AMP in unpwüied tissue extracts was first described by Fain, Pointer and Ward (1972) for rat adipocytes and since applied to other tissues (Forn, Krueger and Greengard 1974, Chase 1975). The assay procedure used in this laberatory was validated as folIows: dose response curves for cyclic AMP and unpurified adipose tissue extracts were parallel; there was complete recovery of known amounts of authentie eyelic AMP added to all incubations immediately prior to boiling; exposure of known amounts of authentie cyclic AMP and boiled adipocyte extracts to cyclic AMP phosphediesterase (Sigma) produced a nearly complete loss of measured cyclic AMP from each sampie. The assay was most sensitive in the range from 0.50 to 10.0 pmoles of cyclic AMP; assay of cell free-medium gave "blank" values equivalent to 0.10 to 0.20 pmoles cyclic AMP. Calcium ion, EGTA, theophylline and epinephrine did not influence the assay for cyclic AMP. The amount of glycerol produced was measured enzymatieally (Wieland 1957). The cyclic AMP and glycerol data are related to the weight of eells present as calculated from the triglyceride content (Lands 1958). For these studies between 40,000 and 60,000 adipocytes were present in each vial.

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196

All data reported herein are the averages of at least 4 replicate experiments, each experiment being done on cells prepared from the pooled tissue of one to four rats, and having three to five determinations for each condition employed.

Results

Exposure of adipocytes to 1.0 pM epinephrine caused cyclic AMP levels to increase from 0.23 to 0.52 nmoles/g 5 minutes after addition of the hormone; 10 minutes later cyclic AMP values were still elevated (Fig. 1). When theophylline was present epinephrine caused greater increases in cyclic AMP levels which also remained elevated for at least 15 minutes. Other

Normal male rats (150-250 gm) derived from the Sprague Dawley strain and pwchased from Zivic-Miller Lab., Inc., Allison Park, Pennsylvania were fasted overnight and sacrificed by cervica1 dislocation. Adipocytes were prepared by collagenase digestion of the epididymal fat bodies as described by Rodbell (1964). In general, 1-4 g of minced epididyrnal adipose tissue was incubated for 30 to 40 minutes studies (not shown) disclosed that 65 to 80% of the in Krebs-Ringer bicarbonate buffer, pH 7.4 (3.0 ml buffer/ total measured cyclic AMP is located within cells at gram tissue) with 1.0 mM calcium, crude bacterial collagen15 minutes of incubation. Maintenance of increased ase, (Worthington, Lot No. CLS 435057, CLS 3B4, CLS I AMP I I ' d' . b d .hr 43Nl11; 1500 Units Enzyme/g tissue), 40 mg/ml bovine secyc ic eve s 10 a Ipocytes 10cu ate wlt IPOrum albumin (Sigma, fraction V) and 1 mg/mI glucose. lytic agents for a comparable period of time has been The adipocytes were harvested and transferred to plastic vials observed by some investigators (larett, Stein er, Smith (14 x 55 mm) containing 1.0 mI Krebs-Ringer bicarbonate and Kipnis 1972, Kono and Barham 1973, Siddle and buffer with 40 mg/mi bovine serum albumin and 1.0 mg/mi Haies 1974), others have, however, reported a rapid glucose and incubated at 37 0 C under an atmosphere of Or CO 2 (95:5 v/v). Epinephrine (Parke-Davis Co.) and theophyl- decline in cyclic AMP levels (Ho and Sutherland 1971, line were present in some vials at the concentrations indiLang and Sehnyzer 1972, Manganiello, Murad and cated in text_ Ascorbate (0.10 mg/mI) was added to all meVaughan 1971). The explanation for the apparent dia. containi~ epinephrine t~ retard o~idation of the ca~echol- variation in the time course of the cyclic AMP reamtne_ Cal~lUm was present 10 so~e VlaiS to produce a f~nal S onse to li 01 tic stimuli remains unresolved alconcentratton of 2.5 mM and omitted from others. Media p . p y '. designated as calcium free contained 1.0 mM EGTA([ethylthough It may. be rel~ted to. the number ~f adlpoenebis (oxyethylene nitrilo)] tetracetic acid). Calcium is a cytes present 10 the 1Ocubahon vessel (Wleser and major contaminant of commercial albumin preparations and Fain 1975). media with no added calcium but containing 40 mg/mi albumin were measwed by Atomic Absorption to have calThe effect of omission of calcium from incubation

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Fig. 1. The time course of cyclic AMP accumulation of rat adipocytes incubated with epinephrine alone or in combination with theophylline. Isolated adipocytes were incubated for 5, 15 or 30 minutes in 1.0 ml of Krebs Ringer bicarbonate buffer, pH 7.4, containing 40 mg/mI bovine serum albumin and 1 mg/mi glucose. Epinephrine (1.0 s,tM) and theophylline (0.05 mM or 1.0 mM) were added to some incubations. Each value represents the mean ± SE of 8 paired experiments; N = 3 for each experiment.

media on lipolysis and accumulation of cyclic AMP is shown in Table 1. Omission of extracellular calcium appeared to reduce the lipolytic responses to 0.25 IlM and 0.50 IlM epinephrine measured after 15 and 30 minutes of incubation; however, the lipolytic responses to 5 IlM and 50 IlM epinephrine were not reduced in the absence of calcium. These data are in general accord with those contained in a previous publication (Schimmel 1973) in which longer incubations were utilized. Epinephrine at concentrations of 0.25 IlM and 0.50 IlM elicited modest increases in cyclic AMP levels in calcium containing media but virtually no increase in cyclic AMP in the absence of calcium. However, the higher (5 llM and 50 IlM) concentrations of epinephrine increased cyclic AMP accumulation to an equal extent in the presence or absence of calcium. Omission of calcium did not appear to alter the time course of cyclic AMP production, nor the distribution of cyclic AMP between cells and medium (not shown). Basal cyclic AMP levels and lipolysis were frequently increased in calcium free media; but this trend was not statistically significant using a paired Student's t-test. Although these data suggested that calcium is necessary for accumulation of cyclic AMP in response to sub-maximum concentrations of epinephrine, caution is advised before drawing firm conclusions from increases in cyclic AMP which are small in relation to basal values which vary in response to the presence or absence of calcium. For these reasons, the foregoing experiments were redone in the presence of theophylline, to permit larger increments in cyclic AMP to be generated (Figures 2 and 3). Because the sensitivity of the assay for glycerol did not permit accurate estimates of the amount of glycerol released

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Fig. 2. The effects of omission of Ca++ on lipolysis and accumulation of cyclic AMP in isolated adipocytes exposed to epinephrine in the presence of theophylline (0.05 mM). Isolated adipocytes were incubated for 15 minutes in 1.0 mI of Krebs-Ringer bicarbonate buffer, pH 7.4, containing 40 mg/mi bovine serum albumin, l.0 mg/mi glucose, 0.05 mM theophylline and varying concentrations of epinephrine in either the presence or absence of ea++. The values for glycerol release and total cyclic AMP represent increments over those measured in the presence of 0.05 mM theophylline alone. Basal values for glycerol release were: 1.1 ± 0.2 llmoles/g/15 min in the presence of ea++; 1.3 ± 0.4 in the absence of Ca++; with 0.05 mM theophylline: 1.2 ± 0.2 in the presence of Ca++; 3.7 ± 0.3 in the absence of Ca++. Basal values for cyclic AMP were: 0.37 ± 0.04 nmoles/g in the presence of Ca++; 0.49 ± 0.03 in the absence of Ca++; with 0.05 mM theophylline: 0.39 ± 0.05 in the presence of ea++, 0.52 ± 0.04 in the absence of Ca++. Each value represents the mean ± SE of 9 paired experiments; N = 3 for each experiment.

after 5 minutes of incubation and since cyclic AMP levels remained elevated for 15 minutes of incubation (Fig. 1), the following studies with theophylline utilized an incubation period of 15 minutes. Incubation of adipocytes in media containing 0.05 mM theophylline did not result in accelerated lipolysis or accumulation of cyclic AMP in the presence or the absence of calcium (Fig. 2). However, both the lipolytic response and the accumulation of cyclic AMP in response to epinephrine were greater than those seen in media containing no theophylline (Table 1). Once again the increases in lipolysis and accumulation of cyclic AMP in adipocytes exposed to concentrations of epinephrine equal to or less than 0.50 IlM were significantly (p < 0.05) decreased in the absence of calcium; the responses to 5.0 IlM epinephrine on the other hand were unaffected under the same conditions. Addition of 1.0 mM theophylline to incubation media increased cyclic AMP levels and lipolysis in the presence and in the absence of calcium. When calcium was present, cyclic AMP increased from 0.42

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