Acta Medica Scandinavica. Vol. 179, fasc. 2, 1966

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

Studies on Adipose Tissue from Obese Patients with or without Diabetes Mellitus

11. Basal and Insulin-stimulated Glucose Metabolism

BY

PER BJORNTORP

I t has recently been reported that adipose tissue in vitro from obese pa- tients with diabetes mellitus released more glycerol and fatty acids per fat cell than controls (4). The high release of fatty acids was probably caused not only by a decreased re-esterification but also by an increased lipolytic activity. Both these defects could be corrected by addition of large amounts of insulin and were therefore suggested to be caused by a deficiency in adipose tissue with respect to insulin level or insulin sensitivity (4).

Ostman (13) recently found that adipose tissue from young, insulin- dependent diabetic patients showed a decreased re-esterification of fatty acids, as measured by incorporation of labelled fatty acids into triglycerides in vitro, and probably also an increased lipolysis, while glucose uptake was not diminished. No differences were found between adult diabetics and controls.

In the present work adipose tissue from obese patients with and without Submitted for publication November 8, 1965.

diabetes mellitus has been compared with tissue from controls with respect to in- sulin effects on certain metabolic proc- esses centered on glucose uptake. Total glucose uptake and the incorporation of l-14C-glucose into carbon dioxide and lipid were selected for investigation, these incorporations being increased by insulin in rat adipose tissue (8).

Material and methods

The material comprised 6 non-diabetic obese patients all more than 25 yo above “desirable weight” (12) and all with a k-value above 0.90 for the intravenous glucose-tolerance test. Six diabetic obese patients were also more than 25 T, overweight, had constant glucosuria and fasting blood sugar above 150 mg per 100 ml. Good control of their diabetes mellitus was obtained by dietary means and/or sulfonylurea drugs. The con- trols were 13 patients operated on for hernia and patients with gall-stone or duodenal ulcer. The material has previously been described in more detail (4).

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TABLE I. Glucose uptake by human subcutaneous adipose tissue in vitro. Mean f SEM

mg/mg DNA/h mg/ 10’ cells/h

No Insulin No Insulin addition (10,000 ,uU/ml) addition (10,000 pU/ml)

Controls 2.lb0.5 2.150.6 1.750.3 1.910.4 Obesity 2.910.7 3.3h0.4 3.710.9 3.8k0.4 Diabetic obesity 4.1h0.6 ’ 3.910.8 3.5b0.5 4.5+0.9

Controls-Obesity p >0.10 p >0.10 p <0.05 p <0.01 Controls-Diabetic obesity p <0.05 p <0.10>0.05 p <0.01 p <0.05 Obesity-Diabetic obesity p >0.10 p >0.10 p >0.10 p >0.10

Adipose tissue from the subcutaneous stores lateral to the umbilicus was excised, dissected into 25-40 m g pieces and incubated in a Krebs-Ringer bicarbonate medium containing albumin and glucose a t final con- centrations of 4 ”/: and 10 m M respectively, as previously described (4). l-14C-glucose (The Radiochemical Centre, Amersham, CFA 204) was added in an amount corre- sponding to about 250,000 cpm. Incubations were performed in cylindrical glass tubes, sealed with a rubber stopper pierced by two glass tubes, closed with rubber membranes as carlier described (2). Six tubes were incubated in each experiment. The first two contained no additions to the basal medium, to another two was added 10,000 niicrounits/ ml of insulin, and two further tubes bere incubated without tissue as a control. Enzymatic activities were stopped after 150 minutes of incubation by injecting 0.2 ml N sulfuric acid through the rubber membrane. Radioactivity in carbon dioxide was then collected and counted, and radioactivity in lipid extracted and counted as described elsewhere (2). Glucose was determined enzymatically (10) in the incubation medium at 0 and 150 minutes of incubation.

Results of radioactive experiments were expressed as glucose converted to carbon dioxide or lipid, calculated from counts found in these products and from the values for the original specific activity of the medium.

The insulin used was mixed crystalline bovine and pig insulin (Nordisk Insulin).

Results Table I gives the results of the glucose- uptake measurements expressed on the basis both of DNA and of the number of adipose tissue fat cells (5). In the basal state the tissue from obese patients with or without diabetes took up more glucose than the controls; on a DNA basis the difference was statistically significant only for the diabetic obese group. The obese groups were not different from each other. After insulin addition, differences between the both obese groups and the controls could again be found on a cell number basis while on a DNA basis the difference was found only as a trend for the diabetic obese group. Insulin did not increase glucose uptake in any of the groups.

In table I1 the values for incorpora- tion of label from l-14C-glucose into carbon dioxide are listed in the basal state and after stimulation with insulin. The non-diabetic obese group incorporat- ed more label into carbon dioxide than the controls or the diabetic obese group. Between the latter two groups no dif- ferences could be demonstrated. After stimulation with insulin, carbon dioxide

ADIPOSE TISSUE FROM OBESE PATIENTS. 11. 231

T A B L E 11. Conversion of 1-14C-glucose into carbon dioxide in human subcutaneous adipose tissue in vitro. Mean & SEM

_____ ~~~

Millimicromolesjpg DNA/ h

No Insulin No Insulin addition (10,000 pUjml) addition (10,000 pU/ml)

Millimicromoles/ 1 O4 cells/h

Controls 0.17 & 0.02 0.31 f '0.05 0.14h0.02 0.25f10.04 Obesity 0.25 k0.03 0.45f '0.07 0.39f0.05 0.58* * 0.09 Diabetic obesity 0.19*0.03 0.26& 0.04 0.18&0.03 0.25f 0.04

Controls-Obesity p < 0.05 p >0.10 p <0.001 p< 0.01 Controls-Diabetic obesity p > 0.10 p >0.10 p >0.10 p >0.10 Obesity-Diabetic obesity p <0.10>0.05 p <0.05 p <0.05 p <0.01

p < 0.05, increase as compared with no addition. p < 0.1 O > 0.05, increase as compared with no addition

TABLE 111. Conversion of l-'*C-glucose into lipids in human subcutaneous adipose tissue in vitro Mean f SEM

Millimicromoles/,ug DNAjh Millimicromoles/ lo4 cells/h

No Insulin No Insulin addition (10,000 pU/ml) addition (10,000 pU/ml)

Controls 0.75 :k 0.14 0.645 0.09 0.59&0.11 0.5410.08 Obesity 0.86 0.13 0.70& 0.12 1.24f0.19 1.38& 0.24 Diabetic obesity 0.60 111 0.13 0.54f 0.08 0.63 f 0.14 0.56f 0.08

Controls-Obesity p >0.10 p >0.10 p < 0.02 p <0.01 Controls-Diabetic obesity p > 0.10 p >0.10 p >0.10 p >0.10 Obesity-Diabetic obesity p > 0.10 p >0.10 p <0.05 p <0.01

incorporation was again higher in the non-diabetic obese group as compared with the controls and the diabetic obese group on a cell number basis, but on a DNA basis a difference was statistically demonstrable only in the comparison between the obese groups. Insulin in- creased incorporation of labelled glucose into carbon dioxide in controls and non-

diabetic obesity, but not in diabetic obesity.

The results of incorporation of label from l-14C-glucose into lipids are given in table 111. The non-diabetic obese group incorporated more label into lipid than the controls and the diabetic obese group on a cell number basis but not on a DNA basis. No difference was

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found between controls and diabetic obesity, After addition of insulin the same results were found. Insulin did not in- crease incorporation into lipid in any group.

Discussion

Since the cell number basis and the DNA basis are not exactly equivalent as reference units for adipose tissue cell activities ( 5 ) , both have been used in the present work. Generally good agreement between results utilizing these reference bases was found, but in some instances differences found between means were statistically significant only on cell number basis.

The biopsies of adipose tissue used in the present work show a relatively small response to hormones as far as the reactions measured here are concerned. Thus only a moderate increase of incorporation of label from l-14C-glu- cose into carbon dioxide and lipid occur- red with large amounts of norepineph- rine (3) , and with large amounts ofin- sulin there was only an increase of incorporation of label into carbon diox- ide. With other types of biopsies, it is possible to get more pronounced hor- mone stimulation (6, 11); but in order to compare the results obtained here with those previously described for fatty acid release and lipolysis of adipose tissue in vitro from the patient groups in question (4), the present type biopsy was used.

Glucose uptake was increased in both the obese groups as compared with the controls. This increase was parallelled by an increase in incorporation of label

from l-14C-glucose into both lipid and carbon dioxide in the obese group without diabetic symptoms. The diabetic group, however, showed a different metabolic pattern. Here the increased glucose uptake could not be explained by an increase in label incorporated from 1-14C-glucose into carbon dioxide or lipid, and has to be explained by an increase in other metabolites not identi- fied.

Insulin added to rat adipose tissue in vitro produces an increased transfer of label from 1 -14C-glucose to carbon dioxide and lipid (8). This is the pattern found in the present work in the obese non-diabetic group, and might thus imply an increased influence of insulin on adipose tissue in vivo demonstrable in vitro. This finding, as well as previously reported increased levels of immunolo- gically determinable plasma insulin in obesity (9), thus suggests an increased influence of insulin on adipose tissue in obesity without diabetes mellitus.

I t seems, however, puzzling that in- sulin added in vitro in large amounts, far above those expected to act on adipose tissue in vivo, increased incorporation into carbon dioxide and lipid only to a limited degree or not at all. This finding is in accordance with reports by Martins- son (11) and Hirsch and Goldrick (6), who also found a limited in vitro insulin response for certain preparations of adipose tissue. Among other factors the trauma during preparation might be responsible for the damage to the delicate insulin response mechanism of the tissue, in accordance with results in rat epididy- ma1 fat pads (1) . However, in respect of the only parameter where insulin re-

ADIPOSE TISSUE FROM OBESE PATIENTS. 11. 233

sponse is measurable in the preparations used in the present work, viz. incorpora- tion of l-14C-glucose into carbon dioxide, no measurable increase was found in the diabetic group, while such a response was found in both non-diabetic groups. The technical disadvantages of the biopsy used, as mentioned, together with the large amounts of insulin added, seem to make it dimcult to argue from the present in vitro system to the possible situation in vivo. The findings might indicate that the insulin effect on the metabolism of glucose in the fat cell in human diabetes mellitus is diminished. ‘This is now further investigated under more “physiological” conditions, with smaller insulin additions, to systems with optimal conditions for insulin response in vitro as worked out by Bjorntorp and Martinsson (6).

Summary

Human subcutaneous adipose tissue from controls, obese and diabetic obese patients was incubated in vitro, and glucose uptake and the incorporation of label from l-14C-glucose into carbon dioxide and lipid were measured in the basal state and after insulin addition. Glucose uptake per adipose tissue cell was increased in both the obese groups as compared with the controls. In non- diabetic obesity this corresponded to an increase of incorporation of label into the products mentioned, a result com- patible with an enhanced insulin effect on adipose tissue. The diabetic tissues, however, showed no increase in these incorporations, nor was there an increase

in incorporation of 1 -14C-glucose into carbon dioxide after insulin stimulation as found for the non-diabetic groups.

Acknowledgements The work was supported by grants T 304, W 253 and Y 595 from the Swedish Medical Research Council. Insulin was a gift from Leo of a Nordisk Insulin preparation.

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