(Department of Pharmacology, Indiana University School of Medicine, Indianapolis, Ind.; andLaboratory of Biochemistry, National Cancer Institute, X.I.H., P.H.S., Bethesda, Md.)
SUMMARY
The metabolic fate of labeled fructose and glucose was compared in normal liverand in hepatomas of different growth rates. For certain biochemical parameters arough correlation with the growth rates of liver tumors was observed.
The metabolic disposition of both fructose and glucose was in the same range in thecontrol tissues, and no strain difference could be discerned.
The fructose uptake was decreased to 48 per cent or less in all hepatomas. The CÛ2production was decreased to 67 per cent or less. Very little glycogen synthesis fromfructose occurred in the slowly growing tumors; however, incorporation was normalor increased in the rapidly growing ones. There was a tendency to decreased glucoseproduction, and all glucose release values were 11 per cent or less of those of the normal.The initial glycogen levels in all tumors were markedly less than those observed inlivers of control animals. Lactate production was less than normal in the slowly growing tumors, with normal or slightly increased values in the rapidly growing ones.
The maximum glucose phosphorylation was decreased in the examined tumors, withthe exception of H-35. The oxidation of C-l as well as C-6 of glucose showed a tendencyto increase parallel with the increase in growth rate of the hepatomas. The C-l/C-6ratio in CÜ2was in the normal range in the slowly growing tumors but was increasedin the rapidly growing hepatomas. The glucose to glycogen ratio also increased withthe increased growth rate. The net lactate production observed with glucose presentas added substrate was normal for H-35, but in the more rapidly growing hepatomaswas markedly increased roughly parallel with the increasing growth rate of the tumors.
Preceding communications in this series exam- suggested that the Xovikoff hepatoma may repre-ined carbohydrate metabolic parameters in terms sent a "full-blown picture of neoplasia," in con-of enzymology (23, 25) and disposition of C14- trast to Morris hepatoma which represents thelabeled substrates (3, 27) in Morris hepatoma least advanced case of the same neoplastic condi-5123, and results were compared with those ob- tion involving liver cells (20). Recent work on atained in Xovikoff tumor (4, 20). The metabolic spectrum of liver tumors of different growth rateslesions and enzymatic alterations in these liver showed that a rough correlation can be observedtumors have recently been reviewed, and it was between growth rates of the investigated hepa
tomas and the behavior of certain carbohydrate* This work was supported m part by grants from the Na- .•... ,.-, __ ,,.,> r™ t ¿i_tional Cancer Institute, National Institutes of Health, U.S. enzyme activities (21, 25, 26). The purpose of thePublic Health Service (CY-5034); American Cancer Society present isotope work is to examine two facets of(No. E-254); and Damon Runyon Memorial Fund for Cancer this problem. First, can the various parameters ofResearch, Inc. (DRG-54Õ). carbohydrate metabolism of the hepatic tumor
t Post-doctoral trainee of the United States Public Health spectrum described be correlated with growth
rate? Second, does the information obtained byReceivedfor publication February 13,1963. isotope methods agree with indications obtained
by enzymatic exploration of the alternate pathways of carbohydrate metabolism?
This paper describes the metabolic routes oflabeled glucose and fructose in a spectrum of livertumors of different growth rates and evaluates theresults in light of the data obtained in Hepatoma5123 and the Novikoff tumor. The isotope investigation showed that, in addition to the behavior ofcertain enzyme systems, a number of other carbohydrate metabolic parameters exhibited a roughcorrelation with the growth rates of the studiedhepatomas. The isotope results in tissue slices werein good agreement with indications obtained byenzymatic methods in homogenates and supernatant fluid.
MATERIALS AND METHODSAnimals.—Adult, male rats of the Buffalo or
ACI/N strains, weighing 150—250gm., were usedas controls in these experiments. The animals wereshipped air express from Dr. H. P. Morris of theNational Cancer Institute, Bethesda, to IndianaUniversity Medical Center, Indianapolis, Indiana.The transplantable tumors used were the slowlygrowing 5123-D and 7800 carried in Buffalo ratsand the Reuber (H-35) carried in ACI/N rats: fortumor with intermediate growth rate the 7288-Cwas used, transplanted in Buffalo rats; the mostrapidly growing tumors in this series were Hepatomas 3924-A and 3683, both transplanted inACI/N rats. The biology and growth properties ofthese tumors were described elsewhere (11, 12),and a more complete spectrum is discussed in apreceding paper of this series (26). Animals weremaintained in separate cages and exposed to foodand water ad libitum until they were sacrificed.
Experimental procedures.—Animals were killedby a sharp blow on the head, decapitated, andexsanguinated. The livers and tumors were removed and blotted on filter paper. Particular carewas exercised in dissecting the tumors free of ne-crotic, hemorrhagic, or nontumor material to assure the selection of viable tumor samples.Immediately following removal all specimens werechilled over ice in a Petri dish. Both liver and tumor samples were sliced on a Stadie-Riggs handmicrotome, and approximately 0.5 gm. wet tissuewas incubated in 6 ml. of Hasting's K 110 medium(7) for 90 minutes. Glucose-1-C14and glucose-6-C14(final concentration, 20 HIM),and fructose-U-C14(final concentration, 30 HIM) were used as substrates. Labeled glucose and fructose were purchased from the Volk Radiochemical Co. In eachexperiment liver slices were incubated concurrently with tumor slices to provide appropriatecontrol values. Five per cent CO»:95 per cent OÃand bicarbonate buffer were used to provide buf
fering at pH 7.4-7.5 in the presence of the tissue(7).
Chemical methods.—Except where noted, allmeasurements were made similarly to those previously reported (4, 27). Glucose in the presence offructose was measured by the glucose oxidasemethod (9). Fructose in the presence of endogenous glucose was measured according to Higashiand Peters (8). Glycogen was prepared accordingto Good et al. (6), and glycogen-glucose was measured by the Nelson method (14). Unlabeled glucose was added to the acid hydrolysate of glycogenfor the preparation and purification of isotopicallylabeled glycogen-glucose as the phenyl glucosazone(10). Lactic acid production was measured colori-metrically by the technic of Barker and Summer-son (5).
Expression of biochemical results.—Therecoveryof labeled carbon in the various metabolic end-products was calculated in terms of the initial specific activity of the labeled substrate (4). Resultsare therefore expressed in terms of ¿¿molesofhexose. Maximum glucose phosphorylation wasestimated by the method of Renold et al. (17).
RESULTSThe metabolism of labeled fructose and glucose
in normal and neoplastic liver is presented inTables 1 and 2. The results are compared withthose obtained previously in Hepatoma 5123-Dand shown for discussion in Charts 1-3.
Metabolism of fructose in normal liver and inhepatomas of different growth rates.—The metabolic
features are given in Table 1.Fructose uptake.—In hepatomas the uptake
ranged from 21 to 48 itmoles/gm as compared withvalues of 74 to 98 /¿moles/gmfor liver. Fructoseuptake was uniformly reduced in all hepatomasstudied and ranged from 28 to 54 per cent of thecontrol liver.
Fructose oxidation.—The recoven,- of fructosecarbon as CC>2(0.9-2.9 /¿moles/gm)was much lessin tumor tissue than in liver (2.9-8.2 /umoles/gm).The reduction in fructose oxidation paralleled utilization by all tumors except H-35.
Glycogen content of tissues.—-Alltumors examined contained very little glycogen (2.3 to 23¿mioles/gm)as compared with normally fed livervalues (122-307 /¿moles/gin).
Glycogen synthesis.—Two of the hepatomas,3924-A and 3683, incorporated labeled carbonfrom fructose into glycogen (2.2—1.2¿imoles/gm)at a rate comparable to that observed in liver. Inthe other tumors, very little fructose carbon wasincorporated into glycogen (0.5 to 0.9 ¿imoles/gm)as compared with liver (2.1-5.6 jumoles/gm).
incubation medium was observed in Hepatomas5123-D (11.5 /¿moles),7800 (12 Mmoles), and H-35(7 /¿moles),and in the more rapidly growing tumors was, for all practical purposes, absent (0 to0.8 ¿mióles).Even though an increase in mediumglucose was observed with three of the tumors, theamount was negligible in comparison with thatproduced by liver slices (73-128 /¿moles).
Lactate production.—The amount of láclate pro
duced by tumor slices ranged from 13.2 to 47.4jumoles/gm, and, in general, more lactate wasformed by the more rapidly growing than by themore slowly growing tumors. In Hepatomas 5123-D, 7800, and H-35 lactate production (13.2-20.4jumóles) was less than that observed in controllivers (27.4-37.8 ¿¿moles),whereas the three morerapidly growing hepatomas, 7288-C, 3924-A, and3683, formed slightly more lactate (33.6-47.4/uñóles).
Metabolism of glucose in normal and neoplasticliver.—The metabolism of glucose-1-C14 and glu-cose-6-C14 in normal and neoplastic livers is com
pared in Table 2.Glucose uptake-maximum glucose phosphoryla-
tion.—The capacity of normal and neoplastic livers
to utilize glucose was estimated by calculating themaximum glucose phosphorylation (17). With theexception of Hepatoma H-35, the neoplastic liversexhibited less glucose phosphorylation (27-44/¿moles/gm) than did control liver slices (49-74timóles).
Glucose oxidation.—With the exception of Hepatoma H-35, all tumors exhibited greater C-l oxidation (1.4-3.5 /umoles/gm) than did control livers(1.0-1.3 ¿imoles/gm). The oxidation of C-6 of
glucose was reduced in the more slowly growingtumors, but in Hepatoma 3683 (the fastest growing tumor studied) the C-6 oxidation observed wasequal to that of the control liver (1.1 jumoles/gm).Both liver and tumor slices exhibited a preferentialoxidation of C-l of glucose (C-l/C-6 > 1), and,since C-l oxidation of tumors was usually increased while C-6 oxidation was reduced, the C-l/C-6 ratio for hepatomas tended to be greater thanthat observed in liver slices.
Glycogen content of tissues.—The initial glycogencontent of all the hepatomas (1.3-14 jumóles)was
very low as compared with that observed in liver(253-307 Amóles).
Glycogen synthesis.—The rapidly growing tumors, 3924-A and 3683, incorporated more C14from glucose into tissue glycogen (2.5-2.9 ¿imoles)than did the more slowly growing tumors (0.5-0.8
/amóles).Lactate production.—With glucose present as add
ed substrate lactic acid production by hepatoma
slices ranged from 21.5 to 91.1 yumoles/gm as compared with liver slice values of 20.9-27.7 /¿moles/
gm. Lactic acid production in the more rapidlygrowing tumors (7288-C, 3924-A, and 3683) wasgreater than that observed in the control livers.
DISCUSSIONFor the purpose of facilitating discussion, re
sults obtained for the various parameters areplotted as percentages of the respective controllivers and summarized in Charts 1-3. Data on glu-cose-C14 metabolism in Hepatoma 5123-D (27) are
also included for comparison.Fructose metabolism in normal liver and in hepa
tomas of different growth rates.—The fructose up
take was uniformly reduced in all liver tumorsstudied and ranged from 28 to 54 per cent of thecontrol livers. Since the reduction in fructose oxidation paralleled the utilization of this hexose, it ispossible that the reduced oxidation reflects mainlythe decreased uptake and does not indicate anyspecific impairment of oxidative mechanisms. Thisis in line with observations that the oxidative potential of tumors is generally in the normal range(28). The glucose production was 11 per cent or lessin the slowly growing tumors and was almost absent in the rapidly growing hepatomas. This is ingood agreement with the decrease and absence ofglucose-6-phosphatase and fructose-l,6-diphos-phatase activities reported in a spectrum of livertumors (16, 21, 22, 24-26). It is also in line with
markedly decreased pyruvate to glucose conversion described in 5123-D (27).
The initial glycogens were extremely low in allhepatomas examined, which observation is in linewith the generally decreased glycogen content oftransplantable and primary liver tumors (4,18, 20,27). The incorporation of fructose into glycogenwas in normal range in the rapidly growing tumors, but was very low in the more slowly growinghepatomas. The net lactate production with fructose as the substrate was decreased or normal inthe slowly growing tumors and showed a tendencyto increase in the more rapidly growing ones. However, no marked lactate production in the presenceof fructose was found in any of these tumors.
Glucose metabolism in normal liver and in hepatomas of different growth rates.—The maximum
glucose phosphorylation was normal or generallyreduced in the liver tumors studied. These calculations are considered as approximations, and theirsignificance is mainly that there is no indication forthe presence of increased glucose uptake in thesetumors. The initial glycogen data further confirmthe almost complete absence of glycogen storage inliver tumors, indicating a failure of this liver fune-
SwEEXEY et al.—Comparative Biochemistry of Ilepatomas. IV 999
tion in hepatomas. The glucose to glycogen formation in slices of slowly growing tumors was low,but it increased in the rapidly growing tumors. Thelow levels of glycogen are in agreement with thedecreased phosphoglucomutase activity observedin these liver tumors (21, 25, 26). However, theoretically, the mutase activity still present in thehepatomas is sufficiently active to mediate thechanneling into glycogenesis.
The net lactate production was in normal rangein the slowly growing tumors, but it was increasedstepwise in the more rapidly growing hepatomas.The potential for channeling G-6-P into lactateformation is present in both livers and hepatomas,since phosphohexose isomerase which routes thishexosephosphate into aerobic glycolysis was in thenormal range in both normal and neoplastic livers(21, 25, 26). The difference observed between lac-
100
tate production with glucose present which paralleled the tumor growth rate and lactate generationwith fructose present which was normal or somewhat increased may lie in the different entrances ofglucose and fructose into the metabolic scheme.These special features of fructose metabolism wereoutlined in detail elsewhere (3).
Both C-l and C-6 oxidation of glucose wasmarkedly decreased in 5123-D, but the oxidationwas gradually increased with the growth rates ofthe liver tumors. In the most rapidly growing tumors the C-6 oxidation reached normal values.However, the C-l oxidation in the rapidly growingtumors was markedly higher than in normal livers.In consequence, the C-l/C-6 ratios were generallyhigher in the tumors, and in 3683 were nearlythree-fold the values found in normal liver. Theobservation that C-l of glucose is recovered in COz
100
il io
NET GLUCOSE
122
100110
35
56
138
gì 114
i^
NET LACTATE
100
INITIAL GLYCOGEN
LIVER5123-
3683 LIVER-3924-A 5123
7288-CH-35
M¡3-DJ j7800-1 I L
H-35
L36833924-A
7288-C
CHART1.—Comparison of the metabolism of fructose in normal and neoplastic livers. Results were calculated as per centof normal; the values of the normal livers were arbitrarily taken as 100 per cent.
CHART2.—Comparison of the metabolism of glucose in normal and neoplastic livers. Results were calculated as per cent ofnormal; the values for the normal livers were arbitrarily taken as 100 per cent. Data on the Morris 5128 were previously reported(27).
MAXIMUMGLUCOSEPHOSPHORYLATION
B S 8 l ' sW « <? CO Tf 00
C4 CO Ci CDp4 H ü St jfin t- co
CHART3.—Comparison of the oxidation of glucose-1-C14and glucose-6-C14in normal and neoplastic livers. Results werecalculated as per cent of normal; the values of the normal livers were arbitrarily taken as 100 per cent. Data on the Morris 5123hepatoma were previously reported (27), and the maximum glucose phosphorylation for this tumor was not measured.
SWEENEYet al.—ComparativeBiochemistry of Hepatomas. IV 1001
in greater yield than C-6 was reported for a number of tumors, including several other hepatomas(30). It is well recognized that the C-l/C-6 ratiodoes not necessarily give an exact measure of theoperation of the pentose phosphate pathway; however, the data may be taken as an indication thatthe direct oxidative pathway is operating in alltumors and probably increased above that in normal liver (1). Such an increase is in agreement withthe elevated levels of glucose-6-phosphate dehy-drogenase described in all liver tumors with theexception of 5123-D, where the enzyme was in thenormal range (21, 23, 26).
Comparison of carbohydrate metabolic parameterswith growth rates of liver tumors.—On the basis of
the tables and charts the question arises whetherone can perceive any clear-cut correlation or definite trend among the carbohydrate parametersstudied in liver tumors of different growth rates.
1. Parameters which do not correlate with growthrate.—The maximum glucose phosphorylation cal
culations indicated that no definite trend could beobserved for this biochemical function in thesestudies.
2. Parameters which show a definite trend, but donot correlate with the growth rate.—The glycogen
content was very markedly decreased in all livertumors. This is in agreement with the decreasedphosphoglucomutase activity in these tumors. Thefructose uptake and the CÜ2production from fructose were also generally reduced in all liver tumors,showing a definite trend to decreased utilizationand oxidation of this hexose.
3. Parameters which show correlation with increased growth rate.—The decreased glucose pro
duction in the slowly growing tumors and theabsence of glucose release in the rapidly growinghepatomas indicate a decrease and complete loss offunction with the increase in growth rates of theliver tumors. In the most malignant tumors studied in this series, as well as in the previously examined Novikoff hepatoma, glucose productionwas absent and glucose-6-phosphatase and fruc-tose-l,6-diphosphatase could not be demonstrated.The depletion and failure of this characteristicliver function can be well correlated with the decreasing activities of the specific phosphatases intumors of increasing growth rates (21, 23, 26).
In contrast to failure of gluconeogenesis, it appears that the direct oxidative pathway may beutilized to an increasing extent in the more rapidlygrowing tumors in this series. Ratios as high asthose in 3683 and even higher were reported in theNovikoff hepatoma (4).
The lactate production with glucose as the substrate is in normal range in the slowly growing tu
mors; however, it is increased in rapidly growingtumors and appears to parallel the increase ingrowth rate. The highest lactate production wasreported in the Novikoff hepatoma (4), which hasthe most rapid growth rate among liver tumors discussed (15, 20).
A note may be made regarding lactate production in neoplastic liver. It has been reported thatin slowly growing liver tumors there is normal lactate production (2, 11, 27, 29, 30), and an absenceof increased aerobic glycolysis is also found in themore slowly growing tumors in the present series.An explanation for reports in the past 30 years emphasizing increased aerobic glycolysis as a cardinalaspect of neoplasia probably lies in the fact thatyears ago only the most malignant tumors wereavailable for biochemical studies, because thesewere transplantable in non-inbred strains. Theslowly growing liver tumors which are availablenow, however, can be transplanted only in inbredrats, which made possible the discovery that thereis neoplastic growth without increased aerobic glycolysis (2, 10, 27). The rapidly growing liver tumors do exhibit an increased aerobic glycolysis.
What conclusions may be drawn from the presented results?—The isotope results outlined in this
paper, with other information accumulated in thisseries in hepatomas of different growth rates on thebehavior of enzymes (23, 25, 26), enzyme-formingsystems (21), and protein synthesis (19), confirmthe expectation that it is possible to establish aframework for a biochemical grading of tumors toserve along with morphological grading (26).
The data examined show the usefulness of studies of a spectrum of liver tumors of differentgrowth rates which contribute to an understandingof the significance of the different carbohydratemetabolic parameters in neoplastic liver. Such aspectrum permits the investigation of the neoplastic phenomenon in terms of different extent ofmetabolic lesions in an analogy to the differentextent of appearance of symptoms and signs in individuals suffering from the same disease. Thisspectrum may point out certain metabolic parameters which correlate with the biological behavior(growth rate) of these tumors and thus draw ourattention to aspects of tumor metabolism wherechemotherapy may be employed. Studies on thebehavior of metabolic pathways under variousconditions in neoplastic livers are now in progress.
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