Lipogenesis from lactate in rat adipose tissue

Biocl~iwicn ( Elsevier

(‘I Biopl~wictr Acrrr. 348 ( I 974) 344-356 Scientific Publishing Company. Amsterdam

Printed

in The Netherlands

BBA 56433

LIPOGENESIS

JOSEPH

KATZ

Cedars-Sinai Cakf:

90029

(Received

FROM

LACTATE

IN RAT

ADIPOSE

TISSUE

and P. A. WALS

Medical (U.S.A.

December

Center.

Researck

Instituie.

475 I Fmntrriu

Avenue,

Los Angeles,

I 3rd.

1973)

SUMMARY

In epididymal fat pad tissue of ad libitum fed rats, glucose greatly stimulates the uptake, oxidation and lipogenesis from lactate. Half-maximal stimulation occurs at about 0.3 mM of glucose, and even when present in great excess glucose does not depress lactate carbon uptake. Pyruvate at concentrations below I mM also stimulates utilization of lactate, but the effect is less than that of glucose and at higher pyruvate concentrations utilization of lactate is decreased. In tissue of fed rats when lactate and glucose are present together at equal concentrations, lactate provides most of the carbon for CO2 and especially fatty acid synthesis, while glucose provides nearly all the carbon for lipid glycerol. At concentrations in the physiological range (IO mM in glucose, 2 mM in lactate) in tissue of fed rats half or more of the fatty acid carbon is derived from lactate. Our findings suggest that in in vivo blood lactate under some conditions may serve as an important source for fatty acids. A balance of production and utilization of reducing equivalents and of ATP in tissue of fed rats has been calculated. In the presence of lactate plus glucose the production of reducing equivalents in the cytosol nearly balances the requirement for the synthesis of lipid. Glucose provides NADPH and some acetyl-CoA, whereas lactate provides the rest of the reducing equivalents and most of the acetyl-CoA. There is an apparent excess of ATP production over its utilization. The bearing of our findings on the role of reducing equivalents and energy balance in the control of lipogenesis are discussed. It appears that the marked difference in the metabolism of lactate and pyruvate and the effects of phenazine methosulfate can not be solely accounted for on the basis of disposal of cytosolic NADH.

INTRODUCTION

The relative utilization of glucose, lactate and pyruvate as substrates for lipogenesis in rat adipose tissue depends greatly on the dietary status of the animal [r-j]. In rats kept on common commercial diets utilization and lipogenesis from lactate is much inferior to that from glucose or pyruvate, but in tissue of rats kept on dietary regimes that stimulate lipogenesis, all three substrates serve equally well for fatty acid synthesis. Comparative studies with these compounds have been of value in

345

studies of the factors controlling lipogenesis in the intact cell, especially of the role of reducing equivalent supply and transport. Most of the previous studies were conducted with adipose tissue incubated with a single substrate. In the present work we used glucose and lactate or lactate and pyruvate together as common substrates. We find that in the presence of glucose lactate incorporation into fatty acids is much increased, and our results suggest that in vivo circulating lactate may be under some conditions an important source of fatty acids of adipose tissue. METHODS

Male rats of the Wistar strain, 180-250 g, were fed a commercial pellet diet or a high carbohydrate high in sucrose (General Biochemicals, Chagrin Falls, Ohio). The rats were either fed ad libitum or had access to food from 8 to IO a.m. (meal fed) or were starved for 2-3 days and refed. The experimental procedures have been in the main as previously described [4, 5f. Essentially r5o-3oo-mg slices of epididymal fat pad segments were incubated in Krebs-Henseleit bicarbonate buffer in an atmosphere of O,-COZ (95: 5, v/v) at 38 “C. The volume of the medium was 2 ml, and at low substrate concentrations, 5 ml. For each substrate combination one substrate was labelled and the other unlabelled or one labelled with 14C and the other with 3H. When tritiated water was employed the substrates were unlabelled. Methods of fractionation, assay of metabolites and radioactivity have been essentially as previously described [4, 51. The one additional procedure was the determination of glycogen. The glycogen was extracted from the defatted tissue residue in the conventional way by KOH digestion and ethanol precipitation, and the glycogen solution (about I ml) was neutralized and an equal volume of 0.5 M acetate buffer, pH 4.5, containing about r mg of amyloglucosidase (Sigma, St. Louis, MO.) was added. After incubation for one hour at 50 “C the glucose was determined enzymatically. The procedure with a purified glycogen sample from Boehringer-Mannheim gave a yield of glucose equivalents 90% of theoretical. Comparison of this procedure with that employing phenol-sulfuric acid and similar calorimetric methods revealed marked discrepancies. Estimates by the colorimetric procedure are higher than the enzymatic one especially with low glycogen content. Calculation of the pentose cycle When glucose and lactate were common substrates the 14C0, production via the pentose cycle was calculated from the 14C yields in COZ and fatty acids from uniformly labelled glucose and lactate. CO* via the pentose cycle = [CO& - [FA],, w

BC

The brackets indicate j4C yields (as Ctatoms of carbon) and the subscripts gl and lac the labelfed substrate. The percent pentose cycle may be obtained from the CO2 yield if total glucose uptake is known. 2 x CO, via pentose cycle o/0pentose cycle = ____I utilized glucose carbon

346 RESULTS

_!@ect of glucose on lactate Epididymal fat tissue of starved rats in vitro can utilize added glucose and pyruvate and synthesize small amounts of fatty acids from these substrates [I -31, but uptake and lipogenesis from lactate is very low. Addition of glucose or of pyruvate was found to increase the 14C yield in CO, and fatty acids [I], but this was not accompanied by an increase in lactate uptake as determined by analysis. There was actually an increase in medium lactate. Thus the 14C incorporation represented exchange between the pyruvate moieties of the common substrates and not net synthesis from lactate. Determination by analysis of the medium of lactate and glucose uptake in conjunction with 14C yields permits distinction between net synthesis and exchange. In our experiments changes in lactate uptake were large enough to measure reliably. The utilization of glucose was often of the order of IO-I 5 7:) of the initial amount and reliable determination by difference was difficult since variability between duplicate flasks is of the same order. In tissue of ad libitum fed rats there is lipogenesis from lactate although to a much lesser extent than from glucose or pyruvate [I, 61. We find that in this tissue the response to added glucose differs from that of starved rats, and glucose stimulated not only 14C yields but net lactate uptake as determined by analysis (Table I). Stimulation of incorporation into fatty acids was most pronounced when the basal yield was low. Thus in one experiment of Table 1, glucose at a concentration of 0.25 mM stimulated lactate uptake 35 times and increasing glucose concentration lo 25 mM further doubled the 14C yield from lactate. The increase in “C incorporation from lactate coupled with the decrease from glucose could be due to exchange of puryvate moieties from the two substrates. While some exchange is likely to have occurred the results of Table I indicate a marked stimulation of lactate and probably a decrease in glucose utilization. The reasons are: (I) adequate agreement between carbon utilization measured by analysis and from 14C yields; (2) total 14C in CO, and lipids from the two substrates exceeds the sum of 14C yields when the substrates are added singly. (3) With the low glucose (0.2mM) the increase in lactate yields is greater than the total added glucose carbon and (4) if there was exchange there should be a marked increase in the 14C incorporation of glucose in lactate. There was some increase but it was less than the depression in the 14C incorporation into CO, and lipids. In Fig. I the effect of glucose concentration is shown in more detail. The addition of glucose not only increased lactate utilization, but changed the pattern of metabolism. With lactate alone the ‘“CO, yield was twice that in fatty acids but with increasing glucose concentrations, the fatty acid yield progressively approached and ultimately exceeded that in CO,, as typical for starved-refed rats. The half-maximal stimulation of fatty acid synthesis appeared to be between 0.2 to 0.4 mM, and above 2 mM glucose there was no further stimulation. In Fig. I the uptake of lactate as measured analytically and the total 14C recovery in CO2 and lipid are shown. They agree well, indicating that 14C incorporation represents closely net uptake of lactate. Tritium from water in fatty acids and lipid glycerol are also shown in Fig. I. The yield in fatty acids was greatly increased by 0.2 mM glucose, a concentration when the contribution of glucose carbon was small.

OF LACTATE

AND GLUCOSE

BY EPIDIDYMAL

2.7

0

5.0 5.0 -

None None None

2

3

5.5 5.5 5.5 -

5.5

None None None None

10

-

0.24 24 24

-

___I__ * Determined by enzymic analysis. ** N.D.. not determined.

4

5.5

IO

-

20

0

0.2

20

-

5.5 5.5 5.5

None None None None

-

2.5 2.5

2.7

-

5.0 5.0

None None None

I

Substrates (mM) Lactate Glucose

Phenazine methosulfate @MI

Expt. No.

-

9.0

IO

I.5 9.5

I2

-

II

II

5.6

N.D.** N.D. N.D.

--

4.0 18

Lactate uptake* (~moles/g)

IO

r4

10

3.6

18

8.0 r4 r4

34 --

22

24 -

I.5

r4 -

IO

-

28 -

I.5 I5

34 3r

-

16

4.5

7.1 16

0.2

r4

16

I2

4.0

48 -

II

2.5 27 -

I

0.9 7.0 14 -

-

20 -

2.1

3.4 20

-

-

20

20

0.4 0.8 0.8

2.1

-

0.5

2.7

-

0.8

0.6

0.3 14 36 -

10

-

36 -

12

1.1

17

4.0 18 31

34

13 27 30 -

34 83 -

5.8 37 48 -

-

4.5 49 84 -

68 77 -

24 68 -

-

3r 89 -

r5r -’ -

-

18

21

0.5

5.5

I2

N.D.

N.D.

N.D.

Lactate from glucose

____-

h for Expts 3 and 4. Lactate and glucose uniformly

1.2

-

2,

._____-. Total Lipid giycerot C0z-t lipid (,a atoms C/g) (a atoms C/g) -Lactate Glucose from Lactate Glucose -1.1 r4 25 77 1.0 9.0 52 9.0 35 -

and

22

-

IO

4.8

-

II

-

IO

Fatty acids (fig atoms CM _ from Lactate Glucose

CO, (qg atoms C/g) from -___ Lactate Glucose

I

FAT PAD SLICES OF FED RATS

200-300 mg of slices incubated in 2 ml bicarbonate buffer. Incubation period 2 h for Expts labelled with 14C. 50 mu/ml of insulin were added in Expts 2-4, and none in Expt. I. _

METABOLISM

TABLE I

t LACTATE

UTILIZATION UTILIZATION

0 GLUCOSE .

.

k IN FATTY CO?

0 +I

IN FATTY

. k

AClDS

APYRUVATC , LACTATE

ACIDS

. “C 0%

‘5

2

10

p

.14C

IN

FATTY

ACIDS

IN GLYCEROL

.

0 t

ACIDS

IN GLYCEROL

30 ;20

UTILIZATION UTILIZATION

IN FATTY

5 0 I!zzzz!:’ 25

1

2

3

4

GLUCOSE

5 mM

10

2

3

PYRUVATE

4

5 mM

Fig. I. Effect of glucose and pyruvate on the metabolism of lactate by rat adipose tissue. 250 mg ot tissue slices of pooled rat epididymal fat pads of 12 rats incubated for 90 min in z ml bicarbonate buffer. Lactate concentration was 5 mM, uniformly labelled with lJC. When “HOH was present lactate was unlabelled. Insulin. IOO mU per flask was present. The same tissue pool was used with glucose (right) and with pyruvate (left). The uptake of glucose. lactate and pyruvatc was measured by enzymic analysis and is shown in the upper curves. The broken curve (right) shows also the uptake of lactate as calculated from the sum of the 14C yields in the products, shown below. Note that lactate uptake decreased with increasing pyruvate concentrations and became negative. that is there was net output of lactate.

In Table I also the stimulation of lactate metabolism by phenazine methosulfate is shown. The effect of glucose at 0.2 mM equalled or exceeded that of the dye at IO LJM, the most effective concentration [7]. Phenazine methosulfate however, but not glucose stimulated lactate incorporation into glycerol. l$kt

qf pyruvate

on lactate

To account for the mechanism of the glucose effect, comparison with pyruvate was of interest. In tissue of starved rats the apparent stimulation of lactate uptake by pyruvate is an exchange reaction with a net production of lactate [I]. However the effect of low concentrations was not tested. The one experiment illustrating the effect of pyruvate on lactate metabolism in tissue of ad libitum fed rats is shown in Table II. The addition of 0.5 mM pyruvate to 5 mM lactate increased incorporation of lactate carbon into CO, and fatty acids and total carbon yield from the two acids was somewhat higher than from the two acids as sole substrates ( 19 vs I 2 /iatoms). The pyruvate stimulation is much less than by similar glucose concentrations. There was in this experiment an extensive exchange as witnessed by the large increase of 14C from pyruvate in lactate (from 2 to 9 {(atoms, over half of the pyruvate incorporation) but there was still a stimulation of net lactate uptake. The effect of concentration is shown in Fig. I, where glucose and pyruvate additions are compared with the same tissue. In this experiment the increase in 14C’ incorporation from lactate in the presence of pyruvate was not accompanied by significant elevation of lactate utilization, as measured by analysis. While with increasing glucose concentration the 14C yield from lactate approaches a plateau, with increasing pyruvate, lactate uptake was depressed. At higher pyruvate concentrations

349 TABLE II METABOLISM OF LACTATE ADIPOSE TISSUE

AND PYRUVATE

AS COMMON

SUBSTRATES

BY RAT

200 mg of epididymal fat pad slices of fed rats incubated for z h in 2 ml of buffer. Results averages of duplicate determinations. .~ Substrates (mM)

Lactate

5

5

0

Pyruvate

0

0.5

0.5

Utilization* (pmoles/g per 2 h) Lactate Pyruvate Total

-9 -to-5 -8.5

+-0.5 -5 -4.5

-14 -3 -17

WZ incorporation (pg atoms C/g per z h) Into CO, from lactate 23 15 Into CO2 from pyruvate 3 16 Into lipid from lactate 9 lnto lipid from pyruvate 3 Into pyruvate from lactate 4 I Into lactate from pyruvate 9 _ -* By enzymic analysis. Values rounded up to

8

26 19

-

3 2

0.5

--

@moles.

there was net production of lactate (Fig. I) and there was a decrease in 14C incorporation. Eflect of glucose on pyruvate

Since the incorporation of lactate into glycerol is very low (Table I) an obvious mechanism for the stimulating effect of glucose would be by supplying glycerophosphate for trjglyceride synthesis. Pyruvate incorporation into glycerol is somewhat higher than that from lactate but much smaller than from glucose, and if glycerophosphate were limiting, lipid synthesis would be stimulated by small amounts of glucose. This was tested in the experiments of Table III. There was an increase in 14C yields in CO, and fatty acids by 0.5 mM glucose but there was little change in pyruvate uptake as determined by analysis. The 3HOH incorporation into fatty acids was approximately additive for the incorporation of the substrates added singly. The high incorporation of glucose into the acids indicates extensive exchange, and it appears that most if not all the increased 14C incorporation from pyruvate is by exchange. Apparently glycerol-P production does not limit pyruvate metabolism, Lipogenesis from lactate at low concentrations

The results of Table I indicated that when glucose and lactate are present at equal concentrations, incorporation of lactate carbon exceeded that from glucose. The lactate concentration in these experiments was higher than commonly found in body fluids, and it was of interest to determine the role of lactate at near physiological concentrations. A medium 2 mM in lactate and 5 mM in glucose was used and to avoid changes in concentration the volume was increased to 5 ml. The glycogen utilization and the incorporation of 3HOH into lipids was also determined. In tissue of rats fed ad Iibitum the glycogen content was negligible. Lactate uptake was much stimulated by glucose and fatty acid synthesis as judged by 3HOH

OF

III

7

10

0

-.

* Lactate equivalents: ** N.D.. not determined.

10

10

0.5

0

0

5.0

5.0

10

IO

-12.9 - r4.0

0.5

10

10

-12.2

0

IO

6

case

ViiE

(

Pyruvatc

GIU-

Pjru-

No.

) uptahe;

IO

-14 - 27 --IS

-

Net

(- ) production.

-: 2.1 -. 1.6

T.2.1

-1 2.2

Laclate

(,nmoles/gf

N.D.

-~ 3.6 -16 ---I7

0

Glucase

17

_.

2.2 12

-

20

2.5 I2

Glucost

26

26

24

25 28

t9

Pyruvate

TISSUE

-.

26

15

9.0

19 26

8.0

Pyruvate

6.0 18

0.3

22

5.0

0.6

case

Gli

Pyruvate

RATS

1.0

1.6 I.6

0.5

t,.5 0.9

Pyruvate _

from

9.0 II

2.0

5.0

4.7

2.0

Glucost

-

N.D.

N.D. N.D. N.D.

‘5 5-o

3.9

Cig)

-

46

47 80

35

76 47

29 50

.-

..~

nith

lAC.

9.4 ‘9 ‘4

6.1

17 I8

N.D.**

IO

acids

__~__

5.5 7.0 9.3

4.2

5.8 7.2

3.8 N.D.

erol

“H from 3HOH (/.lg atoms H/g) Fatty Glyc-

labelled co, I L.ipid t/rg atoms C/g)

uniformly

Acids from Glucose (jrg atoms

and glucose

(,ugatoms C/g)

Glycerol

FED

of insulin.

OF

Fatty acids (j(g atoms C/g) from

I h \vith 20 mU;ml .____ -.

CO, from fjrg atoms C/g)

for

BY ADIPOSE

_. ___

in z ml buffer

METABOLISM

incubated

PYRUVATE

Utilization*

Substrates

Expt

fmM)

ON

fat pad segments

GLUCOSE

zoo mg of epididymai

EFFECT

TABLE

incorporation, was higher from the two substrates than the sum from each substrate added singly. The 14C yield in fatty acids from lactate eceeded that from glucose. The apparent depression of 14C incorporation from glucose in the presence of lactate is in part due to exchange, making quantitation of the contribution of both substrates to fatty acids difficult, but in spite of exchange it appears that lactate supplies a substantial part, and at least half of the fatty acid carbon. In rats fed a high carbohydrate diet or rats meal-fed for about a week the glycogen in the tissue provides a significant amount of carbon, which should be taken in account in establishing a carbon balance. It supports appreciable endogenous lipogenesis, as measured by 3HOH incorporation. As measured by the tritium yield in fatty acids, the synthesis of fatty acids was slightly stimulated by adding lactate to glucose over that from glucose alone. The i4C yields in lactate from glucose did not increase greatly when lactate was added, indicating only limited exchange and it TABLE IV THE ROLE OF LACTATE AND GLUCOSE, WHEN PRESENT TOGETHER CONCENTRATIONS, AS PRECURSORS OF ADIPOSE TISSUE LIPIDS

AT PHYSIOLOGICAL

150-200 mg epididymal fat pad slices incubated in 5 ml buffer, 5 mM in glucose and/or uniformly labelled with r4C or unlabelled with 3HOH. I h incubation.

2

Expt No.

Diet + hormone

Initial glycogen

Substrates

Fattv acids

(wnoWd*

8

9

IO

II

I2

Regular

Regular

Meal-fed

High carbohydrate

High carbohydrate

N.D.**

0.16

2.4

5.2

3.3

insulin

* Glucose equivalent. ** N.D., not determined.

Incorporation

Glucose Glucose + lactate Lactate Glucose Glucose + lactate Lactate None Glucose Glucose $ lactate Lactate None Glucose Glucose + lactate Lactate None

4.5

IO

5

15

4.8

0.7

0.1

-

4.5 5.0

5.5 N.D.

37 57

8.5 IO

IO

18 18

9 I2

0.4

22

17 15

0.2

72 35

0.4

13

28 32

7,5 13

2.7 3.5

8

3.5

0.7

27 36

8.5 12

5.0 6.5

5

I.3 0.5

2.5 9.0

72 107

29 31

9.0 9.2

24

II

6.0 9.5

8 21 36

35 57

9.0 0.3

18

55 42

97 I4

8.5 9.3

8.5 8.0

54 63

0.4

70 +

3.5 6.5

0.2

IO

35

4.5 4.7

0.2

I.5

Glucose Glucose lactate Lactate None

12

Incorporation of 3H in (yg atoms H/g) Fatty Lipid acids glycerol

of 14C (fig atoms C/g) in Lioid Lactate CO, + glycerol lipid

II

5 7

mM in lactate. Substrates

15 0.5

13

13

I5

27

0.6

-

124 68

80 117

38 47

17 17

40

25 14

I2 9.5

141 192

58 63

22 23

31

14

II

II

IO ___

appears that only a part of the depression of 14C yields from glucose in fatty acids by adding lactate is due to exchange reactions. Due to the contribution of glycogen carbon, and of exchange effects, precise quantitative estimates are difficult but it seems that under all the dietary conditions of Table IV, lactate was an important source of fatty acid, providing probably between one quarter to one half of the carbon although the concentration of lactate carbon in the medium was one tenth that of glucose. Reducing equivalent

and energy balance

We have previously shown that in glucose metabolism in adipose tissue [4,8,9], the production of reducing equivalents in the cytoplasm balances closely the reducing equivalent requirement for lipogenesis, so that there is little or no transfer of reducing equivalent between cytosol and mitochondria. This has been also confirmed for adipocytes [IO]. On the other hand with pyruvate all the reducing equivalent for reductive biosynthesis must come from mitochondria. Per mole of acetyl-CoA converted to a twocarbon unit of fatty acids 2 moles of NADPH are required and 2 moles of NADH per mole of pyruvate reduced to glycerol-P. Assuming that malate is the hydrogen carrier 2 moles of ATP per mole of reduced compound are required to carboxylate mitochondrial pyruvate to oxalacetate. Malate oxidized via cytosolic malin enzyme provides NADH and pyruvate is regenerated. With pyruvate there is also a need to dispose of the oxalacetate formed by citrate cleavage and by malate oxidation. It is likely that this occurs via conversion to phosphoenol pyruvate and pyruvate, without a net requirement for ATP. If lactate is the sole substrate only one mitochondrial reducing equivalent per mole of fatty acids is needed and no excess oxaloacetate is formed. With glucose and lactate as common substrates the requirement for reducing equivalent transfer from the mitochondria may be diminished or be absent. Energetically the most efficient synthesis would occur if glucose were to supply about half the NADPH required for fatty acids and the carbon for glycerol synthesis and the fatty acid carbon is derived from lactate. In Table V we have calculated reducing equivalent balances when glucose and lactate were the substrates for four experiments of Table I. The glycogen content ot the tissue was negligible. The production of CO2 via the pentose cycle and NADPH was estimated as described in the Methods section. From this and the carbon balance. the production and utilization of NADH and NADPH was obtained. The results of Table I indicate a close balance of production and utilization of cytosolic reducing equivalents, and there was little or no need for transfer from mitochondria. Glucose provided NADPH and nearly all the glycerol carbon. The NADPH plus the NADH from lactate were sufficient for the biosynthesis of lipids. To calculate a balance in tissue of starved-refed rats a knowledge of the contribution of glycogen, present in significant amounts in this tissue, is required. It appears likely that in this tissue glycogen glucose supplies the glycerol carbon and NADPH. ATP balance

The carbon balance of Table I and the reducing equivalent balance permits under certain assumptions to calculate an ATP balance [5, 81. 2 moles of ATP are formed in the cytoplasm in the oxidation of triose-P to pyruvate. in the mitochondria

353 TABLE V REDUCING EQUIVALENT AND ATP BALANCE IN THE PRESENCE OF GLUCOSE

IN THE METABOLISM

OF LACTATE

Tissue of fed rats, experiments of Table I. Values as ,umoles/g, and are rounded to 0.5 @mole. _ Expt. No. I

3

2

4

Lactate fmM) Glucose (mM) Reducing equivalents formed in cytosol: NADP via pentose cycle NADH via glyceraldehyde-P-dehydrogenase NADH via lactate dehydrogenase Total

r3 3 17.5 33.5

Reducing equivalents used in cytosol: Fatty acids Lipid glycerol Total

28 3.5 31.5

39.5 18 57.5

10.5

20.5

39.5 18 57.5

IO

6.5

9 19

8 14.5

Reducing equivalents formed in mitochondria: Via pyruvate dehydrogenase Via Krebs cycle Total ATP formation : Cytosol Mitochondria Total

5.0

5

2.7

2.5

16

36.5 6 tt5 121

5.5 0.20

37 I 28

66

2.5

2

I

0.5 6 8.5

9 12.5

1.0 I

5.5 0.24

1.5

7 I.5 8.5

2 172 174

37.5 39.5

43 44

2 26 6 34

1.5 12 85 22

ATP utilization: Phosphorylation Fatty acids Reesterification Total

9 47 I6 72

16 IO1 21 138

ATP excess

49

46

2

5.5

I

22

(assuming a P/O ratio of 3) 3 moles of ATP are generated in the oxidation of NADH formed via pyruvate dehydrogenase, and the oxidation of acetyl-CoA via the Krebs cycle generates 2 moles of CO2 and 12 moles of ATP. Thus from the CO, yields of Table V the ATP production in mitochondria is obtained. ATP is consumed in the phosphorylation of glucose and fructose-&P and 3 moles ATP are required for transfer of mitochondrial acetyl-CoA to the eytopIasm and the reduction to a two-carbon unit of fatty acid (one mole each for pyruvate carboxylase, citrate cleavage enzyme, and acetyl-CoA carboxylase). Assuming the fatty acid to be palmitate, 2.87 moles ATP are required per mole of acetyl-CoA. ATP is required for the reesterification of free fatty acids, 2 moles of ATP (one pyrophosphate bond} per mole of fatty acids. The extent of reesterification was estimated from the excess of glycerol carbon over that required for glyceride synthesis of newiy formed palmitic acid. ATP would be required for reducing equivalent transfer but it was negligible in these experiments. The calculations of Table V indicate a sizable excess of ATP production over utilization. In the calculation the conversion of glucose to lactate was neglected and

354

this underestimates ATP production. Apart from the assumptions the calculations are subject to some uncertainty because of experimental errors and cumulative errors in the balance calculations, and caution in quantitatjve interpretation is called for. We have however calculated an ATP excess in practically all experiments and if the assumptions are valid an ATP excess as large as that observed with glucose (see Discussion) is apparent. DISCUSSION

Stimuluiion pf lactatemetabolism by glucose The metabolism of lactate differs from that of pyruvate in several aspects. (I) Utilization and fatty acid synthesis from pyruvate in tissue of starved or fed rats is much better than from lactate, but in tissue of starved-refed rats lipogenesis from both acids is equal. (2) Phenazine methosulfate stimulates greatly utilizatiol~ and lipogenesis from lactate, but not from pyruvate and (3) 2,4-dinitrophenoi at low concentrations depresses oxidation of lactate but stimulates that from pyruvate, and it inhibits fatty acid synthesis from lactate much more drastically than from pyruvate [9]. We have proposed that the oxidation of cytosolic NADH is a rate limiting step in lactate metabolism, and this hypothesis served reasonably well to account for the observations. However the findings that glucose at very low concentrations stimulates lactate metabofism as well or better than the hydrogen acceptor phenazine methosulfate, is hard to reconcile with this theory. An obvious explanation for the stimulatory effect of glucose is the provision of glycerol-3-p for triglyceride synthesis. This explains the efficient lipogenesis in tissue of starved-refed rats high in glycogen, but it is not so obvious why glycerol-P formation limits the metabolism of lactate but not of pyruvate, and does not account readily for the phenazine methosuifate effect and that of dinitrophenol. The synthesis of lipid glycerol from pyruvate has been studied by several investigators [I 1-131. The formation of phosphoenolpyruvate is the key step in this synthesis, since the glycolytic reaction between triose-P and phosphoenolpyruvate are sufficiently reversiblz. Adipose tissue contains phosphoenolpyruvate carboxykinase and the main physiological role of this enzyme has been suggested in providing glycerol-P. In fatty acid synthesis from pyruvate phosphoenolpyruvate is presumably formed from the oxaloacetate (derived via citrate cleavage) and the concentrations of phosphoenolpyruvate in the cytosol may be sufficiently high for efficient glycerol-P synthesis. On that basis the stimulation by phenazine methosulfate would be explained as due to an increase in cellular pyruvate, which then increases glycerol-P forn~atiol~. The defect in lipogenesis from lactate would then be in the low intracellular pyruvate concentration. The hypothesis is not altogether satisfactory. it does not account for the Ending that glucose is a much better activator of lactate metabolism than is pyruvate, and it does not explain the dinitrophenol effects. Other factors in addition to reducing equivalents productions limits lipogenesis from lactate and future work is needed to provide a more satisfactory unifying hypothesis. While there is evidence for net synthesis of lipid glycerol when pyruvate is present at high concentrations it is highly unlikely that this happens under physioiogicdi conditions. In the presence of glucose the incorporation of lactate and pyruvate is much less than that from glucose, and the net flux is from glucose to pyruvate. In

35.5

the presence of glucose the incorporation of ‘“C from the acids in glycerol represents an exchange reaction rather than net synthesis. In vivo pyruvate concentrations rarely exceed 0.2 mM and it is difficult to conceive, even under extreme hypoglycemia, how the glucose is unsufficient to supply glycerophosphate for the synthesis of the low amounts of lipid formed under such conditions. Moreover there is no valid evidence that glycerol-p production is ever rate limiting in lipogenesis in vivo. T/I~ p~ys~olo~~ca~role of lactate as fatty acid precursor

The most interesting aspect of our study is the indication that circulating lactate may have an important role in lipogenesis in vivo. It is well known that in adipose tissue of fed rats with a low glycogen content, lactate as sole substrate is poorly used. Our results establish that provided glucose is present at low concentrations, lactate is a highly eBcient precursor of fatty acid carbon. When lactate and glucose are present at equal concentrations in the range of 5-ro mM the incorporation of lactate carbon into fatty acids exceeds greatly that of glucose, and even if glucose is present in 5-IO-fold excess, the incorporation into fatty acids from lactate equals that from glucose. We have found that in vitro in other tissues lactate is also used preferentially over glucose in fatty acid synthesis. In slices and isolated parenchyma cells of mammary gland of lactating rat lactate (but not pyruvate) in the presence of low glucose concentrations serves as an excellent source for fatty acid synthesis [14]_ In hepatocytes from meal-fed rats Iactate is much superior to glucose as a source of fatty acid carbon, and it stimulates the incorporation of tritium from 3HOH into fatty acids much more than glucose 1151. In vivo the relative roles of glucose and lactate are likely to depend on their concentrations in blood. In the post-absorbtive animal lactate concentrations are in the range of I-Z mM as compared to 5-8 mM for glucose. Our findings suggest that under these conditions lactate may contribute a significant fraction of fatty acid carbon. Lactate concentrations are much increased with anoxia and violent exercise, but these conditions are not conducive to lipogenesis. Circulating lactate levels are however greatly elevated after sucrose feeding [16], up to 8 mM. Extrapolation from in vitro to in vivo studies is not always valid, and it remains to be shown by experiments in the intact animal what role circulating lactate plays in Iipogenesis in the body. ACKNOWLEDGEMENT

This work was supported by U.S.P.H. Grants Nos 5RorAN

1260405, RR

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