The Fatty Acid Release and Lipolysis of Human Subcutaneous Adipose Tissue in Vitro

By PER B J~RNTORP

Human adipose tissue releases free fatty recently described lipase, sensitive acids in vitro and responds to addition epinephrine, also in human adipose of glucose or epinephrine in the same sue is presented. Some evidence, way as rat epididymal fat pads although though not conclusive, is given for with much smaller release per unit presence also of lipoprotein lipase. time. Evidence for the presence of a

for tis- al-

the

I NTENSIVE RESEACH in recent years has elucidated several metabolic activities of adipose tissue. This work to a large extent has been per-

formed on epididymal fat pads from the rat. Work on the human subject in vivo has indicated much the same metabolic pattern as that foutid in rat adipose tissue. Simple model studies on surviving human adipose tissue are, however, sparse. This has not only been caused by the relative difficulty of obtaining samples but also by data which indicated that certain metabolic activities in human adipose tissue in vitro might be difficult to or impossible to demonstrate.‘”

It seems important to probe further the possibilities of establishing simple systems with which to investigate human’ metabolism in health and disease. In the present paper, therefore, optimal conditions for measuring free fatty acid release and lipolysis in adipose tissue in vitro have been investigated.

MATEFUAL AND METHODS

Biopsy specimens were obtained from patients undergoing surgery. General anesthesia, using barbituric acid derivatives, diethy!ether, nitrous oxide and curare, was the rule. Pre- operative injections of morphine OT its analogues were given. The patients were operated upon for various abdominal diseases, although most of them involved the gallbladder and stomach. No patients had bilirubinemia, diabetes or were in a state of severely negative cal- oric balance. The specimens were taken from the subcutaneous fat of the abdominal wall at the beginning of the operation from 36 patients of both sexes aged 41 to 64 years.

The samples were placed immediately into melting ice. Pieces weighing ZOO to 500 mg. and approximately 0.5 cm. thick were incubated for 1 hour at 37 C. as a whole piece; were minced into small pieces with a pair of scissors; or were homogenized in the incubation medium, which consisted of 5 ml. of 4 per cent albumin (Armour, fraction V) in Krebs- Ringer’s phosphate solution having a pH of 7.3. Homogenization was performed in ice in an all-glass Potter-Elvehjem apparatus with the pestle shah attached to a motor. The pestle head was short and fitted loosely into the ground cylinder. These arrangements facilitated the difficult homogenization of the tissue.

In the lipase activity characterization experiments a concentrated homogenate was made using normal saline solution. The homogenate was then centrifuged in the cold at 1,000 Gm.

From the First Me&cd &or& Sweden.

Redoed for pubhztim

Service, Snhlgrenska Siukhuwt, Unhersity of Gdteborg, Giite-

Apr. 22, 1964.

1318

METABOLISM, VOL. 13, No. 11 (NOVEMBER), 1964

LIPOLYSIS OF HUMAN ADIPOSE TISSVE 1319

Table I.-Fatty Acid Release from Whole, Minced and Homogenized Humun Adipose Tissue. Mean + Standard Deviation and Number

of Determinationa (within parentheses)

Tissue No Addition Epinephrine 10-O M

(flEq./Gm./hr.) WWGm./hr.) Glucose lo-? Y

(pEq./Gm./hr.)

Whole 0.34 * 0.11 (11) 0.68 2 0.15 (4) -

Minced 0.61 f 0.20 (26) 0.97 & 0.31 (8) 0.20 + 0.10 (8) Homogenized 1.05 f 0.31 (26) 0.96 * 0.30 (8) 1.07 rt 6.28 (8)

for 10 minutes. The turbid water phase which appeared between the fat cake on the top

and the sediment was then used.

Respiration was measured in a Warburg apparatus at 37 C. with 120 shakings per min- ute. The gas phase was air. Carbon dioxide was absorbed on 0.1 ml. of 20 per cent KOH

in the center well. Temperature equilibration time was 5 minutes. The medium was Krebs-

Ringer’s phosphate buffer with 4 per cent albumin and a final pH of 7.3.

Lipase activity was assayed as follows: equal volumes of enzyme-containing material and a mixture were incubated in a Dubnoff-type incubator at 37 C. to give the following final

concentrations: albumin 4 per cent, Ediol’ 0.8 per cent (triglyceride concentration), Na,HPO,:KH,PO,, l:l, 0.005 M. Fatty acid concentration was determined at zero and 60

minutes, during which period the time-activity curve was found to be linear.

Fatty acids were determined by the method of Dole* and protein by the method de-

scribed by Lowry et al.5

In table 1 are listed the results of determinations of fatty acid release from human adipose tissue in different states, and the effects on these of epineph- rine and glucose. Homogenized tissue gave the highest fatty acid release, followed by minced tissue. The intact tissue gave the lowest release. Both whole and minced tissue responded with an increase in fatty acid release when epinephrine was present in the incubation flask. Minced tissue de- creased its net fatty acid output markedly in the presence of glucose. Homo- genized tissue did not respond to the addition of either epinephrine or glucose.

The mean respiratory rate of minced tissue (QO,) and its standard devia- tion in 7 experiments was 24 * 9 (wet weight). As seen in figure 1 the oxygen uptake curve was approximately linear to about 2 hours. Respiration was not altered by the presence of epinephrine or glucose. Homogenized tissue did not respire.

In individual experiments no obvious correlation was apparent between respiratory rate and fatty acid release. Table 2 shows that fatty acid release was independent of the gas phase in the incubation flask, whether air or nitrogen, and that potassium cyanide did not alter fatty acid release.

The lipase activity of tissue homogenates was next characterized. First, dependence of activity on pH was determined. These curves varied some- what with different experimental material. They often had a broad activity maximum in the pH range 658.5, as exemplified in curve 1 in figure 2. In other samples, however, the pH-optimum was situated in the pH 7 region,

‘Emulsion of 50 per cent w/v cocoanut oil; Riker (Schenley Labs.) Northridge, Cali-

fornia.

1320 PER B JiiRNTORP

Respiration

iJI 02kl

1 2 3 h

4 hours Fig. l.- Respiration of minced human adipose tissue in medium containing no

addition,l glucose (10-Z M) ,a or epinephrine (lo- 6 M) .s Minced tissue (340 mg. in experiment with no addition, 382 mg. in glucose and 402 mg. in epinephrine ex- periment) incubated in 3.0 ml. of 4 per cent albumin in Krebs-Ringer’s phosphate solution. Final pH, 7.3; 0.1 ml. 20 per cent KOH in center well, temperature 3’7 C., temperature equilibration time 5 minutes, gas phase, air. Fatty acid release 0.30, 0.18 and 0.85 pEq./Cm./hour in control, glucose and epinephrine systems, respectively.

as shown in curve 2. In this sample, pooled homogenized biopsy specimens from several patients were used, allowing also comparative determination of pH effect on activity in the presence of 0.2 M NaF (curve 3)) which inhibited the peak at pH 7, and of 0.6 M NaCl ( curve 4), which had no certain in- fluence.

Cofactor requirements for the activity were determined and the results are given in table 3. Albumin was necessary for optimal activity, but the presence of serum in the incubation mixture did not increase activity. OII the contrary, sometimes a slight inhibition was noted. Ammonium sulfate was not necessary, either, for maximal activity. The dependence of phosphate ions varied. Sometimes a marked dependence was noted. On the average, however, the difference was not significant whether or not phosphate ions were present in the incubation mixture.

Different inhibitors were then tested (table 4). Sodium fluoride in 0.2 M concentration invariably had an inhibitory effect. The inhibition of protamine sulfate and NaCl at different concentrations was not constant, however. Usually, at least some inhibition was noted with 0.6 M NaCl, while 0.3 M NaCl and protamine on several occasions demonstrated no inhibitory effect. After inhibition with both NaF and NaCl there was still significant activity remaining.

OIlr some occasions when diluted glucose solutions were administered as vehicles for muscle-relaxing drugs to the patients, it was noted that the m-

LIPOLYSIS OF HUMAN ADIPOSE TISSUE 1321

Table 2.-Fatty Acid Release from Minced and Homogenized Hum& Adipose Tissue under Air or Nitrogen or in the Presence of 2 x lo--” M

KCN. Mean * Standard Deviation and Number of Determinations (within parentheses)

No Addition No Addition KCN 2 x 1O-3 M Air Nitrogen Air

Tissue (aEq./Gm./hr.) (gEq./Gm./hr.) (pEq./Gm./hr.)

Minced 0.53 f 0.14 (6) 0.51 Z!I 0.10 (6)- 0.56 * 0.12 (4) Homogenized 1.21 AZ 0.25 (7) 1.42 Z!I 0.35 (3) 1.26 k 0.32 (3)

hibition of lipase activity by NaCl and protamine was more marked. There- fore, determination of lipase activity characteristics was performed in 3 patients before and after the rapid intravenous infusion of 50 Gm. of glucose, from whom biopsy specimens were taken at these points dvring operation. In the homogenates, lipase activity was determined at pH 6.8 and 8.5, and compared with the activity in the absence of phosphate ions and in the pres- ence of NaF and NaCI. These 3 studies showed consistent qualitative results. A representative study in a man, operated upon for gastric ulcer, is shown in figure 3.

Control activities increased after glucose infusion. This was most marked at pH 8.5. At pH 6.8 the omission of phosphate had no certain effect before or after the infusion. A marked NaF inhibition was seen before infusion, but was not pronounced after it. NaCl inhibition was moderate before and after infusion. At pH 8.5 the omission of phosphate seemed to cause some

Fatty acid release

mvEq/mg prot ./h 4

I a

6 7 0 9 PH Fig. S.-Dependence of lipase activity on pH. Incubation mixture as “complete

system” in table 3, pH changed by addition of 1 M HCl or NaOH. Ionic strength equalized in all incubation mixtures by addition of 1 M NaCI. Curve 1: incubation mixture without addition; curve 2: incubation mixture without addition; curve 3: same sample as curve 2 with the addition of NaF to a final concentration of 0.24 M; curve 4: same sample as curve 2 with the addition of NaCl to a final concentration of 0.6 M.

1322 PER BJijRNTORP

- -

Table 3.-Dependence of Lipape Activity on Different Cofactors. Mean and Range of 6-8 Determinutions

cd Activity

Complete system” 100 Complete system minus albumin 22 (14-42) Complete system minus ammonium sulfate 95(71-112) Complete system minus phosphate 82(30-114) Complete system minus serum 105 (98-119)

‘Complete system: equal parts of enzyme preparation and following mixture to give the final concentrations: albumin, 4 per cent; (NH, )aSO,, 0.05 &f/1; KHsPO,:NaaHPO,

( 1: 1 ), 0.005 M; human serum, 1:20; Ediol triglycerides, 0.8 per cent: final pH. 6.8.

Table 4.--Inhibition of Lipase Actiuity. Mean and Rcmge of 3-8 Detersninatknw. Zncubation Mixture as ‘%ompkte System” in Table 3 __--____

% Inhibition .-.-__. . .___

NaF 0.2 M 6.5 (24-90) NaC10.3 M 14 (O-40) NaClO.6 M 38 (14-62) Protamine sulfate 400 pg./ml. 22 (o-31) NaF 0.2 M + NaClO.6 M 81 (58-100)

increase in activity. NaF inhibition was definite, but was not as marked at pH 8.5 as at pH 6.8 and was noted to be less after infusion than before. NaCl inhibition was more pronounced before glucose infusion than at pH 6.8. After infusion the inhibition was marked.

Since it was demonstrated that fatty acid release in vitro could be stimu- lated by adding epinephrine to whole or minced tissue, lipase activity was determined in tissue homogenates where the minced tissue had been pre- incubated for 1 hour in the presence of 1 pg./ml. epinephrine. Comparisons were performed with control tissues taken from the same patient but pre- incubated without epinephrine. The increase in lipase activity induced by epinephrine (as compared to the controls) was 22 +- 17 (S.D.) (Range 6-52 per cent; 6 experiments. )

DJSCIJSSION

It was found that human adipose tissue in vitro releases fatty acids and that this release is more pronounced in homogenized tissue than in minced tissue, while intact tissue showed the least fatty acid release of the 3 types of preparations studied.

Lipolytic activity of human material seems to be lower per unit wet weight than that of rat epididymal fat pads. Whether or not this is a true species difference or is caused by a comparably greater part of inert material in the human specimens, it is not yet possible to state. The protein content of the

biopsy specimens provides no reliable information in this regard, because of contamination by blood.

In the whole cell preparations it seems reasonable to assume that the dif- ference found in fatty acid release rates is a consequence of the difference in surface area of the different preparations. Equilibration between incuba-

LIPOLYSIS OF HUMAN ADIPOSE TISSUE 1323

FATTY ACID RELEASE

mpEq/mg prot/h 100

T BEFORE GLUCOSE

pH 6.8

100

i i

pH 8.5 50

! O-PQ$3FNacI O-PQNaFNaCl

Fig. 3.-Lipase activity and inhibition at pH 6.8 and 8.5 before and after infusion of glucose. Incubation mixture as “complete system” in table 3. Open bars: control activity. Dotted bars: phosphate buffer omitted. Striped bars: 0.24 M NaF added. Filled bars: 0.6 M NaCl added.

AFTER GLUCOSE

tion medium and central cells seems difficult to attain in biopsy specimen& of considerable thickness. Slicing technics, as usually applied to other tissues, do not seem suited to adipose tissue. These problems are now under further study using various human adipose tissue preparations.6

In general, a wider variance in results was noted iti the present studies on human material as compared with material obtained from the rat.7vs In addi- tion to the reasons mentioned, one must also take into consideration the dif- ficulties encountered in selecting a homogeneous population when working with human material.

The difference in fatty acid release rates between the whole cell prepara- tions and the homogenates could have several causes. Fatty acid release is apparently independent of respiration, because the presence of KCN or the absence of oxygen did not affect fatty acid release iti either whole cell prep- arations or homogenates.

Possibility, which seems difficult to control, is the activation of the hydrolytic

1324 PER B JijBNTOBP

mechanism by the homogenization procedure. Activation of this type, achieved

by disintegrating the cell, has been noted by Rizack!’ when working with rat

adipose tissue.

The net fatty acid output from whole cell preparations of rat adipose tissue is the result of lipolytic acti\.ity and re-esterification. The latter is enhanced

by increased glycolysis .I” It cannot occur in nonfortified homogenates of rat

adipose tissue .l’ It seems reasonable that the increase of fatty acid release in

homogenates of human adipose tissue as compared with whole cell prepara-

tions is caused partly by lack of re-esterification of the fatty acids to triglyc-

erides.

The effect of glucose on net fatty acid output from whole cell preparation’s

is probably also due to its effect on the re-esterification process, causing in-

crease of this because of increased glycolysis. The effect of epinephrine, how-

ever, might be explained by direct action on the lipolytic system, as dis-

cussed below.

In the characterization studies of lipase activity, the turbid water phase,

which occurred between the fat cake on top and the sediment below after

centrifugation at low speed for a short time, was used. Here the greater part

of the activity was collected. The lipase activity measured directly with the

homogen’ized depot fat as substrate was not used for characterization studies,

because it was considered difficult to obtain a reproducible physical-chemical

state of the substrate. Such conditions are important in the kinetics of

lipases.12p’3

In the lipase characterization studies a pH of about 7 was optimal for

demonstrating inhibition by 0.2 M NaF, whereas 0.3 M NaCI and protamine sulfate often showed no inhibitory effect. The NH4+ ion was not tiecessary

for optimal effect, nor was serum. The latter fact might only mean, however, that serum was already present in optimal amount as contamination in the

sample. These data, together with the possibility to increase activity in the

fat by preincubation with epinephrine, seem to demonstrate the presence in

human adipose tissue of the same lipolytic system recently characterized in

rat epididymal fat pads by Rizackg and by Bjiirntorp and Furman.’

The degree of activation of lipase activity in vitro by preincubation with

epinephrine is probably diminished to a varying degree by the endogenous

epitiephrine stimulation which is occasioned by surgery.

The presence of lipoprotein lipase is difficult to evaluate in the prepara-

tions used here. A tendency of the pH-dependence to be maximal in the more alkaline areas in some samples seems to support its presence, as does its in- hibition by 0.6 M NaCl, which was usually found to be present.‘4 Sometimes

inhibition by protamine and lower NaCl ooricentrations was noted, which also seems to speak for the presence of Iipoprotein Iipase. The strongest sup-

port for its presence is probably the ability to activate lipolytic activity by the infusion of glucose, activity which was markedly inhibited by concen-

trated NaCl. It has been shown that heparin elutes more lipoprotein lipase from the epididymal fat pads of fed rats thati from fasting rats’5 and that

the lipoprotein’ lipase remaining in the tissue is also increased in fed rats.’ The general variability of the results in the lipase characterization experi-

LIPOLYSIS OF HUMAN ADIPOSE TISSUE 1325

ments seems explainable on most points by variations in the contribution of the 2 discussed lipase activities to the total activity measured. These variations could be caused by differences in endogenous epinephrine, insulin or glucose levels, conditions which are possible to standardize fairly well in the lab- oratory animal, though not in man.

One of the best ways to establish the presence or absence of lipoprotein lipase in human adipose tissue is to investigate the influence on activity of the serum factor, which activates substrates in the form of emulsions.14 This activation is characteristic for lipoprotein lipase and for no other known lipase activities. It was not possible to investigate this in the preparations reported here, however, because of contamination by serum. The data presented do not of themselves allow the conclusion that it is invariably possible to demon- strate lipoprotein lipase in human adipose tissue in vitro.

The 2 types of lipase activities reported are probably not the only ones existing in human adipose tissue, because in the presence of various inhibitory agents, alone or in combination, a significant level of activity remains.

SUMMARY

Fatty acid release and lipolysis were studied in vitro in specimens of human adipose tissue. Release of fatty acids was slow from whole tissue, accelerated from minced tissue, and most rapid from homogenized tissue. The rate of release was slower than that from rat epididymal fat pads on a wet weight basis. In whole and minced tissue the addition of epinephrine increased fatty acid release, while glucose diminished it. Homogenized tissue showed no response in this regard.

Minced tissue respired, but homogenized did not. No certain effects on respiration by the addition of glucose or epinephrine could be observed. Fatty acid release and respiration seemed to vary independently of each other, because incubation under nitrogen or in the presence of KCN did not alter fatty acid release.

Lipase activity was observed in the turbid water phase of concentrated homogenates. The dependence of the activity on pH varied with different samples. Often the maximal activity was found over a broad pH range. In other samples a distinct optimum was found at about pH 7. This peak could be inhibited by 0.2 M NaF but not by 0.6 M NaCl. The necessity for albumin in the assay mixture was established, but phosphate ions, ammonium sulfate, and serum were not found with certainty to be necessary for optimal activity. NaF invariably inhibited activity, and 0.6 M NaCl usually did so. Lower concentra- tions of NaCl as well as protamine sulfate occasionally failed to produce an inhibitory effect.

After infusion of glucose, lipase activity at pH 8.5 was increased in the tis- sue homogenate and inhibition of the activity by 0.6 M NaCl was more pro- nounced.

Lipase activity is probabIy increased by preincubation in vitro in the pres- ence of epinephrine. This effect varied considerably from one sample to another.

From the results obtained it was concluded that in human adipose tissue

1326 PER BJijRNTORP

a lipase activity is present which has the same characteristics as that recent- ly described in rat epididymal fat pads and which is epinephrine sensitive. The presence of lipoprotein lipase in vitro could not be established with certainty.

ACKNOWLEDGMENTS

This work was supported by grants from the Swedish Medical Research Council. The technical assistance of Miss Majvor Karlsson is gratefully acknowledged,

REFERENCES

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Per Bjtirntorp, M.D., Assistunt Professor of Experimental Medicine, First Medical Service, Sahlgrenska Sjukhuset,

University of Giiteborg, Giitebmg, Sweden.