THE Jonmn~ OF Bromorcn~ CHEMWCRY Vol. 237, No. 11, November 1962

Pnkted in U.S. A.

Fructose Metabolism of Adipose Tissue

I. COMPARISON OF FRUCTOSE AND GLUCOSE METABOLISM IN EPIDIDYMAL ADIPOSE TISSUE OF NORMAL RATS*

E. R. FROESCH AND J. L. GINSBERG

From the Metabolic Unit, Department of Medicine, University of Zurich, Zurich, Switzerland

(Received for publication, March 6, 1962)

During the course of studies on the nature of hereditary fruc- tose intolerance (1,2), it became apparent that, in this syndrome, the ability of the liver to metabolize fructose is greatly impaired. Despite the loss of this liver function, only 10 to 20% of an administered fructose load is excreted in the urine. This sur- prising observation led us to investigate the fructose metabolism of various other tissues. As will be shown in this report, normal leukocytes and erythrocytes metabolize fructose rapidly but only in the absence of glucose. Skeletal muscle metabolizes fructose only very slowly (3, 4). The liver, normally the major site of fructose metabolism in the intact organism (5-7)) utilizes fructose faster than glucose and independently of it. Fructose metabo- lism of isolated adipose tissue proceeds almost as fast as glucose metabolism and with little or no inhibition by glucose. The results obtained with adipose tissue, which are described later, suggest that this tissue may well be an important site of fructose metabolism which so far has been neglected. This report, which deals with certain quantitative and qualitative aspects of fructose metabolism of adipose t,issue, extends observations presented elsewhere (8).

EXPERIMENTz4L PROCEDURE

For each experiment, 12 Osborn-Mendel rats weighing between 110 and 180 g were fasted overnight and decapitated. Each of the two epididymal fat pads was cut into six pieces as nearly equal in weight as possible, yielding a total of 12 pieces per rat. These pieces of tissue were distributed equally between 12 Warburg flasks containing 2.5 ml of bicarbonate Krebs-Ringer buffer, so that each flask contained one piece from each rat, and therefore represented a pooled sample, with an average weight of 360 mg. Care was taken to traumatize the tissue as little as possible during the manipulations and to distribute proximal and distal as well as left and right parts of the fat pads equally among the flasks so that an equal anatomical distribution was assured.

Chromatographically pure, uniformly labeled glucose-CY, uniformly labeled fructose-C14, and, in one experiment, uniformly labeled sorbitol-Cl4 were added to the incubation medium before it was pipetted into the flasks. Each flask contained between 0.11 and 3.1 PC of CY4, and the specific activity of the sugars was kept as nearly constant as possible. The labeled substrates were

obtained from Amersham, England.

* Supported by grants from the Schweizerische Nationalfonds (1923 and A 163) and the United States Public Health Service (A 5387).

In one experiment, 3,5-dinitrobenzoylglucosamine, a competi- tive inhibitor of hexokinase (9) was used.1

Crystalline pork insulin with an activity of 23.9 i.u. per mg was kindly supplied by Eli Lilly and Company, Indianapolis, Indiana. Serial dilutions of insulin were made in bicarbonate Krebs-Ringer buffer containing 200 mg gelatin per 100 ml.

The Warburg flasks were gassed with a mixture of 95 % Or5 % CO2 for 6 to 8 minutes, incubated for 3 hours at 37.5”, and con- tinuously shaken 96 times per minute. The wet tissue weight was obtained by weighing the flasks on a Mettler balance before and after addition of the tissue. rlpproximately 20 to 30 minutes elapsed between the killing of the first animal and the beginning of the incubation. The net gas exchange was measured accord- ing to Umbreit et al. (10). The results in the tables are expressed as microliters of gas taken up or evolved per g of wet tissue per total period of measurement with the flask constant for 02. The COz content of 5% was considered too small to require correction.

Where erythrocytes and leukocytes were used, they were obtained from pooled blood of five normal persons and separated from each other by dextran sedimentation (11). The incubatioh was in bicarbonate Krebs-Ringer buffer, and the conditions were the same as those chosen for the incubation of adipose tissue.

The samples were not pooled when diaphragm incubations were made; the rat diaphragms were simply cut into four pieces as equally as possible and kept on ice until ready for incubation.

Glucose was determined with the cofimercially available glucose-oxidase test combination of Boehringer und Soehne, GmbH., Mannheim, Germany. As a minor modification, the samples were pipetted into the reagent mixture in an ice bath, and the color was allowed to develop for 20 minutes in a water bath at 37.5”. Fructose was measured according to a modifica- tion (12) of the method of Higashi and Peters (13).

C?402 was precipitated as BaCY403, deposited on filter paper by suction through a sintered glass filter, and counted in Tracer- lab and Nuclear windowless flow counters. The total lipids were extracted from the tissue (14), and the fatty acids were released from them by alkaline hydrolysis and subsequent ether extraction. Both total lipids and fatty acids were put on planchets and counted. Glycogen was extracted from the fat- free dried tissue after digestion in 30% KOH and after addition of 5 mg of carrier glycogen per sample to obtain more complete precipitation. The glycogen was reprecipitated five times in cold 70% ethanol after addition of one drop sf 10% Na2S04 to the sample, deposited on filter paper by suction, and counted.

1 We thank Dr. G. Semenza for the preparation of this material.

3317

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3318 Fructose Metabolism of Adipose Tissue. I Vol. 237, No. 11

LEUCOCYTES CY TES )sEdJ-CU GLUC(

ml

-

RESULTS

Fig. 1 shows the behavior of normal human erythrocytes and

x

leukicytes with regard to fructose and glucose metabolism. Whereas fructose oxidation to WOO is almost equal to glucose oxidation when only fructose is present in the medium, it is depressed to approximately 10 ‘% in the presence of equal amounts of unlabeled glucose. In contrast to this, glucose oxidation is not influenced by the addition of fructose to the medium. In

the diaphragm, oxidation of fructose is 22% of that of glucose (Table I). The data presented in Table I show that in thii tissue glucose inhibits fructose oxidation less markedly than in the blood cells and that the incorporation of uniformly labeled fructose-P into muscle glycogen is not depressed significantly in the presence of glucose.

FRUCTI 1M

ERYTHR DSE-U+ Im

f

-

In adipose tissue, the fructose uptake at a hexose concentration of 200 mg per 100 ml ranges between 46 and 89% of that of glucose with a mean of 63.5%. In this instance, glucose barely

GLUCOSE

PO5

w

w3

w2

001

F

GLUCOSE FRUCTOSE FRGTOSE

0.1 into GLVCOGEN

FIG. 1. Metabolism of uniformly labeled fructose-W Cfructose- U-04) of human erythrocytes (EC) and leukocytes (Lc) in the absence and presence of glucose compared to the metabolism of uniformly labeled glucose-c” (gzucose-U-P) in the absence and presence of fructose. Results are expressed as mioromoles of sugar carbon oxidized to 040~ per 100 millions of leukocytes or 1 ml of packed erythrocytes per 3 hours.

TABLE I

Metabolism of uniformly labeled fructose-04 in absence and presence of glucose compared to metabolism of uniformly labeled

glucose-P in absence and presence of fructose by rat diaphragm

The diaphragm of each of 3 rats was cut into 4 pieces. - 1 t Uniformly labeled fruc- ox-Cl’, 100 mg /lOO m

Uniformly labeled glu- I case-CT”, 100 mg/100 ml

Rat

30 fructos e Fructose, 100 mg I

_-

-

To glucose I-

_-

2.75 1.14 10.43 5.18 1.10 1.03 8.95 8.20 2.19 1.01 6.31 5.14 2.01 1.06 8.58 6.17

0.845 0.518 3.08 0.602 0.606 4.28 0.538 0.543 2.99 0.628 0.556 3.45

3.47 5.00 1.79 3.42

1 2 3

Average

1 2 3

Average

co**

Glycogen*

15 SUGAR- UPTAKE

10.

5.

* Micromoles of hexose carbon per g of tissue per 3 hours. I ,

FIG. 2. Additive metabolism of uniformly labeled fructose-C” and uniformly labeled glucose-04 by pooled epididymal adipose tissue of the rat. This figure shows the results of three experi- ments. The first two columns show the metabolism of uniformly labeled fructose-Cl4 and uniformly labeled glucose-04 when the labeled hexose is the only sugar present in the medium. The third column has been constructed by adding the results of the metabolism of uniformly labeled fructose-Cl4 (crosshatched verti- cally) in the presence of glucose to those obtained in the presence of uniformly labeled glucose-W (crosshatched horizontally) and unlabeled fructose. ml.

The hexose concentration was 260 mg per 100

The specific activity of the sugar in the incubation medium was obtained by osazone formation. Uniformly labeled sorbitol- Cl4 was converted to uniformly labeled fructose-04 by treatment with sorbitol dehydrogenase prepared from rat liver according to the method of Blakley (15), and its specific activity was ob- tamed after formation of the fructosazone. All the results in the tables and figures are expressed as micromoles of sugar 04 incorporated per g of wet tissue per total incubation period.

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November 1962 E. R. Froeach and J. L. GYmberg 3319

600 ‘6.0

MO, 6.0

400 6.0

300.30

30 so zm Fructae log.

6lo mgv.

FIG. 3. Net gas exchange of pooled epididymal adipose tiesue of the rat in the presence of glucose or fructoee alone a8 well a8 in the presence of both hexosea. The curve of the net gas ex- change in the preeence of 200 mg of glucose per 100 ml and 10 microunits of insulin per ml represent8 the mean obtained in 30 inaulin assay8 with pooled epididymal adipose tissue.

depresses fructose uptake (-15%). Indeed, the syntheses of fatty acids and of glycogen from fructose are actually enhanced in the presence of glucose. Glucose uptake is only slightly depressed by fructose (-12%). Fig. 2 shows clearly that fruc- tose and glucose metabolism of adipose tissue is additive rather than competitive. The total uptake of hexose when both sugars are present in the medium is nearly the sum of both when each is present alone. As a consequence of increased fatty acid synthesis

from glucose and fructose, the net gas exchange is stimulated well above the level observed with glucose alone, even when its concentration is raised (see Table I I) or when the glucose uptake is stimulated with small amounts of insulin (Fig. 3). The 0’ taken up by the tissue is distributed among the various metabolic moieties in the same proportion, regardless of whether it came from fructose or glucose. The total recovery of the CL’ taken up varies between 63 and 77%; 50% of the C?‘ is recovered in the total lipids. The fatty acids account for 30’%, the BaCO8 for 189& and the glycogen fraction for 1 .l %.

When the glucose concentration is raised to 500 and 1000 mg per 100 ml, with the fructose concentration remaining constant at 100 mg per 100 ml, the fructose uptake is not further depressed, and the distribution of the CY remains unchanged (Table II). The net gas exchange increases from 59 to 160 ~1 per g of tissue indicating that the total fatty acid synthesis from carbohydrate increases with the rising glucose concentration. Glucose uptake is depressed to a somewhat greater degree when the fructose concentration is raised in the same way, and the net gas exchange is more markedly stimulated, i .2. from 51 to 374 pl per g of tissue. Table III shows an experiment in which both hexoses were present in concentrations of 50, 200, and 800 mg per 100 ml; only one, however, carried the label. A measure of the sugar uptake was not attempted because, at high sugar concentrations, the percentage of the sugar taken up is too small to be deter- mined accurately. At 50 mg per 100 ml, the metabolism of fructose, computed from its oxidation to 001 and its incorpora- tion into the total lipids, is 47% that of glucose. At 200 mg per 100 ml, fructose metabolism almost equals glucose metab- olism (93%) and at a concentration of 800 mg per 100 ml, more fructose is metabolized than glucose (164%). The metabolism of both hexoses increases about linearly with the log of the sugar concentration, as shown more clearly in the case of fructose in Fig. 4. A 27-fold increase in the fructose concentration from 30 to 810 mg per 100 ml leads to an 8-fold rise of fructose metabolism.

As shown in Table IV, fructose uptake is almost doubled in the presence of 1090 microunits of insulin per ml, compared to a 3-fold increase of glucose uptake. The insulin effect on fructose uptake is entirely abolished, remaining at the baseline level in

TABLE II

Effect of increuaing concentrations of unlabeled glucose or fructose on melabolism of alfernate labeled ktose kept at constant ccmcenlraiion of pooled epididynal adipose tissue of 12 rata

Mean of the results of duplicate determination8 with their range.

Concentration of added hexosc

m;/100 ml

Unlabeled glucose added to uniformly labeled fruc- toee-04, 100 mg/lOO ml

100 500

loo0

Unlabeled fructose added tc uniformly labeled glucose- Cl’, 100 mg/lOO ml

100 500

1000

-

-

Y

1

-

Huose uptake

7.26 zk 0.36 8.53 zk 0.19 7.72 f 0.61

8.45 f 0.45 8.36 zk 1.19 6.44 i 0.61

59 f 14 120 f 10 160 f 12

511 8 290 f 10 374 f 32

CO¶ I

To-1 lipids I

Fatty acids

pmolw rugor carbon/l uwf lime/3 hours

1.30 * 0.10 1.29 f 0.02 1.35 f 0.02

1.71 f 0.01 1.26 * 0.01 1.26 f 0.01

2.22 f 0.14 2.22 l 0.04 2.20 f 0.07

2.72 zk 0.04 2.41 zk 0.16 2.30 f 0.08

1.08 zk 0.05 1.14 i 0.09 1.24 f 0.03

1.49 l 0.03 1.66 zk 0.07 1.45 f 0.03

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3320 Fructose Metabolism of Adipose Tissue. I Vol. 237, No. 11

TABLE III E$ect of increasing concentration8 of uniformly labeled fructose-0’ compared with that of increasing concentrations of uniformly

labeled gluco8e-C14 on metabolism of pooled epididynal adipose tissue of 12 rat8

Mean of the results of duplicate determinations with their range.

Concentration of hexose

rn~/loo ml

Unlabeled glucose + un formly labeled fructose-04

50 200 800

Unlabeled fructose + uni- formly labeled glucose-C*’

50 200 800

2

1

Irl g&g tissue

mo

-

-100 f 18 82f 4

362 f 14

-81 f 5 1OOf 4 3&l+ 0

-

--

‘5

-

CO2 Total lipids Fatty acids

pmoles sugar carbon/g wcf 1issue/3 hours

Glycogen

0.903 * 0.051 3.01 f 0.11 5.55 f 0.15

1.95 f 0.03 3.04 i 0.13 3.62 f 0.09

P GL’JCO!SE +FRKTGSE 2oOmg%wch

,A GLUCOSE +lOpU MULlt’Umt

50

0 L . 0 15 30 54 90 120 150 min.

Fra. 4. Linear relationship between the log of the fructose con- centration and the uptake and metabolism of uniformly labeled fructose-04 of pooled epididymal adipose tissue of 12 rats. Uni- formly labeled fructose-C’4 and unlabeled glucose were present in the medium in equal concentrat.ions. The incubation lasted 3 hours.

the presence of glucose (Table V). However, when the fructose and glucose concentrations are raised to 810 mg per 100 ml each and maximal insulin stimulation (10,000 microunits per ml) is induced, a significant inhibition of fructose metabolism to 36% of its value in the absence of insulin can be observed. The in- sulin effect on glucose metabolism is in no way altered by fruc- tose.

Addition of 3,5-ditrobenzoylglucosamine to the incubation medium leads to a 68% inhibitionof fructose uptake and metab- olism, whereas glucose uptake and metabolism arc inhibited somewhat less (38 ‘%) (Table VI).

Table VII shows that the utilization of uniformly labeled sorbitol-Cl4 by adipose tissue is very low (at most 10% of that of glucose). Glucose and fructose in the medium have no influence on sorbitol metabolism.

DISCUSSION AND COX!LUSIONS

At no concentration is there a significant inhibition of one hex- ose by the other even at concentrations as high as 1000 mg per 100 ml. If, as is probably the case, both hexoses are phosphoryl- ated by hexokinase, these results would indicate that under the experimental conditions used, in only one instance does phospho- rylation become the rate-limiting step. If glucose uptake be- comes very high as a consequence of high glucose concentrations and maximal insulin stimulation, fructose metabolism is inhibited. This observation may be interpreted in terms of an inhibition at the hexokinase level. Moreover, since it is generally agreed that in cell-free systems, the affinity of hexokinase from various tissues for glucose is at least 5 to 10 times greater than its affinity for fructose, these results would support the view that, under normal circumstances, free glucose is present in the fat cell only in small concentrations or not at all. All the glucose entering the fat cell seems to be readily phosphorylated so that no inhibition of fructose phosphorylation by free glucose can occur. Thus, it would seem that the sugar penetration into the cell is rate-limit- ing until a very rapid transfer has been reached. Morgan et al. (21) have recently shown that this is in fact the case in the heart muscle of the rat, in which no free intracellular glucose could be found, except when the glucose concentration in the medium exceeded 100 mg and large amounts of insulin were simultaneously present. These authors concluded that phos- phorylation becomes rate limiting only under these particular conditions.

The data presented seem to support the concept that with the In the case of the diaphragm, no glucose inhibition of fructose exception of liver, adipose tissue metabolizes more fructose than metabolism was observed (Table I) (22). However, fructose

1.29 i 0.10 4.53 i 0.18

10.63 f 0.90

2.68 f 0.03 5.03 f 0.16 6.22 f 0.02

0.395 f 0.036 3.09 f 0.10 8.27 f 0.42

1.39 i 0.03 3.38 f 0.08 4.78 i 0.10

0.019 i 0.602 0.142 f 0.032 0.325 f 0.035

0.095 l o.ou6 0.206 f 0.023 0.232 i 0.013

other tissues so far examined; tissues of the kidney (16, 17) and small intestine (18) may also be esceptions. The increase of fructose uptake and metabolism with rising sugar concentrations is much greater than that of glucose. The effect of rising glucose concentrations has been studied in greater detail by Jeanrenaud and Renold (19). Ball and Cooper (20) have made the observa- tion that by increasing the concentration of fructose in the medium the net gas exchange of adipose tissue is more markedly stimulated than when the glucose concentration is raised equally. We find that at the low sugar concentration of 50 mg per 100 ml, fructose metabolism proceeds at approximately one-half the rate of that of glucose. At 200 mg per 100 ml, both sugars are me- tabolized equally well, and at 806 mg per 100 ml, almost twice as much fructose is utilized as glucose. by guest on O

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November 1962 E. R. Froesch and J. L. Ginsberg

TABLE IV

Eflect of insulin on uniformly labeled fructose-W and uniformly labeled glucose-0’ metabolism of pooled epididymal adipose tissue of 18 rate

hlean of the results of duplicate determinations with their range.

3321

Net gas exchange I

Total lipids I

Fatty acids

Uniformly labeled glu- cose-C14, 200 mg/lOO ml

No insulin added.. Insulin, 1000 micro-

units/ml. . .

Uniformly labeled fruc- tose-W, 200 mg/lOO ml

No insulin added. . Insulin, 1000 micro-

units/ml. .

16.86 f 0.20

52.95 f 1.80

88fl

488 f 22 2.20 i 0.14 9.60 i 0.16

6.08 f 0.48 36.23 f 3.42

1.43 It 0.06

3.01 r 0.17

3.79 f 0.03

7.10 f 0.64

5.91 i 0.03

30.31 f 2.95

1.92 f 0.14

4.65 f 0.27

7.79 f 0.5i

15.11 f 0.38

17 f 5

166 i 7 - -

TABLE V

Effect of insulin on uniformly labeled fructose-O4 metabolism of pooled epididymal adipose tissue of 12 rats in presence of glucose

h1ean of the results of duplicate determinations with their range.

Fructose uptake I

Total lipids I

Fatty acids h-et gas exchange

Uniformly labeled fructose-W, 200 mg/lOO ml, in presence of glucose, 200 mg/lOO ml

No insulin added.. . . . . . Insulm, miorounits/ml . . .

Uniformly labeled fructose-C”, 810 mg/lOO ml, in presence of glucose, 810 mg/lOO ml

No insulin added.. . . . . . . Insulin, 10,000 microunits/ml.. .

8.50 f 0.56 7.16 i 0.23

75 i 17 444 32 20.0

1.88 f 0.03 1.27 i 0.02

4.28 f 0.02 4.19 f 0.14

2.53 f 0.06 3.20 f 0.15

438 f 18 1120 f 45

3.42 f 0.22 0.935 f 0.012

6.53 f 0.19 2.66 i 0.03

5.86 f 0.52 2.41 f 0.12

-

TABLE VI

Effect of 3,&dinitrobenzoylglucosamine on uniformly labeled fructose-c” and uniformly labeled glucose-04 metabolism of pooled

epididymal adipose tissue of id rats

Mean of the results of duplicate determinations with their range.

Herose uptake

rmolcs:fowe~slslirsuc/3

Uniformly labeled fructose-Cl’, 100 mg/lOO ml

No addition......................... 6.36 i 0.18 Glucose, 100 mg/lOO ml.. . 5.78 i 0.17 Glucose + 3,5-dinitrobenzoylglucos-

amine, each 100 mg/100 ml. . . . . . 2.04 f 0.04

COY

1.37 f 0.18 1.23 f 0.04

0.442 f 0.081

I Total lipids

I Fatty acids

I Glycogen

1.17 f 0.10 1.69 f 0.10

0.344 f 0.011

3.24 f 0.13 2.63 f 0.09

0.887 f 0.019

0.042 f 0.001 0.063 * 0.003

0.012 * 0.001

Uniformly labeled glucose-C*‘, 100 mg/lOO ml

No addition......................... 9.55 f 0.11 Fructose, 100 mg/lOO ml.. . . . 9.63 f 0.28 Fructose + 3,5-dinitrobenzoylglu-

cosamine, each 100 mg/lOO ml.. . . . 5.96 f 0.12

1.72 f 0.14 1.78 f 0.16

1.53 f 0.04

4.27 l 0.11 4.18 f 0.03

2.48 f 0.03

2.45 f 0.03 2.63 f 0.20

1.10 f 0.10

0.111 f 0.015 0.108 f 0.001

0.062 f 0.004

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3322 Fructose Metubolim of Adipose Tissue. I Vol. 237, No. 11

TABLE VII Metabolism of uniformly Labeled aorbitol-0’ by pooled epididymal adipose tissue of 1.8

rats in absence and presence of fructose and glucose

Mean of the results of duplicate determinations with their range.

Uniformly labeled sorbitol-Cl‘, 166 mg/ml No additions ...................................... Insulin, loo0 microunits/ml.. ...................... Glucose, 160 mg/lOO ml ............................ Fructose, 100 mg/160 ml ...........................

Uniformly labeled glucose-Cl‘, 166 mg/lOO ml No additions ...................................... Sorbitol, 166 mg/166 ml. ..........................

-264&S -257-1:m -31 f20 -68 *3

-32 f24 -36 fl

COl I Total lipids

0.135 f 0.011 0.339 f 0.013 0.242 * 0.009 0.492 f 0.022 0.113 f 0.006 0.222 f 0.018 0.166 f 0.966 0.340 * 0.030

2.86 f 0.00 3.92 f 0.07 2.33 f 0.24 3.56 f 0.28

metabolism in this tissue proceeds only at approximately one- fifth of the rate of glucose metabolism (Table I) (4). It would therefore seem, as has been shown in the case of the heart muscle and as we have suggested for adipose tissue, that the transfer of these two hexoses into the cell is also the limiting factor for the diaphragm. The main difference between fructose, metabolism of adipose tissue and that of the diaphragm would then be simply one of the rate at which fructose is entering the cell.

The results with adipose tissue contrast with those obtained in the case of leukocytes and erythrocytes which metabolize fructose readily when glucose is absent, while their fructose utilization is almost completely suppressed in the presence of glucose. Glucose and fructose are known to be present in the free form in these cells, and glucose inhibits fructose phosphoryl- ation most probably because hexokinase is saturated with glucose.

Park et al. (23) have noted that rabbit erythrocytes take up fructose faster than glucose and independently of it, whereas competition between these hexoses for a common transport mechanism has been demonstrated in the case of erythrocytes from other species (24,25) and in the case of ascites tumor cells (26).

On a Lineweaver-Burk plot, the data shown in Table 3 and Fig. 4 yield a straight line with a positive ordinate intercept. The K, of the hexose transport deduced from their metabolism is of the order of magnitude of 2 to 4 x 101 M for fructose and 3 to 4 x 10-* M for glucose, which is close to that found in human and rabbit erythrocytes (23, 27-29). The maximal rate of transport into the adipose tissue cell is three times greater for fructose (30 pmoles per g per 3 hours) than for glucose (10 pmoles per g per 3 hours). These deductions are valid only under the assumption that, under the conditions used, the entry of hexose into the adipose tissue cell is the factor which is limiting for its metabolism.

Fructose uptake and metabolism by adipose tissue is stimu- lated by insulin only in the absence of glucose. When glucose and insulin are present, fructose uptake remains at the baseline level but is not suppressed. Thus, it would seem that fructose enters the adipose tissue cell in two ways, one of which is inde- nendent of glucose and insulin and the other of which is insulin- dependent and subject to glucose inhibition. The insulin- activated transport site seems to have a much higher ffiity for glucose than for fructose. The data presented do not permit

any conclusion as to whether the insulin-activated transport site is identical with the normal glucose transport site or not.

The metabolism of glucose and mannose by adipose tissue has been compared by Wood et al. (30) with strikingly different results. These two hexoses behave in a strictly competitive manner, both seeming to have a similar afhnity for the transfer mechanism with and without insulin stimulation as well as for the phosphorylation process.

No definite conclusions have, as yet, been reached with regard to the mechanisms involved in the further metabolism of fructose in adipose tissue. Several observations favor the hypothesis that fructose is first phosphorylated to fructose g-phosphate by hexokinase. We have observed (Table VI) that 3,5-dinitro- benzoylglucosamine, a competitive inhibitor of hexokinase, inhibits both glucose and fructose phosphorylation in adipose tissue. Fructose metabolism is inhibited somewhat more than glucose metabolism, a finding compatible with the greater afhnity of hexokinase for glucose than for fructose. Hernandez and Sols (31) have successfully isolated hexokinase from adipose tissue and measured its activity. These authors found that the affinity of the adipose tissue hexokinase for glucose is at least 10 times greater than for fructose. They looked for but did not detect any fructokinase activity in adipose tissue. Experiments that we will report at a later date also make it unlikely that fructose is phosphorylated to fructose l-phosphate by a fructo- kinase similar to that present in liver (32). No enzyme attack on fructose l-phosphate as in liver (33, 34) was detected in adipose tissue homogenates whereas fructose 1,6-disphosphate was cleaved readily by this tissue as by others. Furthermore, preliminary results obtained in our laboratory indicate that fructose specifically labeled on the first carbon atom is almost exclusively incorporated into position 1 of the glucose constit- uents of glycogen formed in adipose tissue. This finding may be considered as a proof that the fructose molecule enters the glycolytic pathway as the intact C-6 chain molecule, for if it were cleaved into two triose molecules, the 04 would have been randomly distributed in positions 1 and 6. Moreover, the very similar distribution of the CY of uniformly labeled glucose and fructose among the metabolic products makes it likely that both hexoses enter the glycolytic scheme approximately at the same level.

The possibility of the transformation of fructose into glucose

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or some other sugar before phosphorylation occurs must be con- glucose. In the presence of 206 mg per 100 ml of glucose and sidered. In the accessory glands of the male reproductive tract 1660 microunita of insulin per ml, the fructose uptake remains and in the placenta of certain animal species, glucose is trans- at the baseline level. When the glucose concentration is raised formed first to sorbitol and then to fructose (35). Since the same to 810 mg per 100 ml and maximal insulin stimulation is induced, reactions might occur in adipose tissue in the opposite direction, the fructose metabolism of adipose tissue is depressed. we have looked for the two enzymes involved in these reactions. 4. 3,5-Dinitrobenzoylglucosamine, a competitive inhibitor Whereas a small activity of sorbitol dehydrogenase of question- of hexokinase, inbibits fructose metabolism to a somewhat able significance was observable in adipose tissue homogenates, greater degree than glucose metabolism. no aldose reductase activity could be found. Furthermore, 5. Adipose tissue metabolizes sorbitol only poorly. uniformly labeled sorbitol-Cl4 is only poorly metabolized by 6. These findings are discussed with particular emphasis on intact adipose tissue (Table VII). the mechanism of entry of fructose into the adipose tissue cell

All these findings make it likely that the entrance of fructose and on the metabolic pathways of fructose in this tissue. into the glycolytic scheme involves phosphorylation of fructose to fructose 6-phosphate by hexokinase. The observation that glucose does not interfere with fructose phosphorylation despite

Acknowledgmda-We thank Miss E. Ettinger and Miss S.

its much higher afhnity for hexokinase may then be explained Diem for their expert technical assistance, Dr. P. Bally, Professor A. Labhart, and Professor F. Leuthardt for their valuable advice

with the hypothesis suggested above. As in the case of the heart muscle (21), free glucose would not be present in the cells

and criticism, and Mrs. M. R. Moore for her help with the prepa-

in any appreciable concentration. We would then expect ration of the manuscript.

fructose phosphorylation to be hampered only when the glucose REFERENCES uptake by the cell exceeds glucose phosphorylation, leading to an intracellular accumulation of free glucose. Our finding that

1. FROESCH, E. R., PRADEB, A., WOLF, H. P., AND LABHART, A.,

fructose metabolism is suppressed only when glucose uptake Helv. Paed. Acta, 14, 99 (1959).

becomes maximal in the presence of high glucose concentrations 2. FROESCH, E. R., WOLF, H. P., BAIT~~H, H., PRADER, A., AND

LABHART, A., Am. J. Med., in press. and massive insulin stimulation indeed supports this hypothesis. 3. RENOLD, A. E., AND THORN, G. W., Am. J. Med., 19, 163

The e.xperiments presented indicate that the distribution of (1955).

an increased carbohydrate load among the various metabolic 4. WICK, A. N., SHERILL, J. W., AND DEURY, D. R., Diabetes,

products is the same whether it is brought about by insulin 2, 465 (1953).

5. MENDELOFF, A. I., AND WEICHSELBAUM, T. E., Metabolism, 2, stimulation or by the simultaneous uptake of glucose and fruc- 456 (1953).

tose. It would therefore seem that, under the experimental 6. BOLLMAN, J. L., AND MANN, F. C., Am. J. Physiol., 96, 683

conditions used, the main action of insulin is the acceleration of (1931).

the transfer of glucose into the adipose tissue cell, thus making 7. RENOLD, A. E., HASTIIUX, -4. B., AND NESBE~, F. B., J.

Biol. Chem., 109, 687 (1954). it available for phosphorylation. When the hexose load in- 8. FROE~CH, E. R., GINSBERG, J., AND SEMENZA, G., in Fourth

creases, be it due to insulin or to the simultaneous presence of International Congress of the Znternutional Diabetes Federa-

glucose and fructose, the fatty acid synthesis from carbohydrate tion, Medecine et Hygiene, Geneva, 1962, p. 607.

is increased out of proportion to the incorporation of hexose 9. MALEY, F., AND LARDY, H. A., J. Biol. Chem., 214.765 (1955).

carbon into CO* or into the glyceride-glycerol fraction. De- 10. UYBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., Mano-

metric techniques and related methods for the study of tiesue pending on the concentration of fructose in the medium, the metabolism, 3rd edition, Burgess Publishing Co., Minneapo-

fatty acid synthesis from fructose reaches levels observed with lis, 1957, p. 194.

glucose only under marked insulin stimulation. It will be of 11. SKOOG, W. A., AND BECK, W. S., Blood, 11,436 (1956).

practical interest to know whether adipose tissue from diabetic 12. FROEBCH, E. R., REARDON, J. B., AND RENOLD, A. E., J. Lab.

Clin. Med., 50, 918 (1957). rats behaves in the same manner. If it does, fructose in large 13. HIGASHI, A., AND PETERS, L. J., J. Lab. Clin. Med., 36, 475

quantities might be given a trial in the treatment of diabetic (1950).

ketosis, since this hexose might alleviate some of the major 14. FOLCA, J., LEES, M., AND SLOANE STANLEY, G. H., J. Biol.

metabolic disturbances of adipose tissue metabolism that lead Chem., 336, 497 (1957).

to diabetic ketosis. We are now investigating fructose metabo- 15. BLAKLEY, R. L., Biochem. J., 49, 257 (1951). 16. REINECKE, R. M., Am. J. Physiol., 141,669 (1944).

lism of diabetic adipose tissue. 17. GODA, T., Biochem. Z., 297, 134 (1938). 18. GINSBURG, C., AND HERS, H. G., Biochim. et Biophys. Acta,

SUMMARY 38. 427 (1960).

1. Adipose tissue metabolizes fructose nearly as actively as 19. JEA~RENA~D, b., AND REXOLD, A. E., J. Biol. Chem.. !&4. , .

glucose and independently of it. Fructose is metabolized more 3082 (1959).

20. BALL, E. G., AND COOPER, O., J. Biol. Chem., !&%, 584 (1960). slowly than glucose at low sugar concentrations but faster than 21. MORGAN, H. E., HENDERSON, M. J., REGEN, D. M., AND

glucose at high concentrations. PARK, C. R., J. Biol. Chem., 236. 253 (1961).

2. In the presence of both hexoses, fatty acid synthesis is 22. MACKLER, B., AND GUEST, G. M., Proc. Sot. Ezptl. Biol.

stimulated above the level expected from incubation experiments Med.. 83. 327 (1953).

with each hexose alone. An increased carbohydrate load due to 23. PARK, e. R., POST, k. L., KAL~IAN, C. F., WRIGHT, J. H., JR.,

JOHNSON, L. H., AND MORGAN, H. E., in C. W. E. WOLSTEN-

the simultaneous presence of fructose and glucose in the medium HOLME AND J. S. FREEMAN (Editors). Ciba Foundation

is utilized by normal adipose tissue in the same manner as an Colloquia on Endocrinology, Vol: 9, Littie, Brown &d Com-

increased carbohydrate load due to insulin stimulation. pany, Boston, 1956, p. 240.

3. Insulin stimulates fructose untake onlv in the absence of 24. LEFEVRE, P. G., AND DAVIES, R. I., J. Gen. Physiol., 34,

I 515 (1951).

November 1962 E. R. Frotxh and J. L. Gin&erg 3323

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3324 Fructose Metabolism of Adipose Tissue. I Vol. 237, No. 11

25. WIDDAS, W. F., J. Phyuiol., 125, 163 (1954). 31. HEBNANDEZ, A., AND SOLS, A., Communication auz VZ&mes 26. NIBENBEBG, M. W., AND HOQG, J. F., J. Am. Chem. Sot., 80, Joumbea Biochimiquea L-atines, Geneva, May, lsS1.

4407 (1958). 32. LEUTEABDT, F., AND TESTA, E., Helv. Chim. Act+ 33. 1919 27. LEFEVR~, P. G., Symposia of the Society for Exptl. Biology, (1950).

Vol. 8, Academic Press, Inc., New York, 1954, p. 118. 33. HEW, H. G., AND KUSAKA, I., Biochim. et Biophya. Acta, 11, 28. WIDDAS, W. F., J. Physiol., UO, 23P (1953). 427 (1953). 29. WILBBANDT, W., Modern Problems Paediat., 4, 30 (1969). 34. LEUTEABDT, F., TESTA, E., AND WOLF, H. P., Helv. Chim. 30. WOOD, F. C., JR., LEBOEUF, B., RENOLD, A. E., AND CAHILL, Acta, 38, 227 (1952).

G. F., J. Biol. Chem., 2%. 18 (1961). 35. HERS, H. G., Biochim. et Biophys. Acta, 37, 120, 127 (1960).

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E. R. Froesch and J. L. GinsbergNORMAL RATS

AND GLUCOSE METABOLISM IN EPIDIDYMAL ADIPOSE TISSUE OF Fructose Metabolism of Adipose Tissue: I. COMPARISON OF FRUCTOSE

1962, 237:3317-3324.J. Biol. Chem. 

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