effects of phenazine methosulfate on glucose metabolism in rat adipose tissue

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 147, 405-418 (1971)

Effects of Phenazine Methosulfate on Glucose Metabolism

in Rat Adipose Tissue’

JOSEPH KATZ AND P. A. WALS

Medical Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 90029

Received May 27, 1971; accepted September 1, 1971

Segments of epididymal fat-pad tissue of rats were incubated with glucose labeled uniformly with l4C and in carbons 1 and 6 and with tritium in positions 3, 5, and 6. The effect of phenasine methosulfate (PMS) on incorporation of glucose carbon and tritium into metabolic products and on the formation and utilization of cytoplasmic and mitochondrial reducing equivalents was determined. PMS concentrations below 5 PM have little effect on glucose metabolism. PMS between 5 and 100 pM stimulates the uptake and oxidation of glucose but is without effect on its incorporation into fatty acids and glycerol. Higher concentrations of PMS inhibit glucose oxidation and lipogenesis. The lactate-pyruvate ratio in tissue incubated with glucose ranged from 3 to 10. With PMS the ratio was decreased to less than 0.5. At a PMS ooncen- tration of 50 GX, glucose uptake was increased in tissue of fed rats by 30-507, and CO, production by lOO-200%. The contribution of the pentose cycle to glucose metabolism increased from 15-2301, to 40-50%, and oxidation via the Krebs cycle increased by 50- 300%. In tissue of starved-refed rats with a very high rate of lipogenesis, the stimulation of the pentose cycle and Krebs cycle oxidation was less pronounced. A balance of reducing equivalents was calculated. In the absence of PMS the formation of cytoplasmic reducing equivalents is equal or in a small excess over that needed for lipogenesis and lactate production. In the presence of PMS a large excess is generated. The generation and utilization of ATP was calculated. Without PMS, ATP generation is in excess over that needed for known biosynthetic requirements. The excess is of the order of 23 pmoles/gram/hour for tissue of fed rats, and in tissue of fasted- refed rats, of the order of 40-50 rmoles/gram/hour. PMS increases oxidation via the Krebs cycle in tissue of normal rats but it is likely that the excess oxidation is not coupled to phosphorylation. When tissue was incubated with [5-T] glucose virtu- ally the sole product was labeled water. This label is useful in measuring glucose uptake or turnover in vivo and in vitro by determination of the yield of labeled water. We conclude that lipogenesis is not affected by an increase in cellular pyruvate concentration or the NAD+/NADH and NADP+/NADPH ratios in the cytoplasm, or by a large decrease in the concentration of free reduced pyridine nucleotides.

The addition of hydrogen accept80rs such from carbon 1 of glucose (1). McLean et al. as methylene blue or phenazine met,hosulfate (2) reported the effect of such compounds on (PMS)” to many t’issues increases oxygen up- t’he oxidation of [1-14C]glucose and [6-14C]glu- take and glucose oxidation preferentially case in slices of rat mammary gland and adi-

1 This work was supported by U. S. Public pose tissue. In t,he latter, PMS stimulated

Health Service Grant, AM-12604 and General COz formation from C-l 4-fold and from

Research Support Grant RR-05468. C-6, 1$6 to 2-fold. The effect is thought to

2 Abbreviations: PMS, phenazine methosulfate; be due to catalytic oxidoreduction. The dyes

TMPD, N, N, N’, N’-tetramethyl-p-phenylamine; are reduced by NADPH and NADH and GAP, glyceraldehyde-P; L/P ratio, lactate- reoxidized nonenzymatically by oxygen. pyruvate ratio. We have recently reported (3) that PMS

405

406 KATZ AND WALS

at concentrations of about 5 PM increases greatly lipogenesis from lactate, without much effect on the metabolism of pyruvate and glucose. We report here that PMS markedly alters the redox state of adipose tissue and stimulates glucose oxidation via the pentose cycle and the Krebs cycle without effect on lipogenesis. It appears that the dye uncouples the formation of reducing equivalents in cytoplasm and mitochondria from their utilization in biosynthesis and oxidative phosphorylation. The present paper explores the relationship between these effects and lipogenesis.

METHODS

Labeled sugars were purchased from New England Nuclear Corp. (Boston, MA) and Amer- sham-Searle (Chicago, IL). Phenazine methosulfate, enzymes, and coenzymes were purchased from Sigma Chemical Company (St. Louis, MO) and Calbiochem (Los Angeles, CA) Triton X-100 (purified grade) and phenethylamine were purchased from Packard Instrument Company (Downers Grove, IL) The labeled sugars were purified either by paper chromatography or pas- sage through tandem columns of Dowex 50 (H+) and Dowex 1 (acetate) and the effluent taken to dryness. From 0.5-l &i of “C and 5-10 PCi of tritium-labeled sugars were used per flask. The combination of labels in glucose was U-‘4C, 5-T; lJ4C 3-T, and 6-14C 6-T.

S&e in each expekiment many conditions and types of labels were compared, a pool of tissue from lo-15 rats was used. The tissue (200-300 mg) was incubated for 3 hr in 2 ml bicarbonate buffer, 5 mu or 10 mu in glucose in an atmosphere of 950/o 025% COZ. The separation of the products, determination of lactate and pyruvate, and radioactivity assay have been as described elsewhere (4, 5).

CALCULATIONS

Incorporation of isotopes and of glucose carbon. The yield of carbon into CO*, fatty acids, lipid glycerol, and lactate was calculated by multiplying the amount of added glucose carbon by the fraction of the l4C dose from uniformly labeled glucose (U-IaC), recovered in each compound. In experiments where lactate was not isolated, the carbon and l4C yield was obtained from the analytical lactate determination, assuming equal specific radioactivity of lactate and glucose carbon. Incorporation into pyruvate was estimated from the lactate/pyruvate ratio, determined analytically. The yields of carbon were expressed

as ratoms C per gram tissue (wet weight). It should be stressed that such calculations are generally valid only for uniformly labeled sub- strates. The distribution of l4C and tritium from the various types of label were expressed as specific yields. The isotope yields in all products, which nearly equal the total glucose utilization, were set at 100, and the incorporation into each compound was expressed as a percentage.

Pentose cycle. It was calculated by four methods (6) : (a) from specific l4C yields from [l-‘4C] glucose and [6-‘4Clglucose; (b) the ratio in fatt.y acids from [lJ4C]glucose and [6-‘4Clglucose (7); (c) from the 14C02 specific yields and 7; and (d) from the ratio in fatty acids and lipid glycerol (y and 6) from [1-14Clglucose and [6-14C]glucose in lipid glycerol.

Carbon $0~. The flow of carbon through the various steps was calculated from the incorporation of glucose carbon into products (see above) and the contribution of the pentose cycle (PC). The calculations are summarized below. The rates of production or metabolic flow rates are expressed inI;moles/gram/3 hr, since this was the period of incubation in these experiments.

I.

II. III.

IV.

V-VII.

VIII.

IX.

X.

XI.

Glucose phosphorylation. Utilized glucose carbon batoms C) divided by 6. CO2 via pentose cycle. (I) X 3pc. Phosphofructokinase (amoles of fructose 1,6 diP formed). (I) -PC. amoles of triose-P formed. 2 X (III) + pc or (I) X (2 - pc). Formation of lipid glycerol, medium lactate, and pyruvate. From the incorporation of carbon into each of these compounds respectively, divided by 3. GAP dehydrogenase (Irmoles of GAP converted to pyruvate). (IV) - (V). Pyruvate decarboxylation (total acetyl CoA formed). (VIII) - (VI + VII). Acetyl CoA converted to fatty acids; patoms C in fatty acids divided by 2. CO2 formed via Krebs cycle. Several alternate calculations are available.

(a) Total CO2 - (II) - (IX). @I 2[UX) - (WI.

When oxidation via Krebs cycle is very low, both of these estimates represent small differences and are subject to much error. Calculations based on yield of 14C02 from [6-14C]glucose require in theory corrections for effects on specific activity of C-6 of glucose-6P by the pentose cycle and incomplete equilibration of triosephosphates, and also assumptions of complete oxidation of carbons 1 and 2 of acetyl CoA. However, when oxi-

PMS AND LIPOGENESIS FROM GLUCOSE 407

XII.

d&ion of C-6 is small the CO2 is approxi- mately

Total utilized Specific yield

glucose carbon X in CO2 from x 4.

glucose 6-i%

While this is an overestimate, it is less subject to experimental error as the calculations above, and was usually used. Reesterification of fatty acids formed by lipolysis. This was estimated on the assumption that the fatty acids formed de novo require 1 mole of glycerol to es- terify 3 moles of palmitate or 24 acetyl CoA equivalents, and the rest of the glycerol is used to reesterify palmitate formed by lipolysis. [(V) - (X)/2413.

Reducing equivalents. This is obtained from the above rates, considering that in the cytoplasm 2 moles of NADPH are formed for each mole of COz formed in the pentose cycle (6 per mole of glucose), and 1 mole of NADH per mole GAP oxidized to pyruvate. In mitochondria 1 mole of NADH is formed per mole of acetyl CoA and 2 moles of reducing equivalents per mole of CO* in the Krebs cycle. Reducing equivalents are consumed in the cytoplasm, mole per mole in the synthesis of glycerophosphate for lipid glycerol, and of lactate. The reductive synthesis of fatty acids requires, on the assumption that fatty acids are palmitate, 1.75 moles of NADPH per mole of acetyl CoA.

ATP. Production of ATP in the cytoplasm is 2 moles/mole of glyceraldehyde-P oxidized to pyruvate, and in the mitochondria, assuming a P/O ratio of 3, is 3 moles per mole of pyruvate oxidized to acetyl CoA, and 6 moles/mole of CO2 formed in the Krebs cycle. In addition, ATP will be formed if any reducing equivalents are transferred from cytoplasm to mitochondria.

Utilization of ATP is for phosphorylation of glucose and fructose-6P, and in fatty acid synthesis in t)he reactions catalyzed by pyruvate carboxylase, citrate cleavage enzyme, and acetyl CoA carboxylase. ATP is also required in reesterification of free fatty acids, 2 moles of ATP (one pyrophosphate bond) per mole of fatty acids. Further details of calculation of ATP balance is in Results.

RESULTS

E$ects of PMS on glucose metabolism. The glucose uptake, production of lactate and pyruvate, and incorporation into COZ , fatty acids, and glycerol are summarized in Table I. The incorporation into COZ and lipids was

calculated from the incorporation of glucose labeled uniformly with 14C, In experiments with tissue of rats fed ad libitum, PMS stimulated glucose uptake and COZ production without any increase in the synthesis of fatty acids and glycerol. StimulaGon of glucose upt,ake and oxidation occurs at 5 ~CIM PMS and is maximal at about 50 PM. The increase in glucose uptake was up to 50% and CO2 production 100 %. Concentrations about 100 PM become inhibitory. On the other hand, with lactate as substrate (4) maximal st’imulation of fatty acid synthesis occurs at PMS concentrations of 10 PM or less, and above 25 PM PMS is inhibitory.

The L/P output ratio in tissue from fed rats ranged from 3 to 7 and up to 12 in the tissues of starved-refed rats. At 5 pi PMS the L/P output ratio was lowered to 1 and with higher PMS concentrations, output ratios as low as 0.5 were obtained.

The increased COZ formation in the presence of PMS is mainly due to st’imulation of the pentose cycle. This is brought out in Ta- ble II where t’he specific yields from [1J4C] * glucose and [6J4C]glucose are shown. In the presence of 50 PM PMS incorporation into 14C02 accounts for some 75% yield of 14C from carbon 1 of glucose, as compared to 40- 50 % in controls. The specific yield in fatty acids is depressed from 30 % to about 10 %. This depression reflects the dilution of 14C in glucose-6-P due to the operation of the pentose cycle. As shown in Table I, no change in incorporation of glucose carbon into lipids occurs.3 The pentose cycle contribution to glucose metabolism, calculated from the data of Table I, is presented in Table III. In tissue of fed rats the pentose cycle contribution increases from 13-22 % to 40-50 %. In tissue of starved-refed rats, where the rate of glucose-6-P oxidation to phosphogluconate is 5- 10 times that in fed rat’s, stimulation of the pentose cycle was less, from 20 to about 30%.

The data in Tables I and III permit calculations of carbon flow via the pentose cycle, Embden-Meyerhof pathway, and the

3 These data illustrate dramatically the error of estimating synthesis in tissues with an active pentose cycle from [l-%]glucose incorporation, a still prevalent practice.

408 KATZ AND WALS

Krebs cycle into the major products. The The maximal stimulation due to PMS was: effect of Pm on flow rates for one experi- glucose uptake and phosphorylation, 1.5 ment is shown in Fig. 1. The effects were times; and oxidation via the Krebs cycle, quite similar in all experiments with fed rats. nearly 3 times. On the other hand, the phos-

TABLE I EFFECT OF PHENAZINE METHOSULFATE (PMS) ON GLUCOSE METABOLISM OF ADIPOSE TISSUES

Exp. DO. Diet [nsulin

1 Fed

2 Fed

3 Fed

4 Fed

5 Fed

6 Fed

7 Fed

8 Starved- refed

9 Starved- refed

- -

-

(1

-

PMS M x 10

0 0.5 1 2

0 2 5

10

0 1 5

0 5

20

0 2

0 2 5

0 1 5

0 2 5

0 2 5

-9 GlUCOSe uptake

Pyruvate 3 hr@

LIP ratio

CO1

1

I- a

___ -

24 31 34 40

Lipid slycerol ms C/g

c

%: Ga rtd

b -.- .-

17 18 16 17

Lactate +

PYru- vate?

11) d

ii1

10 11 12 13.

1.5 1.1 0.70 0.30

0.45 0.96 1.2 1.6

3.2 1.1 0.6 0.2

14 4.5 16 5.0 17 6.0 15 5.5

15 2.1 0.60 3.5 33 28 17 19 0.76 1.3 0.6 64 32 16 26 1.2 3.1 0.4 69 29 17 23 1.7 2.9 0.6 70 22 16

6.0 5.5

14 16

14 n.d.d n.d. 35 34 13 7.0 16 n.d. n.d. 39 34 11 8.5 a1 n.d. n.d. 67 33 14 10.0

14 1.5 0.30 6.0 32 31 14 20 0.9 1.1 0.8 67 36 14 11 1.6 1.0 1.6 41 8.6 9

5.5 6.5 8.5

8.5 1.7 0.25 8.5 0.8 0.30

6.7 18 17 7.5 nd. 2.3 23 17 9 nd.

10 1.0 0.16 14 0.7 0.60 18 1.3 1.3

6.7 22 15 13 n.d. 1.4 36 17 16 n.d. 1.0 66 15 15 n.d.

20 2.7 0.50 5.4 55 58 16 nd. 23 1.3 0.65 2.0 66 54 17 n.d. 26 1.3 1.2 1.1 82 53 19 n.d.

102 9.4 1.5 6.3 225 298 51 104 8.1 3.6 2.4 225 285 54 95 9.6 6.2 1.9 204 222 51

56 12.0 1.0 59 6.6 2.2 64 6.6 6.0

12.0 104 137 38 2.6 129 147 32 1.3 129 116 40

-

- Formation Incorporation of glucose carbon

-

a Adipose tissue (ZOO-300 mg) pooled from 10-16 rats was incubated in 2 ml of buffer for 3 hr. In exps. l-7, the concentration of glucose was 5 mM, and in Expet. 8 and 9, 25 mM. Insulin, when present, 0.1 unit/flask. Values shown in bold face type indicate that values were changed markedly from controls.

b By enzymic assay. c i4C counts eluted from Dowex 1. d n.d. = not determined.

PMS AND LIPOGENESIS FROM GLUCOSE

TABLE II

409

EFFECT OF PMS AT SPECIFIC YIELDS FROM [l-W]- AND [6-~4C]G~~~os~-I~~u~~~~~ WITH

ADIPOSE TISSUE

Exp.O no. Label in PMS glUCOSe CM x 10-6)

70 Dose utilized CO,

Specific yield (%)

Fatty acids Lipid Lactate + glycerol pyruvate

Fed rats 1 1-w

6-W

l-‘“C

6-W

1-w

6-W

1-w

6-W

Starved-refed 8 1-w

6W.J

1-w

6-W

0 0.5 1 2 0 0.5 1 2

0 2 5

10 0 2 5

10

0 1 5 0 1 5

0 5

Xl 0 5

20

0 2 5 0 2 5

0 2 5 0 2 5

25 44 28 23 5 28 52 25 17 6 27 59 19 14 8 28 70 13 11 6 25 18 45 24 8 24 29 44 23 9 27 26 44 20 10 30 27 42 23 8

26 50 28 17 5 34 65 18 10 7 48 78 12 5 6 45 81 8 4 7 29 12 59 20 9 36 17 60 17 6 45 12 47 18 23 42 13 41 18 28

47 45 31 13 11 57 52 28 10 10 67 75 13 5 7 40 11 54 15 20 51 13 60 14 23 60 12 49 12 27

40 45 34 15 6 52 61 22 8 9 34 82 6 6 6 43 21 54 16 9 58 27 46 11 16 39 45 21 12 22

48 46 43 7.5 4 46 48 39 7 6 44 54 33 6.5 6 47 2.5 78 11 9 47 3.6 77 10 10 41 5.3 76 10 9

24 45 37 11 7 25 50 35 10 5 22 58 28 9.5 5 23 3.5 69 14 14 23 4.5 71 13 10 22 5.0 70 13 12

. Experiments are the same &8 those in Table I.

410 KATZ AND WALS

TABLE III EFFECT OF PMS ON THE CONTRIBUTION OF THE PENTOSE CYCLE TO GLUCOSE METABOLISM

Eqxa no. PMS (M X 10 5)

2

- - (2?23 )

- -

5 Experiments are the same as those in Table I. Pentose cycle was calculated (from the data of Tables I and II) by the four methods listed in the text, and the average and range are given.

CO2

t R LACTATE PYRUVATE

GLUCOSE FRUCTOSE - Di - P - TRIOSE -P t PYRUVATE

LIPID GLYCEROL

FATTY ACIDS 4 ACETYL CoA

FIG. 1. Flow of intermediates (*atoms/g/3 hr) from glucose in rat adipose tissue. Light- face tvne, no PMS; boldface tvne (encircled), 50 PM phenazine methosulfate (PMS). Data from &p: (2) of Table IV. ” -

phofructokinase and aldolase reactions were not increased by PMS. There was a moder- ate increase in the oxidation of phosphoglyc- eraldehyde, the formation of which is increased via the pentose cycle. In tissue of starved-refed rats t,he effect of P&IS was not so pronounced and only oxidation via the pentose cycle was increased.

Balance of reducing equivalents. We have shown (7, 8) that in rat adipose tissue the

production of total reducing equivalents in the cytoplasm is equal or in slight excess over that required for the synthesis of fatty acids, lipid glycerol, and lactate and that any (small) cytoplasmic excess is t’ransported as malate into mitochondria. Normally the NADPH formed in the pentose cycle provides only part of the required reducing equivalents for fatty acid synthesis-from 40 % to 70 % in the experiments of Table IV.

PMS AND LIPOGENESIS FROM GLUCOSE

TABLE IV

EFFECT OF PMS ON FORMATION AND DISPOSAL OF REDUCED PYRIDINE NUCLEOTIDES

Ex~.~ no.

Fed 1

2

3

4

Starved- refed

8

9

-

-

PMS (aa x lo-9

0 0.5 1 2 0 2 5

10 0 1 5 0 5

20

0 2 5 0 2 5

C

-

I Formed I U&3

1

_-

-

A

A

-- iADH via GAPDH %

(/ C ‘mo1esP3

- LZ

hr) F

-c

59 9 14 15 5.5 2.5 71 13 16 16 6.0 7.5 73 18 16 14 6.5 14.0 78 29 16 15 6.0 24.0 84 18 19 25 9.0 3.0

107 40 20 28 6.0 26.0 129 65 28 25 8.0 64.0 124 70 24 19 8.0 67.0 89 16 22 30 6.5 1.5 93 22 24 32 5.0 9.0

114 50 25 29 6.0 40.0 83 11 21 27 6.5 0.5

113 27 29 32 5.5 19.0 67 28 12 7.5 4.5 28.0

595 126 600 138 525 147 328 65 330 79 318 100

~ -

159 260 161 250 132 195 85 120 86 128 76 101

-

23 2 26 23 20 64 26 5 17 20 19 56

Cytoplasm

(1 Experiments are the same aa those in Table I. b Includes reduced flavin.

The rest is presumed to be formed by trans- hydrogenation via the “pyruvate cycle” (8). In the presence of 20-50 PM PMS, the production of NADPH via the pentose cycle ex- ceeds the requirements for lipogenesis, and total cytoplasmic reducing equivalents are in large excess over that needed for biosynthesis (Table IV). This excess presumably is disposed of by nonenzymatic oxidation via PMS.

PMS stimulates also the formation of reducing equivalents via the Krebs cycle. In tissue of fed rats, the Krebs cycle provides 15-20% of total CO2 and one-third to one- half of the mitochondrial reducing equivalents. Since biosynthesis is not altered and the ATP requirements thus probably remain unchanged, it is likely that the extra reduc-

1

-

-

411

Mitochondria

Dyruvate dehyd.

(

G

Krebs CYCkb

&s/g/3 hr:

H

Total

,

I

12 14 14 14 19 20 23 19 21 20 22 19 25 16

15 27 21 35 22 36 23 37

9 28 15 35 30 53 32 51 12 33 16 36 20 42 15 34 37 62 22 38

159 16 175 147 26 173 117 25 142 23 14 86 75 18 83 65 20 85

ing equivalents are reoxidized directly via PMS, without change in oxidative phosphorylation.

ATP balance. The calculations of ATP formation and requirements are essentially those of Rognstad and Katz (8), and the numerical manipulation is indicated in Table V. The estimate of ATP formation and the utilization for phosphorylation and reesterification of fatty acids formed in lipolysis is straightforward (see Calculations). The ATP requirement for fatty acid synthesis is e&i- mated assuming acetyl CoA transport from mitochondria to the cytoplasm via citrate. If the fatty acid is palmitate, 2.87 moles of ATP are required per mole of acetyl CoA converted to palmityl CoA. However, if there is any formation of oxaloacetate from

412 KATZ AND WALS

TABLE V

ATP BALANCE IN ADIPOSE TISSUE

Reaction: Calculation:c

Exp. no.

HKa

P&b A/WPC)

Required Formed

Fatty acid Reesteritk EXWSS

cation Total Total Formed- synthesis Z.S?b/Z-F c-b/16

Cyt;glasm Mitochondria 3(G + H + F) utilized

~~mol~/dJ hr)

1 18 23 26 67 28 89 117 50 2 25 35 30 90 42 93 145 45 3 27 47 22 96 46 104 150 54 4 26 41 24 91 42 103 145 54 8 177 415 84 685 318 531 849 163 9 97 186 68 341 172 276 448 107

a Hexokinase. h Phosphofructokinase. c The capital letters refer to the columns of Table IV; the lower-case letters to the columns of Table

I. For details of calculations, see text.

the oxidation of ‘Lexcess” malate in mitochondria, the ATP requirement is reduced.

Table V presents an ATP balance for the experiments (without PMS) of Table IV. In tissue of fed rats, ATP requirements range in four experiments from 67 t’o 96 pmoles/g/3 hr, of which 5065 pmoles are for lipogenesis. From one-t,hird to one-half of the latter is used for reesterification of the free fatty acids liberated by lipolysis. ATP synthesis ranges from 117 to 150 pmoles/g/3 hr, with an ATP excess from 45-54 pmoles/g/3 hr.

In the two experiments with tissue of starved-refed rats, ATP requirements were 680 and 340 pmoles/g/3 hr. Less than 10% of t,he ATP was supplied via the Krebs cycle, whereas in the experiments with fed rats the Krebs cycle provided from 25% to 40% of the ATP synthesized. The ATP excess in the two experiments was about, 160 and 110 pmoles/g/3 hr, two to three times higher than in experiments with tissue of rats fed ad libitum.

This calculated excess represents a multi- step addition and subtract’ion and may be subject, to considerable error. In previous experiments with starved-refed rats, we calculated (8) in the presence of insulin a 20% excess of ATP production over utilizat’ion, corresponding to 117 pmoles/g/3 hr, similar to present values. We have calculated, when possible, ATP balances for the experiments done in this laboratory in the last 5 years. While the variability was considerable, there was an ATP excess in all conditions in over 30 experiments. With high glucose utilization

and lipogenesis (glucose utilization from 22 to 102 pmoles/g/3 hr) the ATP excess ranged from 75 to 220 pmoles/g/3 hr. With lower glucose utilizations and lipogenesis, ATP excess range from 20 to 60 pmoles/g/3 hr. While the values of Table V may be taken with caution, the occurrence of an ATP excess appears to be real.

E$ect of PMS on tritium-labeled glucose. Glucose labeled in positions 3, 5, and 6 was used. Results with glucose-5 have not been previously reported either in vivo or in vitro. The tritium in this position will appear on C-2 of glyceraldehyde-P and be labilized by exchange with protons catalyzed by triose-P isomerase (9). Any tritium retained would be lost subsequently in formation of phos- phoenol pyruvate. Thus, the sole product from this tritium is water. Table VI shows that 97-99% of the utilized hydrogen from position 5 appears in water and the rest in glycerol. Actually current experiments (unpublished) indicate most of the activity in glycerol to be due to unknown impurities. The yield in water nearly equals that of total utilized 14C. Hence TzO formation for [5-T]. glucose permits determination of total glucose utilization (excluding conversion to glycogen). Glucose-5-T, which is now commer- cially available, should be useful to follow glucose utilization when this is very small and to follow glucose turnover in vim.

In Table VII, the effect of PMS on the metabolism of [S-Tlglucose and [6-Tlglucose is shown. There was little or no effect on the T/14C ratios from [6-14C, 6-Tlglucose. The


effect of PMS is most pronounced on the on (a) the specific activity of glyceraldehyde- T/14C ratios in fatty acids from [I-14C, 3-T]. 3-P from [1-14C]glucose, (b) the specific activ- glucose (Table VII), which increases from ity of tritium on C-3 of glucose-6-P, and (c) about 1 to 2.3-3.2. This ratio depends mainly on the dilution of NADPT formed via the

pentose cycle by NADPH formed by malic TABLE VI enzyme. The specific activity of NADPT

EFFECT OF PMS ON THF: METIIBOLISM OF formed in the pentose cycle can be calcu- [U-t4C,5-T]G~ucos~ BY RAT ADIPOSE lated. If this were the sole source of hydrogen

Ex~.~ no.

-

(1

-

PMS bI x 10-s

0 2

0 5

0 5

0 20

0 2

TISSUE -

Specific yield - .-

T20

25 97 33 98

32 98 48 99

46 99 63 98

45 98 39 99

55 98 52 98

-

Glycerol

3 2

2 1

1 2

% @ded

utilized

.- 24 31

29 46

47 60

2 44 1 37

2 48 2 46

equivalents for fatty acid reduction, the T/14C ratios could be computed. Comparison of calculated and observed T/14C ratios should thus give an estimate of dilution and NADPH formation by other pathways. The maximal formaGon of NADPH via the pyruvate-malate cycle is limited by the supply of oxaloacetate formed by citrate cleavage. In the last columns, the TJUC ratio was calculated using two alternate assumptions: (a) maximal dilution by NADPH from malate, and (b) no dilution, that is, no decarboxylation of malate. The details of the calculation are presented in the Appendix.

In the absence of PMS, or when PMS stimulation is limited, the calculated values, if NADPH formation from malate is assumed, agree closely with observed ratios.

a Experiments are the same as those in Table I. On the other hand, with high concentrations

TABLE VII

T/W RATIOS FROM [6J4C,6-T]G~ucos~ AND [I-W,3-TIGLUCOSE

Exp.” no. PMS (aa x 10 9

[h-~~C,6-TlGlucose [l-‘“C,3-T]Glucose

Glycerol Fatty acids glycerol Fatty acids

Calculated ratios in fatty acids from cl-‘“C,J-T]glucose

Eq. 4 Eq. 3

1 0 0. 1 2

2 0 2 5

10

3 0 1 5

4 0 5

20

0.95

0.95-1.2 0.21-0.25 1.05 1.1 1.2

0.85

0.86-0.85 0.19-0.21 0.90 0.95 1.00

0.94 1.1 1.1 - 0.96-1.4 0.28-0.35 0.98 1.3 1.2 -

1.1 2.6 1.8 2.7

0.72 0.88 0.86 - 1.0 -1.3 0.23-0.27 0.74 1.2 1.3 -

0.93 2.5 2.3 2.6

1.1 1.1 - 1.4 1.2 - 1.8 1.6 2.2 2.5 1.7 2.4

1.2 1.2 - 1.7 1.8 2.5 2.9 2.4 2.9 3.3 2.8 3.2

(1 Experiments are the same as those in Table I. The calculation from Eq. 4 of the Appendix assumes formation of NADPH from the pentose cycle and from malate. In Eq. 3 it is assumed that the NADPH is formed only via the pentose cycle. For calculations, see Appendix.

414 KATZ AND WALS

of PMS, observed values agree well with those calculated assuming no NADPH formation from malate. The most likely expla- nation is that reduction of oxaloacetate to malate is suppressed.

DISCUSSION

E$ect of PMS on the cytoplasmic redox state. PMS, at concentrations that hardly alter glucose oxidation, already increases the output of pyruvate and lowers the lactate- pyruvate (L/P) ratio. At a concentration of 2 X 1O-5 M, pyruvate output was increased 3- to 5-fold and L/P ratios decreased to one- quarter or less of the control values (Table I). The concentrations of lactate and pyruvate in the medium may differ from those in cells but if we assume that the ratios are similar and cell pH is not changed, some estimates of the cytoplasmic concentration of NADH and NAD+ are possible using the assumptions and equilibrium constants listed by Krebs and Veech (10).

For a L/P ratio of 5, typical for tissue of fed rats, the NAD+/NADH ratio corre- spondsto l/ (5 X 1.1 X 104) = 18OO.Inthe presence of PMS with L/P ratio of 1, the NAD+/NADH ratio would be about 9000. Both these ratios are similar to those reported by Saggerson and Greenbaum (11). According to McLean et al. (la), the content of total NAD+ in rat adipose tissue ranges between 20 and 40 pg and of NADH 5-6 H/g. Since adipose tissue contains 30-40 J/g of intracellular water (13, 14), the concentration in the cytoplasm of total NAD+ is l-l.5 PM and of total NADH 0.2-0.3 PM.

In view of the discrepancies between the ratios obtained by analysis and those calculated, Krebs (1967), assumed that most of the NADH in the cell is “bound” and does not act as a hydrogen carrier in oxidore- ductions. The concentration of the “free” NADH in the cytoplasm may then be calculated to be 0.3 X 5/1800 = 0.8 PM and in the presence of PMS, 0.14.2 PM. These are maximal estimates, because if any of the NAD+ would be in the bound form, or the nucleotides preferentially sequestered in mitochondria, the calculated concentrations would be lower. These concentrations (without PMS) are by two to three orders of mag- nitude less than those of the intermediates

(with the exception of oxalacetate) and adenine nucleotides estimated in adipose tissue (11). These concentrations of NADH are also well below the K, for reductions catalyzed by lactic, malic, and cu-glycerophosphate dehydrogenase, but the activities of these enzymes are very high (11). The formation of lactate and cr-glycerophosphate for lipid glycerol are not impaired at PMS concentrations up to 100 PM (Table I), but reduction of cytoplasmic oxalacetate to malate might be depressed above 20 PM and abolished at higher concentrations of PMS (see below).

While it is likely that changes in the lactate/pyruvate ratio are proportional to the “free” NAD+/NADH ratio, in our opinion the concentrations as calculated above should be accepted with caution. It is difficult to account for over 99 % of the NADH, but little if any NAD+, being “bound,” and there is no evidence to support such extensive binding (15).

McLean et al. (16) measured the adipose tissue content of NADP+ to be 2.4 pg/g and NADPH 10 w/g. Estimates of the NADP+/NADPH ratios from the pyruvate/malate ratios (17) are questionable since it is doubtful that there is complete equilibration by the malic enzyme (decar- boxylating) . Moreover, published values for pyruvatei malate ratio in adipose tissue vary from 0.35 (11) to 0.04 (18).

It is likely that PMS lowers the concentration of NADPH and even a small decrease in NADPH would markedly increase the concentration of NADP+. Saggerson and Greenbaum (11) determined the glucose-6-P (G6P) and 6-phosphogluconate (6PG) levels in adipose tissue incubated with glucose plus insulin, with and without 0.1 mu PMS. The 6PG/G6P ratios were 1.5 without and 0.32 with PMS. While this ratio is insufficient to calculate NADP+/NADPH ratios (17), the ratio of esters is probably roughly proportional to the NADP+/NADPH ratio. Thus, at high concentrations of PMS most of NADP is present in the oxidized form.

Pyruvate concentration and fatty acid synthesis. Our conclusion is that the shift of the redox state toward oxidation with a lowered L/P ratio and increased pyruvate concentration had, over a fairly wide range, no ef-


feet on lipogenesis conflicts with the opinion of Halperin (20) who concluded that pyruvate concentration is rate-limiting in adipose tissue. Halperin (20) and Halperin and Rob- inson (21) studied the effects of N , N , N’ , N’- tetramethyl-p-phenylenediamine (TMPD) on adipose tissue. This compound was reported to enhance oxidation of cytoplasmic NADH by mediating its transfer to mitochondria (22), and like PMS, it increases pyruvate output from glucose, and lowers the L/P ratio, and increases lipogenesis from lactate. They also observed (20, 21) that TMPD increased in tissue of fed rats in the presence of insulin, fatty acid synthesis from glucose by 20% (from 30.8 f 2.1 to 37.0 f 2.0 pg/g/hr with a P < 0.01. Only a single result is reported, which appears in both papers.)4 Our attempts to demonstrate a consistent stimulatory effect of PMS and TMPD on fatty acid synthesis even when using paired tissue from the same rat were unsuccessful, but recently we were able to confirm stimulation of fatty acid synthesis from glucose by PMS in isolated fat cells (unpublished). The effect was only pronounced in the absence of insulin.

The difference in response to PMS from lactate and glucose is very striking. With lactate we find a very marked stimulation of fatty acid synthesis, but not from glucose. Our interpretation of the stimulation of lipogenesis from lactate was that PMS oxidized the excess cytoplasmic reducing equivalents, whose accumulation impeded lactate metabolism. With glucose no such excess exists. On the other hand, Halperin’s interpretation (20) attributed the effect of PMS to the increase in cellular pyruvate concentration. Our results suggest that pyruvate concentration is not a limiting factor in adipose tissue slices or in situ. However, under some conditions fat cells or tissue slices may become depleted of pyruvate and its concentration becomes rate-limiting.

NADH concentration. The conclusion of this study is that decrease in concentrations of reduced pyridine nucleotides, NADH and

4 Results were expressed as ratoms of hydrogen from tritiated water. One patom of hydrogen corresponds closely to incorporation of 1 patom of carbon from glucose.

NADPH does not materially alter the reductive biosynthesis of lipid glycerol and fatty acids, respectively. Saggerson and Green- baum (ll), with 0.1 mM PMS, found a decrease in ar-glycerophosphate concentration to one-third of control values, but no signifi- cant change in the synthesis of fatty acids and lipid glycerol from glucose. It appears that the affinity of a-glycerophosphate dehydrogenase and fatty acid synthetase for the reduced pyridine nucleotides is very high, and within a wide range marked changes in redox state do not affect lipogenesis.

PMS ej’ects on oxidation and glycolysis. The pronounced effect of PMS on oxidation via the pentose cycle is a general phenome- non in cells. Probably the rate of the pentose cycle is determined by the regeneration of NADP to serve as hydrogen acceptor, and the effect of PMS is due to increased reoxidation of NADPH. Since the pentose cycle may be increased 2- to 3-fold, none of the enzymes of this system operate normally at maximal capacities.

The data of Table IV indicate that with PMS glucose uptake can be increased in tissue of fed rats by as much as 50%. This would suggest that glucose transport and glucose phosphorylation are not rate-limiting. There is also an increase in the oxidation of glyceraldehyde-3-P. The “extra” glyceraldehyde represents that formed via the pentose cycle. There is no stimulation of the flow through phosphofructokinase and aldolase.

Oxidation via the Krebs cycle was increased to 2- to 3-fold (Table I and Fig. 1). Increased oxidation with PMS was noted by others (2, 11). Most likely the stimulation is due to reoxidation by PMS of mitochondrial NADH and of reduced flavin, similar to the stimulation of the pentose cycle. PMS thus causes uncoupling of oxidation from ATP formation, but the mode of action is quite different from classical uncouplers such as dinitrophenol, which cause ATP breakdown. Since the flux may be greatly increased, none of the reactions of the Krebs cycle operate at maximal capacity under normal conditions in tissue of fed rats.

The e$ect of PMS on malate reduction. Our results suggest that the decrease in NADH

416 KATZ AND WALS

concent8ration impairs malate formation. The T/14C ratios from [1J4C,3-Tlglucose of Ta- ble VII suggest that at PMS concentrations less than 20 PM, the reduction of oxaloacetate to malate is not much impaired, but at higher concentrations, reduction is decreased and little or no malate is formed above PMS concentrations of 50 PM, when the L/P ratio is below 1. This interpretation of the T/14C ratios is strengthened by the finding that addition of pyruvate to adipose tissue of fasted-refed rats metabolizing lactate nearly abolished the incorporation of tritium from lactate-U-14C-23H into water and fatty acids but not 14C utilization (4).

ATP balance and the regulation of lipogenesis. The calculation of ATP formation in the cytoplasm and mitochondria (Table V) requires no assumptions except for a P/O ratio of 3 for oxidative phosphorylation. On the other hand, the estimation of ATP requirements is based on a number of assumptions of the pathways of lipogenesis and transport between cell compart,ment’s. The calculations of Table V show in tissue of fed rats an excess of ATP of about 45 pmoles/g/3 hr, and with tissue of starved-refed rats, 110 and 160 pmoles/g/3 hr. Obviously the regeneration of ADP must balance the generation of high-energy bonds. The large calculated excess may indicate the existence of unknown energy-requiring reactions, a different mechanism for lipogenesis from that assumed above, or partial uncoupling in mitochondria, with a P/O ratio of less than 3.

Energy is also required for additional bio- symhet’ic reactions to those considered in Table V. The ATP may be used in the synthesis of proteins, glycogen, nucleic acids, and ot,her cell components. The energy estimate for these reactions is difficult, but it is not likely to be large. Since 98 % of the utilized glucose is accounted for in the an- alyzed products, the synthesis of other cell components is limited. Christophe and Wodon (24) have estimated in the presence of glucose protein catabolism to be about 1% of totma cell protein per hour and resynthesis about 0.5 %/hr. It is likely that not more than 5% of total ATP requirement is consumed in the various biosynthetic reactions listed above. A larger requirement of energy is likely to be expended in maintenance of

cell electrolytes, mainly the operation of the sodium-potassium pump. A rough estimate of the ATP requirement is possible from the data of Perry and Hales (25). They measured the ion flux in isolated fat cells to be 12 pmoles/g/hr and about half of the potassium flux to be oubain-sensitive. Assuming then half of the ion flux to be energy dependent, and requiring 1 mole of ATP per 2 moles of cations, the ATP requirements would be lo- 15 pmoles/g/3 hr. Thus, about one-third and possibly half of the energy excess in tissue of fed rats and less than that in tissue of starved-refed rats may be accounted for by synthesis and t,he operation of the sodium- potassium pump.

Flatt (23) has recently called attent,ion to the importance of ATP excess in t’he control of lipogenesis. He pointed out that in adipose tissue, according to accepted pathffays, fatty acid synthesis is accompanied by an excess of ATP formation. He suggested that there is a “basal” ATP requirement of 20-40 pmoles/g/hr, and the formation of ATP in excess of this value limits adipose tissue metabolism. He did not discuss how the “basal” ATP production is used or how the excess is dissipated.

In brown fat, whose major physiological function is heat generat,ion, uncoupling between oxidat,ion and ATP synthesis must be extensive, and represents a key feature of metabolic control (26). The mechanism of this uncoupling is not yet understood. In white adipose tissue some “futile” cycles (for example, recycling of pyruvate catalyzed by pyruvate carboxylase, carboxykinase, and pyruvate kinase) may serve to dissipate the excess of ATP, and such cycles may play a key role in met,abolic regulation. So far these possibilities have received scant attention.

Redox state and regulation of adipose tissue metabolism. Lipogenesis is not affected by large changes in the cell redox state since under proper conditions lactate and pyruvate both serve as good precursors for lipogenesis. Also, high rates of glucose synthesis in liver are obtained from compounds greatly differing in their oxidation state, as glycerol and pyruvate. This stability of lipogenesis in spite of the large changes in the rates of the pentose cycle, glyceraldehyde-P oxidation, and the Krebs cycle is of interest. Our find-


ings suggest that t.he rate of the pentose cycle and the Krebs cycle are determined simply by the rate of reoxidation of the pyridine nucleotides that serve as hydrogen accept,ors.

A large number of factors for control of the key reactions of the pentose cycle and Krebs cycle have been implicated with isolated enzymes and cell-free extracts. Whet,her any of these factors regulates metabolism in the intact cell is a moot question. It is possible that some of the regulatory steps studied in cell-free systems serve to regulate the level (concent,rations) of intermediates in the cell or its compartments, rather than over-all metabolic flow.

Attempts to establish in adipose t’issue a correlat,ion between metabolism and the concent,rat,ion of adenine and pyridine nucleotides, Co-4 derivatives or intermediates of glycolysis and the Krebs cycle have been so far unsuccessful (11, 14, 18, 27-29). Such factors as transfer of reducing equivalents and ATP balance have only recently received attention. The role of these factors in metabolic regulat,ion can be studied only in intact tissue or cells. Agents that affect reducing equivalent balance, such as PMS or TMPD, are valuable tools to examine many of the current theories of metabolic cont’rol.

APPENDIX

Calculation of T/14C ratios in fatty acids from [l -14C, J-Tlglucose. It has been shown by Iiat,z, Landau, and Bartsch (6) that when there is complete isotopic equilibration between GAP and DHAP the relative molar specific activity of the triose-P from [1-14C]. glucose (see below) is expressed as

(1 - PC)

(1 + 2PC)cJ - PC> (1)

and this also would be the relative specific activity of acetyl CoA.

If equilibration is not complete, the relative specific activity of GAP (and acetyl CoA) will be less than that of DHAP. Meth- ods for exact calculation of the relative specific activities have been described (6) but they are cumbersome. Since it can be shown that, in the present experiments deviation from equilibration values ranges from less than 5 % to a maximum of 15 %, the use of

an empirical correction factor of 1.1 to all the data is adequate.

Thus, the specific activity of acetyl CoA from [ 1-14C]glucose is given by Eq. 2 :

Relative sp act. of acetyl CoA from [I - 14C] glucose

(1 - PC) (1 + 2pc)(2 - pc) 1.1 (2)

The relative specific activity of the tritium from [3-Tlglucose in position 3 of glucose-6-P is t,he same as the specific activity of C-l from [lJ4C]glucose, because dilution of this carbon and this t,ritium via the pentose cycle are identical. Two moles of TPNH, one labeled and one unlabeled, are formed per mole of [3-Tlglucose metabolized via the pentose cycle. If there is no other formation of TPNH, the relative specific activity of TPNT will be l/2(1 + 2pc).

If the fatty acid synthesized is assumed to pahnitate, 14 moles of TPNH are required for reduction, or 1.75 moles per mole of acetyl CoA. Thus, the T/l*C ratio in fatty acids would be

T/14C ratio in fatty acids if TPNH formed only via pentose cycle

1.75 x 1.1 x (2 - pc)/2(1 - PC> (3)

The last column of Table VII was calculated by this equation. This equation can be used only if TPNH formed in the pentose cycle is sufficient for fatty acid synthesls.

If TPNH is also formed via malic enzyme, the amount would equal the moles of oxalacetate formed by citrate cleavage in the cytoplasm, and this equals bhe moles of acetyl CoA incorporated into fatty acids. This equals b/2, where b is patoms of carbon incorporat,ed into fatty acids, reported in Table I. Total TPNH formation via the pentose cycle is given in column B of Table IV. Hence, total TPNH production is B + b/2, and dilution of the labeled TPNH is B/ (B + b/2). Multiplying Eq. 3 by t’his factor yields Eq. 4. The next to the last column of Table VII was calculated by Eq. 4.

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effects of phenazine methosulfate on glucose metabolism in rat adipose tissue

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