Acta Medica Scandinavica. Vol. 181, fasc. 3, 1967

From the First Medical Service, Sahlgren’s Hospital, and the Department of Clinical Chemistry, University of Goteborg, Goteborg, Sweden

Conversion of Glucose-14C into Carbon Dioxide and Lipids in Different Specimens of Human

Subcutaneous Adipose Tissue

BY

PER BJORNTORP and ALE MARTINSSON

Nearly all investigations of the glucose metabolism of adipose tissue in experi- mental animals have been performed with fat pads from rat epididymis. A strict standardization of this procedure has proved necessary in order to get reproducible results. Such standardiza- tion has been made in certain respects in investigations of adipose tissue speci- mens from human beings (5, 15). In animal experiments, several factors such as age, sex, breeding, operation and sampling can be made uniform. Such a uniformity is impossible to obtain in human studies. Furthermore, contrary to epididymal fat pads from rats, it is necessary to dissect human adipose tissue intrumentally. This causes disrupted and damaged cells on all surfaces of the specimen as well as other types of trauma during the han- dling of the preparation. Such treatment has been shown to cause considerable derangement of the metabolism in epididymal fat pads from rats (1 4).

Submitted for publication November 24, 1966.

Estimating insulin-like activity in serum by the in vitro oxidation of glu- cose into carbon dioxide, Renold et al. (1 1) found a higher activity in adipose tissue specimens taken from the distal part of the epididymis than in specimens taken from the proximal part. Similar results have also been obtained by Lyngsee

Lyngsee (8) found also that the oxida- tion rate in the presence of insulin was lower in the large specimens than in the small ones but this difference was eliminated by using the square root of the weight, instead of the weight, as a reference basis. Thus, it is important to standardize a number of factors when making adipose tissue preparations. I t seems probable that factors of this kind must be thoroughly investigated before quantitatively reliable data on human adipose tissue can be obtained.

The aim of the present investigation was to study the influence of specimen size on the glucose metabolism in human

(8).

359

360 PER BJORNTORP AND ALF MARTINSSON

Fig. 1. Needle biopsy specimen from subcutane- ous human adipose tissue. Magnification x 20.

subcutaneous adipose tissue in vitro with regard to the conversion of glucose into carbon dioxide and lipids both with and without insulin in the incuba- tion medium. Furthermore, the influence of glucose concentration on the effects of added insulin has been investigated.

T h e results obtained with specimens dissected in the conventional manner by using a pair of scissors were compared with those obtained with specimens aspirated by a needle biopsy instrument. This gave tissue preparations of the same size as the ones obtained with the technique recently introduced by Hirsch and Goldrick ( 5 ) , but the tissue yields were better.

Material and methods The adipose tissue used in the present study was excised from patients operated upon for abdominal diseases, usually cholelithiasis. Patients with acute infections or bilirubinemia were excluded.

The specimens used were prepared in two principally different ways. One type was taken with a small pair ofscissors and dissect- ing forceps. The other type - needle biopsy

Fig. 2. Needle biopsy specimens from sub- cutaneous human adipose tissue. Magnification x 2.

specimen - was obtained with an instru- ment (10) in which specimens wereaspirated into a glass chamber filled partly with buffer solution and connected with a water suction pump by an adjustable valve mechanism. The specimens were collected on a stainless net which was placed between the glass chamber and the valve mechanism. By repeated aspirations and rinsings with buffer solution, small cylindrical adipose tissue specimens which seemed to be macroscopi- cally free from blood and fat droplets were obtained. They were easily collected on the net and could then be transferred to anothrr vessel.

Specimens obtained in this way are shown in figs. 1 and 2. Four groups were used in the series of experiments where the importance of the specimen size was studied. These groups are henceforth referred to as 400 ms, 100 mg, 20 mg and needle biopsy specimens. When working with 20 mg specimens, seven to ten specimens were collected and weighed together. The weight of the specimens obtain- ed by the needle biopsy technique varied between one and five mg.

After surgical excision, the adipose tissue was placed in Krebs-Ringer bicarbonate buffer, pH 7.4, at room temperature and immediately carried to the laboratory for further preparation. The preparation was started within five minutes after the excision and was usuallylfinished after 20 minutes.

LIPIDS IN HUMAN SUBCUTANEOUS TISSUE 361

Preparations by scissors and forceps were performed on a plastic plate moistened with buffer. The specimens were then blotted and weighed on a Mettler B-6 balance after which they were placed in incubation vessels containing 2 ml incubation medium.

The incubation medium consisted of Krebs-Ringer bicarbonate buffer containing radioactive glucose and albumin (Bovine albumin powder, Fraction V, Armour, batch 670) a t a final concentration of 5 per cent. In the experiments in which the effect of the specimen size was investigated, the glucose concentraticn was 10 mM. The total activity of g l ~ c o s e - l - ~ ~ C (The Radiochemical Centre, Amersham CFA 204, sp.a. 20 mCi/ mmol) was 4 x lo5 cpm.

When the effect of glucose concentration was studied, the glucose concentrations were 5 mM, 2.5 m M or 1.0 mM. The total ac- tivity of glu~ose-U-1~C (The Radiochemical Centre, Amersham CFA 16, sp.a. 2 mCi/ mmol) was 3.6 x lo6 cpm. The albumin used was of another batch (HL 1572) but of the same type and manufacture as mention- ed above. The incubation vessels were sealed with rubber stoppers, adapted for collection of “COP in a small glass beaker. This beaker, made of cut Pyrex 10 x 120 mm test tubes, was placed in a wire spiral basket in the incubation vessel.

In the first series of experiments 400 mg, 100 mg, 20 mg and needle biopsy specimens were incubated. In each vessel, the total weight of the specimens was about 200 mg, except the 400 mg group. Two incubations were performed without insulin as controls, and two with insulin (recrystallized mixed bovine and pig insulin, lot 597, Vitrum AB, Stockholm), added to a final concentra- tion of 0.1 I U per ml medium.

In the second series of experiments, investigating the importance of glucose concentration, a biopsy specimen weighing about 100 mg was placed in each incubation vessel. Four incubations were performed at each glucose concentration, two without and two with insulin (recrystallized calf insulin, lot 11 164, Vitrum) as above.

All incubations were carried out a t 37” C

for two hours in an apparatus making about 90 cycles per minute. Then, 1 .O ml Hyamine 10 X (Packard) was injected into the small glass beaker in the incubation vessel. This was immediately followed by an injection of 0.2 ml 0.5 N sulphuric acid into the medium. The vessels were then left for four hours, after which the glass beaker was placed in a counting vial containing 10 ml scintilla- tion fluid (4 g 2,5-diphenyloxazole and 0.1 g 1,4- bis-2- (4-methyl -5- phenyloxazolyl) - ben- zene dissolved in 1,000 ml toluene). This pro- cedure of collecting carbon dioxide is principally the same as that of Snyder and Godfrey (12) and was found to give a recovery of 101 per cent as judged by test with Na

The incubated tissue was rinsed twice in 5 ml physiological saline. The lipids were extracted with methanol-chloroform accord- ing to Folch et al. (2). The chloroform phase was transferred to a glass vial and taken to dryness after which 10 ml scintillation fluid was added.

Radioactivity was determined in a Packard Tri-Carb liquid scintillation spectrometer. The quenching was determined by means of a n internal standard technique. It was found to be 2 1 and 5 per cent for cabon dioxide and lipids respectively.

The carbon dioxide and lipids formed were calculated as glucose by utilizing counts and the original specific activity of the incuba- tion medium. Statistical analyses were per- formed according to Kemp and Nielsen (7).

~ 1 4 ~ 0 , .

Results

The seemingly rectilinear relationships between time and the conversion of gluc0se-1-1~C into carbon dioxide or lipids are shown in two experiments in figs. 3 and 4 respectively. In these experiments, 100 mg specimens were used.

The results of the investigations of the influence of the specimen size on the oxidation of gluco~e-l-~*C into

362 PER BJORNTORP AND ALF MARTINSSON

cpm/g A.T.

3000

2000

1000

A A

0 0

A 0

I

30 90 150 210 mi nut as

Fig. 3. Relationship between time of incubation and the oxidation of g l u c o ~ e - U - ~ ~ C into WO, in 100 mg specimens from human subcutaneous adipose tissue (A.T.) from two patients. The incubations were performed in a medium consisting of 5 mM glucose and 5 per cent albumin in Krebs-Ringer bicarbonate buffer and glucose-U-"C, 1.8 x 10' cpm per ml medium. Each value represents the mean of two duplicates.

cpm /g A.T. 3000

2000

1000

A

0

A

0

A

0 A 0 - 30 90 150 210

minutes Fig. 4. Relationship between time of incubation and the incorporation of glucose-U-"(2 into lipids in 100 mg specimens from human adipose tissue (A.T.) from two patients. The incubations were performed in a medium consisting of 5 mM glucose, 5 per cent albumin in Krebs-Ringer bicarbonate buffer and glucose- U-"C, 1.8 cpm x los per ml medium. Each value represents the mean of two duplicates.

TABLE I. Conversion of gluc0se-l-1~C into "CO, by specimens of different sizes from human subcutaneous adipose tissue in vitro with and without insulin

Specimen No insulin Insulin added

Weight (mymole glucoselg (mymole glucoselg TYQe (mg) n adipose tissue/hr) adipose tissue/hr)

Dissected 400 8 59* 2.8 203 f 40.5 Dissected 100 a a0*6.5 163f27.5 Dissected 20 8 831-9.3 105 f 15.9 Aspirated (needle) - 7 5818 .1 52+ 8.2

Means f S.E.M.

LIPIDS IN HUMAN SUBCUTANEOUS TISSUE 363

TABLE 11. Conversion of glucose-l-"C into l'C-labelled lipids in specimens of different sizes from human subcutaneous adipose tissue in vitro with and without insulin

Specimen No insulin Insulin added

Weight (mymole glucoselg (mymole glucoselg Type (4 n adipose tissue/hr) adipose tissue/hr)

Dissected 400 7 133k11.4 175& 13.3 Dissected 100 7 196 & 24.5 2 0 2 5 18.2 Dissected 20 6 172 k28.3 177519.7 Aspirated (needle) - 5 75& 11.5 51519.1

Means + S .E.M.

W20, are shown in table I. The incorpo- ration of isotopes into lipids is shown in table 11.

Oxidation of glucose-1-14C into 14C0, was significantly higher in the 100 mg specimens than in the 400 mg pieces and in the needle specimens (p < 0.01 and p < 0.025). The oxidation was signifi- cantly higher in the 20 mg specimens than in the 400 mg pieces and the needle specimens (p < 0.025 and p < 0.05 respectively). With respect to oxidation, there was no difference between the 100 mg and 20 mg groups.

After addition of insulin, the l4CO2- production was significantly higher in the 400 mg specimens than in the insulin- stimulated 20 mg and needle specimens (p < 0.025 and p < 0.0025 respectively) but not significantly higher than in the 100 mg specimens.

After stimulation with insulin, the oxidation in the 100 mg pieces was significantly higher than in the 20 mg and the needle specimens (p < 0.05 and p < 0.0025 respectively).

When comparing the stimulating effect of insulin on different preparations the

following results were obtained. There was an almost four-fold increase

in the C0,-production in the insulin- stimulated 400 mg pieces compared with their unstimulated controls (p < < 0.0025). The 100 mg specimens more than doubled their ' basal incorporation (p < 0.005) while the oxidation rate, after stimulation with insulin in the 20 mg specimens, did not differ signifi- cantly from that of the unstimulated controls even if the mean was higher. A lower mean was obtained for needle specimens after addition of insulin but the decrease was not significantly lower than in the controls.

The results of the incorporation of glucose- 1 -14C into total lipids were practically identical for 100 mg and 20 mg specimens and were significantly higher than for incorporation into the lipids of the needle specimens (p < 0.05 and p < 0.01 respectively) as seen in table 11. The incorporation in the 400 mg pieces was significantly higher than in the needle biopsy specimens (p <

After addition of insulin, there was 0.01).

364 PER BJORNTORP AND ALF MARTINSSON

TABLE 111. Conversion of glucose-U-"C to ''CO, and l'C-labelled lipids in the presence of insulin (0.1 IU/ml medium) at different glucose concentrations. Percentage of the unstimulated controls

Specimen Medium glucose

Weight concentration TY Pe (mg) n (mM) CO, production Lipogenesis

Dissected 100 10 1.0 280k3.7 1 5 3 1 4 . 8 Ilissected 100 10 2.5 298*6.6 13014.1 Dissected 100 10 5.0 262 & 5.2 114C3.6

MeansiS.E.M.

practically the same incorporation into the lipids in the 100 mg and the 20 mg specimens. A comparison between the different groups showed that there was no statistically significant difference between the incorporation into lipids between the 20 mg, 100 mg and the 400 mg specimens. After addition of insulin the incorporation into lipids in the needle biopsy specimens was statisti- cally lower than in 400 mg, 100 mg and 20 mg specimens (p < 0.0005 in all cases).

The effect of insulin on the incorpora- tion from g l ~ c o s e - l - ~ ~ C into total lipids was small or none at all in all specimens compared with the unstimulated con- trols.

Table I11 shows the effect of a fixed amount of insulin at various glucose concentrations on the conversion of g l u c ~ s e - U - ~ ~ C into carbon dioxide and on the incorporation into lipids in 100 mg specimens. There were no differences between the effects on oxidation after stimulation with insulin when the glucose concentration varied. The incorporation into lipids, however, was significantly

higher with the lowest glucose concentra- tion (1 mM) than with 5 mhl (p < 0.05). There was no statistical differ- ence between the incorporation into lipids at 2.5 and 5.0 mM glucose concen- trations.

Discussion

I t has been shown that the size and preparation of human adipose tissue specimens are important factors infiuenc- ing the basal and insulin stimulated glucose metabolism. Hirsch and Goldrick (5) recently also studied this problem. Lipogenesis from glucose as well as its stimulation with insulin increased with increasing size of rat epididymal fat specimens. Human adipose tissue specimens above 1 mg generally showed the same glucose lipogenesis. The size of rat adipose tissue specimens was found to exert some influence on the oxidation of glucose into carbon dioxide (Lyngs~e (8)). Similar observations have been made also by others. Variations in metabolic activity with the thickness

LIPIDS IN HUMAN SUBCUTANEOUS TISSUE 365

of adipose tissue preparations have been reported by Vaughan (1 3), who showed that the incorporation of 14C-palmitic acid into triglycerides .varied in different parts of the fat pad from rat epididymis. I n the thicker part, the incorporation was loiver per wet weight unit than in the thinner part. The mechanism behind these phenomena is unknown but the findings stress the importance of defining the size of adipose tissue preparations.

Experimental conditions have varied in different investigations of the metab- olism of human adipose tissue. There- fore, it is difficult to compare the results from different studies. The size of the specimen has varied from 1 mg ( 5 ) , to about 300-500 nig (1) . Different preparation methods have also been used in these investigations.

The needle biopsy technique of pre- paring human adipose tissue specimens was introduced by Hirsch and Goldrick (5), and the effect of insulin in vitro was studied. Both needle specimens and larger specimens of different weights were used. The effect of insulin was absent or small and varying. Hennes (4) also found that insulin has no certain effect on needle biopsy specimens.

In the present study, the needle biopsy specimens converted less g l u c o ~ e - l - ~ ~ C into carbon dioxide and lipids both with and without insulin than other prepara- tions. Several factors may contribute to these differences as earlier discussed (5). A certain degree of trauma takes place during the aspiration of needle biopsy specimens because of tearing when the specimen is loosened from the tissue, as well as by friction against the walls of the biopsy instrument and

by a repeatedly reduced pressure during aspiration.

Whatever causes this lack of response of insulin-stimulation, it must be re- garded as a considerable disadvantage, and at present, it does not seem possible to study the insulin response in vitro with needle biopsy specimens.

The 20 mg or 100 mg specimens show- ed more activity than the needle speci- mens in the parameters measured. A functional optimum in non-stimulated adipose tissue specimens thus seems to be present in specimens weighing 20- 100 mg. I t seems probable that the upper limit of the optimal weight range is equilibration difficulties between central parts of the tissue and the incubation medium. The lower limit is probably dependent on the fact that, in the small specimens, the amount of damaged cells in relation to intact cells is increased.

The insulin effect was more pronounc- ed in the larger specimens. Kahlenberg and Kalant (6) have obtained princi- pally similar results, viz. they found a relationship between a low basal activity and a high activity after insulin stimula- tion in adipose tissue specimens from man.

At a low glucose concentration, 10 mg per 100 ml, Glieman (3) obtained the highest insulin stimulation in isolated fat cells from rats. In the present study, the effect of glucose concentration was studied at three different concentrations. The effect of insulin on the oxidation of glucose-U-1*C did not change at different glucose concentrations. However, the lipogenesis was most increased a t the lowest glucose concentration after insulin stimulation. This indicates that lipo-

366 PER BJORNTORP AND AI.F MARTINSSON

genesis in human adipose tissue in vitro can be stimulated by insulin. This effect is dependent on the concentration of glucose in the medium.

Summary

The influence of size of human sub- cutaneous adipose tissue specimens on the oxidation and lipogenesis from glu- cose was studied. The basal activity was lower in needle biopsy specimens than in larger dissected specimens. There was no effect of insulin in vitro on the needle biopsy specimens. In unstimulat- ed adipose tissue specimens, there was the same oxidation and lipogenesis in specimens weighing 20 and 100 mg.

Oxidation was stimulated by insulin and the response increased with the size of the specimen. Insulin increased lipogenesis when a low glucose concentra- tion was used.

Acknowledgement The investigation was supported by grants from Goteborgs Lakaresallskap.

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