EFFECT OF FRUCTOSE ON LIPOGENESIS FROM LACTATE AND ACETATE IN DIABETIC LIVER*

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1 EFFECT OF FRUCTOSE ON LIPOGENESIS FROM LACTATE AND ACETATE IN DIABETIC LIVER* BY N. BAKER, I. L. CHAIKOFF, AND A. SCHUSDEK (From the Division of Physiology of the University of California School of Medicine, Berkeley, California) (Received for publication, August 18, 1951) Defective glucose utilization in the liver of the glucosefed diabetic rat has been stated to be the result of at least two blocks: one, at an early stage in glycolysis, probably hexokinase; the other, in the synthesis of fatty acids from the Zcarbonlike intermediate. This conclusion was based on the following observations: (1) the conversion of C14glucose to CO2 was depressed in the liver of the alloxandiabetic rat, whereas the rate of CY402 formation from C14fructose proceeds at normal rates (1) ; (2) the incorporation of C14labeled glucose, fructose, lactate, and acetate into fatty acids by diabetic hepatic tissue was markedly depressed (l3) ; and (3) the liver of the alloxandiabetic rat treated with large doses of insulin for 2 to 3 days showed an extraordinary increase in its capacity to synthesize fatty acids from glucose, lactate, and acetate (3, 4). The above experiments were carried out with livers of rats that had been fed a 58 per cent glucose diet. The finding that fructose is more readily oxidized to CO2 than is glucose by the diabetic liver led us to study the effect of previous fructose feeding on hepatic lipogenesis. The incorporation of lactate2cl4 and acetatel,2cl4 into fatty acids was compared in livers of two groups of diabetic rats. One group was fed a diet containing 58 per cent fructose for 4 days before being sacrificed, the other a diet containing 58 per cent glucose for the same number of days. The fructose feeding resulted in a pronounced increase in the capacity of the diabetic rat liver to convert lactate and acetate to fatty acids, an increase, it should be noted, that occurred without the administration of insulin. This fructose feeding did not repair the defective utilization of CY4glucose in the diabetic liver. Our findings thus suggest that impaired lipogenesis from the 2carbonlike intermediate in the liver of the glucosefed alloxandiabetic rat need not be the result of insulin lack per se, but may be secondary to the initial or primary block in glucose utilization. EXPERIMENTAL Treatment of RatsNormal rats of the LongEvans strain were fasted for 18 to 24 hours and then fed ad l&turn for 4 days either the glucose or * Aided by grants from the Corn Industries Research Foundation and Eli Lilly and Company. 435

2 436 EFFECT OF FRUCTOSE ON LIPOGENESIS fructosecontaining diet shown in Table I. This preliminary fasting served to deplete liver glycogen and to stimulate the rats appetites for the high carbohydrate diet. Diabetes was induced in rats by the injection of alloxan monohydrate (Eastman Kodak). The rats that showed a marked polyuria were placed in individual metabolism cages, and their food and water intake, urine volume, and glucose excretion were measured at intervals. The diabetes in the rats was of long standing (Table II), and fasting blood sugars in excess of 200 mg. per cent were observed in all of them about 1 or 2 weeks before their sacrifice. They were fasted for 18 hours and then fed either Diet G or F for 4 days before their livers were excised. Radioactive SubstratesAcetate doubly labeled with Cl* was prepared Diet Glucose Fructose Casein TABLE Per Cent Composition of Diets I sa1tt Cellu flow Liver Vitamin mixture VioBin mixture$ G8 58 None F None * Labco, vitaminfree. t HawkOser mixture (18). $ Each 100 gm. of diet contained 100 mg. of choline chloride, 5 mg. of niacinamide, 100 mg. of inositol, 5 mg. of calcium pantothenate, 1 mg. of thiamine hydrochloride, 1 mg. of riboflavin, 1 mg. of pyridoxine, 1 mg. of paminobenzoic acid, 0.02 mg. of biotin, and 0.2 mg. of folic acid. $ Each diet was supplemented with sardaline (a fish oil containing vitamins A and D) every other day. by the method of Barker and Kamen (5). We are indebted to Dr. B. M. Tolbert for the preparation of Zn lactate2c?*. It was converted to the sodium salt as described by Felts et al. (3). The Cl* content of these two substrates was determined by mounting their sodium salts with essentially zero mass directly on an aluminum disk. The method used to convert counts so obtained to BaCOsfilter paper values has been described elsewhere (6). Radioactive glucose labeled with Cl4 in all of its carbons was made photosynthetically as described by Putman et al. (7). The Cl4 content of this glucose was determined by oxidizing it to CO2 and converting the latter to BaC03, which was then mounted on filter paper (6). The radioactivity of the BaC03 was measured with a thin window GeigerMiiller tube. Incubation ProcedureLiver slices, approximately 0.5 mm. in thickness, were prepared and collected in a Petri dish containing an icecold bicar

3 N. BAKER, I. L. CHAIKOFF, AND A. SCHUSDEK 437 bonate buffer. Approximately 500 mg. of slices were gently blotted on filter paper, weighed, and placed in the main compartment of a flask (8) which contained 4.5 cc. of a bicarbonate buffer (9) and 0.5 cc. of the substrate solution. The concentrations of the labeled substrates used in the experiments are recorded in Tables IV and V. Determination of C1402 and Fatty AcidClThe collection of total CO2 Rat No: Dl D2 D3 D4 D5 D6 D7 D8 DQ DlO Dll D12 D13 D14 TABLE History of AlloxanDiabetic Rats Used in This Study Rat weight Sex hen in When jetted crificed M. I F. M. F. M. F. M. F. gm. Lm. I DUG+ tion of iabetest II Iaily food intaketg (rawe) (2 ) Highest,ine sugar r or 24 hr. SkUllpIe UI f sm. cc. gm my; Blood 3llgar at time of iacrificell * Rats Dl to D7, glucosefed; Rats D8 to D14, fructosefed. t Alloxan was administered intravenously (50 mg. per kilo) except to Rats D6, D7, D13, and D14. The latter received subcutaneous injections (100 to 180 mg. per kilo). $ Determined 1 to 3 weeks before sacrifice. $ Stock diet prior to initiation of high carbohydrate diet. 11 Whole blood analyzed at the end of the experiment and determination of its Cl4 content were carried out as described elsewhere (6). Total lipides were isolated from an alkaline hydrolysate of the entire flask contents. Cholesterol was precipitated with digitonin, and the fatty acids were separated as described previously (3). An aliquot of the fatty acids was oxidized to COZ, and the Cl4 activity of the latter was measured in the form of BaC03. Glycogen Determination500 mg. portions of liver slices were taken from the pool in the Petri dish (see above) and hydrolyzed in hot 30 per cent KOH for 30 minutes. An interval of about 20 minutes elapsed between

4 438 EFFECT OF FRUCTOSE ON LIPOGENESIS the time the rats were sacrificed and the time the liver samples were placed in the KOH solution. Glycogen was isolated by the method of Good et al. (10). It was hydrolyzed to glucose in the presence of 5 N HzS04 as outlined by Sjogren et al. (II), the mixture obtained was deproteinated according to Somogyi (12), and the reducing value of the supernatant was determined by Hassid s ceric sulfateferricyanide method (13). Determination of Total Carbohydrate Content of Liver Slices before IncubationApproximately 500 mg. of liver slices were macerated and heated with 5 cc. of 1 N HCl for 3 hours on a steam bath. The acid hydrolysate was neutralized with a Na2C03 solution, filtered, and made to volume. An aliquot was deproteinated and its reducing value determined as described above. Results Twentythree rats were used, nine normal and fourteen diabetic. They were fed either the 58 per cent fructose or 58 per cent glucose diet for 4 days. The average amount consumed daily by each rat is recorded in Table III. As was to be expected, some variation in the amounts of food ingested by the rats of each group was found, but neither the normal nor the diabetic rats showed a preference for either of the diets. The relative amounts of glycogen deposited in the livers of animals fed glucose and fructose have been repeatedly studied and it appears to be the consensus of opinion that fructose feeding results in greater hepatic glycogenesis than does the feeding of glucose (14). This is borne out by the values for liver glycogen shown in Table III. It should be noted that glycogen was determined on liver samples taken about 20 minutes after the animals were sacrificed. We have confirmed Barfod s finding (15) that glycogen values at this time are about 20 per cent lower than at the time of sacrifice. Experiments with LactateZCl4 and Acetatel,2Cl4 (Table IV) Group 1. Normal Rats Prefed Diet GThe livers of these rats converted from 9.5 to 18 per cent of the lactate2cl4 and from 16 to 21 per cent of the added acetatecl4 to COZ. The fatty acidu4 recoveries for these two substrates ranged from 2.5 to 4.5, and from 3.1 to 7.8 per cent, respectively. Group 2. Normal Rats Prefed Diet FThe Cl402 values were well within the range of those observed with the livers of normal rats fed the high glucose diet. Some of the fatty acidcl4 recoveries were also in the same range, but others were higher. Group 3. Diabetic Rats Prefed Diet GThe incorporation of Cl4 from the lactate and the acetate into fatty acids was greatly depressed, whereas

5 N. BAKER, I. I.. CHAIKOFF, AND A. SCHUSDEK 439 TABLE Carbohydrate and Fatty Acid Contents of Liver from Normal and Diabetic Rats Fed Glucose and Fructose Diets Rat No. Weightt Diet fed previous 4 days I Average daily intake.iver III weighl Composition of liver, per cent wet weight er cent of,dy wei6 :ht Total Total carbo ilycogen fatty t iydrate acids m. Nl 196 N2 210 N3 208 N4 233 N5 236 m. G I 19 ~~ F I gm Average N6 225 N7 192 N8 198 N Average DlS G I D2S D3 I D D5 I D6 I D Average D8$ F D% DlO Dll D D D Average * N, normal; D, diabetic. t Weight of animals at time of sacrifice; see Table II for weights of diabetic rats. $ Lipogenic studies with labeled isotopes not performed on these animals.

6 440 EFFECT OF FRUCTOSE ON LIPOGENESIS the Cr402 values were well within the normal range. Thus lhe results obtained with the livers of these rats are in complete agreement with those reported earlier (1, 3). Group 4. Diabetic Rats Prefed Diet FFructose feeding restored to the diabetic liver the capacity to convert lactate and acetate to fatty acids. The liver slices of diabetic rats that had received the 58 per cent fructose TABLE Conversion of LactateC and AcetateCl4 to Fatty Acids by Livers oj Diabetic and Normal Rats Previously Fed Diets* Containing Either 68 Per Cent Fructose or 58 Per Cent Glucose 500 mg. of liver slices were incubated for 3 hours at 37.5 with 50 FM of either substrate. The gas phase consisted of 95 per cent 02 and 5 per cent COz. The added acetatep contained a total of 10 to 20 X lo4 c.p.m., whereas the lactatecl4 had a total activity of approximately 30 X lo4 c.p.m. Duplicate flasks were incubated and the contents analyzed separately; average values are reported below. Rat No. Nl N2 N3 N4 N5 N6 N7 NS D5 D6 D7 D12 D13 D14 Diet fed previous 4 days G F G 6 F IV Per cent of lactatezc 4 recovered per 500 mg. liver slices ss 3 cent of acetate1,2w *CxoYered per 500 mg. liver slices as Fatty acids co, Fatty acids cot * The constituents of the diets are given in Table I. diet for 4 days incorporated, in 3 hours, from 2.5 to 3.4 per cent of the lactatecl4 and from 2.7 to 4.7 per cent of the acetatec4 into fatty acids. Experiments with F4Glucose (Table V) The livers of normal rats fed Diet G for 4 days before being sacrificed oxidized 4 to 5 per cent of the added glucosecl4 to CO2 and incorporated from 0.3 to 0.4 per cent of it into fatty acids (Table J). The oxidation of glucosec4 to CO2 was markedly reduced in the experiments with diabetic liver slices, and this was the case regardless of whether

7 N. BAKER, I. L. CHAIKOFF, AND A. SCHUSDEK 441 the preceding diet was 58 per cent glucose or 58 per cent fructose. The liver of the glucosefed, diabetic rat lost almost completely its capacity to synthesize fatty acids from added glucose, and previous fructose feeding failed to correct this defect. TABLE Conversion of Glucose04 to CO2 and Fatty Acids by Livers of Diabetic and Normal Rats Previously Fed Diets* Containing Either 58 Per Cent Fructose or 58 Per Cent Glucose 500 mg. of liver slices were incubated at 37.5 for 3 hours with 400 mg. per cent of glucose. The gas phase consisted of 95 per cent O2 and 5 per cent COz. The total activity added to each flask was approximately 70 X lo3 c.p.m. Rat No. Diet fed previous 4 days N3 N4 N5 D3 D4 D7 DlO Dll D14 * The constituents of the diets are given in Table I. G F I DISCUSSION V Per cent of glucosec 4 per 500 mg. liver recovered slices CO8 Fatty acids A major defect in the liver of the glucosefed, diabetic rat is a reduction in its capacity to synthesize fatty acids at the expense of 2carbonlike intermediates (l3). It is shown here that the previous feeding of diabetic rats with a 58 per cent fructose diet yielded livers whose capacities to convert CY4lactate to fatty acids were practically the same as those of normal rats fed a 58 per cent glucose diet. In order to interpret this finding, we at first considered the possibility that fructose stimulated preferentially the secretion of insulin from a small remnant of islet tissue still present in the pancreas of the alloxaninjected rats. Such a possibility, however, seems to be ruled out by the failure of fructose feeding to repair defective utilization of CY4glucose in the diabetic liver. Moreover, the blood sugar values of the rats fed Diets G and F were about the same.l Defective lipogenesis in the diabetic liver can be repaired in two ways: by insulin injections, or by fructose feeding. But the actions of the two 1 Practically no free fructose was found in the plasma of the fructosefed rats (17).

8 442 EFFECT OF FRUCTOSE ON LIPOGENESIS treatments upon lipogenesis are not identical. The hormone repairs lipogenesis not only from acetate, lactate (3), and pyruvate (16), but also from glucose (4). Fructose feeding, on the other hand, repairs lipogenesis from the 2carbonlike intermediate but not from glucose. This difference indicates that the feeding of fructose, by circumventing the initial block in glucose utilization, provides a continuous source of substrates in the glycolytic system. It is possible that a high activity in this system is required for maintenance of lipogenesis in the liver. Support for such a view is found in the rapid decrease in lipogenesis from glucose and acetate2 that occurs in the liver of the fasted rat and in the rat fed a diet totally devoid of carbohydrate. Since insulin increases glucose oxidation in both normal and diabetic livers (4), the action of this hormone in promoting lipogenesis from the C2 unit may, in part at least, be the result of an increased glycolytic activity associated with augmented glucose utilization. The results of the present study bring to light an interesting relationship between diet, enzymatic activity, and a hormone. The surprising response of the diabetic to the mere feeding of fructose suggests that the liver is dependent for fatty acid synthesis upon previous3 activity in the glycolytic system. The repair, by fructose feeding per se, of a metabolic defect hitherto ascribed to an insulin lack throws new light on the lipogenic defect in the diabetic liver. That a mechanism involving adaptation of lipogenie enzymes is concerned with this repair is, of course, one interesting possibility. Experiments designed to elucidate this fructose effect are in progress. SUMMARY 1. Lipogenesis from acetate doubly labeled with CY4, lactate2c14, and CY4glucose was compared in the livers of normal and alloxandiabetic rats that were fed, for 4 days, a diet containing either 58 per cent glucose or 58 per cent fructose. 2. In glucosefed, diabetic rats, hepatic lipogenesis from acetate and lactate was greatly depressed, in some cases to onetenth the normal value. The livers of these rats also lost, almost completely, their capacity to convert glucose to fatty acids. 3. Fructose feeding restored to the diabetic liver its capacity to synthesize fatty acids from acetate and lactate. The fatty acido4 recoveries in the experiments with fructosefed, diabetic rats were in the same range as those observed in the experiments with glucosefed, normal rats. * Unpublished observat.ions of I. Lyon in this laboratory. 3 The mere addition of 400 mg. per cent fructose to the medium does not restore lipogenesis from acetate in liver slices obtained from diabetic rats previously fed the 58 per cent glucose diet (1).

9 N. BAKER, I. L. CHAIKOFF, AND A. SCHUSDEK Fructose feeding failed to correct the defective hepatic lipogenesis from CY4glucose in the diabetic rat. This is in contrast to the action of insulin which has been shown to restore to the diabetic liver its capacity to form fatty acids from glucose as well as from acetate and lactate. 5. Possible mechanisms by which fructose repairs lipogenesis in the diabetic liver are discussed. BIBLIOGRAPW 1. Chernick, S. S., and Chaikoff, I. L., J. Biol. Chem., 188, 389 (1951). 2. Brady, R. O., and Gurin, S., J. Biol. Chem., 187,589 (1959). 3. Felts, J. M., Chaikoff, I. L., and Osborn, M. J., J. Biol. Chem., 193, 557 (1951). 4. Chernick, S. S., and Chaikoff, I. L., J. Biol. Chem., 186, 535 (1959). 5. Barker, H. A., and Kamen, M. D., Proc. Nat. Acad. SC., 31, 219 (1945). 6. Entenman, C., Lerner, S. R., Chaikoff, I. L., and Dauben, W. G., Proc. Sot. Exp. BioZ. and Med., 70, 364 (1949). 7. Putman, E. W., Hassid, W. Z., Krotkov, G., and Barker, H. A., J. BioZ. Chem., 173, 785 (1948). 8. Chernick, S. S., Masoro, E. J., and Chaikoff, I. L., Proc. Sot. Exp. BioZ. and Med., 73, 348 (1950). 9. Krebs, H. A., and Henseleit, K., 2. physiol. Chem., 210, 33 (1932). 10. Good, C. A., Kramer, H., and Somogyi, M., J. BioZ. Chem., 166, 485 (1933). 11. Sjijgren, B., Nordenskjold, T., Holmgren, H., and Mollerstrom, J., Arch. ges. Physiol., 240, 427 (1938). 12. Somogyi, M., J. BioZ. Chem., 86, 655 (1930). 13. Hassid, W. Z., Ind. and Eng. Chem., Anal. Ed., 8, 138 (1936). 14. Deuel, H. J., Jr., Physiol. Rev., 16, 173 (1936). 15. Barfod, B., Acta physiol. &and., 16, 110 (1948). 16. Osborn, M. J., Chaikoff, I. L., and Felts, J. M., J. BioZ. Chem., 193, 549 (1951). 17. Roe, J. H., J. BioZ. Chem., 107, 15 (1934). 18. Hawk, P. B., Oser, B. L., and Summerson, W. H., Practical physiological chemistry, Philadelphia, 12th edition (1947).

10 EFFECT OF FRUCTOSE ON LIPOGENESIS FROM LACTATE AND ACETATE IN DIABETIC LIVER N. Baker, I. L. Chaikoff and A. Schusdek J. Biol. Chem. 1952, 194: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#reflist1