Effect of diabetes on glucose metabolism in isolated fat cells

Effect of diabetes on glucose metabolism in isolated fat cells

217 SHORT COMMUNICATIONS BBA 53153 Effect of diabetes on glucose metabolism in isolated fat cells Previous studies have shown that adipose tissue fr...

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217

SHORT COMMUNICATIONS

BBA 53153 Effect of diabetes on glucose metabolism in isolated fat cells Previous studies have shown that adipose tissue from alloxan-diabetic animals metabolized much less glucose to CO, or lipid than did the tissues from normal animals (refs. I, 2).The extraordinary metabolic activity and sensitivity to a number of hormones of the isolated fat cells that have been reported3 led us to re-examine the problem working with isolated fat cells from epididymal fat tissue. For that purpose normal and alloxan-diabetic rats were prepared as follows: male albino rats from the strain of the Institute, weighing between 120 and 140 g, were fed on rat pellets ad Libitum. Alloxan-diabetic animals were obtained by the rapid intravenous injection of 50 mg/kg of alloxan after a 24-h fast; the animals were not used until at least 6 weeks after the administration of alloxan and unless blood glucose exceeded 300 mg/ IOO ml. The animals were killed by decapitation and the fat pad was removed to isolate the fat cells according to RODBELL'~ procedure. The cells were incubated in the presence of [14C,Jglucose with and without insulin. The incubation conditions and methods for the determination of 14C0, and radioactivity in total lipids were the same as those described elsewherea. The amount of fat cells used in each experiment was determined from the triglyceride ester content by the method of RAPPORT AND ALONZO~. I g of cellswas set equal to I mmole of ester.

TABLE METABOLISM

I OF [14CB]~~~~~~~~~~~~~~~~~ FAT CELLS

Fat cells (25 mg/flask) were incubated at 37” for z h in a final volume of z ml of albuminbicarbonate buffer (pH 7.4) containing 3 pmoles/ml of [r4C,]glucose. Each sample was made in triplicate and the values are expressed as pmoles of glucose per g of cells per z h. The statistical analysis of the results was performed by the paired-samples method. _ _~ E+t. No. Oxidation of[14C,]gZucose to CO, Incorporation of [14C,]gZucose into lipids (voleslg) (vnoles Isl Diabetic Normal Diabetic Normal I 2

3 4 5 6 7

Mean

4.32 3.31 4.27 9.48 5.4’ 3.31 3.32

2.40

3.14 5.07 7.28 4.63 4.56 3.99

4.44 4.77 (no significant difference)

4.80 4.45 3.98 8.40 3.07 7.37

5.51

6.69 9.49 II.43 4.10 17.10

16.23 3.92 10.08 (4) 5.14 (3) (significant difference P < 0.05)

The results of this study are given in Table I. They show that glucose oxidation to CO, was lower in the diabetic cells although the difference was not statistically significant ; however, the conversion of glucose into lipids was unexpectedly higher in the diabetic cells. Additions of insulin (I milliunit/ml) caused a q-fold increase in glucose utilization in normal preparations whereas in diabetic ones the effect was considerable lower (Table II). These results led us to compare the metabolic activity of the isolated fat cells Biochim.

Biophys.

Acta,

152 (1968)

217-219

218 TABLE INSULIN

CO~MU~ICAT~O~S

SHORT

II EFFECT

ON

GLUCOSE

[lpC,]~~~~~~~ + i*CO, +

UTILIZATION

TOTAL

LIPIDS)

Data are given in ymoles/g. Assay conditions for glucose utilization were those described in the legend of Table I. The concentration of insulin when present was I milliunit/ml. The statistical analysis of the results was done by the paired-samples method. -__ - _.___.~_ .______ ..-_. _ Expt. No. NOWUZl Diabetic

.____I____ ~~

--. I 2 3 4 5 6 Mean

--insulin +insulin -&X&Z __.__ -...._. ____~ 9.12 24.31 8.11 7.76 21.10 10.16 8.25 77.30 14.55 17.88 77.00 18.68 8.48 24.43 7.76 10.68 21.66 21.95 10.36 41.01 X3.49 P < 0.05 P < 0.05 .-_ ~ _---_ ~ ~_

-+-in.SUli% 9.61 8.72 18.69 26.~0 II.50 22.45 16.18 -_.

~__

with intact tissue incubating both preparations from normal and diabetic animals simultaneously and under the same conditions. For this study, approx. 80 mg of tissue were cut from the distal portion of each fat pad and the remainder of the adipose tissue was pooled and treated with collagenase. In Table III we can see that the results obtained with isolated fat cells are similar to those described in Table I, i.e., the diabetic fat cells in comparison to normals show lower glucose oxidation and higher incorporation of glucose carbon into lipids. However, in the intact tissue both glucose oxidation to CO, and incorporation of glucose carbon into lipids show a 50% decrease in the diabetic preparations. TABLE

III

COMPARISON INTACTTISSUE

OF

GLUCOSE AND

OXIDATION

ISOLATEDFAT

TO

co,

AND

INCORPORATION

OF

GLUCOSE

INTO

LIPIDS

Isolated fat cells and tissue were prepared as described in text. Values are the means of ments. Assay conditions for glucose metabolism were those described in the legend of The data obtained with isolated fat cells are expressed as ,nmoles of glucose per mmole ceride and those for intact tissue as pmoles of glucose per g of wet weight. The statistical of the results was done by the “t” test for paired samples.

Cells Tissue -.

BY

CELLS

5.94 (no significant difference) 1.85 4.14 (significant difference P < 0.01)

7.67

6 experiTable I. of triglyanalysis

5.69 7.68 (significant difference P < o.og) ;$iiicant

differ::&

P “._~.~~_,___..

It was demonstrated that glucose is transported across the plasma membrane of the fat cell by a “carrier-mediated” diffusion process 5p6.The possibility of an alteration of this property of the cell membrane from diabetic animals due to the treatment with collagenase was considered. The effects of increasing the medium glucose concentration from x.5 to 9 pmoleslml on glucose metabolism can be seen in Fig. I. In both cases the curve maintains the typical shape for the diffusion process mediated by active transport. In consequence, in the present study, isolated fat cells from diabetic animals Biachim. Biq?hys. Acta, rs* (1g68)2x7-219

219

SHORTCOMMUNICATIONS

incubated in the presence of [%,]glucose show a higher incorporation of glucose carbon into lipids compared to normal animals and this effect is exactly opposite to that found with intact tissue. Besides, an effect in vitro of insulin on glucose utilization in these preparations was also observed although its magnitude was considerable lower. The astonishing discrepancy found between the isolated fat cells and the intact tissue opens an interesting problem for discussion. The intrinsic mechanism for the difference observed is unknown. A probable explanation suggested is that in intact tissue the newly formed lipids in the slice are more easily oxidized than in the fat cells, to such an extent that almost all of the lipids formed from glucose during the period of incubation were oxidized before the end of the experiment.

-Normal -----Diabetic

Fig. I.Effect of increasing the medium gluc~e’concentration represents the mean of 2 experiments.

on glucose oxidation.

Each point

An alternative hypothesis to be considered is that, in intact tissue from diabetic animals, a “barrier” located before the cell membrane does not allow glucose to enter the cell. This would not be the case when the isolated fat cell is incubated in direct contact with glucose; the nature of this “barrier” is, of course, unknown. lmtituto de Fisiologia, Facultad de Ciencias MWicas, Universidad National de La Plata, Calle 60 y 120, La Plats (Argentina)

G. E. CHIAPPE DE CINGOLANI

R.R.

I F. X. WAUSBERGER, S. XV. MILSTEI?I AND K. J. RUTMAN, J.Riol. Chem., 2 A. I.WINEGRAD AND A. I%.RENOLD, J.&ok Chsm., 233 (1958) 267. 3 M. RODBELL, J. Bid. &‘&em., 239 (x964) 375. 4 M.M. RAPPORT AND N. ALONZO, J.BioZ. Chem., 217 (1955) 193. 5 0. B. CRAWFORD AND A. E. RENOLD, J. Rid. Chem., 240 (1965)14. 6 M. RODBELL, J. Biol. Chem., 241 (1966) 130.

BRENNER

208 (x954)

431.

Received August r&h, 1967 Biochim.

Niaphys.

Acta,

152 (1968)

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