role of glucose-1-phosphate and glucose-6-phosphate in glycogen synthesis by pigeon liver homogenate

ROLE OF GLUCOSE-l-PHOSPHATE AND GLUCOSE-6-PHOSPHATE IN GLYCOGEN

SY~HESIS BY PIGEON LIVER HOMOGENATE*

ENRIQ~JE FIGUERCIA, IRIS PEIRANO. MARGARITA VEGA AND PATRICIO VEGA

Departamento de Bioquimica, Facultad de Medicina, Universidad de Chile, Sede Norte, Casilla 6671, Santiago 4, Chile

Absbact-I. Glucose-I-C”, G-6-P-l-C’4 and G-1-P-l-C’4 have been incubated with a pigeon liver homogenate. Incorporation of C I4 into glycogen and CO2 were measured.

2. The relative incorporation of C l4 from G-6-P-l-C“’ and glucose-l-C?“ into glycogen and COz together with the relative incorporation of C l4 from G-I-P-I-C?“ and glucose-l-CL4 into glycogen and CO2 were calculated. From these results, it is postulated that G-6-P is not a necessary intermediate in glycogen biosynthesis from glucose; G-I-P would be the first intc~ediate and the metabolic cross that leads to glycogen and CO,.

3. It is suggested that G-l-P is formed directly from glucose through the reactions catalyzed by phosphoglucokinase (EC. 2.7.1.10) and phosphodismutase (E.C. 2.7.1.41).

4. This last hypothesis is supported by the fact that G-l-P and G-1,ddiP stimulated Cl4 incorporation from glucose-l-C’4 inta both CO2 and glycogen. Other minor facts obtained from this work and others from the literature help to sustain the postulations here discussed.

INTRODUCTION Several investigators have presented circumstantial evidence indicating that glucosed-phosphate (G-6-P) is not a necessary intermediate in glycogen synthesis from glucose or alter~tively the existence of more than one pool of G-6-P in the cell (Figueroa et al., 1962; Beloff-Chain ef al., 1964; Figueroa & Pfeifer, 1964; Threllfall. 1966; Landau & Sims, 1967; Antony et al., 1969).

According to the present knowledge of glycogen synthesis, G-6-P is an obligatory intermediate in glycogen synthesis and the reactions take place in one ~rn~r~ent that contains only one pool of G-6-P. Under these conditions, Sims and Landau (1966) pre- dict that glucose or G-6-P must, be incorporated in the same proportion into each of their common prod- ucts. Thus, the ratio of incorporation of Cl4 from G-6-P-Cl4 into glycogen to that from glucose-C’5 into glycogen must be equal to the ratio of incorporation of Cl4 from G-QP-Cl4 into COz to that from glucose-C I4 into CO,.

_c-6-P --+ Glycogen, __.. ..--_. G-6-P -+ co_, Glucose --+ Glycogen = %&%e 4 CO, ’

Sims and Landau (1966) failed to observe idential ratios for these fractions when glucose-C14 and

._..

* Supported by Grant No. 28 of the Oficina T&&a de Desarrollo Cientifico y Creacidn Artistica de la Univer- sidad de Chile.

G-6-P-C’4 were incubated with rat diaphragm. They found a value of 0.029 and 0.15 for the first and second ratio respectively. To explain these apparently conflicting results the authors consider two possihili- ties as the most probable: (a) G-6-P is not an obligatory intermediate in glycogen synthesis. and (b) there exists more than one pool of G-6-P inside the cell.

It is recognized that mammalian liver homogenates are essentially inactive in synthesizing glycogen from glucose, while pigeon liver homogenate will incorpor- ate labeled glucose into glycogen (Nigan & Fridland 1964; Zancan Bi Hers, 1965: Figueroa & Vega, 1968). For this reason most of data dealing with glycogm synthesis have been obtained from experiments done in entire-cell systems. Then, it was considered worthwhile to carry out experiments in pigeon liver homogenate in order to get the above mentioned ratios. They were found dissimilar and are described in this paper. furthermore, experiments that compare glucose-l -phosphate (G- 1 -P) and glucose, that were done in the same system are described. They allowed us to suggest that G-l-P is the first intermediate in an alternative pathway leading to glycogen synthesis bypassing G-6-P in agreement with the hypothesis of Smith et ul. (1967) that proposed a mechanism for G-l-P formation from glucose involving ATP : D - glucose - 1 - phosphate - 6 - phosphotransferase (E.C. 2.7.1.10) that will be referred in this paper as its trivial name phosphoglucokinase and D-glUCOSe- l-phosphate: u-glucose- 1 -phosphate-6-phosphotransferase (E.C. 2.7.1.41), trivial name phosphodismutase.

327

328 ENRIQUE FIGUEROA, 1~1s PEIRANO, MARGARITA VEGA AND PATRICIO VW~A

MATERIALS AND METHODS

Animals and chemicals

Male and female pigeons obtained in the market, weigh- ing between 250 and 350 g were used. Seven hundred milli- grams of glucose per 100 g of body weight in a 25% water solution were injected intraperitoneally 15 min before death in order to ger more reproducible results.

Glucose-l-Ci4, G-6-P-l-C“’ and G-1-P-i-C’4 were obtained from New England Nuclear, Boston, MA. Sub- strates were obtained from Sigma Chemical Co., St. Louis, MO. All other chemicals were of reagent grade purity obtained from several commercial sources.

Preparazjon and incubation of pigeon liver ho~ge~~e

The animals were killed by decapitation and allowed to bleed. Livers were quickly removed and kept in a beaker over cracked ice. A 50% homogenate was prepared in a Potter-Elvehjem homogenizer (glass Teflon) in a solution containing 0.15 M KC1 and 0.05 M glycylglycine_NaOH, pH 7.25. Incubation mixture with a total volume of 1.70 ml, contained 0.70 ml of pigeon liver homogenate; 58 /Imoles ofglycylglycine-NaOH buffer, pH 7.25; 173 pmoles of KCl; 34 pmoles each of glucose and G-6-P or 34 pmoles each of glucose and G-l-P. In this way, both glucose and one hexose-phosphate were in the medium. The flasks were identical in composition except that either glucose or the hexose-phosphate was labeled with Cl4 in carbon l(1 PCi). Three experiments were performed with a mixture of glucose, G-6-P and G-l-P. In this case all the flasks were also identical except that only one of the hexoses was labeled with C14. Any change in the composition of incubation mixture is indicated in the corresponding experiment.

Incubation flasks were ordinary Warburg vessels of 12 ml capacity to which 0.30 ml of 30% KOH and 0.20 ml of 40”/, HCIO, were added to the center well and to the lateral reservoir respectively. Flasks were gassed with pure oxygen during 4 min before incubation and then stoppered and incubated with shaking at 37°C for 5, 10 or 20 min. Every experimental condition was run in duplicate or trip- licate. Two flasks of each experimental condition was removed at the beginning and the other at the end of the incubation period. Incubation was terminated by pouring the content of the lateral reservoir to the incubation mixture.

Determination of rudioactiuity of CO2

After being left half an hour to allow for complete absorption of CO2 by KOH of the center well, the flasks were removed from the incubation bath. C02-Q4 trapped in KOH was transformed in BaC03-C14 to measure its radioactivity according to Figureroa and Pfeifer (1964).

Determination of radioactivity of glycogen

The W~burg flasks were exha~tively washed with 5% HC104 to transfer quantitatively the precipitated protein and incubation medium to a centrifuge tube. It was centrifuged down and the pellet washed two times with 5% HC104. The supematant fluids were joined and neutralized to phenolphtalein with KOH. It was left overnight in the cold room. The precipitated perchlorate was eliminated by ~n~i~~tion. Glycogen was precipi~ted with ethanol from an aliquot of the perchlorate supernatant after adding a small volume of 2% Na2S04. It was left 2.5 hr in the cold room and centrifuged down. The precipitated gly-

cogen was dissolved in 1 ml of water and precipitated again with ethanol. This washing was repeated twice. Finally, glycogen was dissolved in 1 ml of water and an aliquot was plated to count its radioactivity in a window- less gas flowcounter.

RESULTS

Comparison between glucose and G-6-P on CO2 and glycogen formation

We can see in Table 1 (columns 1 and 2) that about 2 or 3 times more Cl4 of G-6-P-1-C’4 than of glu- case-l-Cl4 were converted to CO,, except experiment 4 where the conversion of Cl4 from G-6-P-1-CS4 was 14.3 times that from glucose-l-C14. In contrast, the behavior of these substrates was different with respect to their incorporation into giycogen (columns 3 and 4). Thus, the relationship between incorporation of Cl4 from G-&P-l-Cl4 into glycogen to that from glucose- 1 -C? 4 into glycogen was different with respect to the same relationship for incorporation into CO,.

Comparison between glucose and G-l-P on CO, and glycogen formation

In other series of experiments glucose and G-I-P were compared. It was found that G-l-P has a different behavior than G-6-P. It can be seen in Table 2 that the relationship between incor~ration of Cl4 from G-l-P-1-C’4 into COz with respect of Cl4 from glucose-l-Cl4 incorporated into CO2 is similar to the relationship between Ci4 from G-l-P-1-C14 incorporated into glycogen and Cl4 from glucose-l-Cl4 incorporated, into glycogen. The similarity that was expected for the relationship considered between glucose and G-6-P has been obtained for the same relationship but referred to G-1-P. For both substrates used, G-6-P and G-l-P, the percentage of hydrolysis was about 15% calculated by measuring inorganic phosphate at the beginning and at the end of the

Table 1. Comparison of the conversion of CL4 from glucose-l-C’4 and G-6-P-1-C’4 into glycogen and CO, by

pigeon liver homogenate

GlUCOSe G-6-P GIUCOZ G-6-P

c& c& l0 t0

glycogen” glycogen” Ratios EXP (1) (2) (3) (4) 2, I 413

I 0.220 0.560 1.47 0.95 155 0.65 2 0.103 0.388 3.10 1.76 3.78 OS7 3 0.088 0.199 I.58 1.65 2.26 I 05 4 0.045 0.643 1.02 0.184 14.30 0.18 5 0.151 0.629 1.03 1.71 4.16 1.66 6 0.170 0.480 0.864 I.15 2.82 1.33 76 0.025 0.080 0.078 0.041 3.m 0.53

14” 0.030 0.085 0.085 0.052 2.83 0.62

“Figures indicate pmoles of hexose converted to glycogen and CO1 by the homogenate that corresponds to 350 mg of fresh pigeon liver incubated during 20 min.

’ Homogenate that corresponds to 50 mg of fresh pigeon liver was used in these experiments.

Glucophosp~tcs in glycogen synthesis 329

Table 2. Comparison of the conversion of Cl4 from glucose-14? and G-l-P-l-1C“’ into glycogen and CO2 by

pigeon liver homogenate

GlUCCX G-I-P GIWXe G-l-P to

dl;; to to Ratios

CO; glycoged glycogen”

EXP (1) (21 (3) (4) 2/l 4/3

I 0.220 0.568 I .47 4.08 2.59 2.78 5 0.151 0.542 I .03 4.71 3.60 4.56 6 0.170 0.446 0.864 3.02 2.63 3.50 8 0.528 0.595 2.51 4.06 1.12 1.62 9 0.680 0.520 3.20 3.06 0.77 0.96

10 0.11g 0.397 0.810 2.60 3.37 3.20 II& 0.060 0.110 0.400 0.82 I .R5 205 l2h 0.022 0.054 0.102 0.275 2.46 2.70 13b 0.038 0.08 1 0.515 I.11 2.13 2.15

a Figures indicate pmoles of hexose converted to glycogen and CO1 by the homogenate that corresponds to 350 mg of fresh pigeon liver (for experiments 1,5,6,8,9 and 10) incubated during 20 min.

*Homogenate that corresponds to 50 mg of fresh tissue was used in experiments 11, 12, and 13.

incubation period, (not indicated in the tables). In order to decrease the effect of destruction of the substrates an incubation time of 5 and 10 min (Table 3) or a smaller amount of tissue-homogenate were used (Table 1 and 2). With 5 min of incubation the percentage of hydrolysis lowered to 4”/, and by using 50 mg of tissue to 5%. The characteristics of the ratios under investigation were not changed by these modifications.

EfSects of G-l-P, G-6-P and glucose 1,6-diphosphate (G-1,6-dip) on glucose conversion into CO, and glp cagen

Some experiments were performed in order to investigate the effect of nonlabeled G-l-P, G-6-P and G-1,6-diP on glucose-l-Cl4 conversion into CO, and glycogen. In these experiments the incubation medium always contained glucose-l-C’4 and different concentrations of one of the nonlabeled hexosephosphates indicated above. G-1-P stimulates the incorporation of C i4 from glucose-l-C14 into CO, and

glycogen (Fig. 1). The same optimum concentration- about 6 mM-was observed for both effects. If concentration is increased, an inhibition of incorporation is obtained. Although the inhibition effect of G-l-P on glycogen synthesis is observed at 50 mM (because it was not tested between 20 and 50 mM), in other experiments we have observed an inhibition of 50% at 15 mM and 92% at 20 mM G-1-P concentration accompanied also with an activation effect of 82% at 5.0 mM concentration. G-1,ddiP stimulates also the incorporation of Cl4 from glucose-1-Cl4 into COz and glycogen. The optimum concentration of this ester is about 60 times smaller than that of G-l-P (Fig. 2).

Table 4 shows the effect of adding nonlabeled G-6-P to the incubation medium. It may he seen that this ester inhibits the incorporation of Cl4 from glucose-l-C’4 into CO* and does not modify the incorporation into glycogen.

c- u I / I / I! s 9 t J 11 IO 20 30 40 50

GLUCOSE-l- PHOSPHATE CONCENTRATION CmmM)

Fig. 1. Effect of G-1-P on Cl4 incorporation into glycogen and COa from glucose-l-C l4 by pigeon liver homogenate. Glucose concentration is IO mM. Other experimental conditions as described in Material and Methods, except that G-1-P con~ntrations varied as indicated. Cl4 incorporation is expressed as I.tmoles of glucose equiv/l# mg of

wet tissue.

Table 3. Effect of time on conversion of glucose-l-C“+, G-6-P-1-Ct4 and G-l-P-IX? into CO2 and glycogen

Glucose to G-6-P to G-I-P to R@iOS

Incubation co, Gly co2 Gly co2 Gly E~P txne (min) (1) (2) (3) (4) (5) (61 3/l 412 511 612

15 5 0.039 0.034 0.079 0.006 2.03 0.177 IO 0.084 0.080 0.176 O.OI5 2.09 0.188 20 0.176 0.173 0.378 0.034 2.15 0.197

I6 5 0.060 0.36 0.079 0.052 1.32 I.44 10 O.Il2 0.70 0.161 I.10 I.44 1.57 20 0.2lO 1.32 0.315 2.27 1.50 3.72

17 5 0.017 0.119 0.029 0.180 1.71 1.51 IO 0.031 - 0.055 - 1.77 -...

20 0.057 0.595 0.106 0.823 1.86 I .39

Figures indicate pmoles of hexose converted to glycogen and CO, by the homogenate that corresponds to 350 mg of fresh pigeon liver.

330 ENRIQUE FIGUEROA, IRIS PEIRANO, MARGARITA VEGA AND PATRICIO VEGA

GLUCOSE -1,6- DIPHOSPHATE CONCENTRATlON(mM)

Fig. 2. Effect of G-1,6-diP on Cl4 incorporation into glycogen and CO2 from glucose-l-C’4 by pigeon liver homogenate. Glucose concentration is 20 mM. Other experimental conditions are given in Material and Methods. Cl4 incorporation is expressed as pmoles of glucose equiv/lOO

mg of wet tissue.

EfSect of G-l-P on G-6-P conversion into CO, and glycogen

To investigate this effect the incubation medium had only G-6-P-1-C’4 and the indicated concentrations of nonlabeled G-l-P shown in Table 5. The effect of G-l-P on glucose conversion into CO, and glycogen was also investigated in these experiments in order to compare in .the same liver both effects: the conversion of G-6-P and glucose into CO, and glycogen. G-l-P has an inhibitory effect on incorporation of Cl4 from G-6-P-l-C’4 into CO2 and glycogen in contrast with the stimulatory effect observed on the incorporation of Cl4 from glucose-l-C14 into CO2 and glycogen.

DISCUSSION

Pigeon liver homogenate forms C02-C’4 from G-6-P-l-Cl4 to a greater extent than from glucose- 1-C14. This fact is consistently found. On the other hand it was found that the relative capacity of glu-

Table 4. Effect of G-6-P on the incorporation of Cl4 from glucose-l-CL4 into

COz and glycogen

_ 0.190 0.321 G-6-P (1 mM) 0.188 0.318 G-6-P (2.5 mM 0.171 0.340 G-6-P (7.5 mM) 0.082 0348

“Figures indicate pmoles of hexose converted to glycogen and CO2 by the homogenate that corresponds to 350 mg of fresh pigeon liver incubated during 20 min. The flasks contained radioactive glucose and nonlabeled G-6-P in the concentration indicated.

Table 5. Effect of G-l-P on the incorporation of Cl4 from glucose-l-C14 and G-6-P-l-CL4 into COz and glycogen

GIUCOX G-6-P GlWXe G-6-P to f0 to t0

CO, co, glycogen glycogen

Y Y I _ 0.142 0.385 0.395 0.412 G-I-P (2.5 mM) 0.393 0.314 0.440 0.360 G-I-P (7.5 mM) 0.810 0.289 0.580 0.214

"Figures indicate pmoles of hexose converted to glycogen and COz by the homogenate that corresponds to 350 mg of fresh pigeon liver incubated during 20 min. The values correspond to the mean of 3 experiments. The flasks contained the substances indicated in Materials and Methods plus nonlabeled G-l-P in the concentration indicated.

cose-l-C’4 and G-6-P-1-C’4 to form labeled glycogen varies from one experiment to another. In some cases glucose is more glycogenetic than G-6-P, in others, it is G-6-P, and in others both have the same glycogenetic capacity. As a consequence, the ratio of incorporation of C l4 from G-6-P-1-C14 to glycogen to that from glucose-l-C’4 to glycogen is always striking dissimilar to the ratio of incorporation of Cl4 from G-6-P-1-C’4 into CO2 to that from glucose-l-C14 into COZ. This finding agrees with data in rat liver slices (Figueroa & Pfeifer, 1964) and also to those obtained from rat diaphragm (Beloff-Chain et al., 1964; Sims & Landau, 1966) and from No&off ascites-hepatoma cells (Nigam, 1967).

Since these data do not agree with the present scheme of glycogen synthesis the authors have inter- preted these results supposing the existence of a pathway from glucose to glycogen in which G-6-P is not an intermediary, or alternatively the existence of two separate pools of G-6-P inside the cell. At present available data do not allow differentiation between these two possibilities. It is also not possible to estab- lish the existence of only one of them. The evidence suggests that either one or both may exist. Although the experiments reported in this work have been performed in a cell-free system, double compartmentation of G-6-P pool cannot be ruled out. In fact, a whole homogenate of liver is so complex in struc- ture that the compartmentation phenomena may not be discarded. Notwithstanding, we will work with the first hypothesis, i.e. a pathway from glucose to glycogen without G-6-P as intermediate.

No experimental data have been reported supporting another sequence of reactions that synthesizes glycogen from glucose. However, Smith et al. (1967) have hypothesized a possible pathway in which G-6-P may be bypassed by the direct formation of G-l-P from glucose. This pathway would be a shunt that works by the coupled action of two enzymes: phosphoglucokinase and phosphodismutase. These enzymes would catalyze a small metabolic cycle, dealing to the net synthesis of G-l-P (Fig. 3). Phosphogluco- kinase was found by Paladini et al. (1949) in muscle

Glucophosphates in glycogen synthesis 33r

and yeast. The reaction that catalyses is irreversible. Phosphodismutase was isolated for the first time by Leloir et al. (1949) from micro-organisms and has been studied also in rabbit muscle (Sidbury et al., 1956). The reaction that catalyzes is reversible. Ass~ng that the above postulated scheme is working in our pigeon liver system, all the controversial data presented in this paper may be understood.

The fact that the value of the ratios

G- 1 -P + glycogen G-l-P--+ CO ~._ . -.-.-.._ -_ -2 Glucose --+ glycogen ’ Glucose -+ CO, ’

be essentially equal-in contrast with the results obtained with G-(i-P-suggests that G-l-P could be the metabolic cross that initiates the pathway that leads to glycogen and to CO*. We have found in literature, data that allowed us to calculate these ratios. It is worthwhile to note that every data must be obtained from the same experiment. Beloff-Chat et al. (1964) and Pocchiari (1968) furnished us with the values of the figures to calculate the ratios that compare G-l-P and glucose with respect to glycogen and CO* production (Table 6). Interesting enough, they are equal, in contrast with the values obtained by them in the same experiment for G-6-F and glucose. The data are in agreement with the results reported in this paper.

G-6-P does not inhibit glycogen synthesis from glucose as it is shown in Table 4, a fact that had been reported by Figueroa et al. (1962) in rabbit liver and, under the same conditions that were employed in this work, by Nigam and Fridland (1967) in pigeon liver. On the other hand, G-l-P at low concentration is an activator of glycogen synthesis but inhibits it at higher concentration. These data agree with the assumption that G-l-P, but not G-6-P, is an intermediate in glycogen synthesis from glucose.

That the addition of nonlabeled G-l-P or G-1,6&P to the incubation medium stimulates glycogen and COZ production from glucose is also in accordance with the Smith Taylor and Whelan hypothesis. An examination of the reactions catalyzed by phosphoglucokinase and phosphodismutase shows

ATP A =A,,, G-L-P

11 G-6-P UDPG

1 1 CO" GLYCOGEN

Fig. 3. Proposed new pathway for glycogen synthesis from glucose without glucose-6-phosphate as an intermediate.

Table 6. Comparison of the conversion by rat diaphragm of glucose, G-l-P and G-6-P into CO2 and glycogen

0 h

Glucose - CO2 0.087 0.125 0) Glucose - Bbwen 0.496 0.236 121

G-I-P - co, 0.097 0.217 (3) G-I-P -+ &=wJ 0.453 0.411 (41 G-6-P - co, 0.118 0.291 (5) G-6-P - gIyCOgell 0.042 0.061 (61

Ratios

311 1.11 1.74

412 0.92 1.74

s/t 135 2.33

‘5/2 0.084 0.259

“Data obtained from Beloff-Chain et al. (1964). The figures indicate voles of hexose converted to the indicated product/50 mg fresh tissue after 90 min of incubation in 0.6 ml of phosphate-buffered medium, pH 6.8, at 37°C in Or. The hexose concentration was 1% expressed as glucose.

* Rata obtained from Pocchiari (1968). Figures indicate pmoles of hexose converted to the indicated product/50 mg of wet tissue after 90 min of incubation in 0.6 ml of phosphate medium, pH 7.4, at 37°C in 0,. Concentration of hexose was 1%.

that the overall effect of the system, i.e. G-l-P production, has to be stimulated by the addition of G-l-P or G-l$diP, if the system is not working at its maxi- mum capacity .due to unsaturation of the enzymes, especially, if we consider that G-l-P is being taken up for glycogen synthesis or for CO, production. G-6-P does not stimulate C0,-C14 or glycogen-C14 formation from glucose-l-C’4 supporting that the activating effect of G-l-P and G-l&dip is rather specific (Table 4). A very ~po~nt fact is that G-l-P activates CO* and glycogen production only from glucose and not from G-6-P as it is shown in Table 5. According to this effect the site of action of G-l-P would be in a step that is placed before the metabolic cross that diverges to glycogen and to COZ. However, it seems that hexokinases are not the point of attack, since these enzymes, isolated from pigeon liver are not acted upon by G-1-P (Ureta & Slebe, 1970). Not- withstanding, if G-l-P activates the kinasedismutase cycle, as it was postulated above, these facts fit adequately.

When a large quantity of tissue was used, 15% G-6-P added to the medium was transforms in glucose and Pi by the action of glucose-6-phosphata~. G-l-P undergoes the same process of hydrolysis pre- vious action of phosphoglucomutase, that is then act- ing in our system. As the production of glucose from G-6-P must change the glucose concentration in the medium and alter also its specific activity, the conversion of counts/min into pmoles would be subject to error. To minimize the percentage of hydrolysis of hexosephosphates two modifications were performed: time of incubation of shorter duration and decrease in the quantity of tissue homogenate incubated. With 5 min of incubation the hydrolysis of hexose-phosphate was only 4%. With 50 mg of tissue homogenate

332 ENRIQUE FIGUEROA, IRIS PEIRANO, MARGARITA VEGA AND PATRICIO VEGA

the substrate hydrolysis was also about 5%. Then, although the hydrolysis of the substrate is a factor involved that may alter the results of our experiments, we conclude that it is not significant since the values of the parameters under investigation were apparently not affected by these variables.

It may be considered that glucose-Cl4 incorporation into glycogen by liver homogenates is the consequence of an exchange phenomenon between glucose-Cl4 from the medium and glucose from glycogen, a reaction that is catalyzed by amylo-1,6-glu- cosidase. That this is not the case for glucose-Cl4 incorporation into glycogen by pigeon liver homogenate under our conditions was shown by Figueroa and Vega (1968). After incubation of glucose-C’4 with pigeon liver homogenate they isolated glycogen of the homogenate and treated it with /I-amylase. They found that radioactivity was localized entirely in the external branches of glycogen that were destroyed by /I-amylase, ruling out the hypothesis that glucose incorporation into glycogen is due to a mere exchange phenomenon, in this system.

Some paradoxical effects that have been observed with respect to glycogen metabolism by beryllium administration to rats may be explained by the hypothesis pointed out here. Beryllium inhibits strongly phosphoglucomutase. A 50% inhibition is obtained at 5 PM beryllium concentratien (Thomas & Aldridge, 1966). As is expected, beryllium decreases hepatic flycogen losses induced by fasting but if glucose-Cl is injected intravenously at the end of fasting period, beryllium promotes an increase of glucose-C’4 incorporation into glycogen (Aldridge, 1966). Consi- dering that phosphoglucomutase is on the up and down pathway, it is difficult to understand these find- ings. Nevertheless, with the Smith, Taylor and Whe- lan cycle working in the liver, phosphoglucomutase intervenes in glycogenolysis but not in glycogen synthesis. Thus, the effect of beryllium on glycogen synthesis is secondary to glycogen breakdown decrease caused by phosphoglucomutase inhibition.

Two cases of glycogenesis with an increase of glycogen and a marked deficiency in phosphoglucomutase have been reported. One of them had 16% of glycogen in liver (Brown & Brown, 1968) and the other had 3.7,7.0 and 11.3% of glycogen in 3 different skeletal muscles (Thomson et al., 1963). Phosphoglu- comutase was investigated in liver and muscle respectively. An incapacity to glycolize G-l-P and glycogen was found in both cases. Brown and Brown (1968) tried to conciliate these apparent paradoxical facts, i.e. elevated glycogen and low phosphoglucomutase. They invoked compartmentation phenomena but they were conscious that those assumptions could not be established on the basis of present knowledge at that time. If phosphoglucomutase participates in glycogen degradation and the pathway described here in glycogen synthesis, the paradoxical facts dissolve away.

Antony et al. (1969) obtained evidence in rat liver

slices that G-6-P is involved in the conversion of glucose into glycogen, but not in rat diaphragm. The authors incubated liver slices or diaphragm with pyruvate-l-Cl4 and glucose-6-Cl4 in the same llask. Glu- cose obtained from glycogen and from G-6-P were isolated and the radioactivity in their individual carbons was determined. Pyruvate should label carbons 3 and 4 of both glucose from glycogen and from G-6-P. Glucose should label carbon 6 of both glucose from glycogen and from G-6-P. Then, if G-6-P is an intermediate both from pyruvate to glycogen and from glucose to glycogen, it must be found that the ratio of radioactivity in C-6 to that in C-3 and C4 of glucose from glycogen should be identical with that in the same carbons from G-6-P. This was the case for liver but not for diaphragm. To test the method, galactose-1-C14, a substrate that has not G-6-P as an intermediate and incorporates into glycogen via UDP-glucose was incubated with pyruvate-l-C’4 and rat liver slices. Isolation and analysis of glucose obtained from glycogen and from G-6-P showed that in relation to the activity in carbon 4, there was greater incorporation of galactose carbon measured by incorporation into carbon 1 into glucose of glycogen, than into glucose of G-6-P. This fact means that the conditions utilized by Antony et al. (1969) may be used to detect a pathway from glucose to glycogen without G-6-P as an intermediate. Notwith- standing, the authors accept that the method has a limitation. If there were a rapid equilibrium between G-6-P and an intermediate in the supposed pathway from glucose to glycogen the different distribution of radioactivity between carbon 6 and 4 from glycogen with respect to the same carbon from G-6-P would not be detected. Presumably, this condition does not exist in the case of galactose since its intermediates in glycogen synthesis pathway: galactose-l-phosphate, UDP-galactose and UDP-glucose, do not equilibrate easily with G-6-P. However, in the scheme proposed by Smith et al. (1967) and supported by our experiments, G-l-P is an intermediate and this metabolite would be able to undergo a rapid equilibrium with G-6-P. Thus, the discrepancy found between our experiments in liver homogenate and those of Antony et al. (1969) made in rat liver slices could be explained if that mechanism is working in liver.

Lastly, it is worthwhile to point out that the evidence presented in this paper supports the hypothesis that there exists a pathway from glucose to glycogen in which G-l-P is the first intermediate bypassing G-6-P. Notwithstanding, it cannot be discarded the compartmentation of the pathways of carbohydrate metabolism as a coexisting alternative. If one or the other possibilities predominates in cer- tain circumstances would complicate the facts and would make them difficult to interpret. Thus, to reach a real improvement on comprehension of carbohydrate metabolism at different conditions makes necessary a definitive elucidation of the above mentioned problems.

Glucophosphates in glycogen synthesis 333

Acknowledgement-The authors wish to express their thanks to Dr. Gerard0 Sukez for his valuable advice in the preparation of the manuscript.

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role of glucose-1-phosphate and glucose-6-phosphate in glycogen synthesis by pigeon liver homogenate

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