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Lipid metabolism in kidney and liver tissue from normal and diabetic rats
BIOCHIMICA ET BIOPHYSICA ACTA
307
BBA 55632
LIPID
METABOLISM
AND DIABETIC
BARRY
J. BURNS
IN KIDNEY
TISSUE FROM NORMAL
RATS
AND
J. CLINT
ELWOOD
Department of Biockemistry, Upstate Medical Syrtacecse, N.Y. 13210 (U.S.A.J (Received
AND LIVER
Center,State University
of New York,
June 13th‘ 1969)
SUMMARY
I. Normal and diabetic rats were fed four different diets: laboratory chow, 5% corn oil, 5% hydrogenated coconut oil and a fat-deficient diet. Diabetic liver slices show decreased 0, utilization. The diabetic kidney tissue responded with increased 0, consumption. [rJ4C]acetate labeling of fatty acids was depressed in all the diabetic livers. Diabetic kidney tissue had higher rates of lipogenesis than control kidney tissue. Cholesterol synthesis from acetate was depressed in the diabetic liver except when the diet fed was fat deficient. Diabetes did not influence cholesterol labeling in kidney tissue. 2. Liver from normal and diabetic rats fed hydrogenated coconut oil and fatdeficient diets had increased amounts of monoenoic acids compared to corn-oil-fed animals. Normal liver from animals fed saturated-fat and fat-deficient diets had decreased amounts of stearic and linoleic acid. The diabetic livers had more linoleic acid than normal livers. Kidney tissue from normal animals fed the saturated-fat and fat-deficient diets also had increased amounts of oleic acid and decreased amounts of linoleic acid compared to that from animals fed unsaturated fatty acid. Kidney tissue from diabetic animals had greater amounts of linoleic acid.
INTRODUCTION
There now exists a voluminous amount of scientific literature related to the effect of diet and/or hormonal alterations on many metabolic pathways of rat-liver tissue. Enzyme induction and repression by diet and hormonal regulation have been observed under many circumstances *- d. Some investigators have shown that the level of lipogenesis is dependent on the type of diets-la. Previous studies have also shown that lipogenic pathways are altered in the diabetic animalla-la. Cholesterol biosynthesis has also been reported to be regulated by dietS~8~12~18-22. SIPERSTEIN AND GUESTED showed that feeding a 5% cholesterol diet decreased hepatic cholesterogenesis by inhibiting /I-hydroxy-j?-methylglutaryl-S-CoA reductase (negative feedback inhibition). BiochP’ne. Biofihys. Acta, 187 (1969) 307-318
B. J.
308
BURNS, J. C. ELWOOD
Kidney tissue, on the other hand, has not received an intensive investigation under the same experimental conditions as the liver. Early studies of CHERNICKet al.23 and others2Q-31 have shown that the kidney slice in vitro can synthesize fatty acids and cholesterol from glucose and acetate. The rate of synthesis of fatty acids in the kidney from acetate has been stated to be IO--ZO~!~that of the IiveP. Cholesterol synthesis in the kidneys9 has been shown to be about zsi/, that found in liver. Fasting or cholesterol feedings9 has little or no effect on the rate of cholesterol synthesis in the kidney. Studies of VAN BRUGGENet ~2.30with the immature macaque (Rhesus) monkey have shown that kidney slices have a higher rate of incorporation of [r-laCjacetate and jz-r*C]mevalonic acid into %O, than liver. ELWOODANDVAN BRUGGEN~~have shown that the kidney tissue has an active cholesterogenic process. The following experiments were designed to study the effects of diets containing different types of fat on 0, consumption, [ I-Xjacetate incorporation into fatty acids and cholesterol in the normal and diabetic kidney and to compare these findings with liver tissue response. Also, in the present study, fatty-acid profiles of liver and kidney tissues from normal and diabetic rats were compared to see what changes were elicited by feeding different types of fat in the diet. MATERIALSANDMETHODS
Male adult Sprague-Dawley rats obtained from Carworth Farms, Inc. (New City, Rockland County, N.Y.) weighing 180-200 g were used in these studies. I7pon arrival, all rats were fed Big Red Chow* ad libitum for 4 days, after which time some of the animals were fasted 48 h. The diabetic state was induced in these fasted animals by the administration of a 10% solution of alloxan in 0.9:/o saline solution. Either 60 mg of alloxan/kg body weight were given intramuscularly or 200 mg/kg body weight intraperitoneally. After 14 days, the animals were fasted 24 h and tail blood was collected for glucose determinations (Worthington, Glucostat Method). Only those rats which had fasting-blood-sugar levels greater than 200 mg/roo ml of blood were considered diabetic and used for further study. Diets
Four different regimens, varying in amount and kind of fat, were employed in this study. The four diets were the following: (I) Big Red Iaboratory Chow containing 5% crude fat (Country Best Foods, Agway, Inc., Syracuse, New York). (z) Fat-free diet plus 5% corn oil. (3) Fat-free diet plus 5% hydrogenated coconut oil. (4) Fat-free diet. .-_ _ * Big Red Laboratory Chow. Guaranteed analysis: minimal 24.0% crude protein, 5.0% crude fat
and maximal 5.0 % crude fiber. Ingredients: soybean meal, ground oats, wheat midlings, corn meal, ground wheat, dehydrated alfalfa meal, dried skimmed milk, fish meal, ground limestone, vegetable oil, D-activated plant sterol (“source of vitamin D-2 “), vitamin A, palmitate, salt, MnO, IWO,, Na,SO,, cue, COCO,, zno, CaIO,. Biochim.
Biophys.
Ada,
187 (1969)
307-318
LIPOGENESIS IN KIDNEY AND LIVER
309
Diets 2, 3 and 4 were purchased from Nutritional Biochemical Corporation (Cleveland, Ohio) *. The rats were allowed to eat ad lib&m for a minimum of 6 days, and most animals were used between 6-12 days. The individual animals were not weighed, but the total quantity of synthetic diet consumed was the same in all cases. 2 h before killing by decapitation, food was removed. After sacrificing the rats, the livers and kidneys were quickly excised and placed in cold physiological saline solution prior to slicing. Tissue slices weighing IOO + IO mg were prepared using a Stadie-Riggs slicer. Slices for experiments in vitro were cut after the capsular tissue of the liver and kidney was discarded. Only the cortex portion of the kidney was used in these studies. The IOO f IO mg slices were placed in a r7-ml single-side-arm Warburg flask containing 1.9 ml of phosphate buffer (pH 7.4). A piece of filter paper was placed in the center well of the Warburg vessel, except when using bicarbonate buffer, and impregnated with 0.2 ml of 8 M KOH to collect the CO,. Preliminary experiments were conducted using either substrate levels of [I-%]acetate (12000 nmoles/flask) or tracer quantities (40 nmoles/flask). The changes elicited by feeding, starvation or diabetes were seen whether substrate or tracer amounts of acetate were employed, although the order of magnitude of the changes was different. Therefore, 0.1 ml of radioactive precursor (40-70 nmoles [I-Xlacetate) was added to the Warburg vessel. The Warburg vessels containing phosphate buffer were flushed with pure 0, for 15 set before being placed in the Warburg bath maintained at 37’. The radioactive tracer was added from the side arm after a ro-min equilibration period. The flasks were incubated for I h at 37O. 0, consumption was followed by standard manometric techniques. At the end of the r-h incubation period, the reactions in the Warburg vessels were stopped by the addition of 0.2 ml of a 20% solution of trichloroacetic acid. of lipids The tissue slices were washed with hot distilled water to remove any excess radioactive tracer. The tissue slices were transferred to screw-cap culture tubes to which 5 ml of an 11% alcoholic KOH solution were added. The tissue was saponified for 30 min under N, and then the solution was reduced in volume to approx. I ml. 3 ml of ethanol and g ml of distilled water were added to the tube. To the above alkaline mixture was added an equal volume of petroleum ether and the nonsaponifiable material was extracted 3 times. The remaining tissue digest was made acidic to congo red paper and extracted 4 times with petroleum ether. The extracted fatty acid fraction was dissolved in 5 ml of absolute ethanol, I ml of which was plated on aluminum planchets and the radioactivity was determined as infinitely thin equivalents. The remaining fatty acids were methylated for gas-liquid chromatography. Cholesterol was precipitated from the nonsaponifiable fraction with digitonin, Radioassay
* Fat-deficient diet compositions. “Vitamin-free” casein 21.10% ; Alphacel “cellulose” 16.45% ; sucrose 58.45 o/0 and salt mixture USP XIV 4%. The following vitamin supplements were added : Choline chloride 6oog mg/kg, nicotinic acid 601, inositol 303. vitamin A concentrate (200000 units/g) 99.2. vitamin D concentrate 66.1 (400000 units/g), alpha tocopherol 225, menadione 2.26, thiamine hydrochloride 22.05, riboflavin 22.05, calcium pantothenate 45.2. Biochinz. Bioplzys.
Acta,
187 (1969) 307-318
B.
310
J. BURNS,
J. C. ELWOOD
allowed to stand overnight to insure complete precipitation, washed with acetone times and taken up in methanol and plated as infinitely thin plates. Samples of nonsaponifiable material, without digitonin precipitation, were also radioassayed as infinitely thin samples, but no significant differences were observed. 2
Gas chromatographic analysis was performed with the aid of two different gas chromatographs. One was a Perkin-Elmer-rg4D gas chromatograph having a thermistor (hot wire) detector. The column was packed with Chromosol P (diatomaceous earth) as the stationary phase and coated with diethylene glycol succinate polyester. The column temperature was maintained at 204’. The second gas chromatograph was a F and M Scientific Corporation Model 402 equipped with a hydrogen flame detector. The same type of column support and liquid phase diethylene glycol succinate were employed in the F and M gas chromatograph as in the Perkin-Elmer gas chrolnatograph. Fatty acid ester separations were performed on the F and M by programmed temperature techniques. Programmed temperature runs were from 100' to 200’ at the rate of r”/min. RESULTS
It is well known that bicarbonate buffer stimulates fatty acid biosynthesis as compared to phosphate buffer. In the present experiments it was felt desirable to obtain information as well as lipid labeling data. However, we felt it necessary to show that we could indeed elicit the same responses in fatty acid synthesis with phosphate buffer as has been shown in bicarbonate buffer. Table I presents the data for the comparison of fatty acid labeling from [I-14C]acetate in bicarbonate and phosphate buffer from liver tissue of animals fasted 48 h and then re-fed a fatl-ABLE
I
[I-~%]ACETATE INCORPORATION INTO FATTY ACIDS IN
LIVER
SLICES
OF
NORMAL
ANIMALS in vi&o
Animals were fasted 48 h and then refed a fat-deficient diet for the period of time indicated. Zero day implies a 48-h fast. roe mg of tissue were incubated for 60 min in the presence of 0.5 fimole of [I-Tlacetate (pH 7.4) at a temperature of 37O in a volume of 1.9 ml of buffer. Days on _-cl I 2 3 87 .--
diet
_____.---_I
_-~
76 [W]Acetate
____-
Carbonate
~~lcoyporat~on
buffeer -...-____-
Phosphate
0.03 6.56 5.28 -
0.03 2.72 2.36 1.96
I.90 I.93
0.80 -.
buffer ~--.-
deficient diet for varying time periods. It is seen that bicarbonate buffer stimulated fatty acid synthesis over that obtained in phosphate buffer. However, liver tissue in phosphate buffer responded to the fasting and refeeding routine by a go-fold increase in fatty-acid labeling. The data shows that the increase in synthesis from fasting and refeeding was transient and that after about I week the synthesis rate was stabilized and much lower than the peak rate at about 48 h. However, this Biochim.
Biophys.
Acta,
187 (1969)
307-318
LIPOGENESIS IN KIDNEY AND LIVER
311
rate is still considerably higher than synthesis from liver tissue of animals on laboratory chow. The same observation was reported by SPENCER et a1.32 who showed that the influence of fasting and refeeding was short lived (about 72 h in his experiment). 0, consum$tion The pmoles of 0, utilized by liver and kidney tissue from normal diabetic rats are listed in Table II. There was a statistically significant TABLE 0,
and alloxan decrease in
II
UTILIZATION
BY
LIVER
AND
KIDNEY
TISSUE
Data are expressed as mean 3 S.E. In this table and those to follow, a P value equal to or less than 0.05 is considered to indicate a statistically significant difference. Each number in Tables II-IV represents at least 6 animals in duplicate. pmoles
Diet Laboratory Liver Kidney
5% Hydrogenated Liver Kidney
IOO
mg tissue per h P value
Diabetic
8.1 f 0.43 12.9 * 0.42
6.0 f 16.2 f
0.37 0.93
<0.005 <0.005
6.9 I_t 0.52 14.4 * 0.58
5.1 + 0.33 17.8 * 0.60
to.005 <0.025
coconut oil 6.5 & 0.17 14.9 nk 0.77
5.4 rt 0.25 16.9 k 0.53
<0.005 <0.05
6.5 f 0.24 13.4 k 0.66
4.4 + 0.08 15.5 * 0.66
chow
5% Corn oil Liver Kidney
Fat-deficient Liver Kidney
0, consumed per
Normal
0, consumption by the diabetic livers as compared to controls regardless of the diet fed. This decreased 0, consumption by diabetic liver was reported previously by ELWOOD AND VAN BRUGGEN~’ from animals receiving laboratory chow. In contrast to liver tissue, kidney tissue from diabetic animals had significantly increased 0, consumption as compared to normal kidney tissue. Altering the diet did not change the general picture. Whether increased oxidation by kidney slices from diabetic animals is related to the hyperuremia of the alloxan diabetic rat is unknown. Fatty acid synthesis Fatty acid synthesis in liver and kidney slices of normal and alloxan diabetic rats was followed by the incorporation of [I-lK]acetate (Table III). We have confirmed many reports that acetate incorporation was decreased 3-rz-fold in the diabetic livers depending on the type of diet consumed. Also, acetate incorporation by diabetic liver tissue was increased to lower normal values when the animals were maintained on a fat-deficient diet; but these diabetic liver slices still had decreased synthesis from the normal value of 0.59%. The labeling of fatty acids from [I-Xlacetate in kidney tissue of normal and diabetic animals is also shown in Table III. It is seen that the kidney of the diabetic incorporated more acetate into fatty acids than the normal tissue under all nutritional Biochim.
Biophys.
Ada,
187 (1969) 307-318
B. J. BURNS, J. C. ELWOOD
312 TABLE
III
INCORPORATION INTO FATTYACIDS o/o [14C]Acetate incorporation IOO mg tissue per h
NOWEal
Diet
Laboratory Liver Kidney
per
P value
Diabetic
chow 0.15 + 0.04 0.04 * 0.01
0.01 * 0.08 =
0.47 * 0.02 0.05 5 O.OI
0.04 rt 0.03
0.16 i 0.03
co.025
coconut oil 0.23 & 0.08 0.04 * 0.01
0.02 & 0.01
<0.05
0.03
<0.05
0.59 tr 0.09 0.03 11:0.01
0.12 & 0.03 0.08 11:0.01
5% corn oil
Liver Kidney 5% hydrogenated Liver Kidney Fat-deficient Liver Kidney
0.12
&
0.002
0.02
<0.05
<0.005 <0.005
regimens tested. This is the opposite effect seen in liver tissue. Also, a fat-deficient diet did not stimulate kidney-tissue lipogenesis as was seen in liver tissue. Cholesterol
biosynthesis
The incorporation of acetate into cholesterol was decreased in the diabetic livers from animals receiving chow diet, corn oil or a hydrogenated coconut oil diet (Table IV). However, diabetic animals fed fat-deficient diet had a very marked stimulation in cholesterol labeling from acetate, whereas normal liver tissue had depressed labeling compared to fat-fed animals. CLARENBURG AND CHAIKOFF~~ have reported similar findings while feeding a high-glucose no-fat diet. The incorporation of [r-Xlacetate into kidney cholesterol was not changed by any of the dietary manipulations. A comparison of rate of incorporation between liver and kidney is not valid since the optimum conditions for cholesterol biosynthesis in kidney tissue have not been established. TABLE
I\
“C INCoRPoRATIoN INTO
CHOLESTEROL
~~
0/O1% incorporation per IOO mg liver per h
Dirt
Laboratory Liver Kidney
P value
NOWVLd
Diabetic
I.9 & 0.40 0.07 * 0.05
0.08 5 0.03 0.04 * 0.01
0.46 k 0.12 0.10 k 0.03
0.16 :t 0.06 0.08 i 0.01
0.05
coconut oil 0.25 * 0.05 0.07 _t 0.02
0.05 * 0.02 0.16 k 0.08
0.005
0.13 * 0.03 0.06 f 0.02
0.36 & 0.06 0.08 i_ 0.03
0.005 0.1
chow 0.005 0.1
5% corn oil
Liver Kidney 50/ hydrogenated
Liver Kidney Fat-deficient Liver Kidney
Biochim. Biophys. Ada,
187 (1969) 307-318
0.1
0.1
LIPOGENESIS
IN KIDNEY
313
AND LIVER
Fatty acid corn~os~t~o~ of the diets The percent fatty acid composition of the diets is listed in Table V. The laboratory chow diet contained 5% fat of which 27% was saturated and 73% was unsaturated fatty acids. Long-chain fatty acids (c-18 and over) made up about 70% of the total fatty acids in the laboratory chow diet. TABLE
V
FATTY ACID COMPOSITION
Fatty
acid*
8:o 1o:o
-
12:o
0.2
14:o
I.7 I.4 17.6 3.’ 1.1 5.1 23.6 3r.o 15.2
15:o 16:o 16:1 17:o 18:o ifi:1 18:2
r8:3
OF THE DIETS
0.5 0.1 0.4 0.7
6.7 6.3 37.3 21.4
13.2 0.7 -
II.7 -
2.5 26.5 52.8 2.4
‘3.3 2.4 0.9 -
* Chain length of fatty acid: number of double bonds.
The 504 corn oil diet consisted of 17% saturated and 83% unsaturated fatty acids. Of the total fatty acids in the corn oil diet, 52.8% was linoleic acid. The 5% hydrogenated coconut oil diet contained 96.7% saturated fatty acids and only 3.3% unsaturated fatty acids. Short-chain fatty acids, C-16 or less, made up 83% of the total fatty acids. Laurie acid (x2:0) was the major fatty acid of the diet (37.3yo). Comparison of fatty acid projles from the liver of normal and diabetic rats on four dietary regimens The liver from normal animals fed saturated hydrogenated coconut oil had a slightly increased amount of palmitate (16~0) (42%) compared to the others (z636%) (Table VI). This finding is probably related to the high concentration of shortchain fatty acids in the hydrogenated coconut oil diet. The diabetic liver always contained less 16:o than normal livers. The percent of palmitoleate (16: I) was increased in the normal livers from animals fed hydrogenated coconut oil and in normal and diabetic animals fed a fat-deficient diet. A major decrease was seen in the percent of stearate (18:o) in normal livers from animals fed a hydrogenated coconut oil diet and, to a lesser extent in those fed a fat-deficient diet. This reduction in the percent of r8:o is not seen in the analogous diabetic livers. The percent of oleate (x8:x) did not change in the normal and diabetic livers from animals fed laboratory chow and corn oil. However, there was a very large increase in the percent of 18: I in both the normal and diabetic livers from animals fed hydrogenated corn oil or a fat-deficient diet. As in the normal livers, the diabetic livers from animals fed hydrogenated coconut oil or fat-deficient diets had decreased amounts of linoleate Biochim.
Biophys. Acta,
187 (1969) 307-318
B. J. BURNS, J. C. ELWOOD
314 TABLE FATTY
VI ACID
COMPOSITION
OF LIVER
IN NORMAL
AND
DIABETIC
RATS
Each number represents the results of 3 chromatographs from 3 different sets of pooled samples. Each pooled sample contained at least 5 separate experimental tissues. The areas of the peaks were calculated from planimeter recordings. n.d., not detectable. Fatty acid
Fatty
acid (%)
Laboratory ____.___~
Normal
______.
chow
5%
Diabetic
cwn
._____~_~ 5% hydrogenated coconutoil
oil
Normal
Diabetic
0.4 1.9
12:o
I.1
14:0
2.0
15:o
I.1
16:o
32.0
16:1
2.3 1.7 24.0
36.0 3.3 0.7 23.0
29.0
18:1
3.4 I.3 23.0 15.0
15.0
18.0
14.0
18:2
II.0
IO.0
17:o 18:o
3.6 3.1
x
Y
TABLE FATTY
I.7 3.5 0.6 30.0
6.1
2.2 0.2
42.0 9.7 0.4 9.0
2.4 0.9
3::: n.cl. n.d.
8.1
7.4 4.’ 3.7
5.2
0.5
0.3 0.9 0.3 28.0
I.1
Normal
7.5 9.0
Fat-deficient
Diabetic
_ .._~___. Normal
Diabetic
0.6 0.6 n.d. 35.0 9.1
1.3 4.4 0.9 26.0 3.1 0.9 24.0 27.0
0.6
1.0 0.9 29.0
6.6
0.2
I.6
23.0 30.0 3.3
16.0
37.0 2.4
5.3 3.5 2.5
n.d. n.d.
1.6 1.6
VII ACID
COMPOSITIOK
Fatty acid
Fatty
OF KIDNEY
IN NORMAL
AND
DIABETIC
acid 1%)
Laboratory
5% con%oil
chow -___ Diabetic
Normal
._____~
Normal
Diabetic ___
RATS
~_ 5% hydrogenated coconut oil
Fat-de$cient
h’ovmal
h’ormal
Diabetic-
_.~ Diabetic
I.1
I.7
3.8
1.1
I.5
2.5
0.6
I.2
0.7 0.7 36.0
1.9 I.4 30.0
2.5 I.9 31.0
1.5 0.8 30.0
1.5 I.0 29.0
3.2
1.0
1.2
29.0
2.1
0.6 33.0
I.9 30.0
I.8 1.1
I.4 1.0
2.5
4.4
2.9 2.0
4.1 1.0
3.4 2.0
4.2 0.2
2.1
18:o
33.0
26.0
28.0
28.0
18:1
14.0
17.0
14.0
16.0
12:o
14:o 15:o 16:o 16:r
17~0
7.1 2.1 2.5
18:2
x Y
(IS:
z).
diabetic
6.0 3.4 3.4
10.0
3.6 4.9
31.0 24.0
5.2 6.4 5.9
3.6 2.0 1.0
3.9
26.0
29.0
25.0
14.0
23.0
21.0
6.0 3.8 7.4
3.6 2.2 3.2
5.0 3.1 2.8
Normal livers from animals fed laboratory chow and corn oil and livers from animals all contained two fatty acids, labeled x and y, which were not
identified when these experiments were conducted. The relative retention times of fatty acids x and y were greater than linoleate (18: 2). Normal livers from animals fed hydrogenated coconut oil or fat-deficient diets did not have demonstrable amounts of x and y. However, liver tissue from diabetic animals fed laboratory chow or corn oil contained a total of 11.3 and 14.5% x and y, respectively. Unlike the normal fat-deficient 3%
livers,
diabetic
diets contained
in the animals
corn-oil-fed
Biochim.
tissue from
fed a fat-deficient
animals.
Biofihys.
Acta,
187
animals
6.0 and 3.1%
(1969)
307-318
fed hydrogenated
x and y. Therefore,
coconut
oil and
x and y ranged
diet to a total of 16% in diabetic
from
livers from
LIPOGENESIS
IN KIDNEY
AND LIVER
315
Fatty acid profiles of normal and diabetic kidney from animals on four dietary regimens The percent and diabetic
of palmitoleate
animals
(16: I) was less in the kidneys
fed laboratory
chow than
from animals
from both normal on the other
three
diets (Table VII). Stearate (18: o) levels varied from ~5-33O/~, but there did not appear to be any changes associated with the various diets. Animals fed hydrogenated coconut oil and fat-deficient diets had more 18:1 (23%) than kidneys from animals fed
laboratory
to be highest to kidney
chow
or corn
in tissue
from
tissue from
oil.
As seen with
animals
the animals
fed other
of the lack of 18
: z in the hydrogenated
unlike
the kidneys
diets
the liver,
had an equal
oil and from
laboratory
amount chow.
from
animals
amount
diets.
coconut
tissue, chow
linoleate
(18:~)
tended
and corn oil as compared
This is probably
a direct
reflection
diets.
However,
oil and fat-deficient
fed hydrogenated
of x and y fatty The
liver
fed laboratory
corn oil and fat-deficient
acids compared
to the animals
of x and y in normal
kidney
fed corn
tissue
ranged
3 to 6.80/,.
There was an increased receiving
a fat-deficient
amount
diet. Diabetic
of 18: I in diabetic kidney
kidney tissue from animals
had smaller
percentages
of x and y
fatty acids than the liver. The total percent of x and y ranged from 3% (hydrogenated coconut oil-fed animals) to 12.3~/~ (diabetic, corn oil-fed animals). DISCUSSION
0, consumptio~~ Table VIII schematically shows the results of the data presented in the previous tables. 0, consumed by diabetic liver slices was reduced IS-32% from normal under all conditions tested (Table II). An important factor in the rate of respiration of liver tissue is undoubtedly the availability of substrate, primarily carbohydrate and fat. Insulin is required for glucose utilization by liver. In the diabetic animal
Laboratory chow
5% COYVZ oil
5% hydrogen- Fat ated coconut deficient
PMoles 0, consumed per IOO mg tissue per h Liver 4 Kidney t Incorporation Liver Kidney
of acetate into fatty acid
Cholesterol Liver Kidney
i
$ t
t
J ?
4 _
4 _
J -
4 _
the insulin supply is depressed so that glucose utilization is minimal and this tissue becomes dependent on fatty acid oxidation and gluconeogenesis for energy. The fact that 0, uptake was depressed would seem to imply that the availability of substrate had become rate limiting. However, it is possible that pyridine or adenine cofactors were no longer available in adequate amounts. The diabetic
kidney
utilized more 0, than the normal
kidney
(Tables
Biochim. Biophys. Acta, 187 (1969)
II and 307-318
B. J. BURNS, J. C. ELWOOD
316
VIII). This perplexing problem of different organ responses to the same stimulus is not understood at the present time. Whether this increased oxidative capacity of the kidney was caused by the original toxicity of the alloxan or was a manifestation of diabetes $er se is unknown. However, the kidney differs from the liver in that it is not insulin-dependent. In the diabetic state, glucose concentration in the blood is several fold higher than normal, and glucose is more available to kidney than to liver cells. Therefore, the kidney is not as dependent on fatty acid mobilization from peripheral tissue or gluconeogenesis within the cell for oxidizable substrate. The increased 0, consumption may be related to the increased energy requirement of kidney tissue because of the hyperuremia of the diabetic. Fatty acid studies We have confirmed the decrease in fatty acid synthesis by liver from diabetic animals receiving chow, corn oil or hydrogenated coconut oil diets (Tables III and VIII). Also, the livers from diabetic animals receiving a fat-deficient diet were able to increase their fatty acid biosynthetic machinery 3-Iz-fold. The control of fatty acid synthesis in kidney tissue is not the same as that observed in liver tissue. Kidney tissue from diabetic rats incorporated 2-3 times as much acetate into fatty acids as kidney tissue from normal animals. The fact that a fat-deficient diet can induce enzyme synthesis in one tissue (liver) and not in another (kidney) implies that different controls operate to regulate fatty acid biosynthesis in these two different tissues. Since kidney tissue is relatively insulin-independent, it is possible that the increased blood-glucose levels caused a greater amount of glucose to be utilized by the kidney, thus stimulating fatty acid synthesis. However, TENG~Sreported decreased glucose utilization in rat-kidney slices from alloxan-diabetic rats; he also reported increased production of glucose from pyruvate and other precursors. Therefore, the increased fatty acid synthesis of the diabetic kidney appears to be related to the diabetic state per se. Cholesterol studies As shown previously by others, fat in the diet does stimulate the synthesis of cholesterol and the u~a~nitude of the response depends on the type of fat fed (Tables IV and VIII). Unsaturated fatty acids may increase the incorporation of acetate into cholesterol by stimulating the activity of the rate-limiting enzyme, phydroxy-j-methylglutaryl-S-CoA reductase, since the incorporation of mevalonic acid into cholesterol was unaffected by the type of diet fed to the animals in these experiments (this laboratory). Cholesterol synthesis in the normal kidney was shown in the present experiments to be unaffected by the type of diet fed to the animals. However, the amount of acetate incorporated into kidney cholesterol was only 5% of that observed in the liver. DIETSCHY AND SIPERSTEIN 28 have recently reported on renal synthesis of cholesterol. The cholesterol synthesizing mechanism in the kidney was not changed from normal by the diabetic state, unlike the fatty acid synthesizing mechanism, which was increased over normal levels.
Biochim
Biufihys.
Acta,
187 (1969)
p-318
LIPOGENESIS
IN KIDNEY
AND LIVER
3x7
Fatty acid $rofiles The percent fatty acid composition of liver tissue from animals fed laboratory chow and corn oil diets remained fairly constant (Table VI); the exception is considerably more x and y in the diabetic tissue. However, there was a large increase in the percent monoenoic acids (16 : 1,18 :I) in the liver of normal and diabetic animals maintained on an hydrogenated coconut oil oi fat-deficient diet. These latter results are in agreement with previous studies 3*-38. However, the response of the diabetic animals was less pronounced. This may be due to the effect of decreased desaturation of palmitic and stearic acids observed in the diabetic liver39s40. Normal kidney from animals fed hydrogenated coconut oil and fat-deficient diets showed increased amounts of oleate but no difference in palmitoleate compared to corn oil-fed animals, Only about a z-fold decrease in linoleate was observed compared to a 5-fold decrease in the liver, It is difficult to elucidate the mechanisms of the changes in fatty acid composition of the tissues with the present information. Several factors, however, are present which do play a major role in the determination of tissue fatty acid composition. The first factor is that there is increased desaturation of palmitic and stearic acids due to a fat-deficient or saturated-fat diet34~35J7~5s.Secondly, there is . Thirdly, there is increased mobilizadecreased desaturation in the diabetic state 3gp40 tion of fatty acids from adipose tissue in the diabetic anima141+*2. ~obiIization must play an important role, since the diabetic animal on corn oil and laboratory chow diets nl~ntained the same level of monoenes as the normal animals. The saturatedfat and fat-deficient diets may provide some stimulus for removing inhibitory mechanisms of the desaturation reactions. ACKNOWLEDGMENTS
This investigation was supported in part by Public Health Service Grant No. HE 04567-04 from the National Heart Institute, Public Health Service. This work was done as partial ful~llment of the requirements for the degree of Doctor of Philosophy. REFERENCES I
2
3 4 2 ; 9 IO II 12 ‘3 14 I.5 16
SINGHAL AND S. I<. SRIVASTAVA, Ca~.~.B~o&hem.Phys~oZ., 43 (1965) 1549. H. A. LARDY, D. 0. FOSTER, J. W. YOUNG, E. S~RA~O AND P. D. RAY, J. Ce&lar Gonap. Physiol., 66 (1965) 39. E. J. MASORO, .J. Lipid Res., 3 (1962) 149. N. PER~z,L.C~AR~-T~RRI,E.RABAJILLEANDH.~IEMEYER~ f,Bioa’.Cladm.,239(rg64) 2420. J. D. WOOD AND B. B. MIGICONS~Y, Can. J.Biochem. Physiol., 36 (1958) 433. K. I<.CARROLL, Can. J. B&hem. Physiol., 42 (1gG4) 71. J. D. WILSON AND M. D. SIPERSTETN,A~.~. PhysioE., x96(1959) 599. H. ,X. TEPPER~ZAN AND J. TEPPERMAN, Diabetes, ~(~58) 478. D. W. ALLMANN, D. D. HUBBARD AND D.M. GIBSON, J.LipidRes., 6(1965) 63. S. MUKHERJEE AND R. B. ALFIN-SLATER, Arch. B&hem. Biophys., 73 (1958) 359. R.H~LL,W.W. WEBSTER,J.M.LI~AZASORO AND I. L. ~~AIKoFF,~.~~~~~ Res., I (rg60) 150. W.S. BORTZ,~.ABRAHAM AND I.L.CHAIKOFF, J.Bid. Chem.,z38(rg63) 1~66. R. 0. BRADY AND S. GDRIN, J. Biol. Chem., 187 (1950) $39. S. S. CHERNICK AND I. L. CHAIKOFT,J. Biol. Chem., 188(1951) 389. G. M. TOMKINS AND I. L. CHAIKOFF, .r.Bid. Claem., 196 (IQ~Z) 569. ~,$.VAN BRUGGEN, P.YAMADA,T.T.HUTCHENS AND E. S. WEST, J.Biol. Chem., zag (1954)
G. WEBER,R.L.
Biocbim. Biophys. Acta, 187 (1969) 307-318
B. J. BURNS,
318
J. C. ELWOOD
17 J.C. ELWOOD, A. MARCO AND J.T. VAN BRUGGEN, J.Biol. Chem., 235 (1960) 573. 18 0. WIELAND, I.NEUFELDT, S.NUMA AND F. LYNEN, Biochem. Z., 336 (1963) 455. 19 J. F.MEAD, W. H. SLATON,Jr.AND A. B. DECKER, J.BioZ.Chem., 218 (1956) 401. 20 J. AVIGAN AND D. STEINBERG,Proc. Sot. Exptl. Biol. Med., g7 (1958) 814. 21 J. AVIGAN AKD D. STEINBERG,Circulation, 16 (1957)492. 22 R. CLARENBURC AND I.L.CHAIKOFF,Am.J. Physiol., ZIO (1966) 37. 23 M. D. SIPERSTEIN AND M. J.GUEST, J. Clin. Invest., 3g (1960) 642. 24 S. S. CHERNICK,E. J.MASORO AND I.L. CHAIKOFF,Proc. Sot. Exptl. Biol. Med., 73 (1950) 348. 25 G. MEDES, A. THOMAS AND S. WEINHOUSE, J.Biol.Chem., 197 (1952)181. 26 J. C. ELWOOD AND J.T.VAN BRUGGEN,~.L~P~~ Res., z (1961) 344. 27 J. GANGULY, Biochim. Biophys. Acta, 40 (1960) IIO. 2X E. J. MASORO AND E. PORTER, Proc. Sot. Exptl. Biol. Med., 118 (1965) rogo. 29 J.M. DIETSCHY AND M. Il. SIPERSTEIN,]. LipidRes., 8 (1967) 97. 30 J, T. VAN BRUGGEPI', J,C. ELWOO~, A. MARCO AND W. C. BERNARDS,J. Atherosclerosis Res., 2 (1962)388. 31 J. C. ELWOOD ASD J. T. VAN BRUGGEN, J.Biol. Chem., 235 (1960) 568. 32 A. SPENCER,L. CORMAN AND J.M. LOWENSTEIN,Biochem. J., g3 (1964) 378. 33 C. T. TENG, Arch. Biochem. Biophys., 48 (1954) 415. 34 D.W. ALLMAN APZDD.M. GIBSON,J.Lipid Res., 6 (1965) 51. 35 D. W. ALLMAN,D. D. HUBBARD AND D.M.GIBsoN, j. LipidRes., 6(rg65) 63. 36 H. MOHRHAUERAND R. T. HOLMAN, J.LipidRes., 4(1963) 151. 37 R.REISER,M.WILLIAMS,M.SORVELSANDN.MURTZ, Arch.Biochem.Biophys., 102(1g63)276. 38 H. TEPPERMAN AND J.TEPPERMAN, Am. J. Physiol., 239 (1965) 773. 39 W. BENJAMIN AND A. GELLHORN, J. Biol. Chem., 239 (1964) 64. 40 A. GELLHORN AND W. BENJAMIN,Biochim. Biophys. Actn, 84 (1964) 167. 41 W. M. BORTZ AND F. LYNEN, Biochem. Z., 339 (1963) 77. 42 D. ~.FREDRICKSON AND R. S.GORDON,J~.,Phys.Rev., 38(rg58) 585. Biochim.
Biophys.
Acta,
187 (1969)
307-318
307
BBA 55632
LIPID
METABOLISM
AND DIABETIC
BARRY
J. BURNS
IN KIDNEY
TISSUE FROM NORMAL
RATS
AND
J. CLINT
ELWOOD
Department of Biockemistry, Upstate Medical Syrtacecse, N.Y. 13210 (U.S.A.J (Received
AND LIVER
Center,State University
of New York,
June 13th‘ 1969)
SUMMARY
I. Normal and diabetic rats were fed four different diets: laboratory chow, 5% corn oil, 5% hydrogenated coconut oil and a fat-deficient diet. Diabetic liver slices show decreased 0, utilization. The diabetic kidney tissue responded with increased 0, consumption. [rJ4C]acetate labeling of fatty acids was depressed in all the diabetic livers. Diabetic kidney tissue had higher rates of lipogenesis than control kidney tissue. Cholesterol synthesis from acetate was depressed in the diabetic liver except when the diet fed was fat deficient. Diabetes did not influence cholesterol labeling in kidney tissue. 2. Liver from normal and diabetic rats fed hydrogenated coconut oil and fatdeficient diets had increased amounts of monoenoic acids compared to corn-oil-fed animals. Normal liver from animals fed saturated-fat and fat-deficient diets had decreased amounts of stearic and linoleic acid. The diabetic livers had more linoleic acid than normal livers. Kidney tissue from normal animals fed the saturated-fat and fat-deficient diets also had increased amounts of oleic acid and decreased amounts of linoleic acid compared to that from animals fed unsaturated fatty acid. Kidney tissue from diabetic animals had greater amounts of linoleic acid.
INTRODUCTION
There now exists a voluminous amount of scientific literature related to the effect of diet and/or hormonal alterations on many metabolic pathways of rat-liver tissue. Enzyme induction and repression by diet and hormonal regulation have been observed under many circumstances *- d. Some investigators have shown that the level of lipogenesis is dependent on the type of diets-la. Previous studies have also shown that lipogenic pathways are altered in the diabetic animalla-la. Cholesterol biosynthesis has also been reported to be regulated by dietS~8~12~18-22. SIPERSTEIN AND GUESTED showed that feeding a 5% cholesterol diet decreased hepatic cholesterogenesis by inhibiting /I-hydroxy-j?-methylglutaryl-S-CoA reductase (negative feedback inhibition). BiochP’ne. Biofihys. Acta, 187 (1969) 307-318
B. J.
308
BURNS, J. C. ELWOOD
Kidney tissue, on the other hand, has not received an intensive investigation under the same experimental conditions as the liver. Early studies of CHERNICKet al.23 and others2Q-31 have shown that the kidney slice in vitro can synthesize fatty acids and cholesterol from glucose and acetate. The rate of synthesis of fatty acids in the kidney from acetate has been stated to be IO--ZO~!~that of the IiveP. Cholesterol synthesis in the kidneys9 has been shown to be about zsi/, that found in liver. Fasting or cholesterol feedings9 has little or no effect on the rate of cholesterol synthesis in the kidney. Studies of VAN BRUGGENet ~2.30with the immature macaque (Rhesus) monkey have shown that kidney slices have a higher rate of incorporation of [r-laCjacetate and jz-r*C]mevalonic acid into %O, than liver. ELWOODANDVAN BRUGGEN~~have shown that the kidney tissue has an active cholesterogenic process. The following experiments were designed to study the effects of diets containing different types of fat on 0, consumption, [ I-Xjacetate incorporation into fatty acids and cholesterol in the normal and diabetic kidney and to compare these findings with liver tissue response. Also, in the present study, fatty-acid profiles of liver and kidney tissues from normal and diabetic rats were compared to see what changes were elicited by feeding different types of fat in the diet. MATERIALSANDMETHODS
Male adult Sprague-Dawley rats obtained from Carworth Farms, Inc. (New City, Rockland County, N.Y.) weighing 180-200 g were used in these studies. I7pon arrival, all rats were fed Big Red Chow* ad libitum for 4 days, after which time some of the animals were fasted 48 h. The diabetic state was induced in these fasted animals by the administration of a 10% solution of alloxan in 0.9:/o saline solution. Either 60 mg of alloxan/kg body weight were given intramuscularly or 200 mg/kg body weight intraperitoneally. After 14 days, the animals were fasted 24 h and tail blood was collected for glucose determinations (Worthington, Glucostat Method). Only those rats which had fasting-blood-sugar levels greater than 200 mg/roo ml of blood were considered diabetic and used for further study. Diets
Four different regimens, varying in amount and kind of fat, were employed in this study. The four diets were the following: (I) Big Red Iaboratory Chow containing 5% crude fat (Country Best Foods, Agway, Inc., Syracuse, New York). (z) Fat-free diet plus 5% corn oil. (3) Fat-free diet plus 5% hydrogenated coconut oil. (4) Fat-free diet. .-_ _ * Big Red Laboratory Chow. Guaranteed analysis: minimal 24.0% crude protein, 5.0% crude fat
and maximal 5.0 % crude fiber. Ingredients: soybean meal, ground oats, wheat midlings, corn meal, ground wheat, dehydrated alfalfa meal, dried skimmed milk, fish meal, ground limestone, vegetable oil, D-activated plant sterol (“source of vitamin D-2 “), vitamin A, palmitate, salt, MnO, IWO,, Na,SO,, cue, COCO,, zno, CaIO,. Biochim.
Biophys.
Ada,
187 (1969)
307-318
LIPOGENESIS IN KIDNEY AND LIVER
309
Diets 2, 3 and 4 were purchased from Nutritional Biochemical Corporation (Cleveland, Ohio) *. The rats were allowed to eat ad lib&m for a minimum of 6 days, and most animals were used between 6-12 days. The individual animals were not weighed, but the total quantity of synthetic diet consumed was the same in all cases. 2 h before killing by decapitation, food was removed. After sacrificing the rats, the livers and kidneys were quickly excised and placed in cold physiological saline solution prior to slicing. Tissue slices weighing IOO + IO mg were prepared using a Stadie-Riggs slicer. Slices for experiments in vitro were cut after the capsular tissue of the liver and kidney was discarded. Only the cortex portion of the kidney was used in these studies. The IOO f IO mg slices were placed in a r7-ml single-side-arm Warburg flask containing 1.9 ml of phosphate buffer (pH 7.4). A piece of filter paper was placed in the center well of the Warburg vessel, except when using bicarbonate buffer, and impregnated with 0.2 ml of 8 M KOH to collect the CO,. Preliminary experiments were conducted using either substrate levels of [I-%]acetate (12000 nmoles/flask) or tracer quantities (40 nmoles/flask). The changes elicited by feeding, starvation or diabetes were seen whether substrate or tracer amounts of acetate were employed, although the order of magnitude of the changes was different. Therefore, 0.1 ml of radioactive precursor (40-70 nmoles [I-Xlacetate) was added to the Warburg vessel. The Warburg vessels containing phosphate buffer were flushed with pure 0, for 15 set before being placed in the Warburg bath maintained at 37’. The radioactive tracer was added from the side arm after a ro-min equilibration period. The flasks were incubated for I h at 37O. 0, consumption was followed by standard manometric techniques. At the end of the r-h incubation period, the reactions in the Warburg vessels were stopped by the addition of 0.2 ml of a 20% solution of trichloroacetic acid. of lipids The tissue slices were washed with hot distilled water to remove any excess radioactive tracer. The tissue slices were transferred to screw-cap culture tubes to which 5 ml of an 11% alcoholic KOH solution were added. The tissue was saponified for 30 min under N, and then the solution was reduced in volume to approx. I ml. 3 ml of ethanol and g ml of distilled water were added to the tube. To the above alkaline mixture was added an equal volume of petroleum ether and the nonsaponifiable material was extracted 3 times. The remaining tissue digest was made acidic to congo red paper and extracted 4 times with petroleum ether. The extracted fatty acid fraction was dissolved in 5 ml of absolute ethanol, I ml of which was plated on aluminum planchets and the radioactivity was determined as infinitely thin equivalents. The remaining fatty acids were methylated for gas-liquid chromatography. Cholesterol was precipitated from the nonsaponifiable fraction with digitonin, Radioassay
* Fat-deficient diet compositions. “Vitamin-free” casein 21.10% ; Alphacel “cellulose” 16.45% ; sucrose 58.45 o/0 and salt mixture USP XIV 4%. The following vitamin supplements were added : Choline chloride 6oog mg/kg, nicotinic acid 601, inositol 303. vitamin A concentrate (200000 units/g) 99.2. vitamin D concentrate 66.1 (400000 units/g), alpha tocopherol 225, menadione 2.26, thiamine hydrochloride 22.05, riboflavin 22.05, calcium pantothenate 45.2. Biochinz. Bioplzys.
Acta,
187 (1969) 307-318
B.
310
J. BURNS,
J. C. ELWOOD
allowed to stand overnight to insure complete precipitation, washed with acetone times and taken up in methanol and plated as infinitely thin plates. Samples of nonsaponifiable material, without digitonin precipitation, were also radioassayed as infinitely thin samples, but no significant differences were observed. 2
Gas chromatographic analysis was performed with the aid of two different gas chromatographs. One was a Perkin-Elmer-rg4D gas chromatograph having a thermistor (hot wire) detector. The column was packed with Chromosol P (diatomaceous earth) as the stationary phase and coated with diethylene glycol succinate polyester. The column temperature was maintained at 204’. The second gas chromatograph was a F and M Scientific Corporation Model 402 equipped with a hydrogen flame detector. The same type of column support and liquid phase diethylene glycol succinate were employed in the F and M gas chromatograph as in the Perkin-Elmer gas chrolnatograph. Fatty acid ester separations were performed on the F and M by programmed temperature techniques. Programmed temperature runs were from 100' to 200’ at the rate of r”/min. RESULTS
It is well known that bicarbonate buffer stimulates fatty acid biosynthesis as compared to phosphate buffer. In the present experiments it was felt desirable to obtain information as well as lipid labeling data. However, we felt it necessary to show that we could indeed elicit the same responses in fatty acid synthesis with phosphate buffer as has been shown in bicarbonate buffer. Table I presents the data for the comparison of fatty acid labeling from [I-14C]acetate in bicarbonate and phosphate buffer from liver tissue of animals fasted 48 h and then re-fed a fatl-ABLE
I
[I-~%]ACETATE INCORPORATION INTO FATTY ACIDS IN
LIVER
SLICES
OF
NORMAL
ANIMALS in vi&o
Animals were fasted 48 h and then refed a fat-deficient diet for the period of time indicated. Zero day implies a 48-h fast. roe mg of tissue were incubated for 60 min in the presence of 0.5 fimole of [I-Tlacetate (pH 7.4) at a temperature of 37O in a volume of 1.9 ml of buffer. Days on _-cl I 2 3 87 .--
diet
_____.---_I
_-~
76 [W]Acetate
____-
Carbonate
~~lcoyporat~on
buffeer -...-____-
Phosphate
0.03 6.56 5.28 -
0.03 2.72 2.36 1.96
I.90 I.93
0.80 -.
buffer ~--.-
deficient diet for varying time periods. It is seen that bicarbonate buffer stimulated fatty acid synthesis over that obtained in phosphate buffer. However, liver tissue in phosphate buffer responded to the fasting and refeeding routine by a go-fold increase in fatty-acid labeling. The data shows that the increase in synthesis from fasting and refeeding was transient and that after about I week the synthesis rate was stabilized and much lower than the peak rate at about 48 h. However, this Biochim.
Biophys.
Acta,
187 (1969)
307-318
LIPOGENESIS IN KIDNEY AND LIVER
311
rate is still considerably higher than synthesis from liver tissue of animals on laboratory chow. The same observation was reported by SPENCER et a1.32 who showed that the influence of fasting and refeeding was short lived (about 72 h in his experiment). 0, consum$tion The pmoles of 0, utilized by liver and kidney tissue from normal diabetic rats are listed in Table II. There was a statistically significant TABLE 0,
and alloxan decrease in
II
UTILIZATION
BY
LIVER
AND
KIDNEY
TISSUE
Data are expressed as mean 3 S.E. In this table and those to follow, a P value equal to or less than 0.05 is considered to indicate a statistically significant difference. Each number in Tables II-IV represents at least 6 animals in duplicate. pmoles
Diet Laboratory Liver Kidney
5% Hydrogenated Liver Kidney
IOO
mg tissue per h P value
Diabetic
8.1 f 0.43 12.9 * 0.42
6.0 f 16.2 f
0.37 0.93
<0.005 <0.005
6.9 I_t 0.52 14.4 * 0.58
5.1 + 0.33 17.8 * 0.60
to.005 <0.025
coconut oil 6.5 & 0.17 14.9 nk 0.77
5.4 rt 0.25 16.9 k 0.53
<0.005 <0.05
6.5 f 0.24 13.4 k 0.66
4.4 + 0.08 15.5 * 0.66
chow
5% Corn oil Liver Kidney
Fat-deficient Liver Kidney
0, consumed per
Normal
0, consumption by the diabetic livers as compared to controls regardless of the diet fed. This decreased 0, consumption by diabetic liver was reported previously by ELWOOD AND VAN BRUGGEN~’ from animals receiving laboratory chow. In contrast to liver tissue, kidney tissue from diabetic animals had significantly increased 0, consumption as compared to normal kidney tissue. Altering the diet did not change the general picture. Whether increased oxidation by kidney slices from diabetic animals is related to the hyperuremia of the alloxan diabetic rat is unknown. Fatty acid synthesis Fatty acid synthesis in liver and kidney slices of normal and alloxan diabetic rats was followed by the incorporation of [I-lK]acetate (Table III). We have confirmed many reports that acetate incorporation was decreased 3-rz-fold in the diabetic livers depending on the type of diet consumed. Also, acetate incorporation by diabetic liver tissue was increased to lower normal values when the animals were maintained on a fat-deficient diet; but these diabetic liver slices still had decreased synthesis from the normal value of 0.59%. The labeling of fatty acids from [I-Xlacetate in kidney tissue of normal and diabetic animals is also shown in Table III. It is seen that the kidney of the diabetic incorporated more acetate into fatty acids than the normal tissue under all nutritional Biochim.
Biophys.
Ada,
187 (1969) 307-318
B. J. BURNS, J. C. ELWOOD
312 TABLE
III
INCORPORATION INTO FATTYACIDS o/o [14C]Acetate incorporation IOO mg tissue per h
NOWEal
Diet
Laboratory Liver Kidney
per
P value
Diabetic
chow 0.15 + 0.04 0.04 * 0.01
0.01 * 0.08 =
0.47 * 0.02 0.05 5 O.OI
0.04 rt 0.03
0.16 i 0.03
co.025
coconut oil 0.23 & 0.08 0.04 * 0.01
0.02 & 0.01
<0.05
0.03
<0.05
0.59 tr 0.09 0.03 11:0.01
0.12 & 0.03 0.08 11:0.01
5% corn oil
Liver Kidney 5% hydrogenated Liver Kidney Fat-deficient Liver Kidney
0.12
&
0.002
0.02
<0.05
<0.005 <0.005
regimens tested. This is the opposite effect seen in liver tissue. Also, a fat-deficient diet did not stimulate kidney-tissue lipogenesis as was seen in liver tissue. Cholesterol
biosynthesis
The incorporation of acetate into cholesterol was decreased in the diabetic livers from animals receiving chow diet, corn oil or a hydrogenated coconut oil diet (Table IV). However, diabetic animals fed fat-deficient diet had a very marked stimulation in cholesterol labeling from acetate, whereas normal liver tissue had depressed labeling compared to fat-fed animals. CLARENBURG AND CHAIKOFF~~ have reported similar findings while feeding a high-glucose no-fat diet. The incorporation of [r-Xlacetate into kidney cholesterol was not changed by any of the dietary manipulations. A comparison of rate of incorporation between liver and kidney is not valid since the optimum conditions for cholesterol biosynthesis in kidney tissue have not been established. TABLE
I\
“C INCoRPoRATIoN INTO
CHOLESTEROL
~~
0/O1% incorporation per IOO mg liver per h
Dirt
Laboratory Liver Kidney
P value
NOWVLd
Diabetic
I.9 & 0.40 0.07 * 0.05
0.08 5 0.03 0.04 * 0.01
0.46 k 0.12 0.10 k 0.03
0.16 :t 0.06 0.08 i 0.01
0.05
coconut oil 0.25 * 0.05 0.07 _t 0.02
0.05 * 0.02 0.16 k 0.08
0.005
0.13 * 0.03 0.06 f 0.02
0.36 & 0.06 0.08 i_ 0.03
0.005 0.1
chow 0.005 0.1
5% corn oil
Liver Kidney 50/ hydrogenated
Liver Kidney Fat-deficient Liver Kidney
Biochim. Biophys. Ada,
187 (1969) 307-318
0.1
0.1
LIPOGENESIS
IN KIDNEY
313
AND LIVER
Fatty acid corn~os~t~o~ of the diets The percent fatty acid composition of the diets is listed in Table V. The laboratory chow diet contained 5% fat of which 27% was saturated and 73% was unsaturated fatty acids. Long-chain fatty acids (c-18 and over) made up about 70% of the total fatty acids in the laboratory chow diet. TABLE
V
FATTY ACID COMPOSITION
Fatty
acid*
8:o 1o:o
-
12:o
0.2
14:o
I.7 I.4 17.6 3.’ 1.1 5.1 23.6 3r.o 15.2
15:o 16:o 16:1 17:o 18:o ifi:1 18:2
r8:3
OF THE DIETS
0.5 0.1 0.4 0.7
6.7 6.3 37.3 21.4
13.2 0.7 -
II.7 -
2.5 26.5 52.8 2.4
‘3.3 2.4 0.9 -
* Chain length of fatty acid: number of double bonds.
The 504 corn oil diet consisted of 17% saturated and 83% unsaturated fatty acids. Of the total fatty acids in the corn oil diet, 52.8% was linoleic acid. The 5% hydrogenated coconut oil diet contained 96.7% saturated fatty acids and only 3.3% unsaturated fatty acids. Short-chain fatty acids, C-16 or less, made up 83% of the total fatty acids. Laurie acid (x2:0) was the major fatty acid of the diet (37.3yo). Comparison of fatty acid projles from the liver of normal and diabetic rats on four dietary regimens The liver from normal animals fed saturated hydrogenated coconut oil had a slightly increased amount of palmitate (16~0) (42%) compared to the others (z636%) (Table VI). This finding is probably related to the high concentration of shortchain fatty acids in the hydrogenated coconut oil diet. The diabetic liver always contained less 16:o than normal livers. The percent of palmitoleate (16: I) was increased in the normal livers from animals fed hydrogenated coconut oil and in normal and diabetic animals fed a fat-deficient diet. A major decrease was seen in the percent of stearate (18:o) in normal livers from animals fed a hydrogenated coconut oil diet and, to a lesser extent in those fed a fat-deficient diet. This reduction in the percent of r8:o is not seen in the analogous diabetic livers. The percent of oleate (x8:x) did not change in the normal and diabetic livers from animals fed laboratory chow and corn oil. However, there was a very large increase in the percent of 18: I in both the normal and diabetic livers from animals fed hydrogenated corn oil or a fat-deficient diet. As in the normal livers, the diabetic livers from animals fed hydrogenated coconut oil or fat-deficient diets had decreased amounts of linoleate Biochim.
Biophys. Acta,
187 (1969) 307-318
B. J. BURNS, J. C. ELWOOD
314 TABLE FATTY
VI ACID
COMPOSITION
OF LIVER
IN NORMAL
AND
DIABETIC
RATS
Each number represents the results of 3 chromatographs from 3 different sets of pooled samples. Each pooled sample contained at least 5 separate experimental tissues. The areas of the peaks were calculated from planimeter recordings. n.d., not detectable. Fatty acid
Fatty
acid (%)
Laboratory ____.___~
Normal
______.
chow
5%
Diabetic
cwn
._____~_~ 5% hydrogenated coconutoil
oil
Normal
Diabetic
0.4 1.9
12:o
I.1
14:0
2.0
15:o
I.1
16:o
32.0
16:1
2.3 1.7 24.0
36.0 3.3 0.7 23.0
29.0
18:1
3.4 I.3 23.0 15.0
15.0
18.0
14.0
18:2
II.0
IO.0
17:o 18:o
3.6 3.1
x
Y
TABLE FATTY
I.7 3.5 0.6 30.0
6.1
2.2 0.2
42.0 9.7 0.4 9.0
2.4 0.9
3::: n.cl. n.d.
8.1
7.4 4.’ 3.7
5.2
0.5
0.3 0.9 0.3 28.0
I.1
Normal
7.5 9.0
Fat-deficient
Diabetic
_ .._~___. Normal
Diabetic
0.6 0.6 n.d. 35.0 9.1
1.3 4.4 0.9 26.0 3.1 0.9 24.0 27.0
0.6
1.0 0.9 29.0
6.6
0.2
I.6
23.0 30.0 3.3
16.0
37.0 2.4
5.3 3.5 2.5
n.d. n.d.
1.6 1.6
VII ACID
COMPOSITIOK
Fatty acid
Fatty
OF KIDNEY
IN NORMAL
AND
DIABETIC
acid 1%)
Laboratory
5% con%oil
chow -___ Diabetic
Normal
._____~
Normal
Diabetic ___
RATS
~_ 5% hydrogenated coconut oil
Fat-de$cient
h’ovmal
h’ormal
Diabetic-
_.~ Diabetic
I.1
I.7
3.8
1.1
I.5
2.5
0.6
I.2
0.7 0.7 36.0
1.9 I.4 30.0
2.5 I.9 31.0
1.5 0.8 30.0
1.5 I.0 29.0
3.2
1.0
1.2
29.0
2.1
0.6 33.0
I.9 30.0
I.8 1.1
I.4 1.0
2.5
4.4
2.9 2.0
4.1 1.0
3.4 2.0
4.2 0.2
2.1
18:o
33.0
26.0
28.0
28.0
18:1
14.0
17.0
14.0
16.0
12:o
14:o 15:o 16:o 16:r
17~0
7.1 2.1 2.5
18:2
x Y
(IS:
z).
diabetic
6.0 3.4 3.4
10.0
3.6 4.9
31.0 24.0
5.2 6.4 5.9
3.6 2.0 1.0
3.9
26.0
29.0
25.0
14.0
23.0
21.0
6.0 3.8 7.4
3.6 2.2 3.2
5.0 3.1 2.8
Normal livers from animals fed laboratory chow and corn oil and livers from animals all contained two fatty acids, labeled x and y, which were not
identified when these experiments were conducted. The relative retention times of fatty acids x and y were greater than linoleate (18: 2). Normal livers from animals fed hydrogenated coconut oil or fat-deficient diets did not have demonstrable amounts of x and y. However, liver tissue from diabetic animals fed laboratory chow or corn oil contained a total of 11.3 and 14.5% x and y, respectively. Unlike the normal fat-deficient 3%
livers,
diabetic
diets contained
in the animals
corn-oil-fed
Biochim.
tissue from
fed a fat-deficient
animals.
Biofihys.
Acta,
187
animals
6.0 and 3.1%
(1969)
307-318
fed hydrogenated
x and y. Therefore,
coconut
oil and
x and y ranged
diet to a total of 16% in diabetic
from
livers from
LIPOGENESIS
IN KIDNEY
AND LIVER
315
Fatty acid profiles of normal and diabetic kidney from animals on four dietary regimens The percent and diabetic
of palmitoleate
animals
(16: I) was less in the kidneys
fed laboratory
chow than
from animals
from both normal on the other
three
diets (Table VII). Stearate (18: o) levels varied from ~5-33O/~, but there did not appear to be any changes associated with the various diets. Animals fed hydrogenated coconut oil and fat-deficient diets had more 18:1 (23%) than kidneys from animals fed
laboratory
to be highest to kidney
chow
or corn
in tissue
from
tissue from
oil.
As seen with
animals
the animals
fed other
of the lack of 18
: z in the hydrogenated
unlike
the kidneys
diets
the liver,
had an equal
oil and from
laboratory
amount chow.
from
animals
amount
diets.
coconut
tissue, chow
linoleate
(18:~)
tended
and corn oil as compared
This is probably
a direct
reflection
diets.
However,
oil and fat-deficient
fed hydrogenated
of x and y fatty The
liver
fed laboratory
corn oil and fat-deficient
acids compared
to the animals
of x and y in normal
kidney
fed corn
tissue
ranged
3 to 6.80/,.
There was an increased receiving
a fat-deficient
amount
diet. Diabetic
of 18: I in diabetic kidney
kidney tissue from animals
had smaller
percentages
of x and y
fatty acids than the liver. The total percent of x and y ranged from 3% (hydrogenated coconut oil-fed animals) to 12.3~/~ (diabetic, corn oil-fed animals). DISCUSSION
0, consumptio~~ Table VIII schematically shows the results of the data presented in the previous tables. 0, consumed by diabetic liver slices was reduced IS-32% from normal under all conditions tested (Table II). An important factor in the rate of respiration of liver tissue is undoubtedly the availability of substrate, primarily carbohydrate and fat. Insulin is required for glucose utilization by liver. In the diabetic animal
Laboratory chow
5% COYVZ oil
5% hydrogen- Fat ated coconut deficient
PMoles 0, consumed per IOO mg tissue per h Liver 4 Kidney t Incorporation Liver Kidney
of acetate into fatty acid
Cholesterol Liver Kidney
i
$ t
t
J ?
4 _
4 _
J -
4 _
the insulin supply is depressed so that glucose utilization is minimal and this tissue becomes dependent on fatty acid oxidation and gluconeogenesis for energy. The fact that 0, uptake was depressed would seem to imply that the availability of substrate had become rate limiting. However, it is possible that pyridine or adenine cofactors were no longer available in adequate amounts. The diabetic
kidney
utilized more 0, than the normal
kidney
(Tables
Biochim. Biophys. Acta, 187 (1969)
II and 307-318
B. J. BURNS, J. C. ELWOOD
316
VIII). This perplexing problem of different organ responses to the same stimulus is not understood at the present time. Whether this increased oxidative capacity of the kidney was caused by the original toxicity of the alloxan or was a manifestation of diabetes $er se is unknown. However, the kidney differs from the liver in that it is not insulin-dependent. In the diabetic state, glucose concentration in the blood is several fold higher than normal, and glucose is more available to kidney than to liver cells. Therefore, the kidney is not as dependent on fatty acid mobilization from peripheral tissue or gluconeogenesis within the cell for oxidizable substrate. The increased 0, consumption may be related to the increased energy requirement of kidney tissue because of the hyperuremia of the diabetic. Fatty acid studies We have confirmed the decrease in fatty acid synthesis by liver from diabetic animals receiving chow, corn oil or hydrogenated coconut oil diets (Tables III and VIII). Also, the livers from diabetic animals receiving a fat-deficient diet were able to increase their fatty acid biosynthetic machinery 3-Iz-fold. The control of fatty acid synthesis in kidney tissue is not the same as that observed in liver tissue. Kidney tissue from diabetic rats incorporated 2-3 times as much acetate into fatty acids as kidney tissue from normal animals. The fact that a fat-deficient diet can induce enzyme synthesis in one tissue (liver) and not in another (kidney) implies that different controls operate to regulate fatty acid biosynthesis in these two different tissues. Since kidney tissue is relatively insulin-independent, it is possible that the increased blood-glucose levels caused a greater amount of glucose to be utilized by the kidney, thus stimulating fatty acid synthesis. However, TENG~Sreported decreased glucose utilization in rat-kidney slices from alloxan-diabetic rats; he also reported increased production of glucose from pyruvate and other precursors. Therefore, the increased fatty acid synthesis of the diabetic kidney appears to be related to the diabetic state per se. Cholesterol studies As shown previously by others, fat in the diet does stimulate the synthesis of cholesterol and the u~a~nitude of the response depends on the type of fat fed (Tables IV and VIII). Unsaturated fatty acids may increase the incorporation of acetate into cholesterol by stimulating the activity of the rate-limiting enzyme, phydroxy-j-methylglutaryl-S-CoA reductase, since the incorporation of mevalonic acid into cholesterol was unaffected by the type of diet fed to the animals in these experiments (this laboratory). Cholesterol synthesis in the normal kidney was shown in the present experiments to be unaffected by the type of diet fed to the animals. However, the amount of acetate incorporated into kidney cholesterol was only 5% of that observed in the liver. DIETSCHY AND SIPERSTEIN 28 have recently reported on renal synthesis of cholesterol. The cholesterol synthesizing mechanism in the kidney was not changed from normal by the diabetic state, unlike the fatty acid synthesizing mechanism, which was increased over normal levels.
Biochim
Biufihys.
Acta,
187 (1969)
p-318
LIPOGENESIS
IN KIDNEY
AND LIVER
3x7
Fatty acid $rofiles The percent fatty acid composition of liver tissue from animals fed laboratory chow and corn oil diets remained fairly constant (Table VI); the exception is considerably more x and y in the diabetic tissue. However, there was a large increase in the percent monoenoic acids (16 : 1,18 :I) in the liver of normal and diabetic animals maintained on an hydrogenated coconut oil oi fat-deficient diet. These latter results are in agreement with previous studies 3*-38. However, the response of the diabetic animals was less pronounced. This may be due to the effect of decreased desaturation of palmitic and stearic acids observed in the diabetic liver39s40. Normal kidney from animals fed hydrogenated coconut oil and fat-deficient diets showed increased amounts of oleate but no difference in palmitoleate compared to corn oil-fed animals, Only about a z-fold decrease in linoleate was observed compared to a 5-fold decrease in the liver, It is difficult to elucidate the mechanisms of the changes in fatty acid composition of the tissues with the present information. Several factors, however, are present which do play a major role in the determination of tissue fatty acid composition. The first factor is that there is increased desaturation of palmitic and stearic acids due to a fat-deficient or saturated-fat diet34~35J7~5s.Secondly, there is . Thirdly, there is increased mobilizadecreased desaturation in the diabetic state 3gp40 tion of fatty acids from adipose tissue in the diabetic anima141+*2. ~obiIization must play an important role, since the diabetic animal on corn oil and laboratory chow diets nl~ntained the same level of monoenes as the normal animals. The saturatedfat and fat-deficient diets may provide some stimulus for removing inhibitory mechanisms of the desaturation reactions. ACKNOWLEDGMENTS
This investigation was supported in part by Public Health Service Grant No. HE 04567-04 from the National Heart Institute, Public Health Service. This work was done as partial ful~llment of the requirements for the degree of Doctor of Philosophy. REFERENCES I
2
3 4 2 ; 9 IO II 12 ‘3 14 I.5 16
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