Acta pharmacol. et toxicol. 1964, 21, 139-144.

From the First Medical Service, Sahlgrenska sjukhuset, University of Goteborg, Goteborg, Sweden.

The Effect of a ,!I-Adrenergic Blocking Agent (Nethalidel) in Vitro on the Metabolism of Adipose Tissue

BY

Per Bjorntorp (Received March 19, 1964)

In the search for a P-adrenergic blocking agent, 2-isopropylamine-l- (2-naphthyl) ethanol hydrochloride (nethalide @) was synthesized and found to produce an effective blockage of myocardial adrenergic receptors (BLACK & STEPHENSON, 1962) without adverse physiological effects. The drug was in this investigation found to be free from sympathicomimetic activity.

SCHRODER & BJORNTORP (1964) found a change in free fatty acid pattern after work load in human subject taking nethalide. One possible explana- tion to this was an effect of Nethalide on the metabolism of adipose tissue. Adrenaline and its analogues have pronounced effects on such metabolism. They increase fatty acid output from adipose tissue (GORDON & CHERKES 1958) in spite of an increase in glucose uptake and 14COz production from glucose 6-14C, which gives an increase of fatty acid esterification to glyce- rol-phosphate (CAHILL et al. 1960). The effect on fatty acid output is thus not brought about by a decrease in triglyceride synthesis, but by a true increase in lipolysis, as indicated by an output also of glycerol from adi- pose tissue (LEBOEF et al. 1959). The trigger mechanism for this appear: to be a lipase, whose activity has been shown to be stimulated by adrena. line (RIZACK 1961 ; BJORNTORP & FURMAN 1962; BJORNTORP 1964).

Because of the sensitivity of adipose tissue to adrenaline, it was thought of interest to study the effect of nethalide on the stages of carbohydrate and lipid metabolism in adipose tissue in vitro already mentioned.

1) Imperial Chemical Industries Limited (I.C.I. 38, 174). Also called “Pronethalol” and “Alderlin”.

140 PER BJdRNTORP

Experimental. 200-250 g male rats of the Wistar strain were beheaded, distal parts of the epididy-

ma1 fat pads being then removed and placed in Ringer’s solution at room termperature. They were then immediately cut into pieces, which were randomized, weighed and incubated.

The incubation medium for fatty acid and glycerol release and glucose measure- ment consisted of 5 ml Krebs-Ringer phosphate buffer with 4% serum albumin (Armour, Fraction V). The final pH was 6.8. Glucose, when present, was at a concen- tration of 5.6 mM. Incubations were performed in a Dubnoff type incubator at 37.4”C with air as gas phase. Free fatty acids, glycerol and glucose were determined at 0 and 2 hours.

Lipase activity was assayed in a system consisting of equal parts of enzyme, ob- tained by homogenizing the fat pad in a Potter-Elvehjelm apparatus in Krebs-Ringer phosphate buffer and subsequent collection of the turbid water phase between the top fat cake and the sediment after centrifugation (BJORNTORP & FURMAN 1962), and a mixture of such composition as to give the final concentrations: serum albumin, 4%; KH2P04:Na~HP04(1: 11, 0.005 M; NaCI, 0.6 M; “Ediol”1) 0.8% (concentration of triglycerides). Incubations were at 37T , and fatty acids were determined at 0 and 2 hours.

Incubation for measuring 14CO2 and labelled lipid from glucose 6-14C was per- formed in 2 ml of Krebs-Ringer bicarbonate buffer, pH 7.4, in the presence of 1 I .2 mM glucose. The vessels used were 50 ml cylindrical flat bottom tubes (Hagedorn tubes) sealed with a rubber stopper holding two glass tubes closed by rubber mem- branes. After 2 hours of incubation at 37”C, 0.3 ml Hyaminez) was injected through one of the glass tubes into a 1 ml beaker, held in a stainless steel wire loop. Through the other glass tube 0.2 ml of 10 N H2SO4 was then injected into the medium. After 4 hours, recovery of W O Z in the beaker with Hyamine was complete, as tested in separate experiments with NaH14C03. The beaker was then placed in 10 ml scin- tillation solution (0.4 % PPO3) and 0.01 % dimethyLPOPOP4) in toluene) in a glass vial and radioactivity counted in a Packard Tri-Carb liquid scintillation counter.

Incorporation of label into lipids was measured in the system mentioned. After incubation the tissue was extracted as described by FOLCH et af. (1954). The chloroform phase was washed twice with water, the last washings being practically free from radioactivity. Finally a measured portion of the chloroform solution was evaporated in the counting vial, and 10 ml scintillation solution were added. Counting was then performed as described above.

Glucose was determined enzymatically (LEVIN & LINDE 1962), glycerol by periodate oxidation as described by CARISON & WADSTROM (1959) and fatty acids by the method of DOLE (1956) with Nile Blue as indicator.

Statistical significance was assessed by the (student’s) t-test.

R e d ts

The effects on fatty acid release, lipase activity, glycerol release and glucose uptake at 5 x lO-6M concentration of both adrenaline and nethalide are shown in table 1.

1) Riker Laboratories, Inc. (SchenLabs), Northridge, California, USA. 2) p-(diisobutyl-cresoxyethoxyethyl)dimethylbenzylammonium Hydroxide (Rohm &

3) 2,5-diphenyloxazoI (Packard). 4) 1,4-bis-[2(4-methyl-5-phenyloxazolyl)-benzene] (Packard).

Haas).

NETHALIDE A N D ADIPOSE TISSUE METABOLISM 141

Fatty acid released (uEq/g). . . . .

Glycerol released (uEq/g). . . . .

Lipase activity (uEq FA/g/h)

Glucose uptake (mglg). . . . .

Table I Effects of Adrenaline and Nethalide on Fatty Acid and Glycerol Release, Lipase Activity and Glucose Uptake in Rat Epididymal Fat Pads Incubated in Vitro. Mean f Standard

Deviations. In parentheses Number of determinations.

2.70 50.46 (6)

1.30 10.20 (6)

6.45 50.91 (4)

1.86 50.46 (6)

Net halide 5 x 10-6M

4.32 10.40 (5)

2.60 10.40 (5)

8.52 10.88 (4)

1.60 ~t0.40 (5)

Adrenaline 5 x lO-6M

7.62 10.66 (5)

7.00 i0.53 (6)

10.19 10.62 (4)

4.46 10.61 (6)

idrenaline + Nethalide 5 x IO-6M 5 x 10-6M

8.98 10.66 (6)

6.45 10.52 (6)

10.03 11.33 (4)

2.32 10.44 (6)

Nethalide stimulated fatty acid (p < 0.01) release and lipase activity (p < 0.05) in the absence of adrenaline from the flask. Adrenaline gave a more pronounced stimulation (p < O.OOl), and the addition of nethalide to adrenaline gave a further increase in fatty acid release (p < 0.02), but not in lipase activity.

Glycerol release followed the pattern of lipase activity, viz. an increase after nethalide only (p < 0.01), a further increase with adrenaline only (p < 0.001), and no increase in release by adrenaline after adding nethalide. Glucose uptake showed no significant changes after nethalide. It increased after adrenaline (p < 0.001) and again decreased in the adre- naline plus nethalide flask, compared with adrenaline alone (p < 0.001). These effects are also shown in fig. 1, where the approximate linearity of the time-activity curves for 3 hours is seen.

The effects of increasing concentrations of nethalide on the metabolism of adipose tissue stimulated by a 5 x 10-6M concentration of adrenaline

t l6 t

Fig. 1. Effect of Adrenaline and Nethalide on Rat Epididymal Fat Pads in Vitro. Concentrations of Drugs 5 x IO-aM.

Acta pharrnacologica. vol. 21. fasc. 2. 11

142

Adrenaline 10-6M

PER BJ6RNTORP

Adrenaline (IO-bM) + Nethalide Nethalide

5 x 1 0 - 6 ~ 1 5 x 1 0 - 5 ~ 5 x 1 0 - 6 ~ 1 s x 1 0 - 5 ~

n

~~

coz

Lipid

Adrenaline 0 5.10d 5-10" 5.10% 5.1d6 5.lOd M

~ ~ ~~

10.10 Jr 0.04

0.09 LO.10 10.03

0.15 i 0*09

0.10 0.43 0.26 0.27 ~ 0 . 0 4 10.12 d10.19 - + 0.09

0.44 0.79 0.73 0.33 10.11 50.14 *0.12 ~ t 0 . 0 9

0.39 I

Nethalide 0 0 l*md 5.10d 5.1d5 5sld' M

Fig. 2. Effects of Adrenaline and Nethalide on Metabolism of Rat Epididymal Fat Pads in Vitro. Open columns: Fatty Acid Release.

Hatched columns: Glycerol Release. Solid columns: Glucose Uptake.

are shown in fig. 2. At a 5 x 10-6M concentration of nethalide a decrease in glucose uptake was seen. Here fatty acid release increased. Glycerol release did not seem to be affected, however, before nethalide concentra- tion reached 5 x lO-sM. With this concentration fatty acid release also fell abruptly.

The results of conversion of glucose 6-14C to 14C02 and lipids are seen in table 2.

Adrenaline increased both incorporations (p < 0.0 1). Nethalide in- creased conversion to 14COz (p < 0.02). Incorporation into lipids was significantly increased only at 5 x 10-6M concentration (p < 0.01). Both concentrations markedly blocked the adrenaline effect (p < 0.01).

Table 2. Effects of Adrenaline and Nethalide on Conversion

of Glucose 6-14C to 14CO2 and Lipid 14C by Rat Epididymal Fat Pads. Means & Standard Deviations of 6 Determinations.

NETHALIDE AND ADIPOSE TISSUE METABOLISM 143

Discussion Thus it was found that nethalide diminished both adrenaline-induced

glucose uptake and lipolysis in adipose tissue in vitro. A decrease in glucose uptake diminishes fatty acid reesterification,

affecting fatty acid output, since glucose is the triglyceride-glycerol precursor in adipose tissue (VAUGHAN 1961). Glycerol once liberated from triglycerides does not seem to be able to take part in the re-esterifica- tion of fatty acids, because its activation is not considered possible in adipose tissue in the absence of glycerol kinase (WIELAND & SUYTER 1957). Its diffusion out of the tissue thus reflects lipolysis.

The parallel changes of glycerol release from the tissue and lipase activity seem to support the hypothesis that the lipase in question pro- vides the trigger mechanism for lipolysis in adipose tissue (RIZACK 1961; BJORNTORP & FURMAN 1962).

With these concepts in mind, the nethalide effect on adrenaline sti- mulated metabolism could be interpreted as follows. Glucose uptake is inhibited at somewhat lower concentrations than is lipolysis. This first brings about an increase in fatty acid output and then, at higher concen- trations, lipolysis is also inhibited, and fatty acid output consequently decreases abruptly. Nethalide alone stimulates glycerol release.

This investigation thus tends to show that the increase in glucose uptake effected by adrenaline on adipose tissue can be inhibited at concentrations of nethalide lower than those interfering with the adrenaline lipolytic effect. This seems to indicate that the adrenaline effect on glucose uptake is not necessarily only a consequence of its effect on lipolysis and the accumulation of fatty acids (CAHILL et a1 1960).

FROBERG & ORO (1963) recently studied the effect of nethalide on the adrenaline-induced changes of free fatty acid concentration in the arterial plasma of the dog. They found an increase in free fatty acid concentration after infusion of nethalide and a decrease in adrenaline response thereafter. These results seem to correspond to the invitrofindings in our work of a lipolytic effect of nethalide and a blockage of adrenaline-induced lipolysis.

The changes after nethalide on free fatty acid concentration of human plasma (SCHRODER & BJORNTORP 1964) could possibly be explained from the results obtained. The free fatty acid pattern during and after work load is dependent on lipolysis of adipose tissue (presumably induced by cathecholamines), utilization of free fatty acids and distribution of blood flow. The free fatty acid peak immediately after work is probably caused by a continuing increase in lipolysis despite the cease in utilization (HAVEL et al. 1963). This peak has been observed to diminish after treatment with nethalide at high doses (SCHRODER & BJORNTORP 1964), which could be caused by blockage of lipolysis in adipose tissue, as shown possible with nethalide in vitro.

144 PER BJORNTORP

Summary Nethalide was found to diminish the effects of adrenaline on adipose

tissue metabolism. The increase in glucose uptake was found to be abo- lished at approximately equivalent concentrations of adrenaline and nethalide. At higher concentrations lipolysis was decreased as indicated by a decrease in glycerol release. Fatty acid release at rising concentra- tions of nethalide first increased after the effect of nethalide on glucose uptake and then fell abruptly after the effect also on lipolysis at higher concentrations. This could be explained by interference between glycolysis and lipid metabolism in adipose tissue.

Nethalide alone seemed to excert a small stimulating action on lipolysis.

Acknowledgements.

ledged. The technical assistance of Miss Majvor Karlsson is gratefully acknow-

R E F E R E N C E S Bjorntorp, P. & R. H. Furman: Lipolytic Activity in Rat Epididymal Fat

Pads. Am. J. Phys. 1962, 203, 316-322. Bjorntorp, P.: The Free Fatty Acid Release and Lipolysis of Human Adipose

Tissue In Vitro. Metabolism 1964, in press. Black, J. W., & J . S. Stephenson: Pharmacology of a New Adrenergic Beta-

Receptor-blocking Compound (Nethalide). Lancet 1962, 2, 3 11-314. Cahill, G . F. Jr., B. Leboef, & R. B. Flinn: Studies on Rat Adipose Tissue

in Vitro. J. Biol. Chem. 1960, 235, 1246-1250. Carlson, L. A. & L. B. Wadstrom: Determination of Glycerides in Blood

Serum. Clin. Chim. Acta 1959,4, 197-205. Dole, V. P.: A Relation Between Non-Esterified Fatty Acids in Plasma and

the Metabolism of Glucose. J. Clin. Invest. 1956, 35, 150-154. Folch, J., M. Lees, & G. H. Sloane-Stanley: A Simple Method for Prepara-

tion of Total Pure Lipide Extracts from Brain. Fed. Proc. 1954, 13, 209. Froberg, S., & L. Oro: The Effects of Nicotinic Acid, Phentolamine and

Nethalide on the Plasma Free Fatty Acids and the Blood Pressure in the Dog. A Comparative Study. Acta Med. Scand. 1963, 174, 635-641.

Gordon, R. S. & A. Cherkes: Production of Unesterified Fatty Acids from Isolated Rat Adipose Tissue Inarbated In Vitro. Proc. SOC. Exp. Biol. Med. 1958, 97, 150-151.

Havel, R. J., A. Naimark, & C. F. Borchgrevink: Turnover Rate and Oxida- tion of Free Fatty Acids of Blood Plasma in Man During Exercise: Stu- dies During Continuous Infusion of Palmitate -l-C14. J . Clin. Invest. 1963, 42, 1054-1063.

Leboef, B., B., Flinn, & G. F. Cahill Jr . : Effect of Epinephrine on Glucose Uptake and Glycerol Release by Adipose Tissue In Vitro. Proc. SOC. Exp. Biol. Med. 1959, 102, 527-529.

Levin, K. & S. Linde: Bestamning av glykos i blod, liquor och urin med ett nytt glykosoxidasreagens. Svenska Ldkartidningen 1962, 59, 3016-3026.

Rizack, M. A. : An Epinephrine-Sensitive Lipolytic Activity in Adipose Tissue. J. Biol. Chem. 1961, 236, 657-662.

Schroder, G., & P. Bjorntorp: The Effect of Prolonged P-Adrenergic Blockade by Nethalide in Man on Serum Lipids and Glucose During Rest and Exercise. Acro Med. Scund., 1964, in press.

Vaughan, M.: The Metabolism of Adipose Tissue In Vitro. J. Lipid Reseurch 1961, 2, 293-316.

Wieland, O., & M. Suyter: Glycerokinase: Isolierung und Eigenschaften des Enzyms. Biochem. 2. 1957, 329, 320-331.