hepatic ketogenesis and ketolysis in different species

HEPATIC KETOGENESIS AND KETOLYSIS IN DIFFERENT SPECIES

BY PHILIP P. COHEN AND IRENE E. STARK

(From the Department of Physiological Chemistry, University of Wisconsin, Madison)

(Received for publication, June 20, 1938)

It is generally agreed that in the animal body the liver is the chief site of ketogenesis (l-4). Ketolysis, on the other hand, has been claimed to be chiefly an extrahepatic function (3-9).

Jowett and Quastel (4) reported that rat liver slices were capable of destroying ketone bodies under aerobic conditions, an effect which they found was inhibited by malonic acid. This finding has been confirmed by Edson and Leloir (7) and Stark and Cohen (10).

The monkey, as well as man, develops a ketosis rapidly and readily in contrast to many other species, such as the rat, rabbit, and guinea pig. We were led to investigate the rates of hepatic ketogenesis and ketolysis in the well fed and fasted rat, rabbit, guinea pig, and monkey as a possible basis for explaining this species difference.

Methods

Liver slices from healthy well fed and fasted male white rats, rabbits, guinea pigs, and Macacus rhesus monkeys were used in a Barcroft-Warburg apparatus. The fasting period was 24 hours except in the case of the rabbits which were fasted for 36 hours. The liver slices, 10 to 20 mg. of dry weight, were immersed in a Ringer-phosphate buffer solution of pH 7.4, to which was added the substrate previously neutralized to the same pH. The flasks were filled with pure oxygen and shaken for 1 hour at 38”.

Sodium butyrate made up to give a 0.01 M concentration was used as a substrate for determining the rate of ketogenesis. So- dium acetoacetate, prepared according to Ljunggren (ll), and freed of methyl alcohol, was used as substrate in determining the

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98 Hepatic Ketogenesis and Ketolysis

rate of ketolysis. Acetoacetic and /3-hydroxybutyric acids were determined at the end of the experimental period.

Edson’s (12) method of filtrate preparation and acetoacetic and @-hydroxybutyric acid determinations was used with slight modifications. Control experiments, to which no substrate was added, were run in all cases and treated similarly to the non-control experiments. Sodium acetoacetate solutions were freshly prepared on the day previous and the strength of the solution determined in quadruplicate on the day it was used as substrate. The concentration of acetoacetic acid used as substrate was between 0.006 and 0.008 M in the different experiments. Liver glycogen determinations, by the combined methods of Good, Kramer, and Somogyi (13) and Somogyi (14), were made in order to check the nutritional state of the animals.

Procedure

Four differential manometers and eight flasks were set up to permit measuring the rate of ketogenesis, ketolysis, spontaneous ketogenesis (no substrate added), and oxygen consumption, liver slices from a single animal being used.

At the end of 1 hour of shaking at 38’ the flasks were removed from the water bath and placed in an ice bath for 20 minutes to insure condensation of volatile products. The liver slices were then removed and washed thoroughly with fine streams of dis- tilled water. The washings and the flask contents were combined and treated with Ca(OH)s and Gus04 to give a protein- and carbo- hydrate-free filtrate. Acetoacetic and @-hydroxybutyric acids were determined on these filtrates.

Units A&o, = c.mm. of oxygen consumed per mg. of tissue (dry

weight) per hour in the experimental flask (substrate present) over an equal weight of tissue in the control flask (no substrate present)

QAcOAc = c.mm. of CO2 equivalent to acetoacetic acid formed per mg. of tissue (dry weight) per hour

QBOH = c.mm. of CO2 equivalent to p-hydroxybutyric acid formed per mg. of tissue (dry weight) per hour

QKet = sum of QAcOAc and QBOH QAcOAc (theoretical) = c.mm. of CO2 equivalent to acetoacetic acid

added initially per mg. of tissue (dry weight) -QAcOAc = difference between QAcOAc (theoretical) and

QKet at end of 1 hour

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P. P. Cohen and I. E. Stark 99

Results

In this study seven guinea pigs, ten rats, six rabbits, and three monkeys were used. Tables I through IV are combined protocols of typical experiments on a well fed and fasted animal of each species. Tables V to VII represent a compilation of the averages of all experiments.

The rate of spontaneous ketogenesis (no substrate added) in fasted and well fed animals is seen in Table V. It is apparent that

TABLE I Combined Data from Typical Experiments on Hepatic Ketogenesis and

Ketolysis in Well Fed and Fasted Rat The final concentration of butyrate was 0.01 Y: of acetoacetate, 0.007 M.

Substrate

Butyrate

Acetoacetate

None Well fed

Condition of animal

Well fed -3.91

Fasted -3.04

Well fed +0.65

Fasted -0.84

Fasted

-

-

AQOt

-

c .-

-

-

)AcOAc / QBOH

3.57 3.40 5.88 6.20

12.40 19.25 15.05 13.15 23.40 15.10 0.78 0.90 1.35 1.37 ,

4.24 4.70 2.92 3.10 4.33 4.00 3.70 6.40 5.40 7.16 1.36 2.25 2.49 2.85

-

, QKet

7.81 8.10 8.80 9.30

16.73 23.25 18.75 19.55 28.80 22.26

2.14 3.15 3.84 4.22

-

19.75 3.02 27.44 4.19 21.00 2.25 20.70 1.15 29.91 1.11 22.70 0.44

fasting results in a considerable increase in ketone body formation in all four species.

The rate of ketogenesis from butyric acid in fasted and well fed animals is seen in Table VI. In all four species there was an apparent higher rate of ketogenesis in the case of the fasted animals, although the increase is marked only in the case of the rabbit. This may be related to the fact that the rabbits were fasted for 36 hours, whereas the other species were fasted 24 hours.

Edson (12) has reported increases in QAcOAc of approximately

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double the normal with liver slices from fasted rats in the presence of butyric acid. Jowett and Quastel (15) on the other hand have reported slight but significant increases in QAcOAc in the case of liver slices from fasted guinea pigs in the presence of butyric acid.

It is to be noted that the QBOH values in all instances are approximately equal to the QAcOAc values. This is in con- firmation of Jowett and Quastel (16) and emphasizes the need for

TABLE II

Combined Data from Typical Experiments on Hepatic Ketogenesis and Ketolysis in Well Fed and Fasted Guinea Pig

The final concentration of butyrate was 0.01 M; of acetoacetate, 0.006 M.

Substrate

Butyrate Well fed

Fasted

Well fed Acetoacetate

None

Fasted

Well fed

Condition of animal

Fasted

AQOt

-2.57

-1.91

+0.19

-0.28

-

6

_-

-

!AcOA( QBOH

_ 1.83 1.86 2.28 1.33 6.95 7.35 7.38 7.46

13.80 12.70 11.65 11.25

0.44 0.84 1.46 1.20

2.09

1.73 2.32 3.13 2.69 2.69 2.41 4.07 3.72 4.36 3.98 2.93 0.67 0.76 1.73 1.56

12.29 2.65 12.68 2.64 11.27 1.48 14.36 2.83 17.85 0.33 17.92 0.86 16.80 1.17 14.65 0.47

-

, QKet

3.91 3.59 4.60 4.46 9.64

10.04 9.79

11.53 17.52 17.06 15.63 14.18

1.11 1.60 3.19 2.76

determining both QBOH and QAcOAc where a measure of ketogenesis is desired.

The AQO, values for rat and guinea pig liver in the presence of butyrate are in agreement with those previously reported by Cohen (17), Edson (12), and Jowett and Quastel (15, 16).

The rate of hepatic ketolysis in fasted and well fed animals is seen in Table VII. It is seen from these data that the well fed rat, rabbit, and guinea pig have a considerable capacity for de-

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TABLE III Combined Data from Typical Experiments on Hepatic Ketogenesis and

Ketolysis in Well Fed and Fasted Rabbit The final concentration of butyrate was 0.01 M; of acetoacetate, 0.007 M.

A&O2 JAaOAc QBOH QKet

Well fed -2.17

Fasted -4.32

Well fed -0.52

-0.90

2.72 2.82 5.54 2.91 3.30 6.21 3.80 4.06 7.86 4.29 3.15 7.44 4.89 3.98 8.87 4.82 3.73 8.55 3.92 4.77 8.69 5.70 4.40 10.10

11.10 2.73 13.83 11.03 2.50 13.53

0.74 0.28 1.02 0.73 0.45 1.18 1.54 1.17 2.71 1.67 1.09 2.76

-

-

-

-

-

-

Condition of animal Substrate

Butyrate

Fasted

Well fed

Fasted

Acetoacetate 13.50 4.63 12.58 4.03 14.30 5.61 13.32 3.22 14.90 1.07 14.55 1.02

None

TABLE IV Combined Data from Typical Experiments on Hepatic Ketogenesis and

Ketolysis in Well Fed and Fasted Monkey The final concentration of butyrate was 0.01 M; of acetoacetate, 0.007 M. -

,

.-

-

-

3 1

-

Condition of animal

17.75 0.83 16.80 0.52 11.80 +0.15 11.86 0.53 11.65 0.27 11.60 +0.25

)AcOAI QBOH QKet Substrata AQOa

Butyrate

Acetoacetate

None

Well fed

Fasted

Well fed

Fasted

Well fed

Fasted

2.94 2.72 3.16 2.92

12.80 14.75 6.32 5.97 5.56 6.95 1.21 1.14 0.90 1.10

4.09 7.03 3.01 5.83 4.39 7.55 4.42 7.34 4.12 16.92 1.53 16.28 5.63 11.95 5.36 11.33 5.82 11.38 4.90 11.85 0.77 1.98 1.33 2.47 2.07 2.97 2.24 3.34

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TABLE V Spontaneous Ketogenesis (No Subskate)

1 QAcOAc 1 QBOH

Normal rat. . Fasted “ . Normal rabbit. . Fasted “ . Normal guinea pig. Fasted “ “ Normal monkey. Fasted “

.

0.88 1.52 1.83 2.19 0.76 0.62 1.51 1.02 0.74 0.58 1.43 1.32 1.17 1.05 1.00 2.13

TABLE VI Ketogenesis from Butyric Acid (0.01 M)

Normal rat ....................... Fasted “ ....................... Normal rabbit .................... Fasted “ .................... Normal guinea pig ................ Fasted “ ” ................ Normal monkey. ................. Fasted “ ..................

TABLE VII Ketolysis of Acetoacetic Acid (0.0060 to 0.0080 M)

Normal rat ...................... Fasted “ ...................... Normal rabbit. .................. Fasted “ ................... Normal guinea pig. ............. Fasted “ ‘( .............. Normal monkey .................. Fasted “ ..................

-

-

QKet -~

2.40 4.02 1.38 2.53 1.28 2.75 2.22 3.13

AQOz QAcOAc QBOH ~~ -4.66 4.10 4.27 -3.07 5.10 4.07 -2.61 2.78 2.81 -3.76 4.54 3.45 -2.35 1.99 1.82 -1.91 2.14 2.26 -4.57 2.83 3.55 -4.45 3.04 4.40

AQOz QBOH -QAcOAo ._

+0.46 4.01 3.15 -1.11 7.28 0.80 -0.43 3.88 3.92 -0.65 3.27 0.65 +0.19 3.65 2.30 -0.28 3.75 0.63 -0.10 3.07 0.68 -0.40 5.43 0.10

QKet

8.37 9.17 5.59 7.99 3.81 4.40 6.38 7.44

stroying acetoacetate other than by conversion to p-hydroxybutyric acid. The percentage destruction in these instances is of the order of 14, 25, and 20, respectively, for the above three

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species. These values are in reasonable agreement with those of Snapper and Griinbaum (6) for perfused dog liver, which showed about 15 per cent destruction. The -QAcOAc values for rat and guinea pig are considerably higher than those reported by Edson and Leloir (7) and Quastel and Wheatley (8).

The most striking thing in these data is the inability of liver slices from a well fed monkey to destroy appreciably acetoacetic acid.

In the case of liver slices from fasted animals, there is a marked decrease in ketolytic activity with the rat, rabbit, and guinea pig, while monkey liver shows but a slight decrease. It should be noted, however, that liver slices from fasted rats, rabbits, and guinea pigs show a capacity for ketolysis about equal to that of liver slices from a well fed monkey.

The AQO, values for liver slices in the presence of acetoacetate are very low and in two instances are actually positive. The +AQOZ values imply that the rate of oxygen consumption by liver slices in the presence of acetoacetate is less than that of an equal weight of liver slices without any added substrate. Stijhr (18) has reported a decrease in QO, values in rat liver and kidney slices in the presence of acetoacetate. Earlier work by Wiggles- worth (19) showed an increase in oxygen consumption by liver slices in the presence of acetoacetate. He interpreted this to mean an oxidative destruction of acetoacetate. While Stijhr (18) could not establish a significant difference between the oxygen consumption of liver slices from well fed and fasted rats in the presence of acetoacetate, it would appear from Table VII that there is a small increase in A&O, with liver slices from the fasted animals as compared with the normals. The increase appears to be marked only in the case of the fasted rat.

Table VIII shows the effect of fasting on ketogenesis and ketolysis. The values are the differences between the normal and fasted animals. It appears from Table VIII that in the case of the rat and guinea pig there is a greater increase in ketogenesis on fasting in the absence of substrate than in the presence of butyrate. This would imply that liver slices from fasted rats and guinea pigs oxidize less butyric acid than those of normal animals. The lowered A&O, (Table VI) values in these instances are in keeping with this. It is possible that the rapid production of

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ketone bodies by spontaneous ketogenesis in these fasted animals has an inhibitory effect on the butyric acid oxidation. It is to be noted that the liver slices from the fasted rabbit on the other hand oxidize more butyric acid than the normal. The AQOz is also increased in this case (Table VI). The difference in the ability of liver slices from fasted rabbits, as compared to fasted rats and guinea pigs, to oxidize butyric acid may be due to the longer fasting period in the case of the rabbit. It is conceivable that the liver of the rabbit fasted for 36 hours has used up a larger portion of its metabolites including fat. This is supported by the lower level of spontaneous ketogenesis of the fasted rabbit as compared with the fasted rat and guinea pig (Table V). It follows that butyric acid when added to liver slices from the fasted rabbit will be more readily oxidized both because of the depletion of other

TABLE VIII

Effect of Fasting on Ketogenesis and Ketolysis

AQKet (no substrate) (b%?:%) AQAcOAc

Rat, .............................. +1.62 +0.80 -2.35 Rabbit. .......................... +1.15 +2.40 -3.27 Guinea pig ........................ +1.47 +0.59 -1.67 Monkey ........................... +O.Ql +1.06 -0.58

substrates and the presence of a smaller concentration of spon- taneously produced ketone bodies, which act as an inhibitor of the butyrate oxidation.

In the case of the monkey, the rate of ketogenesis in the presence of butyrate is of about the same order as the rate of spontaneous ketogenesis in the fasted animal. Thus in this case butyrate is oxidized at about the same rate. The A&O, values are in support of this (Table VI).

The decrease in ketolytic power on fasting is seen in the third column of Table VIII. It is apparent that with the exception of the monkey, the decrease in ketolytic capacity on fasting is of such a magnitude as to account for more than the increase in ketogenesis. It is of interest to note that the greatest decrease in rate of ketolysis with fasting (rabbit) is associated with the greatest increase in ketogenesis from butyric acid. In the case

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of the monkey, the decrease of ketolysis with fasting is slight as would be expected from the fact that the normal liver has but a very limited ketolytic capacity.

Table IX gives glycogen values for the well fed and fasted rat and monkey. It is to be noted that at the time of introduction to the flasks, the glycogen value of the liver slices from the well fed monkey is quite high (3.20 per cent). Thus the failure of normal monkey liver to break down acetoacetate cannot be due to a low liver glyeogen.

TABLE IX Per Cent Glycogen in Rat and Monkey Liver

NOiWd Fasted

---___ Immediately on death.. . . . . . . . . . 4.09 5.09 0.55 0.37 At time of introduction of slices to flasks, us-

ually 45 to 60 min. after death. . . 1.97 3.20 0.43 0.33

DISCUSSION

Quastel and Wheatley (8) and Edson and Leloir (7) using liver slices from normal animals have reported much lower rates of hepatic ketolysis than those reported here. The rates reported by these workers in the case of normal rat and guinea pig are of the order reported here for fasted animals. A possible explanation for the differences resides in the fact that these workers employed a bicarbonate-buffer system in their experiments, whereas a phosphate-Ringer’s solution buffer system was employed by us. Ciaranfi (20) studying the metabolism of butyric acid and ketone bodies in guinea pig liver slices reported that in the presence of phosphate-Ringer’s solution there is a considerably greater oxida. tion of butyric acid and breakdown of acetoacetate than in the presence of bicarbonate-Ringer’s solution. On this basis one might speculate that a phosphorylation mechanism may be in- volved in hepatic ketolysis.

The failure of liver slices from a well fed monkey to destroy ketone bodies to any appreciable extent, as compared with the other three species, is difficult to explain on any other basis than

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a species difference. It is well known that there is a marked difference between Primates and the lower species in their susceptibility to a ketosis (21-23). Whether or not this difference in hepatic ketolysis is part of the explanation for the difference in susceptibility to a ketosis between the species is difficult to say. The large amount of work with various techniques showing the extrahepatic tissues, especially the muscles and kidneys (3-9), to be much more active ketolytically than the liver is very con- vincing. However, it is conceivable that hepatic ketolysis can keep pace with hepatic ketogenesis, within broad limits in the case of the rat, rabbit, and guinea pig, and very narrow limits in the case of the monkey, and so serve to keep the level of ketonemia low. The lowering of liver glycogen, as by fasting, would result early in a ketonemia in the case of the monkey, and only late in the case of the other three species, because of the differences in their capacity for hepatic ketolysis.

The relationship between fasting and hepatic ketolysis would seem to implicate liver glycogen as the most important factor. From the data presented here, it would appear that the increase in hepatic ketogenesis (spontaneous) with fasting is more than accounted for by a failure in hepatic ketolysis. The metabolic relationship between hepatic glycogen and ketolysis, however, is still obscure. The mechanism of ketolysis, especially in the kidney, has been studied by Edson and Leloir (7) and Quastel and Wheatley (8). The possible role of the succinate system in the mechanism has been considered by both groups of the above workers but there is no basis as yet for stressing the importance of this system in explaining the ketolytic mechanism. The ob- served action of malonate on inhibiting the breakdown of acetoacetate (4, 7, 10) is probably not entirely due to an inhibition of the succinate system. It is to be hoped that further investiga- tions on the intermediate stages of the ketolytic mechanisms will bring out the relationship between glycogen and ketolysis more clearly.

SUMMARY

1. Liver slices from a well fed monkey have a very low rate of ketolysis as compared with liver slices from well fed rats, rabbits, and guinea pigs.

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2. Liver slices from fasted rats, rabbits, and guinea pigs have very low rates of ketolysis, of about the same order as liver slices from a well fed monkey. Fasting in the case of the monkey results in a slightly lower rate of ketolysis.

3. The difference between the rates of hepatic ketolysis of the monkey and the other three species is discussed with relation to the differences in susceptibility to ketosis between the Primates and the lower species.

BIBLIOGRAPHY

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Philip P. Cohen and Irene E. StarkKETOLYSIS IN DIFFERENT SPECIES

HEPATIC KETOGENESIS AND

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