STUDIES ON KETOSIS Although there is ample evidence in the literature that glycerol is ...

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STUDIES ON KETOSIS
IX. GLYCOGEN FORMATION FROM VARIOUS PURIFIED AND
NATURAL FATS*
BY HARRY J. DEUEL, JR., JOSEPH S. BUTTS, HARRY BLUNDEN,
CHARLES H. CUTLER, AND LESLIE KNOTT
WITHTHETECHNICALASSISTANCE OFWILLARD GOODWIN,CHARLESGOULD,
LOIS F. HALLMAN, ROBERT HELLER, AND SHEILA MURRAY
(From the Department of Biochemistry, University of Southern California
School of Medicine, Los Angeles)
(Received for publication, October 5, 1936)
Although there is ample evidence in the literature that glycerol
is completely convertible to glucose or glycogen when administered
as such, there is no clear cut proof that it follows the same fate
when administered as a constituent of neutral fat. Thus, Cham-
bers and Deuel (1) demonstrated the quantitative conversion of
ingested glycerol to glucose in the phlorhizinized dog, while Catron
and Lewis (2) as well as Shapiro (3) found that it was a ready
source of gIycogen in the rat. Moreover, Voegtlin, Dunn, and
Thompson (4) report that this trihydric alcohol is able to counter-
act the hypoglycemic convulsions following insulin, while Shapiro
noted that it had a strong ketolytic action. However, no one has
been able to demonstrate the formation of “extra sugar” from the
glycerol moiety of fat fed to phlorhizinized dogs or to show that
the deposition of glycogen occurred in the liver as a result of the
feeding of natural fats.
In a recent report from this laboratory (5) it was found that
tributyrin as well as trivalerin was a good glycogenic agent,
although Wesson oil possessedn o ability to serve as a source of
this polysaccharide. In addition we were unable to demonstrate
as great a glycogen formation after the administration of glycerol
and sodium butyrate as occurred after the feeding of an equivalent
dose of tributyrin.
* This work was assisted by a research grant from the Rockefeller Foun-
dation.
119
This is an Open Access article under the CC BY license.
120 Studies on Ketosis. IX
The present experiments were undertaken to investigate further
any glycogenic action of various purified triglycerides and to
determine whether such a glycogenesis could be accounted for
exclusively from the glycerol fragment of the ingested fat. The
investigation was broadened to include the study of glycogen
formation from natural fats of widely varying composition.
EXPERIMENTAL
General Procedure
Male and female rats of 60 to 120 days of age were used. The
animals were fasted 48 hours, following which the substance to be
tested was fed by stomach tube. Female rats were employed for
the tests in which the animals were killed 8 hours after the adminis-
tration of the fats, while male rats were used in the other tests
(17, 24, 42 hours).
In the main group of experiments the purified fats were adminis-
tered in doses equivalent to 0.065 mole per sq.m. of body surface.
Equivalent dosages of the natural fats were calculated from the
average saponification numbers. With the exception of trilaurin,
all of the fats were fed as such with a minimum quantity of water
(0.5 cc.) to remove the last traces of fat from the stomach tube.
Because of the high melting point of trilaurin, it was necessary to
administer it dissolved in Wesson oil (13 gm. of trilaurin made to
25 cc. with Wesson oil) ; the mixture was liquid at body tempera-
ture.
Control experiments with litter mates were made with each
group of experiments. The animals were killed under amytal
anesthesia. The technique for glycogen determination was the
same as that employed elsewhere (6) with the exception that a
single volume of 40 per cent potassium hydroxide equal to the
weight of the liver was used for digestion of the tissue. The use of
1 volume prevents layering, which may result in the loss of some
glycogen.
In order to determine the extent of absorption of the fats, that
remaining in the gastrointestinal tract was ascertained. This
procedure was accomplished by passing 60 to 75 cc. of water heated
to 75” through the whole gastrointestinal tract (including large
intestine and cecum) under sufficient pressure so that the stomach
Deuel, Butts, Blunden, Cutler, and Knott 121
and intestines were dilated. A blunt hypodermic needle was tied
in the esophagus after removal of the gastrointestinal tract and
the water was slowly forced from a large syringe. The acidified
washings were extracted four times with ether. This extract was
dried several days with anhydrous sodium sulfate, after which it
was filtered into weighed extraction flasks and the ether was
removed. We have shown (5) that oil can be satisfactorily re-
covered from the gastrointestinal tract immediately after its
administration by a similar procedure. The higher values obtained
for ether-extractable material in control experiments on fasted
animals in the present tests than those reported earlier is probably
traceable to the fact that we have employed the whole gastro-
intestinal tract here, while earlier only the stomach and small
intestines were used.
The purified fats were Eastman products, the purity of which
we established by the saponification numbers and the specific
gravity. The natural fats used were ordinary commercial products.
The saponification numbers, iodine numbers, Reichert-Meissl
numbers, and specific gravities of these fats were found to be
satisfactory.
Results
The experiments in which the purified triglycerides were fed in
doses of 0.065 mole per sq.m. of body surface are recorded in
Tables I and II. Table I gives a summary of the results of the
experiments in which the rats were killed 8 hours after the admin-
istration of the fats, and those in which 17 hours elapsed between
the period of feeding and the killing of the rats; Table II records
the values for the experiments of 24 and 42 hours duration.
With the exception of trilaurin, the fats were practically com-
pletely absorbed in 8 hours, whereas identical values on the fat
content of the intestinal contents of fat-fed and control rats were
noted after 17 hours. The absorption of trilaurin was very slow
owing to its solidification in the gastrointestinal tract. Even after
42 hours the absorption amounted to only 80 per cent.
The administration of the triglycerides of the fats having an
even number of carbon atoms was followed by a glycogen forma-
tion comparable with that of an isomolecular quantity of glycerol,
TABLE I
Liver Glycogen of Fasting Female (8 Hours) and Male Rats (17 Hours) after
Administration of Various Purijied Triglycerides and Glycerol in Doses
of 0.065 Mole per Sq. M.
Liver glycogen Fat absorbed
Substance fed
Females
- -
m7. pn. cpeenrt cPeenrt cPeenrt mg. mg.
Fasting con- 18 1390.260.070.15 27.4(9)
trols
Glycerol con- 59.3 15 1231.620.150.83 12.37 29.2(7)
trols
Triacetin 141 7 1180.860.320.59 8.00324.2 41.8(5) 95.4
Tripropionin 168 9 1312.590.86 1.41 10.67386.9 33.3(6) 98.4
Tributyrin 195 9 1151.580.270.68 6.78418.6 27.5(6) 00
Trivalerin 222 9 1091.580.641.16 11.48458.6 55.8(7) 93.9
Tricaproin 249 9 1110.890.250.46 5.63531.6 31.3(8) 99.2
Triheptylin 277 8 1041.970.621.37 11.69549.5 87.6(6) 89.2
Tricaprylin 304 9 138 1.680.130.62 3.81732.5 64.8(9) 94.8
Trilauring 402 9 1400.260.080.15 980.0 757.0(6) 25.5
-
-
Males
- -
I I
Fasting con- 28 0.550.040.19 33.3(19)
trols
Glycerol con- 59.: I 15 1890.830.040.31 17.4(3)
trols
Triacetin 141 9 1810.700.060.26 426 27.4(7) 100
Tripropionin 168 10 2002.340.981.63 18.98 517 24.5(9) 100
Tributyrin 195 9 1920.550.060.17 606 27.4(7) 100
Trivalerin 222 10 2022.160.831.46 12.09 673 35.0(7) 99.8
Tricaproin 249 12 2100.380.000.11 762 28.2(8) 100
Triheptylin 277 16 2072.980.431.44 12.27 842 28.8(9) 100
Tricaprylin 304 9 2060.530.140.35 7.04 943 23.3(6) 100
Trilaurins 402 9 2130.590.120.24 1315 914.1(8) 33.0
i
* Ratio of mean difference to probable error of mean difference compared
with the glycogen of the controls.
t Average amount fed only for those animals in which the fat content of
the gut was analyzed.
$ The values in parentheses represent the number of experiments on
which this analysis was made,
5 Trilaurin was fed dissolved in Wesson oil. The average fed is com-
puted for the trilaurin alone, although approximately an equal amount of
Wesson oil was given. The absorption is calculated by considering the fat
recovered from the gut as trilaurin.
122
Deuel, Butts, Blunden, Cutler, and Knott 123
the values for triacetin, tributyrin, tricaproin, and tricaprylin
being 0.59, 0.68, 0.46, and 0.62 per cent compared with a mean of
0.83 for the glycerol controls and one of 0.15 per cent for the fast-
ing controls. All the increases are significant from a statistical
standpoint when compared with the level of the fasting control.
Trilaurin alone fails to elicit a glycogenesis. With the exception
of tricaprylin, the glycogen in the liver has returned to the fasting
level 17 hours after feeding the triglycerides mentioned above.
TABLE II
Liver Glycogen of Fasting Male Rats 94 and 42 Hours after Administration of
Tripropionin and Trilaurin in Doses of 0.065 Mole per Xq. M.
T
1 Liver glycogen 1 Fat absorbed
Substance fed
,?lg. gm. Et A% Et mg. mg.
24 hrs. Fasting controls 5 0.280.050.17 28.9(4)
Tripropionin 168 5 2142.120.241.17 53: 57.0(3) 94.7
Trilaurin 402 4 1960.290.070.16122: 244.3(3) 82.3
42 hrs. Fasting controls 5 0.82 0.070.30 34.9(5)
Tripropionin 168 4 2190.800.110.44 548 30.4(4) 100
Trilaurinl 402 5 2030.490.090.30120~ 296.4(4) 78.4
* Average amount fed only for those animals in which the fat content of
the gut was analyzed.
t The values in parentheses represent the number of experiments on
which this analysis was made.
$ Trilaurin was fed dissolved in Wesson oil. The average fed is com-
puted for the trilaurin alone, although approximately an equal amount of
Wesson oil was given. The absorption is calculated by considering the
fat recovered from the gut as trilaurin.
A much greater increase in liver glycogen followed the adminis-
tration of tripropionin, trivalerin, and triheptylin than could be
accounted for on the basis of the glycerol content. The glycogen
level is somewhat higher after 17 hours than at the 8 hour interval,
the mean values for tripropionin, trivalerin, and triheptylin at the
latter interval being 1.63, 1.46, and 1.44 per cent, respectively,
compared with a level in the fasted animals of 0.19 per cent. The
average glycogen value after tripropionin was still increased over
124 Studies on Ketosis. IX
TABLE III
Liver Glycogen of Fasting Female Rats (8 Hours) after Administration of
Various Fats ia Do-s es Equivalent to 0.065 Mole per Sq. M.
I
Liver glycogen Fat absorbed
M&III
Substance fed body Per
Neight cent
ab-
xbed
-l-l- l-l-
w. gm.
Fasting controls 38 140 0.26 0.06 0.12 25.4(24
Cottonseed oil 759 10 154 0.09 0.06 0.08 1930 742.1(8) 62.9
Wesson oil 761 18 121 0.38 0.03 0.13 1796 684.0(S) 63.3
Linseed “ 777 10 146 0.15 0.10 0.13 1938 767.8(8) 61.7
Peanut “ 780 10 147 0.66 0.06 0.14 1972 762.8(9) 62.6
Coconut “ 586 10 151 0.22 0.12 0.18 1462 540.3(7) 64.7
Butter fat 675 10 145 0.19 0.06 0.09 1680 443.3(7) 75.2
* Average amount fed only for those animals in which the fat content
of the gut was analyzed.
t The values in parentheses represent the number of experiments on
which this analysis was made.
TABLE IV
Liver Glycogen of Fasting Male Rats 1Y Hours after Administration oj Various
Fats in Doses Equivalent to 0.065 Mole per Sq. M.
T
a’ Liver glycogen Fat absorbed
.$ I I
Substance fed
Fasting controls 18 0.720.100.28 36.9(13)
Cottonseed oil 759 11 2720.290.050.20 2730 266.5(11) 91.6
Wesson oil 761 13 2040.620.050.22 2278 lOl.O(lO) 97.2
Linseed “ 777 10 2810.440.070.25 2965 279.9(6) 91.8
Peanut “ 780 10 2890.960.090.31 2920 171.9(7) 95.4
Coconut “ 586 19 2210.700.120.39 3.06 1880 74.6(10) 97.8
Butter fat 675 10 2730.720.060.34 1.07 2435 185.2(4) 93.9
-
For explanation of foot-notes, see Table I.
Deuel, Butts, Blunden, Cutler, and Knott 125
that of the controls after 24 hours (1.17 per cent) but had practi-
cally dropped to the fasting level after 42 hours (0.44 per cent).
In Tables III to V are reported similar experiments on rats with
natural fats at like intervals after the fat feeding.
In distinction to the purified fats, the absorption of the natural
fats was incomplete in every case at 8 hours. This is probably
traceable to the greater bulk fed in the latter case, owing to the
increasing molecular weight. In the experiments with 17 hours
interval, the absorption exceeded 90 per cent. None of the aver-
ages of liver glycogen in the 8 hour test is increased over the level
TABLE V
Liver Glycogen of Fasting Male Rats $4 and 48 Hours after Administration of
Various Fats in Doses of 0.065 Mole per Xq. M.
2
Liver glycogen Fat absorbed
R
8 g %* -
Substance fed “2Z2 ‘3 sg-eCg.2 4; E 45 E d “3. 4 [z0$ f;-;y cPaeben-rt
I 2 ik-2 sorbed
-_ ~-
mg. Qm. cPeenrt cPeenrt cPeenrt "Q. +=Q.
24 hrs. Fasting con 28 1920.890.050.29 31.1(9)
tro1s
Wesson oil 761 15 1880.57 0.06 0.30 2275 83.8(8) i 97.7
Coconut “ 586 30 2031.080.080.37 2.001765 53.9(g) 98.8
Butter fat 675 29 1960.970.060.33 1.062365 59.0(7) 98.8
42 hrs. Fasting con 15 0.820.05 0.21 32.5(10)
trols
Wesson oil 761 12 2070.470.050.17 2364 54.3(9) 99.2
Coconut “ 586 15 2150.710.050.27 1806 36.8(10) 99.8
Butter fat 675 15 2030.360.060.16 2385 30.6(9) 100
For explanation of foot-notes, see Table I.
of the control animals. On the other hand the values of liver
glycogen are slightly but significantly higher 17 hours and 24 hours
after the feeding of coconut oil than in the control animals. A
similar tendency is noted in the experiments with butter fat. All
the other natural fats (Wesson oil, cottonseed oil, peanut oil, lin-
seed oil) are entirely void of glycogenic action.
DISCUSSION
All of the purified triglycerides from triacetin through tricaprylin
are definite sources of glycogen in the fasting rat, while most of the
126 Studies on Ketosis. IX
natural fats such as Wesson oil, cottonseed oil, peanut oil, and lin-
seed oil are entirely without glycogenic ability, although a slight
formation of this polysaccharide sometimes followed the adminis-
tration of similar amounts of coconut oil or of butter fat. The
average percentage of glycogen with fats having fatty acids with
an odd number of carbon atoms usually approximates 1.50 per
cent, although an individual value as high as 2.98 per cent was
noted with triheptylin. Even with those fats with acids having
an even number of carbon atoms, one notes an individual value as
high as 1.58 per cent (tributyrin), although the averages are near
0.5 per cent for the single dose.
The glycogen content of the livers of the rats receiving triacetin,
tributyrin, tricaproin, and tricaprylin can be accounted for on the
basis of the glycerol components of these fats. We now believe
that the inability to record as high values in the control experi-
ments with hydrolyzed tributyrin as with the neutral fat which we
reported in our earlier work (5) is due to the alkaline effect of the
residue resulting from the combustion of sodium butyrate. A
glycogenolysis must occur to furnish the lactic acid needed for
such a neutralization. There is no evidence that the short chained
fatty acids with an even number of carbon atoms contribute to
the glycogen so stored in the liver. Not only have we found (5)
that the sodium salts of butyric, caproic, and caprylic acids were
entirely without potentiality as sources of glycogen but also we
have noted (7) that the ethyl esters of these acids are also in-
effective. In the latter case any possible criticism that the glyco-
genesis is obscured by a possible alkalosis is avoided.
The question arises as to why the glycerol of the lower molecular
fats is able to produce glycogen when that from such fats as cotton-
seed oil, peanut oil, or linseed oil with high molecular weights
cannot do so. It has been demonstrated by.Eckstein that neither
tributyrin (8) nor tricaproin (9) can be stored in the fatty tissues,
while Powell (10) has shown the same to be true with tricaprylin.
On the other hand tricaprin (11) and trilaurin (8, 10) are both
capable of retention in the depot fat, as are the glycerides of the
higher acids of which such natural fats as cottonseed, peanut, or
linseed oil are almost entirely composed.
The fats which cannot be retained in the tissues either are not
resynthesized into the neutral fat after absorption or if resynthes’s
Deuel, Butts, Blunden, Cutler, and Knott 127
does occur, the fat is soon broken down and the fatty acid is either
completely oxidized or built into a new longer chain fatty acid.
In any event considerable amounts of glycerol are left behind and
this is converted into glycogen. In view of the fact that the
glycogen level of the liver after triacetin, tributyrin, tricaproin, and
tricaprylin approximates that of the glycerol controls, it is sug-
gested that the resynthesis of the short chain acids into longer
chain fatty acids (with a new formation of neutral fat) is unim-
portant and that most of the fatty acid must disappear, leaving the
major amount of glycerol free to be transformed into glycogen.
Because of the slowness of absorption of trilaurin, the present tests
do not give absolute evidence about the fate of this fat. However,
the fact that no glycogen whatsoever was noted in the tests as
long as 42 hours after the feeding of this triglyceride (at which
time the trilaurin was 80 per cent absorbed) would seem to indicate
that it was not a glycogen former. This would fit in with the
experimental findings that it can be deposited in the tissues (8, 10).
On the other hand, such natural fats as cottonseed, peanut, and
linseed oils which can be retained as neutral fats in the tissues
have no glycerol available for glycogen synthesis, but it is entirely
stored as a component part of the neutral fat. In none of the tests
could any glycogen formation be demonstrated after the adminis-
tration of these neutral fats.
Coconut oil and possibly butter fat apparently give rise to small
but definite amounts of glycogen. These fats are the only com-
mon ones with an appreciable content of the low weight triglyc-
erides. Although both of these fats contain about the same
amount of triglycerides of fatty acids of 8 carbon atoms and less
(9.08 per cent for butter fat as compared with 7.20 per cent for
coconut oil), coconut oil has an extremely large proportion of
tricaprin and trilaurin (66.40 per cent), while butter fat has prac-
tically none of these triglycerides (12).
A much higher glycogen level followed the administration of the
fats with short chain acids having an odd number of carbon atoms
(tripropionin, trivalerin, and triheptylin) than occurred after the
corresponding even chained acids. This is further support of our
earlier proof that propionic, valeric, and heptoic acids are glyco-
genie (5) when fed as the sodium salts. The close uniformity in
level both at 8 hours (1.41, 1.16, 1.37) and at 17 hours (1.63, 1.46,
128 Studies on Ketosis. IX
1.44) would seem to indicate that tripropionin, trivalerin, and
triheptylin are of equal value as glycogen formers.
SUMMARY
The administration of triglycerides having fatty acids with an
even number of carbon atoms (triacetin, tributyrin, tricaproin,
tricaprylin) to fasting rats was followed by the deposition of
significant amounts of glycogen in the liver. The source of glyco-
gen could be accounted for on the basis of transformation of the
glycerol of the triglycerides.
After the feeding of triglycerides having odd chain fatty acids
(tripropionin, trivalerin, triheptylin) to fasting rats, much larger
amounts of glycogen were found in the liver than could have
originated from the glycerol moiety of the fat. This is further
proof of the convertibility of the odd chain fatty acids into carbo-
hydrate.
Trilaurin and a number of natural fats (Wesson oil, cottonseed
oil, peanut oil, linseed oil) possessed no glycogenic activity,
although significant quantities of glycogen resulted from the in-
gestion of coconut oil and butter fat.
It is suggested that those fats which cannot be stored as such in
the tissues (triglycerides up to and including tricaprylin) are
decomposed, with the result that the glycerol is available for glyco-
gen synthesis; on the other hand, the fats which can be deposited
in the tissues as such yield no glycogen and the glycerol is stored
away as a component of the neutral fat. Coconut oil and possibly
butter fat serve as sources of glycogen in proportion to the amount
of triglycerides of small molecular weight which they contain.
The triglycerides of the fatty acids through caprylic in doses of
0.065 mole are practically completely absorbed in 8 hours, while
the natural fats in similar molecular doses are only about 62 per
cent absorbed (butter fat 75 per cent). The latter are completely
absorbed in 17 hours.
BIBLIOGRAPHY
1. Chambers, W. H., and Deuel, H. J., Jr., J. Biol. Chem., 66,21 (1925).
2. Catron, L. F., and Lewis, H. B., J. Biol. Chem., 84,553 (1929).
3. Shapiro, I., J. Biol. Chem., 108,373 (1935).
4. Voegtlin, C., Dunn, E. R., and Thompson, J. W., Am. J. Physiol., 71,
574 (1924-25).