Acta Medica Scandinavica. Vol. 180, fasc. 1, 1966

From the First Medical Service and Department of Clinical Chemistry, University of Gote- borg, Goteborg, Sweden

The Sucrose Space of Human Subcutaneous Adipose Tissue in Obesity

BY

PER BJORNTORP, BERTIL HOOD and ALF MARTINSSON

The participation of adipose tissue water in the weight increase of obesity is a matter of discussion. Studies of body composition on living persons indicate a considerable increase of total extra- cellular water in obesity, especially in certain clinical entities of this disorder

Morse and Soeldner (12) found no differences in either total water content or sodium space in human adipose tissue from obese and non-obese persons in studies performed in vitro. Bozenraad ( 5 ) , Pawan and Clode (13) and Thomas (14) have, however, found that obese persons have lower amounts of water in adipose tissue. Kahlenberg and Ka- lant (8) investigated the sorbitol space of omental adipose tissue in diabetic and non-diabetic patients and found no differences in the extracellular space.

When working with human subcuta- neous adipose tissue in vitro, the period of time for equilibration of the tissue with the incubation medium was determined for each sample in order to check the Submitted for publication January 13, 1966.

(11).

diffusion during the incubations (4). These data were considered to warrant separate publication since they seem not only to contribute to the question of tissue permeation but also to give values for sucrose (extracellular) space in human adipose tissue a t different body weights.

Material The clinical material consisted of 13 obese patients weighing more than 20 yo over ideal weight (lo), which were compared with 10 control patients. A detailed description of the material has been given elsewhere (4). The patients were classified into the follow- ing four groups:

The diabetic obese group consisted of six patients with constant glucosuria but with- out need for insulin treatment.

The obese group consisted of seven patients with no signs of clinical diabetes mellitus.

The hernia group and the gall-stone group consisted of six and four patients respectively. These 10 control patients were all within 5 13 yo of ideal weight (10).

123

124 PER BJORNTORP ET AL.

sucrose space (%)

T

I b 20 40 60 minutes of

incubation

Fig. 1. Sucrose space after different periods of incubation of human subcutaneous adipose tissue in vitro from different clinical groups. Means f SEM.

Methods Biopsy of adipose tissue was performed as described previously (2). The adipose tissue was cut into pieces each weighing 25- 40 mg and kept at room temperature in 4 yo albumin (Bovine albumin, Armour, fraction V, Batch Hb 1371) in Krebs- Ringer bicarbonate buffer of p H 7.4, containing glucose at a concentration of 1.8 mg per ml. They were carefully blotted with a filter paper and weighed on a torsion balance. Then, thirty minutes after the surgical removal, four pieces were incubated in 2 ml of the albumin-containing buffer with sucrose-UJ4C (The Radiochemical Centre, Amersham, CFB4) corresponding to about 2 x los counts/min. Directly after addition of the piece, and after 20, 40 and 60 minutes respectively, one piece was removed from the incubation medium, rinsed during less than five seconds in about 4 ml of the incubation medium without labelled sucrose, and then transferred to 1 ml water which was brought to boiling.

To 0.1 ml of this extract 0.5 ml Hyamine 10 X (Packard) and 0.5 ml absolute ethanol was added in that order and then 10 ml of 0.4 yo PPO (Packard) and 0.01 yo dimethyl POPOP (Packard) in toluene. Counting was performed in a Packard, Tri-Carb, liquid scintillation counter. Quenching was correct- ed for by internal standard.

TABLE I. Sucrose space of human subcutaneous adipose tissue from different clinical groups. MeansiSEM

Sucrose space’ (YO)

Hernia Gall-stone Diabetic obesity Obesity

20.2*1.2 18.0k 1.5 1 5 2 & 1.1 12.8f 1.8

1 Means of measurements at 20, 40 and 60 minutes (cf. fig. 1).

Since homogenization of the tissue after boiling did not increase the radioactivity of the extract, the above-mentioned proce- dure was considered adequate.

The radioactivity in 10 pl of the incubation medium was also counted in the way men- tioned. The radioactivity in the tissue was assumed to be present in the tissue water in the same concentration as in the incubation medium. The volume of water thus obtained from determination of the radioactivity was expressed as per cent of tissue wet weight and was after equilibration called the sucrose space.

Cell size and deoxyribonucleic acid (DNA) were determined as described earlier (2).

Results

Fig. 1 gives the measured sucrose space at different times after incubation for the groups investigated. I t seems that in all groups, irrespective of the size of the sucrose space, a steady level was reached within 20 minutes.

The sucrose spaces found are listed in table I. The hernia group had a significantly higher space than the obese groups (p < 0.01 for the obese group and p < 0.05 for the diabetic obese

HUMAN SUBCUTANEOUS ADIPOSE TISSUE IN OBESITY 125

group) and the gall-stone group had a higher space than the non-diabetic obese group (p < 0.05).

In fig. 2 the relation is shown between sucrose space and body weight given as the weight index actual weightlideal weight (10). No rectilinear correlation appeared for the whole material, but as judged visually, there seemed to be a levelling off of the rapid initial fall of sucrose space at weight indices above 1. This result further prompted plotting of sucrose space against cell size (fig. 3) and also a chemical expression of this (2), viz. the ratio of triglycerides to DNA (fig. 4). The results seemed to be essentially the same as when weight index was used as abscissa (fig. 2), viz. a levelling off of sucrose space after an early rapid fall at a cell size of about 100 ,u (fig. 3) or, otherwise ex- pressed, at about 3 g triglycerides per mg DNA (fig. 4).

Discussion The sucrose space of adipose tissue in vitro from patients with normal weight was thus found to be higher than that of patients with obesity.

Sucrose is a substance which dis- tributes itself mainly in the extracellular space of the tissue, since it seems unable to pass the cellular membrane as such (7). I t has also been demonstrated that sucrose is not converted into carbon dioxide or lipids in human adipose tissue (9). The sucrose space accordingly will approximate to the extracellular space of the tissue.

Larger pieces of adipose tissue than the ones utilized in the present work seem to increase their total water content during

" ' ( 1 : : = * a ; ,.; ; .

a r l * .

P I LO I 1 It I 3 I' , I I 6 "v.,ghlnd"

Fig. 2. Correlation between weight index and sucrose space in human subcutaneous adipose tissue in vitro (symbols as in fig. 1).

Fig. 3. Correlation between sucrose space and mean cell diameter in human subcutaneous adipose tissue in vitro (symbols as in fig. 1).

'i O D

Fig. 4. Correlation between sucrose space and grams triglyceride per mg DNA in human subcutaneous adipose tissue in vitro (symbols as in fig. 1).

incubation (9). This did not seem to be the case in the tissues used in the present work, since the sucrose space was un- changed at 20, 40 and 60 minutes of incubation. I t is possible that an increase

126 PER BJORNTORP ET AL.

of water content may have occurred when the tissue was kept in buffer solu- tion before incubation for sucrose space measurements, or that water content may increase only in adipose tissue pieces of higher weight or, possibly, that other water spaces, not measured, may increase during incubation.

I t has been shown earlier (3) that weight increase correlates with an in- crease of triglyceride content per fat cell. Taken together with the present findings, this seems to indicate that when adipose tissue mass increases, not only an absolute but also a relative increase of triglycerides occurs since extracellular space, and thus probably total water (9), shows a relative decrease.

Equilibrium between tissue and the labelled sucrose was attained already within 20 minutes of incubation with all tissue samples studied. Thus, even with the comparatively thick pieces of tissue, equilibrium was rapidly ob- tained. It is possible that even larger pieces of tissue equilibrate rapidly enough with the incubation medium to allow measurements of different metabolic activities. These problems are currently under investigation (9).

There did not seem to be a rectilinear correlation for the whole investigated material between different body weights and the sucrose space.The results were essentially the same whether the cell size against which the sucrose space was plotted was measured by morphological techniques or chemically, viz. triglyc- eride amount per unit DNA in adipose tissue. This phenomenon might not be considered as definitely demonstrated because of the limited material, especial-

ly in the region of subnormal weight, but it is of interest to note the agree- ment between this finding and the pre- dicted extracellular space of adipose tissue from the human as deduced by morphological-mathematical techniques by Bjurulf (1).

Even though the data presented seem to be of interest mainly in that they enable comparisons to be made between different clinical groups, they might also be a guide to absolute body-water spaces in vivo. It thus seems probable that a substantial amount of extracellu- lar water is present in adipose tissue in obesity. I n cases of obesity with a small mean diameter of the fat cells and thus a comparatively large extracellular space (cf. fig. 3), this volume of water might a t least partly offer an explanation for the water retention in so-called pathological obesity (1 I).

Summary

Sucrose space was determined in vitro in human subcutaneous adipose tissue from patients of different body weights. The obese patients had a smaller sucrose space than controls. There were higher values for sucrose space at lower body weights, while a t higher body weights this space seemed to level off.

References 1 . BJURULF, P.: The extracellular space per

mms in human subcutaneous adipose tissue. Communications from the Department of Anatomy, University of Lund, No. 6. Lund 1960.

2. BJORNTORP, P. & MARTINSSON, A,: The composition of human subcutaneous adi- pose tissue in relation to its morphology. Acta med. scand. 179: 475, 1966.

HUMAN SUBCUTANEOUS ADIPOSE TISSUE IN OBESITY 12 7

3. BJORNTORP, P., HOOD, B., MARTINSSON, A. & PERSON, B. : The composition of human subcutaneous adipose tissue in obesity. Acta med. scand. 180: 117, 1966.

4. BJORNTORP, P. & HOOD, B.: Studies on adipose tissue from obese patients with or without diabetes mellitus. I. Release of glycerol and free fatty acids. Acta med. scand. 179: 221, 1966.

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7. DEANDE, N., SCHREINER, G. E. & ROBERT- SON, J. S.: The velocity of distribution of sucrose between plasma and interstitial fluid, with reference to the use of sucrose for the measurement of extracellular fluid in man. J. Clin. Invest. 30: 1463, 1951.

8. KAHLENBERG, A. & KALANT, N.: The effect of insulin on human adipose tissue. Canad. J. Biochem. 42: 1623, 1964.

9. MARTINSSON, A. : In preparation. 10. Metropolitan Life Insurance Co. : Statisti-

cal Bulletin 40: 1, 1959. 11. MOORE, F. D., OLESEN, K., MCMURREY,

J. D., PARKER, H. V., BALL, M. R. & BOYDEN, M. C. : The body cell mass and its supporting environment. W. B. Saunders, Philadelphia 1963.

12. MORSE, W. I. & SOELDNER, J. S.: The composition of adipose tissue and the non- adipose body of obese and non-obese man. Metabolism 12: 99, 1963.

13. PAWAN, G. L. S. & CLODE, M. : The gross chemical composition of subcutaneous adipose tissue in the lean and obese human subject. Biochem. J. 74: 9P, 1960.

14. THOMAS, L. W.: The chemical composition of adipose tissue of man and mice. Quart. J. exp. Physiol. 47: 179, 1962.