Estimation of the Glucuronic Acid Pathway Contribution to Glucose

T m J~URNAI. OF BIOLOGICAL CHEMISTRY Vol. 241, No. 3. Issue of February 10, 19G6

Printed in U.S.A.

Estimation of the Glucuronic Acid Pathway Contribution

to Glucose Metabolism in Adipose Tissue and

the Effect of Growth Hormone*

(Received for publication, August 5, 1965)

BERXARU R. LAXDAU,~ GLESN E. BARTSCH, AND HOLLIS R. WILLIA~~S

Frown the Departments of Medicine and Biochemistry, and the Division of Biometry, Western Reserve University, Cleveland, Ohio &$I 06

SUMMARY

A definite contribution of the glucuronic acid pathway to total glucose metabolism by rat adipose tissue in vitro in the presence of growth hormone preparations has not been found. Randomization of 14C of glucose-5-14C into glycerol and glucose-l-14C and -2-14C into glycogen on incubation of rat epididymal tissue indicates that the pathway contributes at most only a few per cent to the over-all metabolism of glucose. Results of incubations with glucose-lJ4C and -6J4 are in accord with metabolism solely via the pentose cycle, Embden-Meyerhof pathway, and Krebs cycle. Thus, essentially all of the 14C utilized on incubation of glucose-lJ4C and -6-14C is isolated in products formed via these pathways. Nonequilibration of dihydroxyacetone phosphate with glyceraldehyde 3-phosphate occurs, and this results in a preferential oxidation to CO2 of carbon 6 as compared to carbon 1 via the Krebs cycle. This coupled with an increase in the fraction of carbon 3 of pyruvate converted to CO2 and a decreased pentose cycle contribution in the presence compared to the absence of the preparations can account for the en- hanced yields of 14C02 from glucose-6-14C as compared to -l-14C that have been reported on growth hormone addition.

Presentation of a growth hormone preparation to adipose tissue in vitro stimulates the oxidation to CO2 of carbon 6 of glucose

* Supported by Grant A-4411 to B. R. L. from the Nat,ional In- stitute of Arthritis and Metabolic Ijiseases, by a grant from the American Heart Association, by the Lee Fund of Western Reserve University for the Study of Diabetes Mellitus and Related Meta- bolic Diseases, and by assistance to G. E. B. through Biometry Training Grant Tl-GM-17 and Grant GM-09310 of the National Institutes of Health. Material supplementary to this article has been deposited as Document No.-8577 with the AD1 Auxiliary Publications Project, Photoduplication Service, Library of Con- gress, Washington 25,J). C. A copy may be secured by citing the Document number and by remitting $3.75 for photoprints or $2.00 for 35.mm microfilm. Advance payment is required. Make checks or money orders payable to: Chief, Photoduplication Service, Library of Congress.

t Portions of this work were doue during the tenure of an Estab- lished Investigatorship of the American Heart Association.

relative to carbon 1, so that frequently yields of 14C02 from glu- case-6J4C are greater than from glucose-lJ4C (I, 2). Winegrad et al. (1, 3) considered this response most likely to be due to a stimulation of glucose metabolism via the glucuronic acid pathway, a pathway by which carbon 6 of glucose, but not carbon 1, is oxidized to COZ. An alt,ernative explanation, considered less probable, was that it was attributable to isotopic nonequilibration of dihydroxyacetone phosphate with glyceraldehyde 3-phosphate, a mechanism by which carbon 6 relative to carbon 1 of glucose would be preferentially oxidized to CO2 in the Krebs cycle. Isotopic nonequilibration has been shown in incubations of adipose tissue in vitro (2, 4-7). LeBoeuf and Cahill (2) presented data interpreted as a stimulation by growth hormone preparations of the metabolism of glucose via the Embden- Meyerhof pathway and its oxidation in the Krebs cycle. They believed these changes and nonequilibration could explain the increase in oxidation of carbon 6 of glucose to COZ. They noted the need for quantitative and qualitative information of a more specific nature to dismiss the possibility of a significant glucuronic acid pathway contribution. Winegrad and Shaw (8) recently introduced additional dat.a in support of the existence of the glucuronic acid pathway in adipose tissue and its stimulation by growth hormone. In particular they noted that (a) glucuronic acid-6J4C is oxidized to 14C0, without incorporation of 14C into fatty acids; (b) some of the enzymatic activities associated with the pathway are demonstrable; (c) nonlabeled glucuronic acid dilutes 14C02 production from glucose-6-14C with a greater dilu- tion in the presence of growth hormone than its absence; and (d) following incubation with glucoseJ4C, 14C-containing material can be identified as glucuronic acid.

There are two objectives of the present paper: (a) to estimate the contribution of the glucuronic acid pathway to total glucose metabolism in adipose tissue, and (b) to measure the extent of isotopic nonequilibration of the triose phosphates in the presence of growth hormone under conditions similar to those of Winegrad et al. (1, 3) and determine if the changes in 14C02 yields from glucose-l-X and -6-14C are in accord with metabolism solely via the pentose cycle, Embden-Meyerhof pathway, and Krebs cycle.

The first objective has been accomplished through the use of glucose-5-V, -2.‘4C, and -1-14C. During the metabolism of a molecule of glucose-5-14C via the Embden-Meyerhof pathway, a molecule of glyceraldehyde-3-P) 2J4C is formed.

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(;lucose-6-P,5-‘% ---f dihydroxyacetone-P + glyceraldehyde-3-P, 2-14C

The metabolism of a molecule of glucose-5-14C via the pentose ~cycle also yields a molecule of glyceraldehyde-3-P) 214C (9).

3 Glucose-6-P,5-14C ---t 3 CO, + 3 pentose-5.P,4-14C

3 Pentose-5-P,4-14C 4 2 glucose-G-P, 5-14C + glyceraldehyde-3-P, 2-l%

Sum: glucose-6-P, 5-% ---) 3 CO, + glyceraldehyde-3-P, 2-W

In contrast, the metabolism of a molecule of glucose-5-14C via the glucuronic acid pathway (10) yields pentose-5-P,5-14C, and the latter’s subsequent metabolism via the pentose cycle yields a molecule of glyceraldehyde-3-P) 3-14C.

3 Glucose-6-P,5-14C 4 3 glucuronic acidm5-14C

3 Clucuronic acid-5-W 4 3 CO2 + 3 D-xplulose-l-14C

3 L-Xylulose-l-14C ---f 3 D -xylulose-5-14C

3 D-Xylulose-5-1% ---f 3 D-xylulose-5-P,5-‘4C

3 D-Xylulose-5-P, 5-14C + 2 glucose-6-P, 6-W + glyceraldehyde-3-P,3-14C

Sum: 3 glucose-6.P,5-W -+ 3 CO2 + glyceraldehyde-3-P,3-14C + 2 glucose-B-P, 6-W

Glucose-5-14C was incubated in vitro with rat epididymal adipose t,issue in the presence of growth hormone and the glycerol formed, presumed to reflect the 1% distribution in glyceraldehyde-3-P, was isolated and degraded. An estimate of the glucuronic acid pathway contribution to over-all glucose metabolism was then made from the ratio of 14C incorporated into C-3:C-2 of glycerol.

From glucose-2-i4C via the pentose cycle, glucose-6-P, 1,2,3- 14C is formed and via the glucuronic acid pathway, glucose-6- P ,2 ,3-14C (9, 10). Via the pentose cycle, carbon 1 of glucose- 1-14C is evolved as 14C02 while via the glucuronic acid pathway it is randomized into carbon atoms 2 and 3 of glucose-6-P. Glu- case-2-14C and -I-14C were incubated with adipose tissue and glucose from glycogen, presumed to reflect the 14C distribution in glucose-B-P, isolated and degraded. Estimates of the glucuronic acid pathway contribution were then made from the relative incorporation of 14C into carbon atoms 1, 2, and 3 of the glucose from glycogen.

To achieve t,he second objective, the pentose cycle contribution to glucose metabolism was estimated from the randomization of carbon 2 of glucose-2-14C during its incorporation int.0 glycogen and glycerol (11) and the extent of equilibration of triose phosphates from the incorporation of 14C of glucose-l-14C and -6-14C into glycerol and fatty acids (6). Parallel incubations in the absence of growth hormone were performed to establish a hormone effect .

Within the limits of these estimates, we conclude that the glucuronic acid pathway contribution to the total metabolism of glucose can be no more than a few per cent. Metabolism of glucose via the pentose cycle, Embden-Meyerhof pathway, and Krebs cycle, in the presence of nonequilibration of triose phosphates, adequately explains the change in 14C02 yields observed with growth hormone preparations.

EXPERIMIZNTAL PROCEDURES

Jlaterials and Incubations--The procedure was similar to that previously described (6). Male albino rats of the Wistar strain, weighing 200 to 250 g and fed ad lib&m on stock chow, were decapitated. Their epididymal fat pads were removed, trimmed, segmented, and distributed so that each of the incubation flasks in an experiment contained one segment from each of the rats killed. Segments with a total weight of 1.0 to 2.1 g, from 8 to 20 rats, were present in a flask. A serum bottle of 30-ml capacity served as the flask. It was closed by a rubber serum stopper from which a vial was suspended. The medium was 2 ml of Mod- ified Krebs bicarbonate buffer (7). Glucose, present at a concentration of 2 mg per ml, was labeled with I-i4C, 2-14C, 5-14C, and 6-W. Glucose-l-i% and -6.14C were purchased from New England Nuclear Corporation. They were purified to remove “carbonate-like” material (12). Glucose-2-14C was purchased from Nuclear-Chicago Corporation. Glucose-5J4C was gener- ously provided by Dr. Lars Ljungdahl. Its preparation and the confirmation of the position of the label by degradation have been reported (13). A bovine growth hormone preparation was added to the medium at a concentration of 1.0 mg per ml. The hormone, a gift of the Endocrinology Study Section of the Na- tional Institutes of Health (Lots NIH-GH-B-7 and B-8), was reported to assay at a potency of about 0.9 unit per mg. A human growth hormone preparation assaying at about twice the potency of the U.S.P. growth hormone reference preparation was a gift of Dr. Olof Pearson (14). It was added at a concentration of 0.2 mg per ml. After gassing with 957, 02 and 5% CO* the medium had a pH of 7.3 to 7.5. Incubation was with shaking for 3 hours at 37”. In each of four experiments, glucose- 1-14C and -6-14C were incubated in the absence and presence of growth hormone. Parallel incubations were performed with glucose-2-‘4C and -5.14C in the presence of growth hormone. A control incubation was performed in which the flask and contents were identical with those w&h 14C except that only nonlabeled glucose was present.

Isolation of Products-Incubations with glucose-l-14C and -6J4C were terminated by injection through the stopper of 0.4 ml of 30% perchloric acid into the medium of each flask and 1 ml of hyamine into each vial. After shaking for an additional hour, each vial was removed and capped.

The medium was withdrawn with a Pasteur pipette to a gradu- ated centrifuge tube. The flask and fat pads were rinsed with water and these washings were combined with the medium (aqueous phase). A negligible quantity of 14C was extracted with petroleum ether from this phase. The tissue was placed in chloroform-methanol (2: 1 by volume), the flask was washed with chloroform-methanol, and the solutions were combined (lipid phase). The tissue residue was removed from the lipid phase and rinsed with additional chloroform-methanol. The rinses were added to the lipid phase.

The aqueous phase was neutralized to phenqlphthalein with potassium hydroxide. After cooling the potassium perchlorate precipitate was removed by centrifugation. The precipitate carried negligible 14C activity. Glucose utilization was determined by preparing glucosazones from the initial solution and the aqueous phase and assaying them for 14C. Glucose in the aqueous phase was also determined with glucose oxidase (15). An aliquot of the aqueous phase was acidified and extracted con- tinuously with ether. Carrier lactate was added to the neu-


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752 Glucuronic Acid Pathway in Adipose Tissue Vol. 241, iSo. 3

tralized extract which was then evaporated and the lactate was purified on a Celite column. The lactate was combusted and assayed for 14C, giving the quantity of 14C incorporated into lactate. The aqueous and initial solutions were also chromato- graphed on Whatman No. 3MIM paper with the upper phase of butanol, water, methanol, and formic acid (320:320:80: 1 by volume), and lactate, glycerol, and glucose areas were identified through guide samples. These areas were eluted and assayed for 1% activity to give the percentage of the added ‘4C incorporated into glycerol, lactate, and glucose. The incorporation into lactate by this procedure was in good agreement with the results obtained by the more complicated isolation and combustion of the lactate. Therefore, the former method was used in all but the first experiment. The determination of residual glucose by this chromatographic method was also in good agreement with that obtained with glucose oxidase or osazone formation. Chromatography was performed with a pyridine, ethyl acetate, acetic acid, water solution (5 :5: I:3 by volume) (16), with a glucuronic acid guide sample, to estimate 14C activity in the area corresponding to glucuronic acid. In addition, chromatography was done with a butanol, acetic acid, water solvent (4: 1: 5 by volume) (17) with xylose as a guide spot to estimate 14C in com- pounds having the mobility of the common pentoses.

An aliquot of the lipid phase was washed with water and the 14C in the water phase assayed. This water-soluble 1% material, considered within the tissue, represented less than 59& of the 1% added to the incubation media. On chromatography, the I%! was present in glucose and glycerol, with the former accounting for 80% or more of the activity. In the tables to follow, this 1% activity in glucose has been included in the estimate of recovered glucose. Another aliquot of the lipid phase was evaporated and the fatty residue hydrolyzed for 12 to 24 hours in 70% ethanolic KOH at 70-80”. The solution was evaporated, acidified, and extracted with heptane. The 14C in the heptane was taken as incorporation into fatty acids and that in the aqueous layer as incorporation into glycerol after correcting for the 14C present as glucose. In two experiments, the solution was extracted prior to acidification to measure the 14C incorporated into nonsaponifi- able lipid. The activity in this fraction was less than 1% of the 14C in the initial medium.

The tissue residue was decomposed in 30y0 KOH. The resulting solution was neutralized to phenophthalein, taken to volume, and an aliquot assayed for l*C activity. This was taken to represent the 14C! in glycogen and supporting tissue. The remaining solution was acidified with HzS04 and heated at 100” for 3 hours to hydrolyze the glycogen. Carrier glucose was added and the glucosazone was made and assayed for 14C in comparison with the osazone of the initial medium, giving the percentage of incorporation into glycogen. Incorporation of 14C into supporting tissue was obtained by difference.

In the incubations with glucose-2-14C and -5.14C the fat pad segments were removed after incubation and immersed in chloroform-methanol. Glycerol and glucose from glycogen were isolated and degraded as previously detailed (7). The specific activity of the glucose-5-14C (2000 dpm per mg) was too low to yield adequate incorporation into glycogen for degradation and assay, so that with glucose-5-14C only glycerol was degraded. As a control glycerol-2-l% was degraded. Lactate isolated from the medium as described above was also degraded. In one experiment, glycogen was isolated and degraded after incubation with glucose-l-14C and -6J4C in the presence of growth hormone.

In a fifth experiment, glucose-lJ4C and -6J4C were incubated in the presence of bovine and human growth hormone and in their absence. The yields of 14C02 established an effect for the hormone similar to that observed in the first four experiments. Glu- cose from glycogen, isolated from the tissues incubated with glucose-P4C and hormone, was then degraded.

Counting ProceduresGlucosazones were plated on stainless steel planchets and assayed in a gas flow, thin window counter. Aliquots from the heptane, aqueous, and hyamine solutions were assayed in a dioxane solution (18) with a liquid scintillation spectrometer. Counting of the final solutions was done simul- taneously with aliquots from the initial solutions so that the percentage of the 14C added to the flask which was incorporated into the products could be calculated directly. Aliquots from the products of the control incubation were added to the initial samples and aliquots of the control initial samples to the product samples to achieve identical quenching (6). Channel ratios were employed to verify equal degrees of quenching.

CO2 obtained from the individual carbon atoms of glucose and glycerol on degradation was usually evolved into ethanolamine which was then dissolved in toluene containing a scintillant (19) and assayed in the spectrometer. In a few instances where 14C activity was very low, samples were assayed in the gas phase.

RESULTS

Incorporation of 14C from Glucose-l-14C and -6J4C into Products -Table I records tissue weights, the percentage of the added glucose utilized, and the recovery in the products of 14C from the glucose-1-14C and -6.l*C. To adjust for the enhancement by growth hormone of the utilization of glucose, more tissue was incubated in the absence than presence of the hormone. An average of 35% of the added glucose was utilized in its absence and 75 y0 in its presence as determined from glucosazones. Utili- zations were similar when determined with glucose oxidase. The percentage of the utilized 14C from glucose-lJ4C oxidized to ‘*CO2 was decreased by growth hormone while the percentage from glucose-6-14C was increased. The percentage of 1*C from both glucose-l-14C and -6-14C incorporated into glycerol was increased by growth hormone. Much more 14C was recovered in glycerol in the aqueous phase. The percentage of the utilized carbon incorporated into fatty acids decreased. The preferential incorporation of carbon 6 of glucose relative to carbon 1 into fatty acids is clearly demonstrated. Except for the last experiment, less than 4% of the utilized 14C was incorporated into lactate. The percentage of 1% in lactate was greater from glucose- 6J4C than -l-14C. Incorporation into glycogen and supporting tissue was a small percentage of utilized 14C and appeared to be decreased by the addition of growth hormone.

The percentage of recovery of the utilized 14C as measured by the summing of the percentage of the utilized 14C found in COs, glycerol, fatty acids, lactate, glycogen, and tissue residue was nearly lOO%, except for Experiment 1 (last column in Table I). Almost all of the 1% in the aqueous phase was accounted for in glucose, glycerol, and lactate, except in Experiment 1 where as much as 20% was not. By paper chromatography (16), 14C was found in an area corresponding to the Rc of glucuronic acid. However, the 14C assayed at no more than 2% of the utilized ‘“C and there was as much 14C in the absence as in the presence of growth hormone. With butanol-acetic acid-water as solvent (17), no 14C activity was detected in the area corresponding to the mobility of xylose and ribose.


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TABLE I

753

Incorporation of I%’ of glucose-l-W and -6-‘%’ into products on incubation with rat adipose tissue in absence and presence of growth hormone

Glucose was at a concentration of 2 mg per ml. Incubation was for 3 hours at 37”. Isolation of the various fractions and their assay is described in the text. In Experiments 1 and 2, a bovine growth hormone preparation was added at a concentration of 1.0 mg per ml. In Experiments 3 and 4. a human growth hormone preparation was added at a concentration of 0.2 mg per ml.

Percentage of 14C of utilized glucose recovered in -

4c la be1 Tissue weight

CO2 Fatty acids Lactate Glycogen E iupporting

tissue Tissue 4queous Total

_- g %

1.4 56.2 1.4 46.7 1.3 87.3 1.3 80.0

36.7 6.9 0.5 7.4 19.0 2.0 0.7 4.1 11.1 10.5 1.1 11.6 35.3 3.2 0.9 3.6 35.2 32.1 2.7 34.8 12.3 2.6 0.3 1.3 26.1 21.3 2.5 23.8 16.8 3.4 0.5 2.0

% 69.9 65.7 86.5 72.6

1 1.5 37.8 45.5 22.7 1.1 23.8 25.4 1.3 0.8 1.9 98.7 6 1.5 25.7 13.2 28.4 0.8 29.2 47.1 3.5 1.9 5.1 100.0 1 1.3 91.3 37.8 31.8 3.3 35.1 20.4 2.0 0.4 0.0 95.7 6 1.3 93.5 16.5 26.7 3.0 29.7 41.1 3.0 0.4 0.5 91.2

1 1.6 39.5 48.4 15.9 1.8 17.7 31.1 0.8 0.3 3.0 101.3 6 1.5 34.3 13.4 17.8 2.3 20.1 57.4 2.3 0.3 3.8 97.3 1 1.0 79.1 33.6 32.9 2.7 35.6 17.4 1.6 0.1 1.1 89.4 6 1.0 60.8 27.8 27.1 2.3 29.4 34.9 3.3 0.2 1.8 97.4

1 2.1 19.3 72.5 33.2 1.0 34.2 14.0 15.5 4.1 4.7 145.0 6 2.1 26.7 19.9 31.5 2.2 33.7 21.3 13.9 1.1 7.1 97.0 1 1.5 51.0 24.7 36.9 7.1 44.0 5.7 6.7 1.0 2.0 84.1 6 1.5 37.5 24.0 38.7 2.7 41.4 7.5 11.2 1.6 2.4 88.1

- I -

Growth hormone

-

+

-

+

-

+

-

+

Experiment

- * Utilized glucose as determined from the difference between the 14C added and recovered in glucose; assayed as glucosazones. t Sum of the percentage of the 14C of the utilized glucose recovered in COZ, glycerol, fatty acids, lactate, glycogen, and supporting

tissue.

TABLE II

Distributions of 14C in glycerol and glycogen on incubation of glucose-P4C and -2-14C with adipose tissue in presence of growth hormone

Incubations were under the same conditions and run parallel to the incubations with growth hormone of Table I. Relative distributions are recorded with carbon 2 = 100. Recovery obtained by totaling the 14C found in each carbon, as a percentage of the 14C in the product on combustion, is recorded to serve as a measure of the adequacy of the degradation procedure.

T Glycerol;* glucose-5-Y as substrate Glucose unit of glycogen; glucose-Z-W as substrate Glycerol;* glucose-2-W as substrate

Experiment I-

c-3

1 2.7 2 0.8 3 41 0.0

Mean 1.2

c-2

100 100

100 100

c-2

100 100 100 100 100

Re- COVSY

% 86 96 93 92 92

C-l Rt?- covery

2.7 2.3

%

85 97

5.3 106 3.4 96

C-5 C-l C-l c-2 c-3 c-4

10.0 100 6.9 0.9 11.4 11.8 100 6.9 0.4 8.2 15.4 100 9.9 1.0 9.2 11.7 100 8.7 1.8 16.0 12.2 100 8.1 1.0 11.2

C-6 Recovery -____

% 1.3 89 0.9 95 1.6 82 3.7 89 1.9 89

c-3

13.8 17.6 11.2 9.7

13.1

--

7.5 10.0

8.3 5.9 7.9

- - - * Glycerol-2-14C, purchased from Orlando Chemical Company, when degraded gave a distribution of 3.3, 100, 1.9 (recovery 94%). t In this experiment distribution in the glucose unit of glycogen on incubation of glucose-2-14C in the absence of growth hormone

was 12.9, 100, 6.5, 1.6, 13.3, 1.2 (99yo recovery).

Randomization of 14C from 14C-labeled Glucoses into Glycogen, degraded (see asterisk footnote of Table II), similar amounts of 14C Glycerol, and Lactate-In Table II, the distribution of l4C on were found in carbon atoms 1 and 3. Carbon 2 of glucose-2.‘4C degradation of glycerol and glucose from glycogen after incuba- was randomized into the other carbon atoms of glycerol and of tion of glucose-2-14C or -5-14C with growth hormone is recorded. the glucose from glycogen and the distributions in the glycerol With glucose-5-14C, 14C appeared in carbon 1 as well as 3 of glyc- and glucose were similar. The distribution in glycogen was simi- erol, but there was less than & as much 14C activity in carbon 3 lar to that observed in one experiment in the absence of growth as 2. When a commercial preparation of glycerol-2-14C was hormone (dagger footnote of Table II). Lactates isolated from


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TABLE III

Distribution of 14C in lactate on incubation of glucose-ii- W and -6-14C with adipose tissue in presence oj growth hormone

Experiments of Tables I and II. The relative distributions are recorded with carbon 2 = 100 with glucose-5-14C as substrate and carbon 3 = 100 with glucose-6- 14C.

Substrate

Glucose-5-i4C Experiment 1. Experiment 2.

Glucose-6-‘*C Experiment I*.

C-l c-2 c-3 Recovery

%

16.8 100 33.1 100 12.4 100 49.1 92

11.6 1 / 100 / 96 19.8

* In this experiment glycerol was also degraded. The relative distribution was C-l = 1.6, C-2 = 3.3, C-3 = 100, with a recovery of 98’%. The distribution in lactate in the absence of growth hormone was C-l = 6.5, C-2 = 16.4, C-3 = 100, with a recovery of 35Y0.

TABLE IV

Distribution of 14C in glucose unit of glycogen on incubation of glucose-l-14C and -6-14C in presence of growth hormone

The l4C of the position in the glucose with the highest 14C concentration has been set equal to 100 and incorporation in the remaining carbon atoms is relative to this carbon. Samples are from Experiment 4 of Table I and Experiment 5.

Substrate

Glucose-l-14C Experiment 4. Experiment 5t. Experiment 51..

Glucose-6-14C Experiment 4.

--

C-l

100 100 100

0.4

c-2 j c-3 c-4 C-5 C-6

4.3* 0 1.3 2.7 1.1 1.2 2.8 0.6

1.0* 2.7

0 0.4 0.4

0

9.7 12.6 10.1

100

:ecovery

%

91 97

107

90

* Sum of the activity in carbons 2 and 3. t Bovine growth hormone. $ Human growth hormone.

the aqueous phase in experiments with glucose-5-i4C and -6-‘*C were degraded (Table III). There was extensive randomization of carbon 5 of glucose into carbon atoms 1 and 3 and of carbon 6 of glucose into carbon atoms 1 and 2 of lactate. A much smaller randomization of carbon 6 occurred during its incorporation into glycerol than lactate (footnote to Table III). In Experiment 4, glucose from glycogen isolated after incubat,ion with glucose-l- l*C and -6-r4C in the presence of growth hormone was degraded (Table IV). r4C of glucose-6-14C was localized to carbon 6 of the glucose from glycogen, but about 10% of the 14C of glucose-l-r4C was randomized into carbon 6. About 4% as much activity was in carbon atoms 2 and 3 as in carbon 1 of the glucose from glycogen with about twice as much 14C in carbon atom 3 as 2 (Experi- ment 5).

DISCUSSION

Estimation of Glucuronic Acid Pathway Contribution

The contribution of the glucuronic acid pathway to over-all glucose metabolism can be estimated from the randomization of the specifically 14C-labeled glucoses. The estimates are inde-

pendent of the extent of equilibration of the triose phosphates except with glucose-5 14C.

Glucose-5-14C-The contribution of the glucuronic acid pathway to glucose metabolism is reflected by the incorporation of 14C of glucose-5-14C into carbon 3 relative to carbon 2 of glycerol. The results of Table II would indicate that this amounted to no more than 30/o of the total glucose utilized (2.7/(100 + 2.7)) in the experiment in which the greatest randomization was 0bserved.l The degradation of glycerol upon which this estimate is based is not completely specific (21). Thus, in a degradation running parallel to the degradations of these experiments, about 3% of the activity in carbon 2 of glycerol-2-X appeared in carbon 3 (asterisk footnote of Table II) and Stjernholm and Wood (21) found 1.2 y0 of the activity of a glycerol-2-r*C sample appeared in carbon 3. Also extensive randomization of carbon atoms 5 and 6 of glucose occurred in the Krebs cycle, as evidenced by the distribution of r4C in lactate on incubation with glucose-5-r4C and -6-r4C (Table III). Possibly 14C in carbon atoms 1 and 3 of glycerol with glucose-5-i4C as substrate was incorporated after metabolism via the Krebs cycle. Randomization of i4C in the Krebs cycle and then incorporation into glycerol has been shown to occur on incubation of specifically labeled pyruvate with mouse adipose tissue (22). A small quantity of 14C from glu- case-6-14C in the absence of added hormone has been found in carbon atoms 1 and 2 of glycerol with Aerobacter aerogenes for degradation (7), and this is again shown in one of the experiments of the present series (footnote to Table III). Therefore, some or all of the small percentage of 1% appearing in carbon 3 of glycerol with glucose-5J4C as substrate is probably due to conditions other than metabolism via the glucuronic acid pathway.

GZucose-$J4C and -1-14C-The randomization of 14C from glu- case-2-14C into carbon atoms 1, 2, and 3 of glucose from glycogen can also be used to estimate the glucuronic acid pathway contribution. As shown in the “Appendix” (Equation 21), in the presence of the glucuronic acid pathway randomization via the pentose cycle should fulfill the relationship previously derived for a system in which the pathway was absent (11, 23), xl/x2 = 2 PC/(1 + 2 PC). x1/x2 is the relative specific activity in C-l: C-2 of glucose-6-P, PC is the fraction of total metabolism via the cycle and the equilibration of fructose-6-P with glucose-6-P is assumed to be complete (E--h+ co). In the absence of the glucuronic acid pathway, x3/x2 = (xl/x,)/(2 - x1/x2) should also hold (II), but in its presence, x3/x2 should be greater than x1/x2/ (2 - zl/r2). Based on the assumption that the glucuronic acid pathway is absent, x3/x2 should be, with the mean value for

1 In this calculation, the rate of the transaldolase exchange reaction (20) is assumed to be zero (in the appendix expressions are derived which do not require this assumption). The incorporation of 14C into carbon 3 of glycerol is assumed to be solely via the ylucuronic acid pathway, where, for each molecule of glucose- 5-14C metabolized in the pathway, a molecule of glyceraldehyde- 3-P,3-14C is formed, while for each molecule metabolized via the Embden-Meyerhof pathway or pentose cycle a molecule of glyceraldehyde-3-P,2-l*C is assumed to be formed. Actually when 1 molecule of glucose-5.l*C is metabolized via the glucuronic acid pathway to CO2 and glyceraldehyde-3-P, 3-14C, 3 molecules of glu- case-6-P,U4C enter the pathway and 2 molecules of hexose6 P,6-‘*C are formed (see introduction). Since this hexose-6-P will be metabolized to triose-P via the pentose cycle and Embden- Meyerhof pathway, 14C will also be incorporated into carbon 3 of glycerol via these pathways. Therefore, in the calculation some of the 14C in carbon 3 attributed to the glucuronic acid pathway is from these other pathways. This results in an overestimation of the glucuronic acid pathway contribution.


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Issue of February 10, 1966 Landau, Bar&h, and Williams 755

Growth hormor

Absent

Present

WI label

1 6

6

TABLE V

Specific yields and ratios of specijk yields

Specific yields*

Fatty acids

%

24 (10-31) 46 (22-59)

15 (7-21) 28 (9-45)

Glycerol

YO

19 (11-24) 26 (18-35)

42 (37-52) 36 (30-47)

Lactate

YO

4.0 (0.8-11) 6.2 (2.4-14)

3.7 (1.8-8) 6.1 (3.3-13)

* Values are the averages with the ranges in parentheses for the four experiments of Table I. The percentage of specific yields of a product equals ((1°C yield in the product)/(l4C in the glucose utilized)) X 100. Since almost all of the 14C of the

xl/x2 of Table II, 0.122/1.878 = 0.065, rather than the observed value 0.081. The greater observed than predicted value may be due to reversal of the pentose cycle (23, 24). It may also be due, at least in part, to contamination of carbon atom 3 by 2 during degradation, since as much as 170 of the activity of glucose-2-W is found in carbon 3 on degradation (12, 25). I f it is assumed to be due entirely to a glucuronic acid pathway contribution, the contribution could account for about 2 % of total glucose metabolism. This is true even though E-h is finite (see “Ap- pendix” for the details of these calculations). Randomization of 14C from glucose-l-14C can also be used to estimate the contribution of the glucuronic acid pathway contribution since carbon 1

of glucose is randomized into carbon atoms 2 and 3 of glucose-6-P by this pathway, but not by the pentose cycle. From the data of Table IV a contribution of about 3.5% is estimated (see “,p- pendix”). Reversal via the pentose cycle will also make this an overestimation. Furthermore, the small incorporation of 14C into carbon atoms 2 and 3 approaches the limits of the degradation procedure.

Assumptions-The above estimates of the glucuronic acid pathway contribution depend upon the assumption that glucose utilization can proceed only via the Embden-Meyerhof pathway, pentose cycle, glucuronic acid pathway, and Krebs cycle. The recoveries in Table I would support this assumption since, except for a small quantity of 14C incorporated into glycogen, essentially all of the 14C utilized appeared in products of these pathways. The estimates also require that the glucuronic pathway be complete, that is that the carbon atoms of glucose entering the pathway, other than carbon 6, are metabolized via pentose 5-phosphate. If intermediates of the glucuronic acid pathway accumulated, randomization of l*C of the %Xabeled glucoses into glycerol and glycogen would not reflect the quantity of glucose entering the pathway. The essentially complete recoveries of 14C in the products isolated (Table I) and the results of paper chromatography are evidence for the nonaccumulation of intermediates. Therefore, the increase in the oxidation of carbon 6 of glucose to COZ in the presence of growth hormone cannot be attributed in more than a small measure to metabolism

via the glucuronic acid pathway.

Pathways followed by Glucose Carbon in Presence oaf Growth Hormone

The increase in the oxidation of carbon 6 by growth hormone,

as well as the decrease in the oxidation of carbon 1, relative to

CO2 Fatty acids -

%

49 (46-52) 16 (13-20)

0.51 (0.45-0.55)

37 (2941) 28 (18-36)

0.60 (0.47-0.80)

Ratio of specific yields

Glycerol Lactate

0.75 0.51 (0.61-0.85) (0.31-0.79)

1.19 0.61 (1.11-1.33) (0.5330.64)

glucose utilized was recovered in fatty acids, glycerol, lactate, and COZ, for simplicity of calculation the sum of the 14C incorporated into these products has been substituted in the equation for the 1% in the glucose utilized.

glucose utilization is in accord with metabolism solely via the Embden-Meyerhof pathway and the pentose and Krebs cycles in the presence of incomplete equilibration of the triose phosphates. The quantity of glucose carbon converted to the various products required to produce the yields of 14C given in Table I and the randomizations patterns in Tables II to IV can be estimated. This is done by the use of the model of Katz, Landau, and Bartsch (6). The transaldolase reactions (20), which are omitted, do not affect the results if pathways which do not form triose phosphate, i.e. non-triose-P pathways, are absent. As already noted, in adipose tissue the major non-triose-P pathway is glycogen formation and its contribution is too small to affect the estimations. Equilibration of the hexose B-phosphates is assumed to be sufficiently extensive to be considered complete. This is the case for adipose tissue unstimulated by hormone and the similar patterns of 14C randomization in carbon atoms 1, 2, and 3, and carbon atoms 6, 5, and 4 of glucose from glycogen, and carbon atoms 3, 2, and 1 of glycerol (Table II) indicate this is also so when growth hormone is active (7, 20).

The estimation procedure parallels that already used for data on the effect of growth hormone on adipose tissue from rats fasted and then refed (6). The yields from glucose-1-i4C and -6-14C in Table I have been converted to specific yields and the ratio of the specific yields from glucose-l-14C to -6-14C for fatty acids, glycerol, and lactate calculated (Table V). Not,e the similar ratios for fatty acids and lactate and the increased difference between the glycerol and fatty acids ratios produced by growth hormone. From the data in Table V, the pentose cycle contribution, the relative rate of triose-P isomerization in both di- rections, the fraction of glucose metabolized to glycerol and pyruvate, and the specific activities of dihydroxyacetone-P and glyceraldehyde-3-P relative to that of glucose have been estimated.2 The pentose cycle contribution calculated from the specific yields of 14C02 averaged 19% (range 18 to 20%) in the absence of hormone and 8% (range 2 to 14%) with hormone. Under similar experimental conditions with no hormone present, estimates based on the randomization of 14C of glucose-2J4C into glycogen averaged only 9% (7), and this is in agreement with

2 Intermediate expressions in these derivations are omitted from the text. Details of the more complex derivations, as well as the calculations resulting in the estimations of Table VI, can be obtained under Document 8577, from Chief, Photoduplication Ser- vice, Library of Congress, Washington 25, D. C., upon remittance of $2.00 for microfilm (35 mm) or $3.75 for photoprints.


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TABLE VI Carbon balance and possible contribution by pentose cycle of hydrogen atoms for fatty acid synthesis*

Percentage of the carbons of utilized glucose metabolized to

Growth hormone

Absent

Present

co2 Utilized glucose

Fatty acids Glycerol Lactate Pentose cycle Pyruvate

decarboxylation Krebs cycle

27.8 29.3 27.1 6.3 10.0 20.4 6.8 (17.4-33.6) ; (13.3-37.9) (17.7-35.8) (1.6-16.4) (9.2-10.8) (14.5-24.4) (5.1.-7.3)

69.9 I 16.8 43.1 5.3 4.0 16.5 14.4 (33.6-90.0) / (5.9-25.9) (39.0-52.6) (2.8-11.1) (0.9-7.0) (13.0-18.3) (7.5-19.6)

Percentage of hydrogen atoms

required for fatty acid synthesis available from

the pentose cycle

(58?79 )

(35Zl)

* Averages with the ranges in parentheses are recorded for the four experiments of Table I. Utilization is in microatoms of carbon per g of tfissue per 3 hours of incubation. The absolute quantities of the carbons of the utilized glucose converted to each product have been calculated.

the distribution observed in Experiment 4 (dagger footnote of Table II). The rates of triose-P isomerization relative to glucose utilization were less with hormone than without (relative rates averaging 1.4 compared to 2.5). However, as previously noted (6), increased utilization with growth hormone makes the absolute rates similar. The specific activity of glyceraldehyde-3-P is calculated to be lf times the specific activity of dihydroxyacetone-P relative to that of glucose-6-i4C with growth hormone present (averaged 1.18 compared to 0.84). With glucose-l-14C, the activity of dihydroxyacetone-P is higher than that of glyceraldehyde-3-P (averaged 1.00 compared to 0.72). The differences in specific activity are less when growth hormone is absent (re- spective values 1.17 compared to 1.00 and 0.74 compared to 0.60). These estimates require that the triose phosphates once formed do not recombine to hexose-6-P in significant quantity. Evi- dence that this latter requirement is fulfilled for adipose tissue in the absence of hormone addition has been detailed (7). The failure of i4C from glucose-6-i% to appear in carbon 1 of glucose from glyrogen (Table IV) is evidence for fulfillment in the presence of growth hormone. The incorporation of i4C of glucose- 1.i*C into carbon 6 of glucose from glycogen can be attributed to the transaldolase reactions (7, 20).

With these calculations, the absolute quantities of the carbon of the glucose ut,ilized which were converted to fatty acids, glycerol and lactate carbon, and COS, during the incubations have been estimated (Table VI) (6).2 Similar quantities of carbon were converted to fatty acids in the presence of growth hormone, as in its absence, despite the greater utilization of glucose in the presence of hormone. Growt,h hormone increased glycerol carbon formation relative to glucose utilization (43.17, of glucose carbon utilized compared to 27.1%), while the quantity of glucose carbon to lactate was a similar percentage of the glucose carbon utilized. The quantity of CO% formed in the pentose cycle was similar with or wit,hout hormone although the percentage of the glucose carbon converted to COZ was less with growth hormone (4% compared to 10%). CO2 formation by decarboxylation of pyruvate paralleled glucose utilization, but with growth hormone a greater percentage of glucose carbon utilized was oxidized to CO2 in the Krebs cycle. In the estimates of COz formed in the Krebs cycle, the specific yield of 14C02 from glucose-6-14C has been adjusted for the quantity of 14C02 formed during the decarboxylation of pyruvate. This has been done by

assuming the distribution of i4C in pyruvate to be the same as in lactate (Table III) so bhat approximately one-thirteenth of the 14C of glyceraldehyde-3-P is assumed to have been randomized to the carboxyl carbon of pyruvate. The quantity of TPNH formed via the pentose cycle could provide only one-third to two-thirds the hydrogen required for reduction of acetyl-CoA to the two-carbon unit of fatty acids. A similar result has been obtained under different conditions and with other hormones (6). Growth hormone also decreased the pentose cycle contribution in the experiments with fasted-refed rats (6). The percentage of carbon converted to lactate is less in the present than these previously reported experiments (6).

Other Evidence for Incomplete Equilibration of Triose Phosphates and Small Glucuronic Acid Pathway

Contribution in Presence of Growth Hormone

The isotopic nonequilibration of triose-P in the presence of growth hormone, shown in these experiments, is in accord with the observations of Winegrad et al. (1). Thus, while incorporation in glycerol was not measured in their experiments, about twice as much 1% from glucose-6-14C as -1X! was incorporated into fatty acids (Table VII of Reference 1). Since the 14C in their petroleum ether-extractable material was probably almost completely in triglyceride, the equal incorporation of carbon atoms 1 and 6 into this fraction would support a greater incorporation of carbon 1 relative to carbon 6 into glycerol. The measurements of glucose uptake and incorporation of carbon atoms 1 and 6 of glucose into COZ, glyceride-glycerol, fatty acids, and glycogen are in good agreement with those of LeBoeuf and Cahill (2) done under similar conditions. Their conclusions that growth hormone preparations decreased the pentose cycle contribution and increased the Embden-Meyerhof contribution to glucose metabolism and oxidation in the Krebs cycle were based on a calculation in which recycling was not considered.

Other data of Winegrad et al. (3), while not accompanied by measurements of glucose and glucuronic acid utilization and probably dependent on the concentrations of substrate selected, suggest that in the absence of added hormone the glucuronic acid pathway makes a relatively small contribution, at most, to over-all glucose metabolism in adipose tissue from fed rats. Thus, their yield of 14C02 on incubation with glucuronic acid- 6-14C was only 0.075 pmole per mg of tissue nitrogen while with


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Issue of February 10, 1966 Landau, Bar&h, and Williams

glucose&% under similar conditions it was 0.24 pmole per mg of tissue nitrogen. Every molecule of glucose-6-14C metabolized via the glucuronic acid pathway yields a molecule of 14C02. All of carbon 6 of glucose metabolized via the Embden-Meyerhof pathway and pentose cycle appears in triose-P, but only a small fraction of carbon 3 of this triose-P is oxidized to COz (Table VI). Therefore, much more glucose-6-14C would have to be metabolized via the Embden-Meyerhof pathway and pentose cycle than glucuronic acid pathway to produce the same yields of ‘4CO2.

Other hormones showing lipolytic properties in adipose tissue also stimulate the oxidation of carbon 6 relative to carbon 1 and under these conditions, nonequilibration of triose-P occurs (2, 6, 24,26). Lynn, MacLeod, and Brown (5), in their balance studies with adrenocorticotropic hormone, with a phosphate buffer, concluded that the glucuronic acid pathway makes at most a negligible contribution to over-all glucose metabolism, since 90% or more of the 1°C utilized was converted to products other than intermediates of the pathway. They did not eliminate, as we now have done, metabolism occurring via the pathway to pentose phosphate and then subsequently via the pentose cycle.

Adequacy of Growth Hormone Preparations

The conditions of the present experiments were designed to approximate those of Winegrad et al. in their original studies (1). Large concentrations of the growth hormone preparations have been used. As stated by Winegrad and Shaw (8) the changes in metabolism observed on addition of the growth hormone preparations may be due to contaminants. Jungas and Ball (27) have considered thyrotropic hormone to be a possible contami- nant causing the effects seen with growth hormone preparations. In a single experiment, under conditions similar to those employed with the growth hormone preparations, the effect of thyrotropic hormone on the metabolism of glucose-l-i%, -2-‘4C, and -6-14C in adipose tissue were studied.3 A pattern of response similar to that seen with the growth hormone preparations was observed in that there was an increase in the oxidation of carbon 6 of glucose relative to carbon 1, an increase in the percentage of incorporation of 14C into glycerol, a decrease in the percentage of incorporation into fatty acids, and a decrease in the pentose cycle as measured by the randomization of carbon 2 of glucose-2.14C into glycogen. Thus, the effects observed with the growth hormone preparations may not be due to growth hormone.

APPENDIX

Expressions are derived for estimating the contribution of the glucuronic acid pathway to glucose metabolism in the intact cell with the use of a model in which non-triose-P-forming pathways, the pentose and Krebs cycles, and the Embden-Meyerhof pathway are also active.2 These expressions are applied to the data for adipose tissue just presented. Estimates from the extent of randomization of 14C from glucose-2-14C or -1-14C into glucose- 6-P or its derivative prove possible. These estimates are probably maximal since reversal of the pentose cycle is absent in the model. Reversal, i.e. the conversion of glucose-6-P to pentose- 5-P via fructose-6-P, can produce the incorporation of r4C into carbon atoms 1, 2, and 3 of glucose-6-P (23, 24) which is attributed by the model to the glucuronic acid pathway. Esti-

3B. R. Landau and J. Katz, unpublished observations.

fructose-6-P

glyceroldehyde-3-P

co2 lactate fotty acids other products

FIG. 1. Model of glucose metabolism. The model is identical with that presented in an accompanying paper (20) except that rate V4 is introduced. This represents the rate of conversion of glucose-6-P carbon atoms to CO, and pentose-5-P via the glucuronic acid pathway. As in the pentose cycle, $ of the carbon atoms entering the pathway are oxidized to COZ. The remainder are metabolized to glyceraldehyde-3-P and converted to fructose- 6-P. The fraction of hexose metabolized via the pathway is then Q VdVo.

mates with glucose-5-14C depend upon the relative rate of the transaldolase exchange reaction (20). To estimate this rate, data with glucose-2-l4C and glucose-l-14C and -6-l4C are required. With adipose tissue, where randomization of UC is not marked, particularly from glucose-l-14C and -5-i%, and where incorporation into carbon atom 3 of glucose-6-P of small quantities of 14C from glucose-2-14C is a determinant, experimental error severely limits the precision of the estimates, but not the confidence to be placed on their magnitude.

The model (Fig. 1) depicting the pathways of glucose metabolism is identical with that of an accompanying paper (20) except that provision is made for the contribution of the glucuronic acid pathway to over-all nletabolisnl.4 Glucose-6-P enters this pathway at the rate V4 atoms of carbon per unit of time. + V4 is oxidized to CO2 and the remainder is converted to pentose-5-P via pentose. Further metabolism is via the pentose cycle with the return of 8 V4 carbon atoms per unit of time as hexose-6-P and with the conversion of the remaining carbon atoms at the rate % V4 to glyceraldehyde-3-P. The fraction of glucose metabolized in the glucuronic acid pathway, designated by GA, is then + V4/V0.

The sizes of the pools of glucose-6-P, fructose-6-P, dihydroxyacetone-P, and glyceraldehyde-3-P in atoms of carbon are designated by M,, Mf, Md, and M,,, respectively. The sizes of these

4 In the model the glucuronic acid pathway is depicted as pro- ceeding from glucose 6-phosphate (10). I f glucose is converted to glucuronic acid without glucose 6-phosphate as an intermediate, the glucuronic acid pathway contribution to over-all glucose utilization would be overestimated.


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758 Glucuronic Acid Pathway in Adipose Tissue Vol. 241, n-o. 3

(‘our pools are assumed to remain unchanged and hence the following relationships hold.

vo + -t-a = va + VI + 173 + v*

va + Q (VI + V4) = V-h + VP

a VP + vt + t (VI + V4) = v-t + 8,

The specific activities of glucoseB-P, fructose-B-P, dihydroxyacetone-P, and glyceraldehyde-3-P are denoted by z, y, U, and z, respectively, and those of the individual carbon atoms of these molecules are indicated by adding a subscript corresponding to the number of the carbon atom. For example, x6 denotes the specific activity of carbon 6 of glucose-B-P, and z2 denotes that of carbon 2 of glyceraldehyde-3-P. The specific activity of glucose as substrate is designated by G with a subscript corresponding to the position of the carbon bearing the label.

Gkose-WY? as Substrate-Equations 1 to 18 express the c:hanges in **C in each of the four pools.

dM,xl ~ = V&Y1 - (VI + v3 + viq + Vd 21 dt

dM,x, -- = V,,Gp + V-hyz - (VI + V3 + V4 + Vn) x2

dt

- = V&Y, - (VI + v3 + v4 + v/J x3 c!t

dM,x4 -- = dt

vq”y* - (VI + 113 + v4 + Va) x4

dM,X j ~ = v-i_hys - (V* + v3 + 1’4 + Va) x5

dt

dM,xs __ = V--hyG - (VI + v-3 + v4 + Va) X6

dt

dMjys ~ = v*x2 + $ VI23 + f V&2 - (V2 + v-dY2

dl

__ = V)&XS + + VI (x2 + 23) + + v4 (Xl + x2) dt

- (V, + V-h) Y3

dMfy4 ~ = vax* + 5 VI (x4 + 21) + + V4 (23 + 4 dt

+ 2 VT.21 - (VP + v-a + 2 VT) Y4

__ = VhXS + g VI (x5 + 22) + f v4 (x4 + 4 dt

+ 2 lip22 - (Vz + V-a + 2 VT) Y5

dMfvo

dt - Vf&XO + 5 VI (X6 + 23) + f v4 (25 + 23)

+ 2 VT.23 - (V, + v-a + 2 VT) Y/6

dhfm __ =

dt + vzys + V-tz1 - (Vi + V,z) u1

dMduz ~ = dt

4 vzy, + V-t%? - (Vt + V,z) u2

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

~ = 4 v2y, + V-&t - (Vt + Voz) UP dt

(15)

dM,aa __ = + v2y* + + VIZ4 + f v4x3 + VTY4 + v&h

dt

- (i VI + 6 v* + VT + V-t + V,) 21

(16)

- = : vzys + 5 VlX5 + a VlX4 + VTY5 + VI% dt

- ($ VI + B v4 + VT + V-t + VP) 22

(17)

dM,&3

dt - g v,ys + g VI26 + + v4x5 + VTYG + VIU3

(18)

- (B VI + B 84 + VT + V-6 + VP) 23

For isotopic steady state conditions t’hese equations are equated to 0. Eliminating y1 from Equations 1 and 7 and replacing Vh by (V, + 17Ch - VI - Tr3 - V4) and VZ by (V, - 5 VI - s v4 - V3)

Xl 2/3V1 -= (19) X2 V. + 2/3Vl + (v, - 1/3v1 - 1/3v4 - v~)vcd’-~

Denoting PC = + VI/V,, G1 = 4 V~/VO, NTP = VJVo, and ,lph = Vph/Vo, the expression for XL/X* becomes

Xl 2PC - (20) X2 1 + 2 PC + (l-PC-GA-NTP)/EL,

When equilibration of the hexose A-phosphates is complete (E-h-’ 00 ) , Equation 20 simplifies to

Xl 2 PC -=~ 1+2pc X2

(21)

This is t,he result previously derived for a model in which no provision was made for the presence of the glucuronic acid pathway (11). Eliminating y1 from Equations 1 and 7 and y3 from Equa- tions 3 and 9, the quot,ient of the two resulting equations yields, following rearrangement of terms, an expression for the relative contribution of the glucuronic acid pathway to that of the pentose cycle for any value of K-h (E-h # 0), NTP (NTP # I), PC (PC # 0), and GA (GA # 1).

GA 2X3/21 - 1 - x,/x, -= PC 1 -I- Xl/X, - 2x3/22

(22)

I f the equilibration of the hexose 6-phosphates is assumed to be complete or nearly so, Equation 21 yields an estimate of PC and its substitution in Equation 22 gives an estimate for GA.

Following algebraic manipulations with Equations 1 and 3 to 18, an expression for estimating E--h for the case of GA # 0 is obtained.

E-h + 1 =

(x,/x, - x3/x*) x3,x1 _ x,,x, + (X5/X6 - x*/x5) (X4/% - x3/51)

(23) (1 - x,/x,)

X4/26 - x3/x5 1 Equations 20, 22, and 23 allow estimates of PC, GA, and E--h. The estimates are independent of the relative rate of the transaldolase exchange reaction, Ex 1V, where ExT = VT/Vo, and of V,i/Vt. qf the three equations, only Equation 20 is a function of NTP. The results of their application to the data of Ta- ble II, assuming NTP = 0, are given in the first three columns


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of Table VII. I f E-h is assumed to approach m, rather than TABLE VII

being finite, the remaining values in the table are changed little Estimates using glucose-2-V except in Experiment 4 where E-lL is negative. With E-h -+ m E-h, PC, and GA are calculated from Equations 20, 22, and 23 for this experiment, PC = 0.066 and GA = 0.028. with the data of Table II. The upper bounds on the estimates of

After algebraic manipulations of Equations 1, 6, 12, 15, and 18, V,l/V, and Exr are obtained from Equations 35 and 24 with the an upper bound on the estimate of E’x* is individual specific yields of fatty acids (from Table V). The

values for each experiment as well as the average value are re- 1 + GA

ExT < (l-PC-GA-NTP)xJz6 _ (2-Pc + 2GA) corded.

1 + V,zlVt (24) Experiment E-h PC GA VdVt JQ!r

- l/2 (PC + GA)

The inequality given by Equation 24 is valid only if the denominator of the first term is positive. In its use to obtain an upper bound on Ex, an estimate of V,/Vt or its upper bound must be available. The estimates of GA and PC can be those with Equa- tions 20, 22, and 23. Expressions for estimating the upper bounds on Vg~/Vt with the ratio of specific yields of 14C in a

1 6.6 0.063 0.020 0.81 0.49 2 2.7 0.089 0.009 1.23 0.25 3 6.8 0.103 0.020 1.09 0.42 4 -0.6 -0.056 -0.024 0.14 1.02

Average 3.9 0.050 0.006 0.82 0.54

GA = x3 + 1/2x2

(29) glyceraldehyde-3-P derivative, as fatty acids or lactate, from

Xl + x2 - 2x3

glucose-1-‘4C and -6-14C are presented next. Since the estimate Under the assumption of complete equilibration of the hesose of VgL/Vt is an upper bound, its substitution in Equation 24 B-phosphates, the application of Equation 29 to the results of accentuates the inequality for the upper bound on the estimate Experiment 5t of Table IV yields GA = 0.035 and to those of of Exr. The results of the application of Equation 24 to the Experiment 55 GA = 0.036. data of Tables II and V, assuming NTP = 0, are presented in hgain following algebraic manipulations of the modified Equa- the last two columns of Table VII. tion 2 and Equations 3 to 18, an expression for estimating E-h

Glucose-l-X’ and -6X7 as Substrates-VOG1 is added to Equa- when GA # 0 can be obtained and is tion 1 and VoGz is deleted from Equation 2. These two modified equations and Equations 3 to 18 represent the changes of r4C in (9 _ 5) each of the four pools when glucose-lJ4C is substrate. Equa- tions similar to Equations 19 to 23 can be derived.

Eliminating y2 from the modified equations, 2 and 8, and replacing Vh by (VO + V-h - VI - V3 - V4) and Vz by (V, - 5 Vl - 5 v4 - V,)

The four equations denoting the changes of K! in each of the four pools when glucose-6-14C is substrate are given by Equations

X2 2/3V1 (25)

31 to 34. -= X3 T'o + 2/3V1+ (Vo - 1/3V1 - 1/3V4 - Va)Vo/V.a dM,x 6 __ = VoG6 + V-Q/C - (Vh + VI + Vs + Va) 26

Denoting PC = 4 VI/Vo, GA = f Vb/Vo, NTP = Va/V,, and lit (31)

&, = V-h/VO, the expression for xJx3 becomes dMfY6 - = VhX6 + f v1 (X6 + 23) + B VA7 -t 2 VTZQ X2 2 PC dt (32) -= (26) - (V-h + v2 + 2 VT) Y6 23 1 + 2PC + (l-PC-GA-NTP)/E-h

dMdua When equilibration of the hexose 6-phosphates is complete - = v-&z3 - (Vi + Vod ut

dt (33)

(E-h--+ 00 ). Equation 26 reduces to dM,&

X2 2 PC - = vzys + t VlX6 + 2 VTY6 + 2 VI% - -=-

1+2pc (27) dt (34)

X3 (5 Vl + f v4 + 2 V-t + 2 VP + 2 VI-1 23

Eliminating y2 from the modified equations, 2 and 8, and y3 from An expression for estimating the upper bound on V,l/V, in terms Equations 3 and 9, the quotient of t.he two resulting equations of GA, NTP, and y can be derived, where y denotes the ratio of

yields, following rearrangement of terms, an expression for the the specific yields of 14C in a glyceraldehyde-3-P derivative with

relative contribution of the glucuronic acid pathway to that of glucose-l-i4C and -6-14C (6). From Equations 31 to 34? an ex- the pentose cycle for any value of E-h (E-h # 0), NTP (NTP # pression for the specific yield with glucose-6J4C is obtained and

I), PC (PC z 0), and GA (GA # 1). from modified Equations 1 and 2 and Equations 3 to 18, an ex-

2x2 - x2(x* + x3) pression for glucose-l-14C. Then an upper bound for the case in

GA -= which the denominator of the right side is positive is PC x2(x1 + x2) - 22223

(28)

1 + VgflVt I 1

I f the equilibration of the hexose B-phosphates is assumed to be ?(l - 3 GA-NTP) Gs/Gl - GA (35)

complete or nearly so, Equation 27 yields an estimate of PC and its substitution in Equation 28 yields Application of Equation 35 has been presented in Table VII

759 Issue of February 10, 1966 Landau, Bar&h, and Williams


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with the ratio, y, of the specific yields of fatty acids given in 4, respectively. I f E-h is assumed to approach m the respec- Table V. The estimates of V,l/Vt are similar with the specific tive values for GA are unchanged. The contribution of glucuro- yields of lactates. Although the estimate of V,l/V( may have nit acid pathway to total glucose metabolism is therefore esti- little intrinsic interest, it is required to obtain the bounds on Exr. mated at no greater than 1.7%. If x6/x5, as well as U&Z, had The latter is required for estimating the glucuronic acid pathway been determined, a lower estimate of the upper bound on GA contribution from data obtained when using glucose-5-i4C. could be estimat,ed with Equation 44.

Glucose-5-14C as Xubstrate-Equations 36 to 43 represent the changes in 1% in each of the four pools. Acknowledgments-The approaches to this study were de-

Cl&X5 veloped in conversations with Drs. Joseph Katz and Harland G.

~ = VoGs + v.-ays - (vh + 8, + va + v,) 56 dt

(36) Wood. Dr. Wood suggested the use of glucose-5-14C. Their assistance is gratefully acknowledged.

dM,xs ~ = V-nys - (Vh + Vl + vs + V,) x.5 (37) REFERENCES

czt

dMfY5 ~ = (V, + f Vl)XS + 8 (VI + V&z + 2 VT.22

dt - (V.---h + VB + 2 VT)YS

dMrys ~ = (VA + B V1)X6 + 5 v4x5 + f (Vl + V&

dt + 2 VT& - (V-h + vz + 2 VT)YS

dMduz __ = La - (V, f V,z)u,

dt

(38)

(39)

(40)

1. WINEGRAD, A. I., SHAW, W. N., LUKENS, F. D. W., STADIE, W. C., AND RENOLD, A. E., J. Biol. Chem., 234, 1922 (1959).

2. LEBOEUF, B., AND CAHILL, G. F., JR., J. Biol. Chem., 236, 41 (1961).

3. W;NE&AD, A. I., SHAW, W. N., LUKENS, F. D. W., AND STADIE, W. C.. Am. J. Clin. Nub.. 8. 651 (1960).

4. CAHILL,‘G. F., JR., LEBOEU~, B., ANI) R&TOLD, A. E., J. Biol. Chem., 234, 2540 (1959).

5. LYNN, W. S., MACLEOD, R. M., AND BROWN, R. H., J. Biol. Chem., 235, 1904 (1960).

6. KATZ, J., LANDAU, B. R., AND BARTSCH, G. E., J. Biol. Chem., 241, 727 (1966).

dMciua ~ = V--t23 - (Vt + V~Z)UB

dt

dM,,zz - f VIZ5 + (3 v2 + VT)YS + Vtuz

dt - t (Vl + V&z - (V-t + v, + VT)22

dMgaa ~ = f vlx6 + 5 1/4x5 + (a VZ + VT)yI + vtu3

dt - 2 (VI + V&3 -- (V-t + v, + VT)28

7. LANDAU, B. R., AND KATZ, J., J. Biol. Chem., 239, 697 (1964).

(41) 8. WINEGRAD, A. I., AND SHAW, W. N., Am. J. Physiol., 206, 165

(1964). 9. KATZ, J., AND WOOD, H. G., J. Biol. Chem., 235,2165 (1960).

10. HORECKER, B. L., AND HIATT, H. H., New Engl. J. Med., 258,

(42) 225 (1958). 11. LANDAU, B. R., BARTSCH, G. E., KATZ, J., AND WOOD, H. G.,

J. Biol. Chem., 239, 686 (1964). 12. MERLEVEDE, W., WEAVER, G., AND LANDAU, B. R., J. Clin.

(43) Invest., 43, 1160 (1963).

13. LJUNGDAHL, L., WOOD, H. G., RACKER, E., AND COURI, I)., J. Biol. Chem., 236, 1622 (1961).

For isotopic steady state conditions, these 8 equations are set 14. DAMM, H. C., DOMINGUEZ, J. M., PENSKY, J., AND PEARSON,

equal to 0. Equation 42 can be expressed in terms of x5, .r6, and 0. H., Endocrinology, 74, 366 (1964).

15. WYNGAARDEN, J. B., SEGAL, S., AND FOLEY, J. B., J. Clin. z2 by solving Equation 38 for y5, Equation 40 for uz and sub- Invest., 36, 1395 (1957). stituting these expressions in Equation 42. Similarly, Equation 16. FISCHER, F. G., AND D~RFEL, H., 2. Physiol. Chem., 301, 224

43 can be expressed in terms of 25, ~6, and z3 by solving Equation (1955).

39 for y6, Equation 41 for uI and substituting these expressions in 17. PARTRIDGE, S. M., Biochem. J., 42, 238 (1948).

Equation 43. Denoting GA = + V4/V0, PC = f V1/VO, and 18. WERBIN, H., CHAIKOFF, I. L., AND IMADA, M. R., Proc. Sac.

Exptl.. Bioj. Med., 102, 8 (1959). ExT = VT/V0 and noting that PC 5 1, the inequality obtained 19. KORNBLATT. J. A.. BERNATH. P.. AND KATZ. J.. Intern. J. Appl.

’ ’ _ _

from these expressions follows Radiation’lsotopes, 15, 19i (1964). 20. LANDAU, B. R., AXD BARTSCH, G. E., J. Biol. Chem., 241, 741

GA< u3 ( )

- 5 (1 + EXT) (44 (1966).

u2 x5 21. STJERNHOLM, R., AND WOOD, H. G., Arch. Biochem. Biophys.,

78, 28 (1958).

Hence, if the ratio of activity in C-3:C-2 in dihydroxyacetone 22. CHRISTOPHE, J., JEANRENAUD, B., MAYER, J., AND RENOLD,

phosphate or its derivative, glycerol, is known, an estimate can A. E., J. Biol. Chem., 236, 648 (1961).

23. WOOD, H. G., KATZ, J., AND LANDAU, B. R., Biochem. Z., 338, be made of the upper bound on the contribution of the glucuronic 809 (1963).

acid pathway. No assumptions of the contribution of the pen- 24. KATZ, J., in H. SCHWIEGK AND F. TURBA (Editors), Radioac-

tose cycle and of the non-triose-P pathways and the rates of tive’ isotopes in physiology, diagnostics and therapy, Vol. I,

isomerizations of the hexose B-phosphates and of the triose phos- Springer-Verlag, Berlin, 1961, p. 705.

25. BERNSTEIN, I. A., LENTZ, K., MIGNON, M., SCHAMBYE, P.. phates are required. AND WOOD, H. G., J. Biol. &em., 215,.137 (1955).

When the inequality given by Equation 44 is applied to the 26. LANDAU. B. R.. AXD KATZ. J.. in A. E. RENOLD AND G. F.

data obtained with glucose-5-]4C! (Table II with E-h and ExT as CAHILL, JR. (Editors), kanhbook of physiology, Section 5,

given in Table VII), GA is found to be no greater than 0.040, American Physiological Society, Washington, D. C., 1965,

0.010, and 0.000 (average 0.017) for Experiments 1, 2, and p. 253.

._ 27. JUNGAS, R. L., AND BALL, E. G., Endocrinology, 71, 68 (1962).



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Bernard R. Landau, Glenn E. Bartsch and Hollis R. WilliamsMetabolism in Adipose Tissue and the Effect of Growth Hormone

Estimation of the Glucuronic Acid Pathway Contribution to Glucose

1966, 241:750-760.J. Biol. Chem.

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Estimation of the Glucuronic Acid Pathway Contribution to Glucose