FAT METABOLISMIN DIABETES MELLITUS1

By WILLIAM C. STADIE(From the John Herr Musser Department of Research Medicine of the

University of Pennsylvania, Philadelphia)(Received for publication August 7, 1940)

Hypotheses of fat metabolism in diabetes mellitus

The development of thought concerning themetabolism in diabetes mellitus since the classicalexperiments of v. Mehring and Minkowski hasbeen formulated into two contrasting theories.Evidence of crucial character bearing upon thesetheories is still being actively sought for by ex-perimenters, but it appears that the totality of thisevidence is not yet sufficient to bring completeconviction to all in one direction or the other.

Briefly, these opposing theories may be statedas follows:

1. Under-utilization hypothesis. The major, ifnot the sole, defect in the intermediary metabolismin diabetes mellitus is that the peripheral tissue(i.e. chiefly muscle) cannot, either at all or insufficient measure, oxidize carbohydrate withoutthe catalytic intervention of insulin.

2. Overproduction hypothesis. Fatty acids areconvertible into carbohydrates by the liver. Thefunction of insulin is to control, directly or indi-rectly, the extent of this conversion; its action inthe periphery, which is to catalyze the oxidationof carbohydrate, is either nil or of minor im-portance.

According to the first of these hypotheses, thecomplete diabetic subject must lose all of the en-ergy derivable from carbohydrate and most of thatfrom protein; hence he must fall back upon fatsas the chief source of his energy requirements.According to the second hypothesis also, there isin the diabetic a profound disturbance of thenormal course of fat metabolism in the liver. Ittherefore becomes necessary to examine the theo-ries of fat metabolism which developed simul-taneously with the growth of the above hypotheses.

There are two possible chemical mechanisms bywhich fats can be utilized in the periphery. Theseare:

I Aided by grants from The American PhilosophicalSociety and The Rockefeller Foundation.

I. Fats are utilized directly by the periphery,i.e., oxidation is initiated and completed in themuscles themselves.

II. There must be a preliminary partial oxida-tion in the liver to diffusible substances which areoxidizable by the muscles.

The possibility, discussed later, that both I andII are operative at the same time must also beconsidered. However, differences of opinionarose over the detailed mechanism of II whichwere expressed in the alternative hypotheses:

II-A. Fats are preliminarily oxidized in theliver to ketone bodies plus a two-carbon compound(acetic acid). In the normal subject these arecompletely oxidized in the periphery.

II-B. Fats are initially converted by the liverto carbohydrates as well as ketones for peripheralutilization.

II-C. Fats are completely converted in the liverto ketone bodies only.

Early in the literature the emphasis was placedupon one of these alternatives (II-A) almost tothe complete exclusion of the others. The reasonsfor this were threefold: the ascendency of theunder-utilization school, the strong position of theKnoop hypothesis of successive beta oxidation offatty acids, and the development of the hypothesisof obligatory coupling of ketone body-carbohy-drate oxidation in the periphery. The relation ofthese to the problem of fat metabolism in thediabetic will be briefly discussed.

Hypothesis of successive beta oxidation of fattyacids

The original experimental work of Knoop (1)upon which this hypothesis was based was con-fined to the study of phenyl-substituted fatty acidsin which the side chain contained 5 carbons orless. When these acids were fed, the nature ofthe phenyl residue excreted clearly showed thatthese short fatty acids were oxidized at the carbon

843

WILLIAM C. STADIE

atom which was in the beta position to the ter-minal carboxyl group. In the case of phenylvaleric acid, the longest fatty acid studied, Knooplimited his conclusion to the statement that therewas beta and delta oxidation but asserted nothingabout the possible paired splitting off of aceticacid or other oxidized two-carbon compound. Nordid he state that the type of oxidation which heshowed for these short fatty acids was a generalbiological reaction for the oxidation of longerfatty acids.

Dakin (2a), however, continued work withphenyl-substituted fatty acids and became con-vinced that "the evidence obtained " . . . (indi-cates) . . . "that five acids of the type Ph.C.-C.C.C.COOH undergo oxidation in the body insuch a way that four carbon atoms are removedfrom the side chain in two pairs. . . ." Thisprocess "may be termed successive beta oxida-tion " and he saw " no reason to suppose that it isnot a general biochemical reaction." For then itbecame clear " that the catabolism of a fatty acidgroup, CH3. (CH2)2.COOH, is effected by thesuccessive removal of two carbon groups at atime." It would necessarily follow that eachmolecule of fatty acid would be degraded througha succession of fatty acids each shorter by twocarbon atoms than its immediate precursor.Finally one molecule of butyric acid would resultwhich in its turn would be oxidized to one mole-cule of acetoacetic acid or beta hydroxybutyricacid.

Of the nature of the two-carbon compound splitoff little was known. It was most generally as-serted to be acetic acid and as such was incor-porated in schemes of fatty acid oxidation. Thedifficulty, pointed out by Friedmann (3), thatacetic acid was markedly ketogenic in perfusedlivers was generally ignored. For then the fattyacids with odd numbers of carbon atoms, whenoxidized by successive beta oxidation, should like-wise produce acetic acid and therefore should beketogenic. But they are not. Dakin (2b), rec-ognizing the difficulty, avoided it by postulatingthe possible formation of other two-carbon oxida-tion products of the type of glyoxylic acid. How-ever, nowhere in the literature are there anyreports of the isolation or identification of anyof these hypothetical two-carbon compounds.

Hypothesis of obligatory coupling of ketone-carbohydrate oxidation

The striking ketone body excretion by the dia-betic subject and its equally striking recessionfollowing resumption of carbohydrate oxidationremained to be explained. Although there wereisolated appeals for a simpler hypothesis (Cf.,for example, von Furth (4); Raper and Smith(5)), the hypothesis of obligatory coupled oxida-tion of ketone bodies and carbohydrates, initiallyfounded upon an aphorism and not much more,dovetailed so neatly with the Knoop hypothesisthat it became the almost unchallenged theory ofthe metabolism of fats in the diabetic for a periodof forty years. The statement of this hypothesisas it became fully developed was as follows: thelong even-numbered fatty acid chains (C => 16)present in natural fats are oxidized by successivebeta oxidation in the liver. A two-carbon com-pound-presumably acetic acid, which is easilyoxidized by the peripheral tissues of the completediabetic-is split off. The fatty acid molecule isthus reduced step by step to the four-carbonbutyric acid. This in turn is oxidized to aceto-acetic or beta-hydroxybutyric acid which cannotbe further utilized by the diabetic and is excretedin toto.

If the diabetes is " incomplete " or if insulin isgiven, carbohydrates can be oxidized and thereoccurs in the periphery the chemical reaction ofcoupled ketone body-carbohydrate oxidation, viz.:

Ketones + carbohydrate + 02= CO2+ H20.

This is the sole mechanism by which ketone bodiescan be oxidized in normals or diabetics.

The reasons for the ascendency of this com-bination of hypotheses are easy to discern:

1. It permitted the under-utilization school ofdiabetic students to explain how the complete dia-betic, who was losing about 70 to 80 per cent ofthe energy from protein as carbohydrate or ketonebodies, and 100 per cent of that from carbohy-drate, could still exist. The calculations in TableI show that there was still a possible 27 to 62per cent of the energy of the original fat in theform of a hypothetical two-carbon compound splitoff by the beta oxidation available for the energyrequirements of the periphery. The balance waslost to the periphery either iiVhe process of pre-

844

FAT METABOLISM IN DIABETES

TABLE I

The heats of combustion of oxidized derivatives of fatty acidsand the caulcked energy availabk to the periphery

1 gram mole of fat (triglyceride) = 870 grams = 8200calories (typical fatty acid = C1H,202, palmitic)

Total fatSubstrate formed in liver Moles/ oalories

by partial oxidation Calories/Moles Mole of available Glucogenicof fatty acid fat to

l________periphery

per amnAcetic acid .209 3X6* 46 NoAcetaldehyde ........... 281 3X6 62 NoGlyooilic acid ............ 167 3X6 37 See footnoteGlycolaldehyde .......... ? 3X6V8 footnoteGlyoxylic acid ........... 125 3X6 27 8S footnoteFormi .63 3X12* 27 oBeta-hydroxybutyrio acid. 488 3X4t 72 NoAoetoacetic acid ......... 3X4t NoGluos ................ 673 3X2.67t 68 Yes

* By successive beta oxidation hypothesis: 1 mole fattyacid = 1 mole ketone + 6 mole substrate.

t By multiple alternate oxidation hypothesis: 1 molefatty acid = 4 mole substrate.

$ Hypothetical maximum yield per mole fat.§ No information found that these compounds are

intermediates in fat catabolism. None have been clearlydemonstrated to be glucogenic.

liminary oxidations in the liver or in the form ofunoxidizable ketone bodies. The position of thecomplete diabetic was indeed quite precarious, butit was conceivable that his energy requirementscould be met by such a mechanism. Curiouslyenough, as is seen from the table, the formationof acetic acid or glyoxylic acid, which are thosemost frequently advocated, would yield the leastamount (27 to 46 per cent) of the energy of theoriginal fat to the periphery.

2. It allowed this school to avoid postulatingthe formation of carbohydrate from fats, since acompound other than carbohydrate, but presum-ably oxidizable by the diabetic, was formed in theliver.

3. It allowed of a ready explanation, whichappeared to be quantitative, of the marked in-fluence of carbohydrate utilization upon urinaryketone body excretion.

The position of the over-productionists was lessclear. Obviously, they could hardly espouse theobligatory coupling hypothesis, for then ketonuriawould never occur, since by assumption abundanceof carbohydrate is oxidized in the periphery. Itwas natural, then, that they concerned themselveswith experiments designed to show that the ketonebodies were oxidized in the periphery withoutcoupling with carbohydrate oxidation (6, 7). The

abnormal fat metabolism in diabetes could moreeasily be explained by postulating that there oc-curred in the liver the reaction:

Fatty acids + oxygen = ketone bodies +glucose + (two-carbon compound?)

The exact stoicheiometric proportions of this re-action wer-e never clearly defined. The importantassumption was made that this reaction was con-trolled directly or indirectly by insulin. In dia-betes mellitus, therefore, it ran to excess, resultingin " over production " of ketone bodies and glu-cose. The effect of what the under-utilizationistscall a " resumption of carbohydrate oxidation inthe periphery " in causing a recession of ketonuriaand glycosuria could just as readily be explainedby asserting that under the same conditions theabove reaction was inhibited. However, despitethe fact that the hypothesis of successive betaoxidation was the accepted explanation of themechanism of fat oxidation in the liver, certainof its implications were ignored, viz.:

1. All the conceivable two-carbon oxidized com-pounds derivable from fatty acids (Table I) haveeither been shown to be aglucogenic or else havenever been shown to be intermediaries in thecatabolism of fat. It was necessary to fall backupon the assumption that the four-carbon residuesof the oxidation of fatty acids were the immediateprecursors of glucose, although there was no con-clusive evidence that this was so. Butyric aciditself, which is known to be glycogenic in the liver(8b), has never been shown to be an intermediatein the hepatic catabolism of fat. Nor is there anyevidence that the ketone bodies themselves areglycogenic. The recent experiments of Weil-Malherbe (9) showing that acetoacetic acid isconverted into glucose by the kidney have notbeen confirmed (10).

2. Aside from this, there is another difficultyif only the residuaJ four-carbon compounds offatty acid are convertible to glucose. For thenboth the ketone bodies and glucose originatingfrom fatty acids must come entirely from theseresidues and the maximum grams (G) of glucosewhich could be obtained from the catabolism of

845

86WILLIAM C. STADIE

F grams of fat when K grams of beta-hydroxy-butyric acid are excreted:

180 70-( X104)

orG =0.41F -1.15 K.In other words, there should be in the diabetic

an inverse relation between glucose and ketoneexcretion, and in the limit, according to the equa-tion, glucose excretion from fats should be zerowhen the grams of urinary ketones is approxi-mately % of the grams of total fat catabolized.But no such relations have ever been remotelysuggested in innumerable metabolic studies of dia-betes mellitus. These considerations logically re-quire that the over-productionists abandon thehypothesis of successive beta oxidation of fattyacids as incompatible with their own views andpropose another, although to date no convincingsubstitute has been suggested.

Ezidence against the combined hypotheses

A retrospect of the literature shows that, longbefore outspoken doubts began to appear, therewas evidence which was completely at variancewith the successive beta oxidation and obligatorycoupling hypotheses when these were carried totheir logical conclusions. For example, it waslong known that the partition of fats in the liversof fasting normal animals who must be subsistinglargely on fats failed to reveal any of the lowerfatty acids which were supposed to be formed bydegradation of the higher ones. The major por-tion of the fatty acids contained carbon atoms=> 16. Small amounts (1 to 2 per cent) ofmyristic (C14) and lauric (C12) were found, butessentially no acids with fewer carbon atoms. Inparticular, butyric acid should have been formedin large amounts by the livers of diabetic subjectswith severe ketosis and, being freely diffusible,should have appeared in the urine. There, asHurtley (11) pointed out, its presence shouldlong ago have been revealed by its odor alone ! 2Still more convincing was the evidence in theworking diabetic, particularly the exercising de-

2 Butyric acid at pH 5 to 7.4 is easily detectable by itsodor at 0.01 M. This is approximately 1o of the urinaryconcentration of ketone bodies frequently observed insevere ketosis.

pancreatized animal.8 Since these animals werepresumably oxidizing no carbohydrate, they shouldoxidize no ketone bodies. Hence, during exercise,the excess ketone body excretion over the basalexcretion could easily be calculated from the ex-cess fat metabolism. But there was no doubt,as found by many observers, that there was es-sentially no increase of ketone body excretionduring exercise despite large increases of fatmetabolism. This is clear indication that largeamounts of fats were completely utilized withoutcoupling with carbohydrate oxidation. This onefact alone was completely at variance with theobligatory coupling hypothesis and should haveforced its abandonment at once.

The more systematic attack upon the Knoophypothesis may be said to have begun with Hurt-ley's paper (11). The absence of the lower fattyacids in the livers of diabetics dying of coma ledhim to reject the Knoop hypothesis and proposeinstead the hypothesis which became known asthe multiple alternate oxidation hypothesis (II-C).According to this, fatty acids are oxidized simul-taneously along the whole length of the carbonchain at alternate carbon atoms with completedisruption into ketone bedies according to thescheme:

CH$ . . . CH2.CH2.CH2.CH2.CH2.CH2.CH2.CH2... COOH+02= CH8. . . CO.CH2.CO.CH2.-CO.CH2.CO.CH2 ... COOH+ 02=4CH8.CO.-CH2.COOH.

A typical fatty acid such as pahnitic (C16H8202)would accordingly yield not one but four mole-cules of ketone bodies. Jowett and Quastel (13)determined ketone body formation by liver slicesin the presence of various fatty acids. The yieldof ketone bodies from the higher fatty acids(C410) in comparison to that from the lower onesbrought them to the conclusion that the oxidationwent by way of multiple alternate oxidation.Deuel, Hallman, Butts, and Murray (14) fed theethyl esters of fatty acids up to C18 to fasting rats.Comparison of the rates of excretion of ketonebodies makes it appear unlikely that successivebeta oxidation alone is responsible for the degrada-tion of the fatty acids. For example, in the case

8 See for example Barker's (12) recent experiments onexercising depancreatized dogs.

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FAT METABOLISM IN DIABETES

of caprylic acid (C8), they concluded that deltaoxidation as well as beta oxidation occurred sothat two acetoacetic acid molecules would result.In the case of palmitic and stearic acids their re-sults were more difficult of interpretation, butthey concluded that three or possibly four aceto-acetic acid molecules might result from one mole-cule of fatty acid. Their conclusion was that inthe case of the higher fatty acids (C8-C18) theiroxidation proceeded according to the hypothesisof multiple alternate oxidation.

Blixenkrone-Moller (8a) perfused the livers ofdiabetic cats and compared the total oxygen con-sumption with the ketone formation. He foundthat it was possible to explain the low oxygen toketone ratio only by assuming that four moleculesof ketones were formed per molecule of fattyacid oxidized. Stadie, Zapp, and Lukens (15a)studied the formation in vitro of ketone bodies byslices from the livers of diabetic cats. They com-pared the experimentally observed ratio of oxygenuptake to ketone formation with the theoreticalones calculated according to the Knoop hypothesisand the multiple alternate oxidation hypothesis.They found (Table II) that, when oxygen uptake

TABLE II

The calculatd and observed ratios of hepatic oxygen uptaketo ketone formation in the diabetic animal (15a)

Equation for oidation of a typicalHypothesis higher fatty acid (paimitic) Ratio

02: ketonesKnoop C1Hs202+602

C4H80s+6CH,COOH 6 :1Multiple

alternateoxidation C1,H5202+502=4CH sOs 1.25 : 1

Observed in 6diabetic cats 1.1 10.12

was corrected for carbon dioxide formation andfor the oxygen required for the deamination ofamino acids, the ratio did not differ significantlyfrom that calculated by the latter hypothesis.Stadie, Zapp, and Lukens (15b) added furtherevidence at variance with the hypothesis of suc-

cessive beta oxidation which predicts that some

oxidized two-arbon compound, presumably aceticacid, should be formed from the higher fattyacids. Acetic acid formnation should be approxi-mately six times the ketone body formation.

Nevertheless, in slices from the livers of diabeticcats they could find no trace of acetic acid forma-tion, although the ketone body formation waslarge. In addition, they found that with liverslices actively producing ketone bodies, the molecu-lar ratio of ketone body increase to fatty aciddecrease was in accordance with the hypothesis ofmultiple alternate oxidation rather than that ofsuccessive beta oxidation ( 10).

Present position of the successive beta oxidationhypothesis

The totality of the evidence cited above appearsto be convincing proof that the major portion offatty acid oxidation in the liver occurs in such away that the entire fatty acid molecule is com-pletely oxidized to ketone bodies. It is not in-tended to imply that beta oxidation does not occur.Indeed, the original experiments of Knoop andof Dakin are strong proofs that it does. Morerecently Stetten and Schoenheimer (16), usingdeuterium, have shown that palmitic acid (C14)can be degraded to a small extent step-wise tolauric (C14) and myristic (C12) acids. The pointto be emphasized is that the original implicationof the successive beta oxidation hypothesis,namely, that large amounts of intermediary fattyacids down to acetic acid are formed in the liverby oxidation of the long-chain fatty acids, is nolonger in conformity with the experimental facts.Successive beta oxidation, if it occurs, must ac-count for only a small part of the total fatty acidoxidation in the liver; most of it appears to beaccounted for by multiple alternate oxidation.

Concerning the mechanism by which multiplealternate oxidation is brought about, nothing canbe said. It is worth while remarking, however,that the conception that there is an enzyme whichwill " fit " a large triglyceride molecule and causeits complete disruption into ketone bodies withoutthe formation of intermediates is not a priori im-probable. An analagous case would be that ofglycogen whose molecule is much larger (16-18glucose residues) and yet is rapidly broken downto glucose-l-phosphate by a specific enzyme with-out the formation of intermediary polysaccharides(17).

847

WILLIAM C. STADIE

The production of carbohydrates from fats by theliver

The conversion of fatty acids into carbohy-drates by the liver would supply the muscles withan oxidizable derivative of fat other than ketonebodies. The evidence pro and con has been re-cently reviewed by Mitchell (18) and Soskin(19). Evidence in favor of this hypothesis hasbeen, as a rule, indirect and circumstantial. Suchdirect proof as is in the literature has usuallyfailed of confirmation. Stadie, Zapp, and Lukens(15a) moreover, showed that this conversion didnot occur in the livers of diabetic cats. Withliver slices they found that the summation of therespirations for the main oxidative process oc-curring in the liver was essentially equal to thetotal observed oxygen uptake.

TABLE III

The oxidive metabolism of liver slices from6 depancreatied cats (1Sa)

Meanoxygen uptake perOxidative process gram liver per hour

micromolesDeamination of amino acids. 8.2 1.2

Carbon dioxide formation . 28.0 + 5.0

Ketone body formation ........ . 63.0 t 4.0

Sumof known oxidations......... 99.2 4 6.5

Total observed oxygen uptake.... 87.5 4 4.0

Difference unaccounted for. 7 7.6*

* Difference not significant.

It is obvious that fatty acid (e.g. palmitic) re-

quires oxygen for its conversion to carbohydrate.The maximum4 yield would be according to theequation:

C1,H8202 + 7.002= 2.67C6H1206.Thus 1 uM of 02 would suffice for the productionof 0.38 uM of glucose. Now, the average excre-

tion of glucose in Stadie, Zapp, and Lukens' seriesof cats ( 1 to 4 hours before the actual experimentwith the liver slices) was 1100 + 167 uM per

4 It is frequently stated in the literature, apparentlyupon entirely hypothetical grounds, that the reaction forthis conversion has a respiratory quotient of 0.3, viz.,

C1oHga20 + 1002 = 2.2C.H.0, + 3CO, + 3H20.

Then 1 mole of oxygen would be equivalent to only 022mole of glucose. The difficulty in balancing the oxygen

uptake in the liver against the hypothetical glucose for-mation would become even greater.

kgm. cat per hour. Or, since the average weightof the liver is 30 grams per kgm., there would berequired approximately 97 uM of oxygen pergrams of liver per hour to produce this amount ofglucose from fatty acids. But there was no oxy-gen whatever available in the metabolism of thediabetic liver slice for this conversion. This ex-periment alone, even if there were no others avail-able, is convincing proof that the conversion offatty acids to glucose does not occur in the dia-betic liver. In the case of fasted phlorhizinizedrats in which there was marked glucose andketone-body excretion, they found the same oxy-gen balance with liver slices as in the diabetic cats(10). They also showed by direct analysis thatthere was no gluconeogenesis attributable to fatby liver slices from diabetic cats (10).

Direct utilization of ketone bodies by the diabeticThe above discussion makes it clear that the

chief oxidation products of fatty acids in the liverare the ketone bodies. The calculations in TableI show that more than 70 per cent of the originalenergy of fat still resides in these ketone bodiesand the question arises as to whether this energyis available to the peripheral tissues of the diabetic.The evidence for the answer that the diabetic canabundantly utilize ketone bodies in the peripheryis completely convincing. Stadie, Zapp, andLukens (15a) reviewed the evidence on this prob-lem and added their own. Table IV gives calcu-lations from data in the literature of the utilizationof ketone bodies by the peripheral tissues of nor-mal and diabetic animals. An inspection of thistable shows the following:

1. Under basal conditions the diabetic animalcan utilize sufficient ketones to furnish a largepart, if not the whole, basal energy requirements.

2. The potential capacity for utilization underconditions of (a) work or (b) with high bloodketone concentration (induced by injections ofketones) is 4 to 6 times the basal utilization.

In other words, it is apparent that, provided theproduction of ketones from fatty acids by partialoxidation in the liver is sufficient, the diabeticcould subsist practically entirely upon ketonebodies, even under conditions of severe exercise.Furthermore, this oxidation of ketone bodies pre-sumably is completely independent of carbohydrate

848

FAT METABOLISM IN DIABETES

TABLE IV

Cakulations from data in the literature of ketone bodystilization by the peripheral tissues of the normal and,diabetic animal

Mean ketone bodyutilization per kgm.

per daymilhinsoes

Chaikoff and Soskin (6)By measurement of rate of disappearance

of injected ketones from blood of:Diabetic eviscerated dogs ............ 29 1 1.0Normal eviscerated dogs .......... . 34 4 5.5

Friedemann (20)By measurement of ketone body excretion

in eviscerated dogs when injected withvery large amounts of acetoacetate... . Maximum 120*

Blixenkrone-MOller (8)By comparison of ketone formation of

perfused diabetic cat livers with priorketoneexretion .......................39 4 8.0

By measurement of rate of disappearanceof ketone bodies from blood perfusedthrough:

Resting hind limbs of normal and dia-betic cats..... 27 1 3.6

Working hind limbs of normal and dia-betic cats ...................... 130 k 6.7*

Dye and Chidsey (21)Injection of acetoacetate at high rate into

nephrectomized-depancreatized dogs... Maximum 180*Stadie, Zapp, and Lukens (lSa)

(1) By comparison of ketone formationby diabetic cat liver slices withprior urinary ketone body excretion 27 1 3.6

(2) By measurement of ketone bodyutilization of normal and diabeticmuscle mince in presence of addedacetoacetate.................... 22 - 6.3

(3) By measurement of ketone bodyutilization by diabetic cat musclemince simultaneously equilibratedwith diabetic cat liver slice .... . 50 :1 10.0

(4) By measurement of the portal-hepatic ketone body difference ofdiabetic cats.30 9.8

Mean basal ketone utilization .. . 32 :& 3.1Equivalent in grams of fat .2.3 i1 0.2

* Excluded from basal mean.

oxidation. The hypothesis of obligatory couplingof ketone-body-carbohydrate oxidation becomes,therefore, not only unnecessary, but indeed diffi-cult, if not impossible, of retention.

The calculation of the ketogenic-antiketogenicratio as support of the obligatory

coupling hypothesisThere still remained in the minds of the stu-

dents of diabetes the possibility that there is adefinite molecular ratio between carbohydrate oxi-dized and ketone bodies oxidized. This idea

stemmed from numerous calculations in the lit-erature of the so-called ketogenic-antiketogenicratio in diabetes mellitus. It remains,' therefore,to re-examine the calculations and the assumptionsupon which they are based.

The assumptions are clearly stated by Shaffer(22): ". . . The hypothesis states that antiketo-genesis in the human subject is based upon a. . .ketolytic reaction in the body between aceto-acetic acid, the first formed of the acetone bodiesand a derivative of glucose (or of other antiketo-genic substances), the compound being furtheroxidized, but that failing to react with ketolyticsubstance, acetoacetic acid is resistant to oxida-tion, accumulates, and . . . is excreted. . . . Thefact that one finds at the threshold of ketosis anapproximately constant ratio between the numberof molecules of the precursors of acetoacetic acidand of glucose in the metabolic mixture, mustmean that the further oxidation of acetoacetic acidconstantly taking place under normal conditions isaccomplished through a chemical reaction with aderivative of glucose.

The best way to test the hypothesis by meansof the quantitative data which are available is toset it up in the form of an equation. It has beenfrequently emphasized in the literature that theinterrelations of ketones and carbohydrates in th{eintermediary metabolism of the diabetic can onlybe properly evaluated if the amounts of fat, pro-tein, and carbohydrate in the metabolic mixtureare determined by calorimetric methods. Let thepartition of the metabolic mixture be:

K=Total mM. of ketone bodies formed fromtotal fat and protein catabolism. (Theketone bodies derived from fat are calcu-lated assuming 1 molecule of ketone bodyper mole of fatty acid.)

U mM. of ketone bodies utilized.A mM. of " antiketones " oxidized.E mM. of ketone bodies excreted.r " ketogenic-antiketogenic" ratio: a small

whole number ( 1 or 2).

According to the hypothesis, there occurs in theperiphery as the sole mechanism by which ketonescan be utilized the reaction:

Ketones + antiketones + 02= CO2+ H20.

849

WILLIAM C. STADIE

The total number of ketone body moleculesformed in the liver by partial oxidation of fattyacids which can be utilized in the peripheral tissuesof either the normal or the diabetic subject cannever exceed r X A mM. Accordingly, if ketonebodies are produced in excess of this value theywill be excreted. These relations are expressedby the equation:

U=K-E=r X A.

There are three ways in which the data obtainedfrom subjects with diabetes mellitus can be ap-plied to the equation:

1. By the selection of conditions where E isjust a little more than zero. Then it may be as-sumed (practically) that

Ker X A

and r may be calculated when K and A are knownfrom the metabolic mixture. This method wascalled the "measurement of the ketogenic-anti-ketogenic ratio at the ketone body threshold."However, the definition of " ketone threshold " issomewhat arbitrary and the values of r obtainedby different observers using this method or by thesame observer in different cases were found va-riant. This was explained away by supposing an" unequal distribution " of metabolites so that insome places glucose molecules were oxidized with-out encountering ketone bodies and hence were" wasted " as ketolytic agents, while in other cellsor localities ketone bodies formed without thepossibility of encountering glucose molecules andhence accumulated.

2. Patients with definite excess of K, as indi-cated by marked ketonuria, were selected, and theobserved ketone body excretion compared withthat calculated by the equation (r being assigneda value of 1 or 2):

E=K-r X A.Unfortunately E is the small difference of two

large numbers. Hence the approximate agree-ment of the small observed and calculated Evalues was considered sufficient even though, aswas frequently found, they differed from eachother by several hundred per cent.

3. In patients with definite excess of K thestatistics of the equation

U=rXA (1)

can be calculated. This is by far the best methodsince the statistical calculations would constitutean objective test of the equation and the hypothesisupon which it is based. Oddly enough, it appearsnever to have been used.

Fortunately, there is in the literature a sufficientamount of the necessary data for re-testing thishypothesis in subjects with diabetes mellitus. Thecases are all classical ones, reported in the litera-ture before the advent of insulin when markedketonuria was, of necessity, a frequent accom-paniment of the disease. Similar data on humandiabetics will in all probability never be obtainedagain, since it is unlikely that patients with markedketonuria will be allowed to remain untreatedover long periods of time.

The assumptions made in calculating the dataare:

1. The metabolic mixture as represented by thecalories of protein, fat and carbohydrate calculatedby the original authors is assumed to be correct.

2. The conversion of calories into ketones and"antiketones " is made by the use of the factorsgiven in Table V.

3. In testing the obligatory coupling hypothesisas expressed by equation (1), the original assump-tion that one fatty acid molecule yielded one ketonebody molecule was adhered to. The employmentof the 4:1 ratio as required by the multiple alter-nate oxidation hypothesis, however, would makeno difference in the conclusions drawn from theanalysis of the data.

TABLE V

Factors converting calories of metabolic mixture intomM. of ketones and "antiketones"

FactorKgm. calories of: Ketosenic Antiketogenic

Fat on basis of 1: 1 ratio ..0.363 0.121Fat on basis of 4: 1 ratio ..1.45Protein . 0.442 1.25Carbohydrate....... 2.96

Factor converting mM. of ketone bodies intograms of original fat = 0.0726 (on basis of 4moles of ketone per mole of fatty acids). (Aver-age molecular weight of fat = 870.)

Presentation of data1. Cases of diabetes meUitus with marked ke-

tonuria (i.e.=> 10 mM./day). In this typeof case, since there is an excess of ketone forma-

850

FAT METABOLISM IN DIABETES

1B

4

'IaII2c

5*ANTIKETONE:S OXDIZED, mM/KG/DAY

30 35 40TOTAL FAT CATABOLISM IN

.AETONE EQUIVALENTS, mM/K/ A

FIG. 1. DLABETES MELLITus, CASE No. 740 (JosuN, 1915)A. Ketone utilization as a function of "antiketones" oxidized. Correlation = 027 0.17; K: A ratio = 027 4-

0.30; intercept constant = 8.9 0.5 mM./kg./day.B. Ketone utilization as a function of total fat catabolisnL Correlation = 0.99 + 0.04; k = 0.75 + 0.04; Uo =

28.0 + 2.1 mM./kg./day.

tion, it is possible to determine whether the dataare in accord with the equation:

U=K-E=r X A.

In order to present the data briefly, the obser-vations have been plotted for each individual case.

The statistical analyses accompany each plot andfor convenience of comparison these statistics are

collected in a summarizing table.Correlation coefficients, regression coefficients,

etc., together with their standard errors, are cal-culated according to standard methods (Cf. Dunm,H. A., Physiol. Rev., 1929, 9, 215).

Case number 740, Diabetes mellitus (23), (Figure 1A).Moderate ketonuria (= > 5 grams beta hydroxybutricacid per day). The dashed line is calculated by the equa-tion U= 2A. The agreement of this line with the ob-served points is specious since the correlation coefficientis 027 + 0.17 (not significant) and the value of r

027 0.30 (i.e. without significant relation to the hypo-thetical value of 2). Moreover, U when A = 0 (i.e.when no antiketones are oxidized) is 8.9 + 0.5 mM. perkgm. per day instead of 0. The hypothesis gains no

support from this case.

Bessie B., Diabetes mellitus (24), (Figure 2A). Ke-tonuria slight to moderate (beta hydroxybutyric acid 1

to 17 grams per day). The dotted line is theoretical for

U= 2A. It bears no significant relation to the observedpoints. There is no significant correlation (coefficient =

-0.40 0.30) and the statistical (heavy) line shows a

negative value of -0.43 0.35 instead of a value of 1

or 2. There is an appreciable utilization of ketones(13.8 + 1.7 mM. per kgm. per day) when the " anti-ketones " oxidized are zero.

Kramer, Diabetes mellitus (22), (Figure 3A). Severeketonuria (28 to 102 grams per day of beta hydroxy-butyric acid). There is no significant correlation(-0.10 0.29). The theoretical lines for U= A andU = 2A have no significant relation to the observedpoints. The statistically calculated value of r is 0.10029 and has no relation to the hypothesis.

Cyril K., Diabetes mellitus (25), (Figure 4A). Mod-erate to severe ketonuria (beta hydroxybutyric acid = 11to 88 grams per day). Of all the cases in this group thedotted line for the theory U= 2A shows the best ap-

parent agreement to the "eye." But this agreement isfound to be specious when the statistical analysis is con-

sidered. For then the true equation for the observationsis found to be

U =5.6 12= (0.72 0.39)A

rather than

U= 0 + 2A.

The statistical value of r = 0.72 0.39 is significantlydifferent from the supposed value of 2. Moreover, Ushould be 0 when A (antiketones oxidized) is 0. But it

1A

22

ti 9Sz

V8

3

* % /or/

*~~ /

/* 0#

851

852 ~~~~~~~WILLIAM C. STADIE

2A

2 14E

I2 1

'g

"ANTIh

2B

2 4 8. 35 40 45 s0

'LTONES" OXKDZED, aM/KG/DA TOTAL FAT CArABOUSMI NK~ETONE EQUIVALENTS, mM/KG/ DAY

FIG. 2. DiABETEs MELLiTus, BESSIE B. (WiWDER, BOOTHBY, AND BEELER, 1922)A. Ketone utilization as a function of " antiketones " oxidized. Correlation 0.40 ±'- 0.30; K: A ratio = - 0.43 4-

0.35; intercept constant = 13.8 -+ 1.7 mM./kg./day.B. Ketone utilization as a function of total fat catabolism. Correlation 0.95 ±+ 0.03 ; k = 0.75 ±- 0.09; Uo =

34.5 ±+ 4.6 mM./kg./day.

3A

'-4N

Li

3B

0 1 2 3 4. 40 so0. s"'ANTIK~ETONES" OXIDIZED, TOTALjbA FAT CADIABOUSMIN

mMkWA'KETONEEQUIVALENTS, unM/KG/DOV'FIG. 3. DIABEEmsMELIiTUS, KRtAMERt (SHAFFER, 1922)

A. Ketone utilization as a f unction of " antiketones " oxidized. Correlation = - 0.10 ±1 0.29; K: A ratio-- 010 -+ 0.29; intercept constant = 5.3 -'- 3.1 mM./kg./day.

B. Ketone utilization as a function of total fat catabolism. Correlation = 0.91 -+' 0.05; k = 0.28 4- 0.04; Uo =

34.5 ±4 1.4 mM./kg./day.

852

FAT METABOLISM IN DIABETES

4A 4B

2 4 6 40 50 60

ANTIKETONES OXIDIZED, mM/KG/DAY 'TOTAL FAT CATABOLISM IN~KETONEEQUIVALENTS, M/ KG/ DAY

FIG. 4. DiABETEs MELLITUS, CyRiL K. (GEPHART, AuB, Du BoiS, AND LUSK, 1917)A. Ketone utilization as a function of " antiketones " oxidized. Correlation = 0.67 + 027; K: A ratio = 0.72 +

10.39; intercept constant = 5.6 + 12 mM./kg./day.B. Ketone utilization as a function of total fat catabolism. Correlation = 0.97 + 0.03; k = 41 + 0.05; UO=

35.4 1.3 mM./kg./day.

5A SB

LO 7IJ

6

li

NJosjc

2 4 6 8 10

*ANTIKETONES!OXIDIZED, mM/KG/DAY

25 30 35TOTAL WCATABOLISM IN

KETONEEQUIVALENTS. mM/KG/DAYFIG. 5. DIABETES MELLITUS, JERvis B. (RICHARDSON AND LADD, 1923)

A. Ketone utilization as a function of "antiketones" oxidized. Correlation =- 0.99 + 0.01; K: A ratio =

-021 -+- 0.05 intercept constant = 12.3 + 0.1 mM./kg./day.B. Ketone utilization as a function of total fat catabolism. Correlation = 0.97 + 0.04; k = 0.84 + 0.14; Uo =

232 + 1.9 mM./kg./day.

5 .e

z0

8

wU _

853

WILLIAM C. STADIE

6A 6B

2 3 .40 50 60

ANTIKETONESr OOXIZED, .M/WDAY TOTAL SM IN

ANTII~ET0NE/KG' AETONE EQUIVALENTS, MM/KG/ DAVFIG. 6. DIABETES MELLITUS, E. W. (MOSENTHALAND LEWIS, 1917)

A. Ketone utilization as a function of "antiketones" oxidized. Correlation= 0.45 + 0.33; K: A ratio=-3.6 -+ 2.4; intercept constant = 11.3 + 0.8 mM./kg./day.

B. Ketone utilization as a function of total fat catabolism. Correlation =-0.89 + 0.08; k= 0.38 + 0.08;UO= 46.6 3.5 mM./kg./day.

is found to be 5.6 12 mM. per kgm. per day. In otherwords, it is practically certain (prob. = > 0.999) thatthere is utilization of ketones when there is no oxidationof "antiketones." The observations in this case, there-fore, do not support the hypothesis.

Jervis B., Diabetes mellitus (26), (Figure 5A). Mildketonuria (2 to 12 grams per day). There is no sig-nificant relation between the observed points and thedashed lines calculated for r = 1 or 2. There is a nega-tive correlation (- 0.99 0.01). The statistical lineshows a greater utilization of fat as the carbohydrateoxidation ("antiketones ") is diminished.

E. W., Diabetes mellitus (27), (Figure 6A). Severeketonuria (65 to 11 grams of beta hydroxybutyric acidper day). There is a negative correlation (- 0.45 +0.33) giving a negative r = -3.6 + 2.4, a value whichhas no meaning with respect to the hypothesis underdiscussion. The theoretical lines for r = 1 or 2 bear nosignificant relation to the observed points. When ketoneformation is calculated on the old 1: 1 basis, there isfound in this case on two occasions a ketone body ex-cretion which exceeded the calculated ketone body forma-tion. A similar observation was found in the case ofKramer.

Hypothesis of fat metabolism in the diabetic

In summary, it is apparent from this analysisthat there is no significant relation between theketone bodies utilized and the " antiketones " oxi-

dized. The hypothesis of obligatory coupling ofketone body-carbohydrate oxidation in the diabeticreceives no support from the quantitative data inthis series of cases of diabetes mellitus. It re-

mains, therefore, to formulate an hypothesis whichwill be in confornity with the observations.

The following appears to fuffill these require-ments:

Up to a certain level fat metabolism is completand there is no ketonuria. Beyond this level fatmetabolism is incomplete and part of the fat cata-bolized is excreted in the form of ketone bodies.

The relation of carbohydrate to fat metabolismis an inverse one: the greater the carbohydratemetabolism, the less is the fat metabolism. Thereis no fixed relation in the sense of a definitemolecular ratio of ketogenic: antiketogenic sub-stances.

The hypothesis is elaborated in the form of a

diagram (Figure 7) and an equation:Let F= total fat catabolized in grams, mM., or

equivalent mM. of ketone bodies.U= total fat utilized in grams, mM., or

equivalent mM. of ketone bodies.UO= maximal aketonuric fat utilization.

I

E

0!ZN

:3hi

854

FAT METABOLISM IN DIABETES

N~~~~~~~~~~~~~~

z

FI- _ _ __ _ _

F-TOTAL FAT CATABOLISM. GM/KG/DAYFIG. 7. SCHEMATICREPRESENTATIONOF FAT METABOLISM IN

DIABETES MELLIrUSThe figure embodies the hypothesis discussed in the text. The equations relat-

ing fat utilization and ketonuria to total fat catabolism are shown in the graph.F = total fat catabolized; U = total fat utilized; Uo = maximal aketonuric fatutilization; E = total urinary ketone bodies; k = coefficient of excess fat uti-lization.

E =total urinary ketone bodies in mM. ofketone bodies or the equivalent grams ofmM. of " original " fat.

k = " coefficient of excess fat utilization."

In any given calculation all of the above termsmust be expressed in the same units. The con-

version factors necessary are:

1 gram of fat

17000 1.15mM. of fat.

-870 X 12=- 13.8 mM. of ketone bodies.

1 8701 mM. of ketone bodies 12 10c0

0.0726 grams " original " fat.=0.0836 mM. of " original " fat.

870 = molecular weight average fat (triglycer-ide).5

The balance of the total metabolism not repre-sented by carbohydrate or protein is, of course,represented by fat metabolism. Part or all ofthe total fats catabolized undergo a preliminarypartial oxidation in the liver to acetoacetic or betahydroxybutyric acids. The ketone bodies (or fatitself) are utilized in the peripheral tissue by bothnormal and diabetic subjects without chemicalcoupling with carbohydrate oxidation. Up to acertain point, catabolism keeps exact pace withthe body's need for energy from fat and there isno ketonuria. We may express this by sayingthat U = F and represent it on the diagram (Fig-

5 In accordance with the multiple alternate oxidationhypothesis, these conversion factors are calculated on theassumption that 1 molecule of fatty acid yields 4 mole-cules of ketone bodies.

855

WILLIAM C. STADIE

ure 7) as the first or aketonuric phase of fatmetabolism. When, however, at a given state ofactivity the need for fat metabolism exceeds a

certain maximal aketonuric utilization value (U0)fat is catabolized in excess of utilization. The ex-

cess of fat catabolism over the aketonuric level isdivided into two parts: (1) the kth part is utilizedas extra fat utilization; (2) the (1 - k)th part isexcreted in the form of urinary ketone bodies.Therefore, in this second or ketonuric phase thefat metabolism is represented by the equation:

U=F- E Uo+ k(F-Uo) (2)

and the urinary ketone bodies are given by theequation:

E ( 1-k) (F- Uo). (3)

Application of the equation to the cases

The cases cited in Group I all had moderate tosevere ketonuria. In other words F > UO.Equation 2 may then be applied to the data. Inthe figures (1 to 6) (1) already given, the B seg-

ments show the data plotted in this way. In theconversion of the fat of the metabolic mixture toequivalent amounts of ketone bodies the ratio offour molecule of ketone bodies per molecule offatty acid was assumed in accordance with themultiple alternate oxidation hypothesis. The sta-tistical calculations are shown in the figures. Ina series of cases (Group II) in which ketonuriawas relatively low (=- < 5 grams per day) or

where there are but a few observations, the valueof UO has been calculated directly (Equation 2).These values have been included in the summariz-ing table (Table VI).

Inspection of Figures lB to 6B 6 and of TableVI shows the following:

1. The cases of Group I show in every case a

high correlation between UOand F, indicating that6 The labelling of the ordinates in these figures as

"Ketone utilization" and "Total fat metabolism in ke-tone equivalents" does not imply that 100 per cent ofthe fat catabolized goes through the ketone body stage.The proportion which does is not known (Cf. section on" Direct and indirect fat metabolism "). The terms " Fatutilization " and " Total fat catabolism " could just as

well have been used as they are in Figure 9. The presentterms are used here to emphasize the relation betweenfat catabolism expressed as ketones and potential ketoneutilization.

TABLE VI

Summary of maximal basal aketonuric fat utilizationin cases of diabetes mellitus

Coeffi-Maximal aketonuric cient of

Case Reference ~~fat utilization in excessCase Reference equivalents of ketone fat

bodies utiliza-I

-I I tion

Group I

JCyril K...

1Bessie B...

Kramer. ...

4740.......J E. W.....JJervis B...

Group II

Ray H....Chris. Q...Harold J. .

George H..Frank B...K. A......

Gephart, Aub, DuBoisand Lusk (25)

Wilder, Boothby andBeeler(24)

Shaffer (22)Joslin (23)Mosenthal and Lewis (27)Richardson and Ladd (26)

Richardson and Ladd (26)Richardson and Ladd(26)Richardson and Ladd(26)Richardson and Ladd (26)Richardson and Ladd (26)

McClelland, Spencer, andFalk (28)

mM. per kgm. per day

3534

3528

(47) *23

372637373740

+0.41+0.75+0.28+0.75-0.38+0.84

Mean (11 cases) 34 h 1.6 (S. E. of mean)Equivalent in grams 2.5 1 0.12

of fat

* Excluded from basal meanon account of fever (1020 F.).

utilization of fats by the diabetic is a function ofthe total fat catabolism.

2. The values for UO, the maximal basal ake-tonuric fat utilization, are all concordant (exceptE. W., v. infra). The mean value was found tobe 2.5 + 0.12 grams of fat per kgm. per dayequivalent to 34 + 1.6 mM. of ketone bodies perkgm. per day. This value is in essential agree-ment with the mean basal ketone utilization (32± 3.1 mM. of ketone bodies per kgm. per day,Table IV) found in the experimental animal bydifferent workers and by different methods.

These findings are considered to constituteproof that the hypothesis concerning fat metabo-lism in diabetes mellitus already stated is in con-formity with the quantitative data available in theliterature.

Significance of the coefficient of excess fatutilization

It will be observed (Table VI) that the valueof k, which we have called the coefficient of excessfat utilization, varies considerably among the sixcases with sufficient data for its calculation. Thesignificance of this is that some subjects (withhigh value of k) are able to utilize excess fatwithout an undue increase of ketonuria. Those

856

FAT METABOLISM IN DIABETES

with low k values are only able to increase theirtotal calories from fat at the cost of a relativelyheavy increase of ketonuria. Whether this divi-sion of diabetics into two classes on the basis oftheir k values has any bearing on the problem ofhigh fat versus low fat in the dietary cannot besaid.

Fat metabolism in febrile severe diabetic (Mosen-thal and Lewis' E. W.)

This case had three features which are in con-trast to those of the other cases:

1. Fever, 1020 F. (Infected foot ulcer.)2. High aketonuric fat utilization level, 46.6 + 3.5

mM. per kgm. per day ketone bodies.3. Severe ketonuria (approximately 8 to 20 mM.

per kgm. per day of beta hydroxybutyricacid).

4. Negative value of k, the coefficient of excessfat utilization.

Since there is only one case in the series show-ing these peculiarities, discussion is speculative,but it may be that this case represents an extremetype of fat metabolism, possibly characteristic ofthe febrile severe diabetic. The well known clin-ical fact that infection superimposed upon severediabetes leads to sharp increase of ketonuria couldthen be reasonably explained as follows: unlike theafebrile diabetic who can increase his fat utiliza-tion above the aketonuric level (k positive), thefebrile diabetic has a decreased " tolerance " forfat (k negative) as well as carbohydrate. Inconsequence, without appreciable change in totalfat catabolism there is a sharp fall of fat utiliza-tion below the aketonuric utilization level accom-panied necessarily by an increase of ketone bodyexcretion.

Fat utilization in exerciseFor basal conditions, U0 per unit of body weight

will be constant and within limits should be thesame for different subjects. As discussed in theintroduction, the peripheral tissues are capable ofoxidizing ketone bodies at a rate equivalent to 5to 7 times the basal metabolic needs. It has alsobeen well established that in the completely dia-betic depancreatized animals or in severe diabetesmellitus, there is essentially no change of ketonebody excretion during exercise.

From equation (3) the ketone excretion is:

E= (1 -k) (F- UO).Therefore, for two states of activity, basal and

working, if we assume that k does not change,we get:

AF=AUo.The meaning of this is clear: during exercise

the increase of utilization keeps pace with in-creased fat catabolism. This conclusion seems tobe a simple explanation of the non-increase ofketonuria in the diabetic during exercise; whereas,as has been previously pointed out, this non-increase is entirely incompatible with the obliga-tory coupling hypothesis.

Ketonuria in diabetes mellitusWehave preferred to discuss the problem of

fat metabolism in diabetes mellitus in terms ofutilization rather than in terms of urinary ketonebodies in order to emphasize ketone body oxida-tion rather than excretion. However, it is equallypossible to show that the data on ketonuria indiabetes conform to the hypothesis. By the trans-formation of the equation (3) we obtain:

E- (1-k)(F- UO).

By way of illustration, the data in one case areplotted in Figure 8 and the points may be com-pared with the line calculated, using the constantsalready found. It is obvious that the relationshitherto described predict the course of ketonuriain the diabetic with precision.

It might also be emphasized that the informa-tion obtained from observations on ketonuria perse is limited. For ketone body formation mayvary within wide limits up to utilization = UOwithout any ketonuria. Above this value theexact relation between formation and excretioncannot be determined unless the value of the" coefficient of excess utilization," which varieswidely from subject to subject, is also known.

Direct and indirect fat metabolismThe proportion of the total fat metabolism rep-

resented by (a) preliminary partial oxidation inthe liver to ketone bodies followed by oxidationof the ketone bodies in the periphery, and (b)

857

WILLIAM C. STADIE

I5

10

gX

0-35 40 45 50 55 60

TOTAL FAT CAMkBOLISM IN KETONE EQUIIALENTS, mMI/KG/DAYFIG. 8. DumTEs MEU.Trus, KRAMER(SHAFFER, 1922)

Urinary ketone body excretion as a function of the total fat catabolism. E = (1 - k) (F - UO).Correlation = 0.99 + 0.01; k = 0.28 + 0.03; Uo = 35.3 ± 1.3 mM./kg./day.

direct oxidation initiated and completed in theperiphery, remains undetermined. There appearsto be no doubt that ketone body utilization in theperiphery may be sufficient to account for 4 to 6times the basal requirements of the animal. Butwhether the liver does in fact partially oxidize suchlarge amounts of fat is doubtful. For example,Stadie, Zapp, and Lukens (15a) found, with liverslices from depancreatized cats, ketone body for-mation equivalent to 1.3 mM. per kgm. per bodyweight per hour. But the total basal metabolismof the depancreatized cat, which must be largelythat of fat, when expressed as ketone bodies isequivalent to approximately 4.5 mM. per kgm. perhour (calculated from data of Ring and Hampel(29)). Apparently only about 30 per cent ofthe total fat metabolism could be accounted forby the mechanism of preliminary oxidation toketone bodies in the liver followed by completeoxidation in the periphery. In the normal fasted(2 to 4 days) cats they found still lower values,

viz., about 10 per cent. In a series of fasted

phlorhizinized rats, hepatic ketone body formationwas found to represent not more than 10 to 15per cent of the estimated total basal metabolism(10).

The relation of hepatic and muscle respiratorymetabolism

Another difficulty in the problem of fat metab-olism which has not been sufficiently emphasizedin the literature arises from a consideration ofthe oxygen requirements of the liver for the pre-liminary partial oxidation of fats to muscle sub-strates such as acetoacetate. From the observa-tions of Barcroft and Kato (30) on the oxygenuptake of the gastrocnemius of the dog in theresting state and during stimulation at the rateof 60 times per second, the data shown in tableVII have been calculated.7

7Barcroft and Kato's results may be too high. (Com-pare, for example, Himwich and Rose (33).) However,cutting their values in half would not influence the mainargument here.

858

FAT METABOLISM IN DIABETES

TABLE VII

Observed muscle and calculated hepatic metabolismof resting and exercising dog

For case: 100 per cent preliminary hepatic partialoxidation of fats to acetoacetic acid

CalculatedCalculated hepatic oxygen

Observed equivalent uptake requiredoxygen uptake amount of for production

by muscle* acetoacetate of acetoacetatebymucle * oxidized by from fat bymuscle partial oxidation

in liver

per gram per kgm. per gramof musde body weight of liver

micromoles millimoles micromolesper hour per hour per hour

Resting... 46 4.6 268Exercising .. 166 16.6 968

Difference.. 120 12.0 700

Conversion factors:For total combustion in muscle: 1 mole 02 = 1/4 mole

acetoacetate.For partial oxidation of fat in liver: 1 mole aceto-

acetate = 1.75 moles O2.Assume:

400 grams of muscle per kgm. body weight.30 grams liver per kgm. body weight.

* Original data of Barcroft and Kato (30).

The table illustrates the expectation in the hy-pothetical case in which the entire muscular me-

tabolism is dependent upon indirect fat utilization,i.e., one in which there is a preliminary partialoxidation of fat by the liver to acetoacetate. Itwill be noted that the increase, due to exercise, ofthe oxygen uptake per unit weight is some sixtimes greater in the case of the liver than in thecase of the muscle. Furthermore, these calcu-lated hepatic respirations both in the resting andworking state are greatly in excess of observedoxygen uptakes. For example, the observationsof Staub (31) on perfused livers of dogs give a

mean value of about 70 micromoles per gram ofliver per hour for the oxygen uptake. In theintact cat Barcroft and Shore (32) found some-

what lower values. Innumerable observations ofliver slices in sitro give a value of 75 to 100 uM.per gram per hour. These values are about 1/3

of the basal requirements and about Mo of therequirements under exercise calculated in the table.It is, of course, impossible to say that these in-tense hepatic respirations cannot be attained bythe liver in vivo, but the marked discrepancy be-tween the observed values (unless these are con-

sidered to be in gross error) and those calculatedon the basis of complete indirect fat metabolismmust be taken into consideration in interpretingthe problem of fat metabolism.

The difficulties discussed in the last two sec-tions can be overcome by supposing that a con-siderable part of the fat metabolism of muscleoccurs directly, i.e., without preliminary hepaticpartial oxidation to ketone bodies. The evidenceon this point is not unequivocal. For example,Gemmill (34), who reviews the recent literatureon this subject, was unable to find any diminutionof fat in the working muscle of the normal orphlorhizinized rat, and he concluded that when fatis used to supply energy for work in mammalianmuscle, it is used indirectly. However, in viewof the many observations of respiratory quotientsranging from 0.7 to 0.8, in the case of musclewith intact blood supply, or isolated perfusedmuscle (33), or muscle equilibrated in vitro, thepossibility of direct utilization of fat cannot beexcluded. The extreme view, held by somephysiologists, that carbohydrate only can be oxid-ized by muscle appears no longer tenable.

Schematic representation of total metabolism inthe diabetic

The -relation of ketonuria to the fat metabolismand the total metabolism elicited by the study ofthese cases of diabetes mellitus can be incorporatedinto a scheme (Figure 9) which is intended toshow the interrelations of these factors only in ageneral way. It is not a nomogram for applica-tion to specific cases. For simplicity, assume acase to be without glycosuria deriving 20 per centof the caloric needs from protein. Then

0.8 Total calories= Fat calories + Carbohydrate calories.

The figure is based upon this equation and thenecessary conversion factors. Several points ofinterest are shown by the diagram:

1. Ketonuria is avoided if the fat catabolized isequal or less than about 2.5 grams per kgm. perday. It must be remembered that this value is forthe resting state and may be higher during statesof activity.

2. When the caloric metabolism is low, keton-

859

WILLIAM C. STADIE

DIABETIC DIETS

HIGH FAT

aN

i.-

u

n

0N

2

hli

I.. 0

NO

0I-

PER CENT OfCALORIES AS:

/ ^ 20 40 60 80 FAT60 40 20 0 CARBOHYDRi

FIG. 9. SCHEMATIC REPRESENTATIONOF CARBOHYDRATEAND FAT METAOLISMIN

DIABETES MELLITUS AND THEIR RELATION TO KETONEBODY EXCRETION

uria may be absent even when the carbohydratemetabolized is quite low.

3. The greater the caloric needs, the greater

must be the proportion of carbohydrate in themetabolic mixture in order to avoid ketonuria.

4. The diets which through clinical experiencehave been recommended in the literature are, withrespect to their proportions of carbohydrate andfat, essentially of the type which avoid ketonuria.

SUMMARY

1. The problem of fat metabolism in diabetesmellitus and its relation to ketonuria is reviewedand discussed.

2. Evidence favoring the replacement of thesuccessive beta oxidation hypothesis of fatty acidsby the multiple alternate oxidation hypothesis ispresented.

3. It is shown that the diabetic animal utilizesketone bodies in the periphery abundantly andindependently of carbohydrate oxidation.

4. The hypothesis of obligatory coupling ofketone body-carbohydrate oxidation in diabetesmellitus is not supported by the quantitative datain a series of cases.

5. A simple hypothesis of fat metabolism in

diabetes mellitus is presented and shown to con-

form to the observations in these cases.

6. Other significant problems in fat metabolismare discussed.

BIBLIOGRAPHY

1. Knoop, F., Der Abbau aromatischer Fettsauren imTierkorper. Beitr. z. chem Phys. u. Path., 1904,6, 150.

2a. Dakin, H. D., The mode of oxidation in the animalorganism of phenyl derivatives of fatty acids.Part V. J. Biol. Chen., 1909, 6, 221.

b. Dakin, H. D., Physiological oxidations. Physiologi-cal Rev., 1921, 1, 394.

3. Friedmann, E., Zur Kentniss des Abbaues der Kar-bonsiuren im Tierkorper. XVII. Ueber die Bild-ung von Acetessigsiure aus Essigsiure bei derLeberdurchblutung. Biochem. Ztschr., 1913, 55,436.

RANGEOFl

LOWFAT

5'

4

0

I-

f--'a2.

RANGEOF MAXIMAL BSAl,FAT UTILIZAON

860

I

~fE

FAT METABOLISM IN DIABETES

4. von Furth, O., The Problems of Physiological andPathological Chemistry of Metabolism. Transl.by Allen J. Smith. Lippincott, Philadelphia, 1916,p. 439.

5. Raper, H. S., and Smith, E. C., Insulin and the pro-duction of acetone bodies by the perfused liver.J. Physiol., 1926, 62, 17.

6. Chaikoff, I. L, and Soskin, S., Utilization of aceto-acetic acid by normal and diabetic dogs before andafter evisceration. Am. J. Physiol., 1928, 87, 58.

7. Mirsky, I. A., Nelson, N., and Grayman, I., The uti-lization of acetone bodies. I. The influence offeeding and of glucose in nephrectomized femalerats. J. Biol. Chem., 1939, 130, 179.

8a. Blixenkrone-Moller, N., Respiratorischer Stoff-wechsel und Ketonbildung der Leber. Ztschr. f.Physiol. Chem., 1938, 252, 117.

b. Blixenkrone-Moller, N., Kohlenhydrat- und Keton-k6rperbildung aus Fettsauren in der kunstlichdurchstr6mten Katzenleber. Ibid., 137.

9. Weil-Malherbe, H., The formation of glucose fromacetoacetic acid in rat kidney. Biochem. J., 1938,32, 2276.

10. Stadie, W. C., Zapp, J. A., Jr., and Lukens, F. D. W.,J. Biol. Chem. (In press, Dec. 1940).

11. Hurtley, W. H., The four carbon atom acids of dia-betic urine. Quart. J. Med., 1916, 9, 301.

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