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STUDIES OF TISSUE PERMEABILITY

II. THE DISTRIBUTION OF PENTOSES BETWEEN PLASMA AND MUSCLE*

BY ERNST HELMREICHt AND CARL F. CORI

(From the Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri)

(Received for publication, July 23, 1956)

While it is comparatively easy to deal experimentally with the permeability of single cell preparat.ions (see Paper I (l)), it is much more difficult to analyze the penetration of sugars into the tissues of t,he living animal. The following major factors must be considered in the distribution of an intravenously administered, non-utilizable sugar in the body of a nephrectomized animal: (a) the rate of penetration t?hrough capillary walls; (b) the rate of distribut,ion in extracellular fluid space; and (c) the rate of penetration into different tissue cells. In general, as indicated by the initial rate of disappearance of the sugar from the blood, (a) is a rapid process. The rat,e of (b) will depend on t.he cross section of open capillaries, on blood pressure, and on rate of blood flow. Recause of unequal rates of (c), different tissues will attain a st.at.e of equilibrium at different rates.

There are only a few instances in the literature in which the distribution of sugars between blood and tissues has been measured under controlled conditions. In view of the complexity of the situation, it may or may not. be correct t,o draw conclusions from measurements in the blood alone as to how the distribution of sugars between blood and tissues is affected by various experimental procedures. Levine and Goldstein (2) reported t.hat the injection of insulin or muscular stimulation in eviscerated nephrectomized animals caused a decrease in the blood level of cert.ain non-ut,iliz- able sugars, owing to an accelerated rate of transfer from t,he extracellular to the intracellular compartment of muscle. The effect was regarded as specific, since the distribution of only those sugars appeared to be affected which had the same conf$urat,ion on the first three carbon atoms as glucose. The further implication was that these sugars (n-galactose, L- arabinose, and n-xylose) served as models for glucose.

It has since been shown that the effect described by Levine and Gold- stein is not specific for a particular configuration of the sugar molecule.

* This work was supported by a grant from the Rockefeller Foundation. t Foreign FeIIow from the University of Munich, Germany, sponsored by the

National Academy of Sciences, jointly with the International Cooperation Adminis- tration, Washington, D. C.

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664 PERMEABILITY OF MUSCLE

Thus Park (3) reported, after both blood and tissue analyses, that the distribution of mannose and fructose was increased by injection of insulin. A similar result was obtained when t,he effect of muscular work or insulin on the distribution of the remaining pentoses of the D series (lyxose, arabinose, ribose) was examined (4). An at.tempt is made in this paper to appraise the significance of these observations.

EXPERIMENTAL

Most experiments were performed on non-fasted rats, 300 to 400 gm., nephrectomized by a dorsal approach under Amytal anesthesia. Sugars in 10 per cent solution were injected into a femoral vein, and unless other- wise stated the amount was 100 mg. per 100 gm. of body weight. Blood was withdrawn with a heparinized syringe from the vena cava and the plasma obtained by centrifugation. A protein-free filtrate was prepared by precipitating the plasma in a 1: 20 dilution with barium hydroxide and zinc sulfate (5).’

The gastrocnemius muscle was dissected free and cut at its insertion while being lifted by the achilles tendon, thus avoiding contamination with blood. After being weighed, the muscle was cut into small pieces in the glass container of a high speed homogenize? and homogenized for 10 minutes in 10 ml. of water. Barium hydroxide and zinc sulfate solution was added in amounts calculated to give a final dilution of muscle of 1: 10, including the water content of muscle, which was assumed to be 76 per cent of the muscle weight. The mixture was homogenized for another 5 minutes and filtered. This procedure insured a complete extraction of the sugars.

In some experiments the whole bodies of nephrectomized rats of about 100 gm. of weight, were analyzed. After being cut into pieces, the tissues were first heated in water in order to facilitate passage through a meat grinder, which was fixed in an inclined position in order to avoid loss of fluid. The ground tissue was extracted with hot water by stirring for about 20 minutes; this was followed by three to four additional extractions. Between each extraction the residue was squeezed dry on a Biichner funnel. The combined extracts, about 1 liter, were measured, and an aliquot was deproteinized with barium and zinc, followed by centrifugation and filtra- tion. In the same group of experiments some rats, previously fasted for 24 hours, were eviscerated prior to the injection of pentuse.

For stimulation of the gastrocnemius, needle electrodes connected with

1 It was found that the barium-zinc precipitation caused an appreciable loss of inulin. In experiments with this carbohydrate, plasma was deproteinized by sul- furic acid and sodium tungetate.

* VirTia 45, E. Ma&l&t and Son, New York.


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E. HELMREICH AND C. F. COB1 665

an electrodyne stimulator were inserted through the skin, and three supra- maximal stimuli per second were applied for from 30 minutes to 2 hours. The muscle lifted a weight of 75 gm. over a pulley during each contraction, and the excursion of the wheel served as a rough measure of the extent of contraction. In some experiments, both hind legs were st,imulated by applying elect.rodes over the spinal cord.

Diabetes was produced by injecting fasting rats either intravenously or intraperit,oneally with freshly prepared 5 per cent solut.ions of alloxan. Only t,hose animals which had a fasting blood sugar level of 300 mg. per cent or more 3 to 4 days after the injection were used for experiments. In experiments involving the injection of insulin, 1 to 2 units per 100 gm. were given intraperitoneally.

The following technique permits the effect of temperature to be determined in the living animal: One hind leg of the anesthetized, nephrectomized rat was wrapped up to the knee joint in a silver foil, the end of which was dipped in ice water. The temperature inside the cooled muscle was measured by two copper-constantan thermocouples, soldered into the top of a 20 gauge inject.ion needle, one needle being inserbed through t.he skin into the proximal and t,he other into the distal part of t,he muscle. A third thermocouple served to measure the temperature of the control muscle. The reference junctions were kept in ice water, and the thermocouples were connected through a three-way switch with a sensitive low resistance galvanometer. Temperatures could be read to 0.1”. The rest of the animal was covered, if necessary, and kept at a normal temperature which was controlled by rectal measurements. The temperature of the control muscle was close to 37” in all experiments.

With the device described above, it is possible to cool one muscle to 25’ or below in about 10 minutes and to keep it at a given temperature, with minor fluctuations, for as long as 2 hours. Temperature adjustments are made by changing the extent of surface contact between the silver foil and the skin of the leg. If the end of the silver foil is dipped into a cooling mixture below O”, the muscle can be cooled rapidly to temperatures below 15’, but t,his is not recommended because the blood flow through the cooled muscle is thereby markedly reduced. In order to determine the effectiveness of circulation, raffinose was injected and its concentration determined in the control and the cooled muscle (see below).

Methods and Materials

Pentoses were determined by the method of Roe and Rice (6), with standards prepared from t,he actual sugar under investigation. This met.hod, in comrast to orcinol methods, gives no blank in blood and tissue extracts deproteinized with barium and zinc and is not interfered with


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by hexoses, except when present in amounts exceeding that of the pentose. In some experiments, in which glucose was t,he interfering sugar, it w&s first removed by treatment with glucose oxidase (7) before the pentose method was applied. In experiments with galactose, reducing power was determined by the Nelson procedure (8) before and after treatment of the barium-zinc filtrate with glucose oxidase. In addition, a muscle which had been removed before the injection of galactose was analyzed for reducing substances after treatment with glucose oxidase. From these three determinations t,he amounts of galactose and glucose present could be calculated by difference. In some cases glucose, in the presence of other sugars, was determined by means of the hexokinase-glucose-6-phosphate dehydrogenase reaction.

The extracellular space was measured by injection of r&nose, except in some preliminary experiments in which inulin or sucrose was used. The fructose in these carbohydrates was analyzed by the method of Roe et al. (9), with corresponding standards.

3-Methylglucose was obtained from Ayerst, McKenna and Harrison, Ltd. All other sugars were products of the Pfanstiehl Chemical Com- pany, except glucose, analytical grade, which was obtained from the Mal- linckrodt Chemical Works. Glucose oxidase was obtained in the form of a powder from the Sigma Chemical Company.

CuZcuZalions--The concentration of free sugar in mg. per 100 ml. of plasma water (C,) and per 100 ml. of total muscle water (C,) was calculated by assuming a water content of 94 and 76 per cent of plasma and skeletal muscle, respectively. Concentration of sugar in mg. per 100 ml. of intracellular water of muscle (CJ was then calculated as follows.

ci p KS - (G..E)l (1.0 - E)

E is the ratio of distribution of injected raffinose (C,:C,) as a measure of the extracellular space of muscle (see below). The calculation is analo- gous to that used for the concentration of a substance in the erythrocytes when hematocrit value and concentrations in whole blood and in plasma are known.s

Result-s

Determination of Extracellular Space-Knowledge of the amount of sugar present in the extracellular space is essential, if the intracellular distribu-

8 In order to avoid ambiguities, a numerical example is given for a value of E of 0.2. From pentose as determined, 282 mg. per 100 ml. of plasma and 114 mg. per 100 gm. of muscle, one calculates C, = 300 mg. per 100 ml., CI = 150 mg. per 100 ml., and Ci = 112.5 mg. per 100 ml. For calculation according to weight, E has the value of 0.15 and the divisor is (0.76 - E), corresponding to an intracellular water content of 0.61 ml. per gm. of muscle.


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E. HELMREICH AND C. F. CORI 661

tion of the sugar is to be determined. The intracellular distribution can be determined only relative to some reference substance which can be shown to remain extracellular under the experimental condition8 employed.

After trials with other sUbBtanCe8, raffinose was chosen for the following rt3Z&BOnB. As shown in Fig. 1, rafhnose leaves the blood stream rapidly, and 20 minute8 after injection reaches a plasma concentration which remains virtually constant for the next 2 hours, the period of time over which most experiments extended. Experimental procedures, such a8 stimulation of both hind leg8 or injection of insulin, which caused a decrease in the plasma level of other sugars had no effect on that of raffinose.

. 7 ’ ’ ‘. ‘. . . .

(1) (6) l - D-RAFFINOSE

.

(1) U) --s_

z 40. (3,-----o---,,

z (8, ----- ---.- -Dz_XYLOSE

0 5 60.

(IO, ---------. .

(1) . m

!! 80. 6 s

100~

MINUTES 20 40 60 80 100 120 140 I60 180 FIG. 1. Time-curve of distribution of n-r&nose and n-xylose in the body after

injection of 100 mg. per 100 gm. The number of experiments are given in parentheses.

By contrast, it may be seen (Fig. 1) that n-xylose, after a rapid initial fall, shows a steady decrease in its plasma level which continues as long as the experiment lasts. It will be shown later that this decrease is made up of two components, slow penetration into the tissues and slow utilization.

The concentrations of raffinose in plasma and muscle water were determined in twelve separate experiments 2 hours after injection of 106 mg. per 100 gm. of nephrectomized rat. The average value8 and standard deviation were as follows: plasma, 564 =t 2.8 mg. per 100 ml.; muscle, 115 f 9.6 mg. per 190 ml.

From these values a distribution of sugar between muscle and plasma ((115/564) X 100) of 20 per cent was calculated which was assumed to represent the extracellmar (rafhnose) space in terms of the ffuid volume of muscle. According to weight, the extracellular space of muscle would be 15 per cent, whereas 18 per cent, according to weight, would be the


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distribution of raffinose in the body as a whole. The distribut,ion of inulin in the body as a whole was about 10 per cent smaller than that of raffinose, and that of sucrose was a little larger.

Rafflnose also remained extracellular in a muscle which had been stimulated or subjected to t,he action of insulin. In a number of experiments, raffinose was injected wit,h other sugars t,o serve as an internal st.andard for the ext,racellular space.

Distribution between Plasma and Erythrocytes-RaEnose did not penetrate the red cell membrane, whereas the pentoses penet.rated slowly (Ta- ble I). When pentoses are determined in whole blood rather t.han in plasma, as was done by Levine and Goldstein (2), their rate of disappearance from t.he blood is underestimated, and in general values are obtained which are inaccurate for calculation of di&ibution between blood and

TABLE I Distribution of Sugars between Plasma and Erythrocytes

The amount of sugar injected was, in each case, 100 mg. per 100 gm. of nephrectomized rat.

Sugar injected Time after injection

D-Xylose ......... D-Arabinose. ..... D-Xylose. ........ D-Raffinose .......

min. mg. fier cent mg. per cent per Cenf

40 294 196 41.7 120 222 166 41.7 180 174 140 41.7 120 532 256 41.8

concent*=- Cmcentm tion in plw-

I I tion in Hemstocrit

ma whole blood

-

t Concentra- Distribution ion in myth- between cells

rocytes and plasma

mg. per cent per cent

57.5 20 88.0 40 93.5 54 0 0

tissues. Thus, in Table I, xylose in plasma between 40 and 180 minutes after inject,ion disappeared t.o the extent of 120 mg. per cent, whereas in the same e&mate from whole blood t.here is a disappearance of only 5G mg. per cent. Whether such factors as muscular stimulation or injection of insulin change the rate of penetration of the pentoses into the erythrocytes has not been determined.

Utilization of PentosesOne of the reasons for t.he steady disappearance of the pent,oses from the plasma (Fig. 1; Table I) might be utilization in the tissues. In order to detect such utilization, the whole bodies of raOs were analyzed (a) without injection, (b) immediately after injection, and (c) 2 hours aft.er injection of 100 mg. of pentose per 100 gm. of weight. In (a), the extract of whole rats, prepared as described under %fethods,” gave no detectable blank with the pentose method.4 In (b), the pentose

4 A similar method used previously (10) was based on determination of sugars by copper reduct,ion, and gave a large blank without injection of sugar. The uncer- tainty introduced by this blank made the estimate of the disappearance of injected sugar less accurate.


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injected with a calibrated syringe was nearly quantitatively recovered (0 to 6 per cent loss). The values obtained under (c) are recorded in Table II, and it may be seen that each of the pentoses disappeared to some extent, while D-raffinose did not disappear at all. An estimate of the rate of disappearance of glucose at approximately the same plasma levels is included for comparison. This estimate is made from a determination of the intravenous tolerance rate in normal and in insulinized rats (11).

Injection of insulin (1 unit per 100 gm.) significantly increased the disappearance of all pentoses with the exception of D-arabinose, and an even greater effect was achieved when both hind legs were stimulated for 2 hours. The enzymes which cause a disappearance of pentoses in animal

TABLE II Utilization of Sugars in Nephrectomized Rats

The whole body was analyzed 2 hours after injection of 100 mg. of sugar per 100 gm. of weight. C, control; D, diabetic; E, eviscerated; I, insulin; M, stimulation of both hind Iegs.

Sugar lost per 100 gm. weight

Sugar injected

C

mg.

n-Arabinose . . . . . . . . . . . . . . . . 9 n-Arabinose. . . . . . . . . . . . . . 20 D-Lyxose.................:.. 13 D-Ribose.................... 46 D-Xylose.................... 20 D-Raffinose.................. 0 D-Glucose................... 500*

-

--

-

-T-

E

mg. 6

16

40 19 0

D

mg.

56 28

-

-- I

mg.

12 32 23 64 33

600*

M

mc. 17 41

83 42

* Estimated from intravenous tolerance rate.

tissues have not been investigated in detail, except t,hat a ribokinase is known to be present (12). This enzyme, by transferring phosphate from adenosine t,riphosphate to carbon 5 of the furanose form of ribose, leads to a product which can be oxidized over the pat.hway described by Hor- ecker et al. (13) or can be incorporated into nucleotides via phosphoribo- mutase (14) and nucleoside phosphorylase (15). The question as to how muscular work or insulin might increase the activity of ribokinase will be discussed later.

In two cases, severely diabetic rats were analyzed, but a decrease in the rate of utilization of t,he pentoses could not be detected, thus foreshadow- ing a result to be described later; namely, that a high glucose level in blood does not inhibit t.he penetration of the pentoses into the tissues, the latter being a prerequisit,e for t.heir utilization.

Upon comparison of the amount of pentoses lost in nephrectomized rats


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with that which disappeared in nephrectomized eviscerated rats, one can see that utilization of pentoses takes place to a considerable,extent in the extrahepatic tissues. There appears to be no advantage in the current use of eviscerated preparations in investigations concerned with tissue permeability.

Distribution of Pentoses in Body-It seems clear that, in order to calculate the per cent distribution of the pentoses in the body (Fig. l), a correction has to be applied for the amount of pentose which disappears because of utilization. In Table III, Column 2, the average plasma level of

TABLE III Plasma Concentration of Sugars

The values were obtained 2 hours after injection of 100 mg. of sugar per 100 gm. of weight and are expressed in mg. per 100 ml. of plasma water. C, control; I, insulin; M, stimulation of both hind legs. The number of experiments is given in paredtheies.

Sugar injected C

- I

(1) (2) D-Arabinose. ........ 242 (7) L-Arabinose ......... 233 (6) D-Lyxose ............ 224 (8) D-Ribose ............ 114 (5) D-Xylose. ........... 224 (10) D-Galactose ......... 230 D-Raffinose .......... 564 (13)

%- (3)

234 198 198 76

188

I

Found

(4)

212 (7) 139 (4) 171 (4) 72 (4)

142 (12) 140 (3)

M

(5)

230

177 59

131

1

-

-

-

?er cent distribution in body*

C

(6) 38 34 39 47 36

18

42 49 45 50 47

* Corrected for utilization of sugars aa follows: (mg. of sugar recovered per 100 gm.)/(mg. of sugar per ml. of plasma water).

four of the pentoses and of galactose 2 hours after injection was very similar, ranging only from 242 to 224 mg. per cent. The plasma level of the more rapidly utilized ribose was considerably lower, about one-half of that of the other pentoses. When compared with raffinose, which remains extracellular and distributes itself in 18 per cent of the body weight, all of the pentoses showed an intracellular distribution. In Table III, Col- umn 6, this distribution, when corrected for utilization, varied between 34 and 39 per cent of the body weight for four of the pentoses, and ribose differed again by showing a higher distribution. Since it had not been determined how much galactose disappeared through utilization, the per cent distribution in the body could not be calculated.

InsuIin or muscular work, in the form of stimulation of both hind legs,


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E. HELbIREICH AM) C. F. CORI 671

caused a decrease in the plasma level of the pentoses and of galactose (Table III). When compared with the controls (Column 2 ver.suS Col- umn 4), the differences were statistically significant (p < 0.1 per cent) in the case of L-arabinose (s.d. = 17.6), n-lyxose (s.d. = S.S), and n-xylose (s.d. = 3.7) and marginally significant (p = 1.3 to 2.0 per cent) in the case of D-ribose (s.d. = 12.6) and D-arabinose (s.d. = 10.9). Column 3 refers to the plasma values which should have been found, had the only effect of insulin been an increased utilization of the pentoses of the magnitude shown in Table II. Insulin appears to have an additional effect (Table III, Column 3 versus Column 4) which is statistically significant only in the case of n-xylose (p < 0.1 per cent) and marginally significant

TABLE IV Distribution of Sugars in E&a- and Intracellular Compartments of Body

Calculated from the data of Tables II and III for an extracellular space of 18 per cent of the body weight.

Sugar pet loo gm. bodyweight

Sugar injected Control

Extracellular Intracellular Ratio, I: E

w. m. D-Arabinose. . . . . . 43.5 47.6 1.09 L-Arabinose . . . . . . 41.9 38.1 0.91 D-Lyxose . . * . . . . . . 40.3 46.7 1.16 D-Ribose . . . . . . . . . 20.5 33.5 1.63 D-Xylose. . . . . . . . . 40.3 39.7 0.98

T _- I

--

-

mb.

38.2 25.0 30.8 13.0 25.6

I .-

-

Insulin

ntmcellular Ratio, 1:E

m.

49.8 43.0 46.2 23.0 41.4

1.31 1.72 1.60 1.77 1.61

(p = 1.5 per cent) in the case of L-arabinose and n-lyxose. If the additional insulin effect were to consist of a wider distribution of the sugars in the tissues, the values shown in Columns 6 and 7, Table III, could give an indication of the magnitude of this effect. The question was whether one could detect these changes in the distribution of sugar by direct tissue analyses.

In Table IV the amount of pentose present in the extracellular and intracellular compartments of the body has been calculated. It may be seen that, under the experimental conditions chosen, insulin produced a barely significant increase of pentose in the intracellular compartment of the body as a whole. In the case of D-ribose, there was actually a decrease. The possibility remained that insulin increased the pentose concentration in a particular tissue, e.g. muscle, corresponding to 40 per cent of the body weight, and that this occurred at the expense of the pentose present in the intraceliular compartment of other tissues. Such a change


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in the distribution of pentose within the intracellular compartment would imply that some tissues attain equilibrium with the external pentose concentration much more rapidly than others. The former would then yield pentose as the external pentose concentration falls because of increased withdrawal of pentose by the latter.

When liver and muscle were compared (Table V), a marked difference in the rate of attainment of equilibrium was indeed observed. In liver the ratio, intracellular to extracellular pentose, approached unity 30 minutes after the injection. On the other hand, the penetration of pentose into resting muscle was a slow process, even slower than the penetration into the erythrocytes, as shown by the fact that the above ratio

TABLE V

Distribution of Sugar between PEama, Mqscle, and Liver The extracellular space determined with rafhnose in t,his experiment was found

to be 20 per cent for muscle and 29 per cent for liver. These values were used for calculation of intracellular concentration. The animal was fasted for 48 hours. The mvater content of the liver of a fasting rat was assumed to be 70 per cent of the fresh weight.

Sugar injected

L-Arabinose . . . . . . . . . ‘I . . . . . . . .

Time after injection

min. 30

120

Intracellular Total ntose, mg. per pentose, mg. per 100 m . of tissue water p” 100 ml. of tissue

water

374 70.5 346 0 335 236 61.4 17.6

Ratio of distribution

Muscle Liver to

plasma p&a --

had reached a value of only 0.08 some 2 hours after the injection. With this background concerning the dynamics of distribut,ion, the effect of insulin and of muscular work on the penetration of the pentoses into muscle will be examined. The results will be expressed in terms of concentration in the intracellular water of muscle, since this is presumably t.he concentration in contact with the enzymes.

Intracellular Distribution of Pentoses in Muscle-The values for the control muscles in Fig. 2 indicate that each of the pentoses showed an intracellular distribution 2 hours after the injection. This was also found to be the case in muscle of alloxan-diabetic ram. The order in the rate of penetration under these basal conditions was D-lyxose > D-xylose > D-arabinose > n-ribose > n-arabinose.

After injection of insulin, the distribution of each of the pentoses in muscle was increased. The effect was statist,ically significant (p < 0.1 per cent) in all cases (s.d. r,-arabinose = 9.3, D-lyxose = 9.7, D-ribose =


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E, HELMRFJCH AND C. F. CORI 673

2.9, D-XJ’lOSe = S.O), except for D-arabinose (s.d. = 11.7). Though the actual concentrations in muscle after insulin injection were nearly the same for L-arabinose, n-lyxose, and n-xylose, those of D-arabinose and D-ribose were considerably lower. In the latter case, one must consider that two- t.hirds of the injected sugar disappeared by utilization. The effect of insulin on L-arabinose appears to be greater than on the other pentoses, since L-arabinose shows the slowest rate of penetration in the absence of insulin injection. The highest distribution ratio (0.98) between the aque-

260

240 _-_ -__. __ .._.. .._.-._ __. _... _

220. _.___--_. 200.

2 ISO-

8 160-

- 140. ._ __. . __ -

_ ______._ ___-.. ___

D-ARABINOSE D-LYXO! L-ARABINOSE I GR~BosE

D-XYLOSE

FIG. 2. Intracellular concentration of pentoses in muscle 2 hours after the injection of 100 mg. per 100 gm. C, control; I, insulin; S, stimulated; D, diabetic. The dotted lines represent the corresponding plasma values. The number of experiments are given in parentheses.

ous phases of muscle and plasma was reached in the case of D-ribose after insulin injection.

In order t,o determine the effect of insulin on the rate of pentose penetration, experiments of short duration, not shown in Fig. 2, were undertaken. The initial rate of penet,ration of n-xylose was again faster than that of r,-arabinose. Thus, 10 minutes aft,er the pentose injection into rats treated with insulin, the ratio of distribution was 71:258 = 0.29 in the case of the former, and 31:311 = 0.1 in the case of the latter pentose.

The effect of stimulation could be investigated by stimulating one gas- t.rocnemius muscle6 and comparing it with the resting gastrocnemius in

6 Stimulation of a small muscle such as the gastrocnemius, in contrast to stimulation of both hind legs, has no demonstrable effect on the pentose level of plasma.


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the same animal. Fig. 2 shows that stimulation caused a significant increase in the intracellular concentration of each of the pentoses and that this effect, like that produced by insulin, was not dependent on a particular configuration of the pentose molecule. The effect of stimulation appeared to be stronger than that of insulin. Stimulation plus injection of insulin did not produce any greater effect than stimulation alone (4). The highest distribution ratio (0.75) was reached by n-lyxose in st,imulated muscle.

In stimulated muscle, 10 minutes after the sugar injection, the order in the rate of penetration was n-lyxose (152) > n-xylose (94) > D-ribose (66). The values in parentheses represent mg. of pent,ose per 100 ml. of intracellular water. From these values, in conjunction Tvith those for resting muscle in Fig. 2, one can arrive at the rough estimate that stimulation may increase the rate of penetration lo-fold or more over that in resting muscle.

In a small series of experiments wit,h alloxan-diabetic rats (Fig. 2), t,he effects of stimulation and of insulin injection were the same, except that the values tended to be lower in the muscles of the diabetic than in those of normal animals.

In other experiments, not shown in Fig. 2, a muscle denervated for 2 days could be made more permeable to pentoses by direct stimulat,ion, provided that it performed sufficient work. This indicat.es that intact innervation is not needed to demonstrate the effect of muscle work.

The following experiment was performed to see whether a humoral agent is involved in the effect of st.imulation on permeability of muscle, as has been suggested by Levine and Goldstein (2). The muscles of both hind limbs were stimulated for 2 hours through the spinal cord, except for one gastrocnemius, the nerve of which had been cut. The denervated muscle did not show an increased pentose uptake. This does not support the above suggestion.

It is of particular interest that the effect of muscular work on permeability was found to persist for as long as 30 minutes after the stimulation. Use was made of this fact in the experiments with cooled muscle (see below).

The question may be asked whether the effect of insulin on muscle is of suflicient magnitude to explain the fall in plasma pentose. For this pur- pose, one multiplies the difference in plasma concentration (Table III, Column 3 versus Column 4) with the original ratio of distribution, e.g. for L-arabinose 59 X 0.34 = 20.1 mg. of sugar, which would be the amount taken up by 40 gm. of muscle in a 100 gm. rat when insulin is injected. From the difference in the bar diagrams in Fig. 2, one may calculate an increase in the intracellular L-arabinose content of 19.8 mg. per 40 gm. of muscle when insulin is injected. A similar calculation for n-lyxose yields


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10.5 versu.s 12.6 mg. and, for D-xylose, 16.5 WKW.S 12.7 mg. It would thus appear that the principal insulin effect on the distribution of pentoses takes place in muscle.

E$ect of Glucose and S-Methylglucose on Pentose and Galactose Penetra- tion-As the animals receiving insulin were not injected with glucose, they developed hypoglycemia, which is shown by determination of plasma glucose with the hexokinase-Zwischenferment reaction. The mechanism by

TABLE VI Effect of Glucose and S-Methylglucose on Penetration of Other Sugars into Muscle The values in parentheses are those obtained under the same conditions, but

when no glucose or 3-methylglucose was in, jetted.

Pairs of sugars injected Tim.?. after injection

mg. per 100 gm.

D-Arabinose, 100 3-Methylglucose, 400 D-Xylose, 100 3-Methylglucose, 200 D-Xylose, 100 3-Methylglucose, 250 D-Arabinose, 100 D-Glucose, 400 D-Xylose, 100 D-Glucose, 400 D-Gsiactose, 100 D-Glucose, 700 D-Xylose, 100 D-Glucose, 200

min. 120

60

120

20*

120*

120*

w

* Insulin injected at zero time. t Muscle stimulated for 30 minutes.

-

t

.-

I

-

ng. )ar loo ml.

290 2060

273 1595 224

1631 276 982 126 178 174 519 361 600

Concentration in muscle water

m*. pa 100 ml.

63.1 (37.9)

19.0 (23.6)

42.5 (40.9)

86 (67)

33.6 (92.7)

112 (83.2)

156 (127)

which pentoses enter the intracellular compartment of muscle is not known, but, if it were the same as that for glucose, this sugar should inhibit the penetration of the pentoses in proportion to its concentration in the extracellular fluid. In consequence, the effect of insulin on the distribution of the pentoses could have been due to a lowering of the glucose concentration. In this case the effect of insulin would disappear if enough glucose were injected to prevent hypoglycemia.

Experiments in which this possibility was tested are presented in Ta- ble VI. 3-Methylglucose was chosen as a non-utilizable glucose analogue which has been shown to be “actively” absorbed from the gastrointestinal tract (16). In the first three experiments the penetration of neither


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D-arabinose nor n-xylose into resting muscle was inhibited by a 6- to 7-fold excess of 3-methylglucose in the blood stream. When glucose was present in excess, it did not inhibit the penetration of n-xylose or L-arabinose into insulin-treated or stimulated muscle and did not prevent the decrease in the pentose level of plasma after insulin injection. The penetration of

TABLE VII E&et of Temperature on Penetration of Sugars into Muscle

The dose of insulin was 3 units per 100 gm., injected 70 to 120 minutes before the termination of the experiments. “Stimulated muscle” refers to the preceding stimulation of both gastrocnemius muscles for 30 minutes at the rate of 3 per second. In each case, one muscle had been cooled to the indicated temperature before sugar (100 mg. per 100 pm.) was injected. With one exception, the averages of two experiments are tabulated. --

Experi- ment No.

1 2

3 4 5

-

-

Sugar injected

D-Raffinose D-Xylose +

D-raffinose D-Xylose

‘I I‘ +

D-raffinose D-Xylose

L-Arabinose + D-raffinose

D-Xylose + D-raffinose

10 60

120

10

30

70

- -

1 htracellular pentose

w. ‘n JOI

ml.

266 25 26

258 74 0 171 128 56 157 105 72

358 94

360 204

294 137

13

71

111

- -

t

-

“C.

17 24

22 24 23

20

21

25

-

t

T

Total ra5nose in

Remarks

w. ICI 101

ml.

141 124

132

130

146

96 129

132

115

137

Insulin Resting mus-

cle Insulin

‘I ‘I

Stimulated muscle

Stimulated muscle

Stimulated muscle

galactose could not be inhibited either, even when the final plasma glucose level exceeded that of galactose S-fold.

E$ect of Temperature-The distribution of pentose in the extracellular space of muscle is generally rapid enough so as not to be rate-limiting for the intracellular penetration. Among the factors which influence the rate of extracellular distribution, the cross section of open capillaries and the rate of blood flow play an important role. Cooling results in vaso- constriction and decreased rate of blood flow, and the question was to what extent this might influence extracellular distribution and hence intracellular penetration. Raffiose was injected in order to determine the rate of distribution of sugar in the extracellular space. It was found that,


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when muscle was cooled below 20”, the rate of distribution was decreased. In Experiment 1, Table VII, analyses were made 10 minutes after injection, at which time the extracellular space of the control muscle had come to equilibrium with the plasma rafhnose concentration corresponding to a distribution in 22 per cent of the fluid volume of muscle. The muscle cooled to 17” contained considerably less rafhnose and had reached only 68 per cent of the equilibrium value of the control muscle. In four additional experiments in Table VII, analyses were made 30 to 120 minutes after injection; in these, no significant difference in the raffinose content between the control muscles and muscles cooled to 20-25” could be detected.

Experiment 2, Table VII, shows that the slow basal penetration of pentose into muscle does not have a measurable temperature coefficient. After injection of insulin (Experiments 3 to 5, Table VII), the cooled muscle contained in each case less intracehular pentose than the control muscle. Whereas the distribution of pentose in the control muscle had reached its maximum 60 minutes after the injection, the penetration of pentose into the cooled muscle continued for 120 minutes. For this reason the difference in the pentose content of the two muscles became smaller with time. An effect of temperature on the rate of penetration is also seen in previously stimulated muscle (Experiments 6 to 8, Table VII), and here again the difference becomes smaller with time (see above). The stimulated and cooled muscle is still more permeable to the pentoses than resting muscle at normal temperature. This was to be expected since an approximate lo-fold increase in the rate of xylose penetration as the result of stimulation would not be abolished by a decrease in temperature of 16” unless the temperature coefficient was very large. A rough estimate would indicate that the temperature coefficient (&) of the accelerated penetration after stimulation is between 2 and 3.

DISCUSSION

The conclusion of Levine and Goldstein (2) that insulin or muscular work lowers the blood level of pentoses and of galactose because of a greater distribution in the body tissues is confirmed, but the correlation of this phenomenon wit,h a particular structure of the sugar molecule is not substantiated. This does not exclude the possibility that the cell membrane shows specificity with respect to other structural changes, e.g. re- placement of the reducing group by a primary alcohol group. This point needs further investigation.

The first question to arise is whether the pentoses can serve as model substances for glucose. This would depend on the mechanism by which they penetrate into the interior of the muscle cell as compared to glucose. It has been shown in this paper that glucose does not inhibit the penetra-


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tion of the pentoses; this could mean that glucose and the pentoses enter muscle by two different mechanisms, both of which are influenced by insulin or muscular work. It should be pointed out that t.hus far it has not been established how glucose enters muscle. This point needs to be set- tled before further discussion becomes profitable.

The second question to arise is how insulin or muscular work increases the utilization of the pentoses. In the case of stimulation of both hind limbs, the utilization of n-arabinose, L-arabinose, D-ribose, and n-xylose is approximately doubled. At the same t,ime the concentration of the pentose rises in the intracellular water of muscle, for example (Fig. 2) in the case of n-iylose from 2.7 X 10-a M in resting muscle t.o 6.2 X 10ms M

in insulin-treated muscle and to 8.5 X 1O-3 M in stimulated muscle. One might assume that this represents the substrate saturation curve of an enzyme. A corollary of this mechanism would be that the rate of penetration of sugars controls the rate of metabolism through the degree of saturation of an enzyme.

An alternative explanat,ion would be that the internal concentration remains the same while the volume of distribution changes. Thus ribose in Fig. 2 would occupy 31 per cent of the intracellular volume in resting muscle and 70 per cent in stimulated muscle. If it is assumed that ribokinase is uniformly distributed in the different “compartments” of muscle, the approximate doubling of ribose utilization in stimulated muscle would be accounted for. In this case muscular activity or insulin would remove some intracellular barriers, permitting access of substrate to a larger amount of enzyme. Therefore, the rate of utilization would depend on the total amount of enzyme in contact with the substrate.

The third question is how muscular work or insulin could change the cell membrane or some inner cell structure (according to whether one favors the first or second hypot.hesis) in such a manner that pentoses can penetrate at a higher rat,e than normal. The fact that t,he change in permeability after insulin or muscular work is influenced by t,emperature may seem of primary importance. Actually, this does not tell us what kind of “active” process is involved. One might be measuring the effect of temperature on a “carrier” mechanism, but it is equally possible that an energy-yielding reaction is required to keep the “membrane” in a stat.e of increased permeability. These and other questions will be more fully discussed in subsequent papers.

SUMMARY

1. The distribution of five aldopentoses, of galactose, and of raffinose between plasma and tissues has been determined in nephrectomized rats at rest, after injection of insulin, and after stimulation of muscle. The


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E. EELMREICH AND C. F. CORI 679

whole body was extracted in order to determine how much pentose disappeared through utilization. Individual tissues analyzed for pentose were erythrocytes, liver, and muscle.

2. Each of the pentoses was utilized at a slow rate (from 2 to 10 per cent of the rate of glucose), and the rate was increased by injection of insulin or by stimulation. of both hind legs. Raffinose was not utilized.

3. When compared with raffinose, which distributed itself in an extracellular space corresponding to 18 per cent of the body weight, each of the pentoses showed an intracellular distribution 2 hours after injection in normal as well as diabetic animals. The relative rates of penetration of the pentoses into the intracellular compartments of individual tissues were liver > eryt,hrocytes > muscle.

4. After injection of insulin or muscular activity, the pentose level of plasma decreased, partly because of increased utilization and partly because of increased distribution which took place mainly in muscle. The rate of penetration was also increased. The effect was not specific for a particular configuration of the sugar molecule.

5. The penetrat,ion of pentoses and of galactose into muscle at rest, after injection of insulin, or after stimulation could not be inhibited by the injection of large amounts of glucose or 3-met,hylgIucose.

6. The basal rat,e of penetration of pentose was not decreased in a muscle cooled to 22”, whereas the increased rate of penetration after insulin or muscular activity showed a definite temperature dependence with a &lo of about 2.

BIBLIOGRAPHY

1. Crane, R. K., Field, R. A., and Cori, C. F., J. Biol. Chem., 224, 649 (1957). 2. Levine, R., and Goldstein, AT. S., Recent Progress Hormone Res., 11, 343 (1955). 3. Park, C. R., Ciba Foundation colloquia on endocrinology, London, 9, 240 (1956). 4. Helmreich, E., and Cori, C. F., Ciba Foundation colloquia on endocrinology,

London, 9, 227 (1956). 5. Somogyi, M., J. Biol. Chem., 160, 69 (1945). 6. Roe, J. H., and Rice, E. W., J. Biol. Chem., 173, 507 (1948). 7. Keilin, D., and Hartree, E. F., Biochem. J., 42, 230 (1948). 8. Nelson, N., J. Biot. Chem., 168, 375 (1944). 9. Roe, J. H., Epstein, J. H., and Goldstein, N. P., J. Biol. Chem., 178, 839 (1949).

10. Cori, C. F., and Cori, G. T., Proc. Sot. Exp. BioZ. and Med., 26,432 (1929). Cori, C. F., 1st International Congress of Biochemistry, Cambridge (1949).

11. Cori, C. F., and Cori, G. T., J. BioZ. Chem., 73, 597 (1927). 12. Agranoff, B. W., and Brady, R. O., J. BioZ. Chem., 219, 221 (1956). 13. Horecker, B. L., Gibbs, M., Klenow, II., and Smyrniotis, P. Z., J. BioZ. Chem.,

207, 393 (1954). 14. Guarino, A. J., and Sable, H. Z., J. BioZ. Chem., 216, 515 (1955). 15. Kalckar, H. M., in McElroy, W. D., and Glass, B., The mechanism of enzyme

action, Baltimore, 675 (1954). 16. Wilson, T. H., and Vincent, T. N., J. BioZ. Chem., 216,851 (1955).


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Ernst Helmreich and Carl F. CoriBETWEEN PLASMA AND MUSCLE

II. THE DISTRIBUTION OF PENTOSES STUDIES OF TISSUE PERMEABILITY:

1957, 224:663-679.J. Biol. Chem.

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