Biochimica et Biophysicu Acta, 337 (1974) 3 I 3-3 I 7

i”l Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 56404

ATP BALANCE AND THE EFFECT OF 2,4-DINITROPHENOL ON

FATTY ACID SYNTHESIS

JOSEPH KATZ, P. A. WALS, and ROBERT ROGNSTAD

Cedars-Sink Medical Research Institute, Cedars-Sinai Medical Center, Los Angeles, Crdif go029 f U.S.A.)

(Received October 8th, 1973)

SUMMARY

The effect of 2,3-dinitrophenol on glucose metabolism and fatty acid synthesis by rat adipose tissue slices, adipocytes, and parenchyma cells from mammary gland of lactating rats was investigated. 2,4-Dinitrophenol at the lowest concentrations that caused uncoupling, depressed fatty acid synthesis. I % albumin abolished the effects of 2,4_dinitrophenol at concentrations below 0.2 mM.

INTRODUCTION

Calculation of energy balance during lipogenesis from glucose by rat epi- didymal fat pad slices indicates an excess of ATP formation over its utilization [r-4]. The excess was estimated to be of the order 10-20 ,nmoles ATP/g per h with moderate rates of lipogenesis and up to 70 ,nmoles/g per h in tissue of starved re-fed rats with high rates of fatty acid synthesis [3]. Flatt [4] has proposed that the production of excess ATP is limiting lipogenesis and that energy balance had a major role for the regulation of lipid synthesis. The concept of Flatt is of great interest, but it has at present little direct experimental support. According to Flatt’s hypothesis, mild partial uncoupling of oxidative phosphorylation should enhance fatty acid synthesis, but Rognstad and Katz [2] found that z,4_dinitrophenol at low concentrations de- pressed lipogenesis in adipose tissue slices. Recently Saggerson [5] has claimed that in rat adipocytes low concentrations of 2,4_dinitrophenol and other uncouplers stimulate fatty acid synthesis by about 20%. In view of the discrepancy and the significance of Saggerson’s results in supporting Flatt’s theory, we have re-examined the problem. Our conclusion is that 2,4_dinitrophenol does not stimulate fatty acid synthesis, and that no uncoupling occurred in Saggerson’s experiments.

METHODS

Male rats (200--250 g) were kept on a commercial high carbohydrate diet for 5-8 days. The procedures used with epididymal fat pad slices (except for the modi- fication in the presence of albumin, see below), have been previously described [2, 31. Adipocytes were prepared by collagenase treatment essentially according to Rodbell

314

(ref. 6). The washed adipocytes, 0.15-0.2 ml packed cells, were incubated for I h in 2 ml Krebs-Henseleit buffer with I ‘%; bovine albumin, in an atmosphere of O,/CO, (95: 5, by vol.), in siliconized glass Erlenmeyer flasks. The reaction was terminated by the addition of 0.5 ml 0.5 M H,SO, and the flask content transferred and made to volume (IO ml) in a centrifuge tube. The aqueous phase below the floating fatty layer was sucked off and used for the determination of lactate and glucose. The fat was extracted with chloroform-methanol, the extract washed to remove albumin and the chloroform evaporated in tared tubes. The weight of the fat was obtained and used to calculate the yields. Other procedures were as with slices.

Mammary parenchyma cells were prepared by collagenase treatment from glands of lactating rats I 2-15 days after parturition. The procedure will be described in detail elsewhere (Katz, J. and Wals, P. A., unpublished). The yield from one rat was 4-5 ml packed cells. The cells are stable and do not require the addition of albumin. 0.1-0.15 ml packed cells were incubated as described for adipocytes. The reaction was terminated with H,SO, (see above) which does not precipitate the albumin. The flask contents were transferred and made to volume (IO ml) in a tared centrifuge tube. The precipitate was extracted first with 80% acidified ethanol to remove albumin, then with chloroform-methanol and the fat free residue dried and its weight obtained. The alcohol-chloroform extracts were washed, evaporated and treated as with adipose tissue. Methods of separation, analysis and isotope assay were as previously de- scribed [3].

Results are expressed for adipose tissue/g fat per h. With slices, fat content was either measured as for adipocytes for each flask, and in other experiments it was assumed that So”/; of the fat pad is lipid. With mammary gland cells, results are ex- pressed per IOO mg defatted dry weight, which approximates the protein content of the preparation.

RESULTS AND DISCUSSION

With adipose tissue segments incubated without albumin, 2,4_dinitrophenol at a concentration of 40 /tM depressed slightly fatty acid synthesis from glucose (Table I), and at a concentration of IOO /IM glucose oxidation was depressed by 35 y,;> and lipogenesis by 60%). This inhibition is somewhat higher than that reported (at somewhat different conditions) by Rognstad and Katz [2]. The most sensitive index for the uncoupling effect was lactate production, either measured analytically or by the 14C yield. The increase in lactate was apparent at 20 /IM a,4_dinitrophenol, without a significant effect on glucose oxidation or lipogenesis. The inhibition of 14C02 production at levels of 2,4_dinitrophenol above 40 ,uM is due to the marked depression of the pentose cycle and to a lesser extent of pyruvate decarboxylation. Rognstad and Katz [2] found in adipose tissue segments that 2,4-dinitrophenol at levels of 0.1-0.3 mM greatly depressed 14C0, formation from [ t-r4C]glucose, but stimulated several-fold 14C02 production from C-6 of glucose. They calculated a 2- to 4-fold increase in oxidation via the Krebs cycle at such 2,4-dinitrophenol levels. The presence of I “/) albumin abolished the effects of 2,4-dinitrophenol on lactate production at concentrations less than IOO PM and significant inhibition of lipo- genesis occurred only at 400 AIM (Table I). The stimulation of lactate production in the presence of albumin at 200 /tM was nearly that with 20 HIM 2,4_dinitrophenol

3’5

TABLE I

EFFECT OF 2,4-DINITROPHENOL ON GLUCOSE METABOLISM OF RAT ADIPOSE

TISSUE SEGMENTS AND ADIPOCYTES

Segments and adipocytes from epididymal fat pad of rats kept on high carbohydrate diet incubated in Krebs-Henseleit buffer for I h. (200 mg segments in Expt I, 300 mg in Expt 2, about 0.2 ml adipocytes in 2 ml final volume). Glucose 5.5 mM, labelled uniformly with 14C Insulin 200 munits/ml in seg- ments, 20 munits/ml in adipocyte experiments. Albumin when present, IO mg/ml.

Expt Prepa- Analytical lJC incorporation (llatoms C/g*per h) in

Fatty Lipids Lactate No. ration 2,4-Di-

nitro-

phenol

I Segments 0

20

40 100 200

400 0

20

40 100

200

4oo

2** Segments 0 10

40 100

200

4oo

Cells 0

IO

40 100

200

400 -___- ~~~

Albu- min

_

_

I-

f I-

-t + .i~

_

+

i 1.

+-

-t +

Lactate production

(Itmoles/ g*per h)

Sum acids Lipid-

glycerol

3.2 43 55 7.0 ‘05 4.4 48 55 12 ’ ‘5 7.0 42 40 18 100

17 27 21 SO 98 19 15 8 51 74 8.8 4.0 2 21 27 2.0 45 53 7.0 I05

2.5 4’ 49 6.0 96 2.0 46 53 6.5 106

2.4 5’ 58 7.0 I 16

4.9 50 56 ‘5 121

17 39 3’ Ii] 121

2.1 29 34 4.0 7.2 74 3.1 34 37 4.0 IO 85

8.7 35 32 3.3 25 95 19 24 15 3.2 54 96 20 15 5.5 I.7 55 77

8.5 4.3 0.6 0.4 20 25

7.1 24 22 4.3 22 72

6.4 21 23 4.0 18 66

5.7 13 12 2.7 18 46

4.5 12 II 2.7 17 43 6.8 14 12 2.6 23 52

23 17 II 3.0 68 99 _ * Result expressed per g fat.

** One fat pad from each rat pooled for adipocyte preparation, the other for segments.

without albumin, suggesting that at these concentrations (200 PM 2,4-dinitrophenol, I “//o or 0.15 mM albumin), So-go”/, of the 2,4-dinitrophenol is bound.

The ability of albumin to tightly bind a great variety of compounds is well known. It has the capacity to bind 22 moles/mole of methyl orange and 24 moles/mole of m- and p-nitrophenols [7]. The capacity for 2,4_dinitrophenol should be similar, so that a I % albumin solution can bind up to 3.5 Llmoles/ml of the acid. Actually the binding will depend on the ligands adsorbed on the albumin preparations, and on the fatty acids and other ligands present in the medium, and the adsorption will vary for different albumin batches and experimental conditions, but obviously the active concentration of uncouplers in Saggerson’s experiments [5] must have been very low and not likely to have induced any uncoupling. Our findings indicate the advantages

316

TABLE II

EFFECT OF z,4-DINITROPHENOL ON GLUCOSE METABOLISM BY RAT MAMMARY GLAND CELLS

Cells (8-12 mg defatted dry weight) incubated in 2 ml Krebs-Henseleit buffer, IO mM in glucose. uniformly labelled with r4C for I h. Gas phase Oz/COz (95: 5, by vol.). Insulin 20 munits/ml. Albumin when present, IO mg/ml. Maximal glucose uptake was 38 74. N.D., not determined.

Expt Analytical* “C incorporation (/(atoms C/loo mg** per h)

No. z,4-di-

nitro- phenol

(PM) ___~~~~~

t 0

20

40 100 200

4oo 0

20

40 too 200

4oo

2 0

5 10

20

0

20

100

-90 -80

--48

- -26

N.D. N.D.

ml -73

~1 -97 tm -97 -t --94 + ~-67

-I- -39

N.D. N.D.

_ N.D. N.D.

I- N.D.

i N.D.

I N.D.

l-19 186

t 21 189

i.65 84 f5o 20

i-36 8.0 I 28 6.0

--29 ‘59 I-33 185

-: 33 191 -3’ 162

-60 I21

+72 42

+ 8.5 202

+ 9.0 212

/ II 226

720 2’5 I- 8.0 181

Cl0 198 II 212

Albu- Glucose Lactate in

min (pmoLes/ IOO mg**per h CO2 Fatty Lipid- Lactate

acids glycerol

1st

‘4’ 9

‘. I ,:I -c 1 ‘40 167 169

’ 30 34

7

2’5 211

210

205 180

195 200

_

40 55 21 69

4.5 187

2.5 136

I.0 70 I .o 65

29 97 25 96 26 95 22 80

13 165 3.5 218

25 N.D. 26 N.D.

29 N.D. 32 N.D.

26 N.D. 28 N.D.

29 N.D.

Sum

432

420

285 ‘59

80

73

425 473 481

394

333 270

* (-) Indicates uptake, and (-1-j production. ** mg defatted dry weight.

of adipose tissue slices, which do not require albumin for this type of study. Adipocytes lyse rapidly without albumin and it is essential to maintain their function.

In Table II effects of 2,4_dinitrophenol on lipogenesis in mammary gland cells is shown. The cells are very stable, not requiring albumin and show a high rate of glucose utilization and lipogenesis. Also the activity was reproducible from animal to animal (Katz, J. and Wals, P. A., unpublished). Without albumin there is an in- hibition of 90% of lipogenesis with 40 LLM z,q-dinitrophenol, but in its presence inhibition becomes only significant at between IOO and 200 PM. In the second ex- periment of Table II, concentrations of 2,4_dinitrophenol (without albumin) of 5- 20 PM were explored. These concentrations showed no evidence of uncoupling as indicated by increased lactate formation, and they were also without effect on lipo- genesis.

It was of interest that with mammary gland cells, the addition of albumin (with no 2,4-dinitrophenol) suppressed lipogenesis by I 5 ‘A, and low concentration of 2,4_dinitrophenol restored lipogenesis to levels obtained without albumin. This de- repression cannot be attributed to uncoupling. It is possible that 2,4_dinitrophenol displaces some hormones or ligands from albumin which somewhat increase lipo-

317

genesis. This effect was not observed by us in adipocytes, but it may possibly explain the small (15-20 %) stimulation observed by Saggerson [5]. He provided no evidence

(such as increase in lactate production) for the occurrence of any uncoupling in his system.

Our work indicates that even the mildest uncoupling of oxidative phosphoryl- ation does not stimulate lipogenesis. While the experiments do not support Flatt’s hypothesis, they do not disprove it and it deserves further examination.

ACKNOWLEDGMENT

This work was supported by United States Public Health Service Grant No. NIH 5 Ror AM-12604.

REFERENCES

I Rognstad, R. and Katz, J. (1966) Proc. Natl. Acad. Sci. U.S. 55, I 148-r 156 z Rognstad, R. and Katz, J. (1969) Biochem. J. III, 431-444

3 Katz, J. and Wals, P. A. (1971) Arch. Biochem. Biophys. 147, 405-418 4 Flatt, J. P. (1970) J. Lipid Res. II, 131-143

5 Saggerson, E. D. (1972) Biochem. J. 128, 1069-1078 6 Rodbell, M. (1964) J. Biol. Chem. 239, 375-380 7 Haurowitz, F. (1963) in The Chemistry and Function of Proteins, pp. 239-241, Academic Press,

New York