lipid mobilization from human subcutaneous adipose tissue in vitro

Acta Medica Scandinavica. Vol. 182, fasc. 6, 1967

From the First Medical Service, Sahlgren's Hospital, University of Goteborg, Giiteborg, Sweden

Lipid Mobilization from Human Subcutaneous Adipose Tissue in Vitro

BY

PER BJORNTORP

In the rat epididymal fat pad the utilization of glycerol is low, and usually the glycerol produced originates from the complete cleavage of triglycerides (24) . Glycerol production is therefore probably a good measure of adipose tissue triglyceride hydrolysis (24).

These reactions are only partially know in human adipose tissue. Glycerol utilization during basal conditions is low (14) . Glycerol is released, but the extent to which it originates from the complete hydrolysis of triglycerides, partial glycerides, or even f i om non-glyceride sources, is not known. Furthermore, the lipase acitivity which is believed to be mainly responsible for triglyceride hydrolysis in rat adipose tissue j5, 13, 18, 25) has only been qualitatively described in human adipose tissue ( 1 ) .

Studies of these reactions in human adipose tissue seem worthwhile not only because species differences of adipose tissue metabolism have been repeatedly noticed (8, 21). but also because clas- Submitted for publication May 29, 1967.

sification is needed of the abnormally elevated glycerol release from subcutaneous adipose tissue in human diabetes mellitus described previously (6, 27).

I n the present work these reactions have been studied in human subcutaneous adipose tissue and compared with corresponding reactions in the rat epididymal fat pad.

Material and methods Human subcutaneous adipose tissue was nbtaint,d from patients operated on for different abdominal diseases. They were fasting for 18 hours before operation. T h e specimen \ \ a$ taken from the upper part of the abdominal subcutaneous fa t layer, placed in Krebs-Ringer bicarbonate buffer, pH 7.4, a t rooni ternperaturc, brought to the labora- t r q and ininipdiately prorrswd.

Rat epididyrnal fat pads from 250-350 g. Spraguc-Dawley rats fed ad libitum were oh- lai'ned as previously dcsrribed ( 3 ) .

Total adipose tissue glycerol and free fatty arid production were determiried in incubation systems consisting of 3 nil Krebs-Ringrr bicarhonatc buffer containing- 4 % SCTUIII al-

717

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humin (Bovine serum albumin, Fraction V, Armour, hatch KC 2271) . Glucose was present at a Goncentration of 10 mM. Samples of human or rat tissuc (100-200 mg) in duplicate flasks were first incubated for 30 nlin. to allow equilibration between the medium and tissue glycerol. From the medium the first aliquots for glycerol (15) and fatty acid determinations (2, 9 ) were then taken, and the tissues from two flasks taken for the determination of tissuc fatty acid conccntratiun. Thcreafter additions of insulin (Crystalline pig plus bovine insulin, Nordisk Insulin, Gentofte, Denmark) ( 10,000 pU/ml) or norepinephrine (50 pg/ml) were made, and incubation continued for another 60 min. The coincentrations of glycerol and fatty acids in the medium and 'fatty acid concentration in the tissues were then determined again.

In cxpcrinients wherc glycerol utilization was measured, three picccs of human adiposc tissue, each weishing about 200 ing, wcrc incubated as described above but without glucose. The final free fatty acid concentration of this incubation medium was 0.15 mM. Glycerol was added to all flasks to a final concentration of 5 niM and approxirnatcly 200,000 co'tints/min. of l-I4 C-Glycerol (The Radiocheniical Centre, Amersham, England, CFA47). Two flasks were incubated without further addition and two flasks with 10,000 pU/ml of insulin. In a similar serics run in parallel the albumin was prepared so' that the final incubation mcdium contained a concentration of 0.85 mM of frce fatty acids. This was accomplished by the addlition to a 20% albumin solution, with thorough mixing, of a suspension of palmitic acid in water, brought to pH 7.4 with KOH. Incubations wcrc pcrformed in 50 ml cyhindrical flat- bottoni tubcs oscillating 120 timesimin. in an incubation bath a t 37.0" C. The final pH was 7.4 and the gas phase was 95% 0 2

and 5 % COz. Radioactivity in carbon dioxide, fatty acids and glyceride-glycerol was determined as described previously (4 ) , and calculated as glycerol from t,he initial spccific activity of the incubation medium and the counts obtained.

When incorporation of glucose into glyceride-glycerol and free glycerol was nicasur- d, the incubation medium was the same a s that described with addition of glucose to a final concentration of 10 ntM and also approximately 200,000 countdmin. of U-14C- glucose (The Radiochemical Centre, Amers- ham, England, CFB35). After incubation for 60 min. glyccridc-glyccrol radioactivity was determined, 1 nil of the medium added to 1 ml acetone and the protein precipitate ccntrifuged. Aliquots of the supernatant were applied on silicic acid thin-layer chroniato- plates (Kicselgel nach Stahl, Merck, Darm- stadt, Grrniany) and run in chloroform: acetone: 5N ammonia (10 : 80 : 10) (20) until a suitablc running distance was obtain- cd. Glycerol and glucose were run separately as standards. Duplicatc aliquots from an incubation mediium and standards were run on cach plate. Aftcr running for a suitable distance the plates were dricd a t about 100" C until all smell of ammonia ha'd disappeared. The area of the plate on which standapds had been applied was sprayed with developing agents (20) with the other part of the plate covered. Areas corrcspond- ing to glycerol (R, = 0.3-0.4) and g l i i c o . ~ iR, = 0.1) were then scraped into count iy vials and countcd as described by Snyder and Stcphcns (19) .

Lipase activity was determined as described prrviously ( 2 ) . Human adipose tissue nccded to he homogenized in homogenizcrs with a somewhat loosely fitting pestle ( 1 ) . The whole hoinogenate was assayed. Since i he amount of the endogenous lipids was suf- ficient to give saturation, no addition o,f sub- strate was nndc unless stated otherwise.

In experiments whcrc hydrolysis of partial glyccrides was measured, adipose tissue was taken directly after excision and homogeniz- cd with 2 ml 0.15 M ,potassium chloride and 0.1 g of glycerylmonooleate (Hopkins and Williams), or 0.1 g of glycerylmonostearate (BDH), or 0.1 g of glyceryl-I, 2-dipalmitate (melting point 59.2-60.5") or as a control in cach series of expcriments, 0.1 g of human adipose tissue lipids. These were obtained by extraction of human adipose tissue in

LIPID MOBILIZATION FROM HUMAN SUBCUTANEOUS ADIPOSE TISSUE 7 19

TABLE I. Balance between fatty acid and glycerol production in human and rat adipose tissue

Human Rat n - 8 n = 8

~~~~ ~

Tissue fatty acid conccntrations 0’ (pEq/g) 1.15&0.04 3.28 10 .20

60’ (pEq/g) 1.02 50.05 1.4810.20 60’ minus 0’ (pEq/g) -0. I3 *0.04 - 1.80+0.20

60’ minus 0’ (pEq/g) 1.18 10.08 1.2 7 +O. 18 Medium fatty acid concentration

Total fatty acid production

Glycerol production (pEq/g Per 60’) 1.05.1-0.07 -0.53 *0.21

(pmoles/g per 60’) 0.37 50.10 1.01 i 0 . 0 8

Fatty acids “reesterified”* 0.06 1 0 . 1 5 3.56 1 0 . 2 1

(PEqlg Per 60’)

Means & S.E.M.

* Calculated as glycerol production x 3 minus total fatty acid production (cf. (24) and Discussion.) Times given are after 30’ preincubation.

methanol : chloroform, partition by the method of Folch et al. (10) and evaporation of the chloroform phase under nitrogen. One ml of the homogenate, 0.2 ml of 20 % albumin, pH 7.0 and 0.1 ml of 0.25 M phosphate buffer, pH 7.0, were thcn incubated and the free fatty acid conrcntration determined at 0 and 30 min. All flasks wcrc incubated in duplicate.

Results Balance of glycerol and fa t t y acid production Balance of glycerol and fatty acid production, measured essentially in accord- ance with Vaughan (24), is shown in table I.

In the basal state total fatty acid production was higher in human than in rat adipose tissue. Rat epididymal fat pads showed a net uptake of fatty acids in spite of the fact that glycerol pro-

duction was higher in these tissues than in human tissues. Insulin in a high dose caused a decrease in net fatty acid production as seen in table 11, and in some cases a net uptake could be demon- strated. Glycerol release appeared to be unchanged. When norepinephrine was added, glycerol and frce fatty acid production were increased.

Metabol ism of glycerol In order to explain the higher total fatty acid production in relation to glycerol release in human adipose tissue, and its lower glycerol production, factors deter- mining the turnover of the free glycerol pool were studied.

Glycerol utilization was first investigated. Table I11 shows that when the fatty acid concentration of the incubation medium was low, a small but significant

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TABLE 11. Balance between fatty acid and glycerol production in human adipose tissue. Effects of insulin and nor-epinephrine

No addition Insulin Nor-epineph- (10,000 yU/ml) rine

(50 Llglml)

Tissue fatty acid concentrations ‘0’ (/IEq/g) 1.09+0.04 I ,0910.04 1.09 50.04

60’ minus 0’ (yEq/g) 0.22 &0.03 -0.2310.05 1.03 60 .09 60’ (flEq/g) 1.31 50 .05 0.85 10 .04 2.1250.10

Medium fatty acid concentration

Total fatty acid production

Glycerol production

60’ minus 0’ (yEq/g) 0.86 1 0 . 0 7 0.31 h0.07 1.15 10.09

(PEq/g Per 60’) 1.08 f 0.10 0.08k0.06 2.18k0.14

(ymoleslg per 60‘) 0.41 .k0.04 0.3810.04 0.71 50 .04

Fatty acids “reesterified”2 ( P W g per 60‘) 0 .1510. I 1 1.0610.05 - -0.05&0.13

Means & S.E.M. n = 8

* Calculated as glycerol production x 3 minus total fatty acid production (cf. (24) and Discussion). Times given are after 30’ preincubation.

TABLE 111. Metabolism of l-’aC-glycerol in human adipose tissue

co, Glyceride Total (mpmoles/g/hr) (mymoles/

Fatty acids Glycerol d h r ) (mpmoles/g/hr) (mymoles/g/hr)

Low FF.4 in medium 0 12.4 h0 .7 36.5 +4.4 1 7.8 :i 4.8 66.7 &5.9 Insulin 10.9*0.9 35.5 1 4 . 2 16.2 1 4 . 2 62.6+5.1 (10,000 pU/ml)

0 19.412.9’ 45 .017 .3 17.4h5.3 81 .816 . l a Insulin 20.8 5 1 .g3 36.5 1 4 . 4 23.4*3.9 80.754.72 (10,000 ,uU/ml)

High FFA in medium

Means S.E.M. n = 4 Low FFA concentration: 0.15 m M High FFA concentration: 0.85 m M All comparisons with low FFA in medium, 0

a p <0.05 p<O.IO> 0.05

p <0.02

LIPID MOBILIZATION PROM HUMAN SUBCUTANEOUS ADIPOSE TISSUE 72 1

amount of glycerol from the incubation medium was incorporated into the measured metabolites. As reported previously (141, this amount is about 1/10 of the incorporation from 1-I4C glucose under similar conditions. With higher fatty acid concentrations in the medium, incorporation into carbon dioxide was almost doubled while labelled fatty acid arid glyceride-glycerol did not change. Insulin had no influence.

Non-lipolytic glycerol rrlease C-14 from glucose was incorporated into glyceride-glycerol while no significant incorporation into the released free glycerol could be found. These results accord with those obtained in the rat ( 7 ) and assuming a small a-glycerolphospate pool in human adipose tissue, provide evidence that in human adipose tissue, glycerol cannot be released directly from the a-glycerolphosphate pool.

Lipolytic activity in hornog~nates

Since it had been shown previously that the hormone-sensitive lipase of the rat epididymal fat pad can be found in human adipose tissue ( 1 j, an assay system optimal for this type of activity was used ( 2 5 ) . These results are given in table IV. Glycerol release was approximately doubled by the addition of a large amount of norepinephrine, and addition of ascorbic acid to avoid oxi- dation of norepinephrine caused only a small further increase. Lipase activity increased slightly but significantly after norepinephrine with or without ascorbic acid. The activity was small compared with that in the rat ( 3 ) .

4-673002. Acta Med. Scand. Vol. 182' 6

T.\BLE IV. Nor-epinephrine activation of glycerol release and lipase activity in human subcutaneous adipose tissue

Addition to incubation Glycerol Lipase mixture release' activity

(ymolesl (pEq 9 . b ) FFA/g/

30')

0 1.04 0.30 Nor-epinephrine (50 pg) 2.36 0.39 Nor-epinephrine (50 pg) + ascorbic acid (2.5 pmole) 2.47 0.45

~~ ~ ~

Means. n = 3 1 Measured without 30 min pre-incubation.

The discrepancy between these results from human adipose tissue and those obtained with rat adipose tissue prompted a direct comparison between these two. Such a comparison a t different times after removal of the tissues, is given in fig. 1. In the rat, lipase activity and glycerol release tended to decrease after removal of the tissues and then increased by a factor of 2-3 after norepinephrine addition. These parameters seemed to follow each other but on different activity levels a5 previously described ( 3 ) . In the human tissues glycerol release was somewhat lower than in the rat tissues. A twofold increase was found after norepinephrine stimulation. Lipase activity was low and amounted to only about 1/5 of that in the rat. This difference was still more pronounced after the small norepinephrine stimulation of human lipase activity, which was only about 1/10 of that in the rat.

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Cp rnotes/g 1 (peq . FFA/g per 30’)

2.0

1.0

Minutes

Fig. 1. Glycerol release from and lipase activity in human (open) and rat (striped) adipose tissue at different times after incubation, Values are means of three experiments. Nor- epinephrine (50 pgjml) added at 0’. G = glycerol release. L = lipase activity.

Hydrolysis of partial glycerides The higher ratio of net fatty acid production/glycerol release in human than in rat adipose tissue (table I ) could be explained by qualitative differences be-

tween the lipolytic process in human and rat adipose tissue. Hydrolysis of triglycerides to partial glycerides would thus be expected to produce such a high ratio. Therefore the ratio of fatty acid pro-

TABLE V. Fatty acid and glycerol release in homogenates from human subcutaneous adipose tissue and rat epididymal fat pad

Human Rat

Fatty acid release (ml /g /30 ‘ ) 0.40 + 0.08 4.27 0.41 Glycerol release (,umoles/g/30’) 0.15 & 0.03 1.54 3 0.21 Fatty acid release

Glycerol release 2.7 2.8 ~ _ - _ _

TABLE VI. Lipase activity in human subcutaneous adipose tissue homogenate after addition of different glycerides

Fatty acids released (&l/g/30’)

Human adipose tissue triglyccrides (0.1 g) 0.43 10.04

Monoolein (0.1 g) 10.0&1.2 Monostearin (0.1 g) 4.24*0.8 1,2-dipalmitin (0.1 g) 1.0410.06

Means & S.E.M. n = 6 . Means S.E.M. n = 6.

LIPID MOBILIZATION FROM HUMAN SUBCUTANEOUS ADIPOSE TISSUE 723

duction/glycerol production was measured in homogenates from rat and human adipose tissues. As seen in table V no difference was found, indicating that if accumulation of partial glycerides in homogenates of these two tissues occurred, it was the same.

When lipolysis was followed in human adipose tissue homogenates, containing added partial glycerides, an increase in fatty acid production was noted when glyceryl- 1,2-dipalmitin was added, but the effect was greater when the oleic and stearic acid monoglycerides were added. These results are set out in table VI.

Discussion

In the rat epididymal fat pad, triglyceride hydrolysis to diglycerides seems to be the rate-limiting step in complete cleavage of triglycerides in adipose tissue. Triglyceride lipase activity is much lower than that of di-or mono-glyceride lipase when assayed in a cell-free system, and partial glycerides do not seem to accumulate under normal conditions in the rat epididymal fat pad ( 2 2 ) .

As with rat adipose tissue ( 7 ) , the measurement of glycerol as an end prod- uct of lipolysis in human adipose tissue is probably not invalidated by glycerol production from non-lipolytic sources such as a-glycerolphosphate. Further- more, likewise in accord with results obtained with the rat epididymal fat pad, utilization of free glycerol was small during the present experiments with

human tissues, indicating that glycerol liberated by the lipolytic process would disappear only to an insignificant degree.

Insulin did not increase glycerol utilization by adipose tissue, indicating that the net decrease in glycerol production caused in human adipose tissue by insulin (4, 6), is not attributable to an increased utilization, hut rather to a decreased release of glycerol.

Accumulation of partial glycerides probably did not occur in homogenates from human adipose tissue. Furthermore, hydrolysis of partial glycerides in homogenates was faster than that of triglycerides. Taken with the finding of Hirsch et al. ( 1 2 ) that human adipose tissue contains no partial glycerides, this seems to indicate that partial glycerides do not ordinarily accumulate during the lipolytic process. As with the rat epididymal fat pad, this does not, however, exclude that in certain circumstances, for example when triglyceride lipase is activated, partial glycerides could accumulate as discussed by Strand et al. (22) .

If it were accepted that no partial glycerides accumulated during the lipolytic process in human adipose tissue, the production of one mole of glycerol would correspond to the production of three equivalents of free fatty acids (cf. Vaughan, 24). The finding of a high ratio of free fatty acid production/glycerol production in human adipose tissue would then actually be an indication of a lower capacity for re-esterification of free fatty acids than in the rat epididymal fat pad. A direct estimation from the data of table I indicates that little or no esterification had occurred in the human adipose tissues. Ostman (27) has

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shown, however, that radioactive fatty acids are readily incorporated into the glycerides of human adipose tissue in vitro. Even if such techniques do not seem to allow quantitation of the re- esterification process, due to unknown isotope dilution (26)) Ostman's ( 2 7 ) data taken together with those of table I probably indicate that an unknown amount of fatty acids was released without glycerol production. Although indirect this seems to be an indication of accumulation of partial glycerides in human adipose tissue during lipolysis. The still higher ratio of fatty acid production/glycerol release during norepinephrine stimulation (table 11), found independently by Martinsson (16), could be an indication of further accumulation of partial glycerides during these conditions.

'The patients from whom the adipose tissue was taken were fasting for about 18 hours, but the rats were fed ad libitum. This could mean a decreased re- esterification in the human tissues as compared with tissues from non-fasting subjects. Comparisons with the rat tissues are still pertinent, since Vaughan (24) has shown that, even if the rats had been fasted as long as these patients, re- esterification would still not have been impaired.

Recent data presented by Have1 (1 1 ) indicate a ratio of fatty acid turn-over rate/glycerol turnover rate in plasma higher than three in the human, partic- ularly during exercise with increased lipid mobilization. These data accord with the findings in tables I and I1 and seem to strengthen the validity of these data for in vivo conditions.

In human adipose tissue the possible contribution of free fatty acids from partial hydrolysis of triglycerides could be very small. In this case a re-esterification deficiency would be relatively large since only a small part of the released fatty acids had then been re-esterified (cf. table I ) . If partial hydrolysis of triglycerides made a very large contribution to the free fatty acid pool, the re- esterification process would also be insuf- ficient to retain the free fatty acids within the adipose tissue. Human adipose tissue lipid mobilization therefore seems to be more influenced by a deficient capacity of the re-esterification process than that in rat adipose tissue. Re- esterification in the rat thus keeps pace with lipolysis after fairly long fasting (24) and a-glycerolphosphate synthesis is not decreased early in uncontrolled alloxan diabetes ( 2 3 ) . Lipolytic activity in human adipose tissue is also low as compared with the rat, as reflected by the low glycerol and fat ty acid release after lipolytic hormones (17) and the low lipase activity in homogenates. This could mean that human lipid mobilization depends not only on lipolysis but also to some degree on restricted re- esterification, while in the rat the lipolysis predominates.

The probably lower rate of glyceride breakdown in human adipose tissue as compared with that in the rat epididymal fat pad, and also the probably lower rate of re-esterification in human tissues, indicate a lower turnover rate of human adipose tissue triglycerides. This implies a more static subcutaneous fat depot in the human as compared with the rat epididymal fat pad.

LIPID MOBILIZATION FROM HUMAN SUBCUTANEOlJS ADIPOSE TISSUE 725

Summary Lipid mobilization from the rat epididymal fat pad is known in considerable detail, while the reactions responsible for lipid mobilization in human subcutaneous adipose tissue are only partially known.

A comparison between the balance of glycerol and free fatty acid production in human and rat adipose tissue revealed that the ratio of free fatty acid production/glycerol production was higher in the human tissues. Norepinephrine increased this ratio in human tissues while insulin decreased it.

The reason for this discrepancy was investigated, It was found that glycerol release is probably a measure of lipolysis in human tissue as it is in rat adipose tissue. It was found that glycerol released from human subcutaneous adipose tissue probably did not originate directly from the a-glycerolphosphate pool and that utilization of free glycerol was insignificant.

The ratio free fatty acid release/glycerol release was the same in human and in rat adipose tissue homogenates, sug- gesting that partial glycerides do not accumulate more in human adipose tissue than in rat adipose tissue. Further- more, lipolytic activity against partial glycerides was high in human adipose tissue homogenates. These results, taken together with previous data showing that no partial glycerides are present in human adipose tissue, do not suggest the accumulation of partial glycerides in human adipose tissue during the lipolytic process.

The high ratio of free fatty acid production/glycerol production was there-

fore considered to be due mainly to a relative incapacity of the re-esterification system of adipose (issue from fasting humans as compared with that in rat tissue. The possibility of accumulation of partial glycerides in human adipose tissue during the lipolytic process in certain conditions was also considered.

The results indicate a lower turnover rate of adipose tissue triglycerides in the human than in the rat, implying perhaps a more static fat depot in the human.

Acknowledgement Supported by the Swedish Medical Research Council (Project No. 19X-251-0 3A).

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