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Metabolic control of enzymes in normal, Diabetic, and diabetic insulin-treated rats utilizing 1,3 butanediol
Metabolic Control of Enzymes in Normal, Diabetic, and Diabetic Insulin-treated Rats Utilizing 1,3 Butanediol By MYRON A. MEHLMAN, RICHARD B. TOBIN AND JAMES Diets containing 1,3 butanediol (BD) as a replacement of carbohydrate were fed to normal, diabetic, and diabetic instdintreated rats for two weeks. The diabetic animals fed BD survived longer than the diabetic animals on essentially the same diet without BD. The activities of mahc enzyme in 105,000 X g. (1 hour) supernatant fractions of liver were determined. The PEPcK activity in liver was increased by 320 per cent in diabetic and by 240 per cent in diabetic rats fed the BD diet. Insulin decreased the PEPcK activity toward normal. In liver and adipose tissues malic enzyme activity was greatly decreased in diabetic and increased in diabetic insulin treated rats. The incorporation of radioactive bicarbonate into organic acids by liver mitochondrial pyruvate carboxyiase and the formation of intermediates for gluconeo-
B. JOHNSTON
genesis were examined. The concentration of metabolites in liver was also examined and all changes were found to be minimal. OraI administration of BD greatly increased blood ketone levels and the ratio of p-hydroxybutyrate to acetoacetate. Normal animals fed BD had significantly lower blood glucose levels. Liver perfused with BD also showed decreased glucose production from lactate. Analysis of metabolites from livers perfused with BD showed a large increase in the lactate to pyruvate ratios (from 13.1 to 81.0). Malate and aspartate were increased, whereas pyruvate, PEP, 2 PGA, and 3 PGA were decreased. It is concluded that BD exerts its hypoglycemic effect at the conversion of malate to oxalacetate and hence to PEP. (Metabolism 20: No. 2, February, 149-167, 1971)
INABILITY OF ANIMALS to utilize dietary carbohydrates, mainly glucose, under conditions of insulin deficiency, such as diabetes, leads to enhanced utilization of lipids as the primary energy source and increased synthesis of endogenous carbohydrates. 2 Fatty acids are known to stimulate glucose production from different substrates in perfused livers and tissue slices.“-” Previous studies showed that when part of the dietary carbohydrate was replaced by 1,3-butanediol (BD) as a dietary energy source for the rat, changes in tissue lipid composition were observed.lO-l2 Because in diabetes there is an impairment in the utilization of glucose due to the lack of insulin,
T
HE
From the Department of Biochemistry, University of Nebraska Medical Center, and V.A. Hospital, Omaha, Nebr., and from the Institute for Enzyme Research, University of Wisconsin, Madison, Wis. Supported by NIH and Celanese Corp. grants. MYRON A. MEHLMAN, PH.D.: Associate Professor of Biochemistry, University of Nebraska College of Medicine, Omaha, Nebr. RICHARD B. TOBIN, M.D.: Professor of Medicine and Physiology, University of Nebraska College of Medicine: Associate Chief of Staff and Associate Chief, Medical Service, V.A. Hospital, Omaha, Nebr. JAMES B. JOHNSON, PH.D.: Institute for Enzyzme Research, University of Wisconsin, Madison, Wis. METABOLISM, VOL. 20, No.
2 (FEBRUARY), 1971
149
150
MEHLMAN, TOBIN AND JOHNSTON Table l.-Composition
of Diets Basal
Casein Glucose monohydrate Dextrin Sucrose Lard Corn oil Cellulose Minerals* Vitamins? Choline chloride (50% ) 1,3 Butanediol (BD)
-___
Per Cent
22.0 13.0 13.0 13.0 22.5 7.5 3.4 4.0 I.0 0.4
~~~
.~ ~~~~.._ 1.3 Butanediol
22.0 4.1 4.1 4.0 22.5 7.5 13.4 4.0 1.0 0.4 18.0
* Mineral mix contained (in Gm./Kg. of mix) CaCO,. 4.295; KH,PO,. 343.1; NaC< 250.6; MgSO,, 7 Ha0 99.8; CaHPO,.2H,O; ferric citrate, 6.223; CuSO,, 1,558: MnSO,eH,O, 1.209; ZnCl,, 0.200; KI. 0.005; (NH,),MoO,,.4H,O, 0.0025; and Na,SEO,. 0.015. t Vitamin mix contained (in I.U./Kg. diet) vitamin A, 5,000; vitamin D, 500: Dl-a-tocopheryl acetate, 100; and (in mg./Kg.). Menadione, 5: thiamine HCl, 10: riboflavin, 20; niacin, 50; ascorbic acid, 200; pyridoxine, HCl; p-aminobenzoic acid 100; biotin 0.5: Ca pantothenate, 50; folic acid, 2; inositol 200; and vitamin B,, 0.05.
and the utilization of BD does not require insulin, part of the dietary carbohydrate was therefore replaced by BD. The purpose of the present study is to report results of investigation on feeding a BD-containing diet to normal, diabetic, and diabetic insulin-treated rats, concerning enzyme activities involved in gluconeogenesis and lipogenesis, tissue metabolite levels, mitochondrial pyruvate metabolism, perfused liver metabolite levels, and synthesis. MATERIALS AND METHODS Male albino rats of the Sprague-Dawley strain weighing between 170 and 210 Gm. were housed in individual cages and kept at 25 + 2’ C. Alloxan diabetes was produced by injecting intraperitoneally 120 mg. of alloxan per Kg. body weight into rats previously fasted for two days. Rats were fed Purina chow diet for two weeks before use. During this period about 50 percent of the animals died. Ten diabetic rats were given 4 units of insulin daily by intraperitoneal injection and were maintained on their respective diets for the duration of this experiment. A total of 30 animals ( 10 normal and 20 alloxan diabetic) were divided into six groups of five rats each and fed two diets: (1) 30 percent fat, and (2) 30 percent fat with 18 percent 1,3-butanediol (BD), substituted isocalorically for carbohydrate. The composition of these diets is shown in Table 1. Group 1 (normal). Group 2 (alloxan diabetic 1.and Group 3 (alloxan diabetic insulin-treated) were fed the 30 percent fat diet. Group 4 (normal), Group 5 (alloxan diabetic), and Group 6 (alloxan diabetic insulin-treated) were fed the 30 percent fat diet containing 18 percent BD. The animals were supplied with water and their respective diets ad libitum for two weeks. Food consumption and body weights were measured daily. The animals were killed by cervical dislocation after a 12-hour fast.
Liver and Adipose
Tissue Enzyme
Analysis
A portion of liver or total epididymal fat pads was suspended in 9 volumes of cold 0.25M sucrose, homogenized and centrifuged for 1 hour at 105,000 X g. The supernatant fractions were used for enzyme assays. Phosphoenolpyruvate carboxykinase (PEPcK) was analyzed by the procedures described by Young, Shrago, and Lardy. IX Malic enzyme was assayed by procedures described by Ochoa.l4sis
METABOLIC
CONTROL
Table 2.-Body
Weight and Food Consumption in Normal, Diabetic, and Diabetic Insulin-treated Rats Fed 1,3 Butanediol in Diet C0atKll Without With BD (5)* BD (5)
Parameters Examined Initial Final Food Food
151
OF ENZYMES
body weight, Gm. body weight, Gm. consumed, Gm. efficiencyt
195 2 14t 261&20 209538 31.5
189? 11 229?21 146k-12 35.0
Diabetic With BD (5) OF% 175+-11 168rt20 142+22 -
193c15 189r20 119?19 -
Diabetic with Insulin With BD (5) :FG? 199?8 281?15 213~26 38.4
204+ 12 255216 189220 37.0 -
* Number of animals in parentheses. t Mean + SE. $ As Gm. gain/Gm. food intake x 100.
Tissue Preparation and Metabolite Analysis Part of the liver was removed and quick-frozen in a tissue press that had been precooled in liquid nitrogen. The metabolites were extracted with 6 percent HCIO,, centrifuged, neutralized with 5 percent K&O, to remove perchlorate as previously described, and the clear supematants were frozen and stored at - loo C until analysis. Analysis for metabolites was performed individually on neutralized extracts by enzymatic procedures described in Bergmeyer.
Isolation of Mitochondria
and Analysis for Tricarboxylic
Cycle Acids
Liver samples were pooled from 5 animals in each group, except in Group B diabetic rats fed the 30 percent fat diet, where three animals were used. All experiments were carried out with intact washed liver mitochondria isolated according to the methods of Schneider18 as modified by Johnson and Lardy.1” The methods for studying pyruvate metabolism, processing of samples, 14COs fixation and analysis of tricarboxylic acid cycle intermediates have been previously described.so-2s
Protein and Blood Glucose Determination The mitochondrial nitrogen content was determined by the biuret procedure of Gornall et al.24 The protein on the 105,000 X g. supernatant fraction was analyzed by the biuret procedures described by Layne.25 The blood glucose was analyzed by the glucose oxidase procedure (Calbiochem) .
Liver Perfusion The techniques used in perfusing the liver with lactate, for preparation of extracts and assays of metabolites and glucose levels were described in detail by Veneziale et al.26 The NaH14C03 was obtained from Tracerlab and was diluted with unlabeled KHCO, to give a final concentration of 0.4 pc./pmole. The specific radioactivity of the solution used was determined as previously described .ss The enzymes were purchased from Boehringer Mannheim Corporation. All other reagents were of the highest purity commercially obtainable. RESULTS
Food Consumption, Levels
Body Weight, Adipose
Tissue Weight, and Blood Glucose
The average body weight, food consumption and food efficiency of normal, diabetic, and diabetic insulin-treated rats fed diets containing BD are presented in Table 2. The results show that normal and diabetic insulin-treated rats fed BD consumed less food and had lower body weight gain. These results are
MEHLMAN.
152
CONTROL
DIABETIC
TOBIN
AND JOHNSTON
DIABETICWITH INSULIN
Fig. l.-Adipose tissue weight. Analyses were performed on four normak and five animals where BD was added to the diet. The levels of significance were P < 0.025 (T = 3.14). The levels of significance were n.s. (T = 1.32). No significant differences for adipose tissue weight in diabetic animals (T = 1.33) were noted. Analyses were performed on five diabetic insulin-treated and five diabetic insulin-treated where BD was added to diet. The levels of significance were P < 0.025 (T = 3.17).
in agreement with those in previous publications,l”-l2 which showed that addition of BD to diets leads to a decrease in body weight gain and food consumption. The diabetic rats fed BD also consumed less food and continued to lose weight. The food efficiency was 10 per cent higher in animals fed BD and approximately 20 per cent higher in diabetic insulin-treated rats fed both diets. The adipose tissue weight was decreased in normal rats fed BD and was considerably lower in diabetic rats (Fig. 1). In the diabetic insulin-treated rats the adipose tissue weight was higher than in normal rats and there was no difference between BD-fed rats. In rats without BD there was a 61 per cent decrease in adipose tissue weight of diabetic animals and a 10.3 per cent increase in the adipose tissue weight of the diabetic insulin-treated rats, above normal levels. In rats fed BD the decrease in the adipose tissue weight in diabetic rats was not significantly different from the normal rats. A significant increase above normal in the diabetic insulin-treated rats fed BD was observed. There is a large difference in the adipose tissue weight loss between the diabetic fed and the normal on the BD diet. The diabetic rats fed the BD diet lost considerably less adipose tissue lipid than normal rats. Since adipose tissue is highly re-
METABOLIC
CONTROL
153
OF ENZYMES
250
200
ae P sl 8 3 u
150
8 $
100
50 CONTROL
DIABETIC
DIABETIC WITH ,lNSULlN
Fig. 2.-Blood glucose concentration. Analysis was done on four normals and five animals where BD was added to the diet. The levels of significance were P < 0.01 (T = 3.98). No significant differences for diabetic (T = 2.34) and diabetic insulintreated rats (T = 0.27) where BD was added to the diet were noted. Blood glucose levels for diabetic were 264 i- 19 mg. per cent and for diabetic where BD was added to the diet were 218 + 6 mg. per cent.
sponsive to changes in the physiological condition of the animal, the lipids from adipose tissue are utilized as energy. It is probably of physiological importance that there was less loss of lipids from diabetic BD fed rats as compared with the animals without BD in their diet, since diabetic rats are dependent on fatty acids as their primary energy source. Results presented in Fig. 2 show the average blood glucose levels of normal, diabetic, and diabetic insulin-treated rats fed BD. The blood glucose levels were significantly lower in normal rats fed BD. All other changes in blood glucose levels resulting from BD ingestion were minimal. Phosphoenolpyruvate
Carboxykinase
and Malic Enzyme
Activities
The activities of enzymes are greatly influenced by both dietary and physiological conditionslz*l” and can therefore be used as indicators of metabolic
154
MEHLMAN,
TOBIN
jg
W It hod
m
With B.D.
AND JOHNSTON
B.0
30 CONTROL
DIABETIC
DIABETIC WITH INSULIN
Fig. 3.-Liver phosphoenolpyruvate carboxykinase activity. Analysis was done on four normals and five animals where BD was added to the diet. The level of significance were P < 0.001 (T = 7.43). No significant differences for diabetic (T = 0.23) and diabetic insulin-treated rats (T = 1.30) where BD was added to the diet were noted.
processes. Results in Fig. 3 show that there was a large increase in liver PEPcK activity in diabetic rats and that in the diabetic insulin-treated rats PEPcK activity was suppressed. The normal rats fed the diet containing BD had an increase of 43 per cent in PEPcK activity. There was no significant difference in PEPcK activity of diabetic rats or in the diabetic insulin-treated rats with and without BD. Malic enzyme activity in the adipose tissue was greatly decreased in diabetic rats and increased over normal level in diabetic insulin-treated rats (Fig. 4). No difference in the enzyme activity were observed in rats fed BD. The activity of malic enzyme in liver is shown in Fig. 5. There was a small decrease in malic enzyme activity in normal rats fed BD. In diabetic rats there was no difference between the BD fed rats and a 37 per cent decrease in diabetic insulin-treated rats fed BD. Malic enzyme activity was greatly suppressed in diabetic rats and was increased in diabetic insulin-treated rats to slightly below control values. In adipose tissue the malic enzyme activity was considerably higher than in liver.
METABOLIC
CONTROL
155
OF ENZYMES
q
Without
T T
T
CONTROL
DIABETIC
B.D
mWithB.D.
DIABETIC WITH INSULIN
Fig. 4 .-Adipose tissue malic enzyme activity. Analysis was done on four normals and five animals where BD was added to the diet. No significant difference for normal (T = 0.47), diabetic (T = O.?l), and/or diabetic insulin-treated rats where BD was added to the diet were noted,
Tissue Metabolite
Concentrations
The concentrations of hepatic metabolites in the pathway of gluconeogenesis in normal, diabetic, and diabetic insulin-treated rats fed BD are shown in Fig. 6. The pyruvate concentrations were decreased in controls (30%) and in diabetic and diabetic insulin-treated rats fed BD; however, all changes were minimal. The concentrations of 3 PGA were decreased in control, diabetic, and diabetic insulin-treated rats fed BD; however, these changes were not significant. The 2 PGA levels were increased in diabetic and diabetic insulintreated rats fed BD. The malate levels were significantly lower only in normal rats fed BD. The concentrations of lactate in liver of normal, diabetic, and diabetic insulin-treated rats fed BD are presented in Fig. 7. The liver lactate concentrations of rats fed BD were decreased in controls by 40 per cent. No other changes were significant. The lactate to pyruvate ratios in liver (Fig. 8) were increased in control ( 12% ) , diabetic (29% ) , and diabetic insulin-treated rats (55%) fed BD.
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MEHLMAN,
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32
m
Without
5
With B.D.
B.D.
0
CONTROL
DIABETIC
DIABETIC WITH INSULIN
Fig. S-Liver malic enzyme activity. Analysis was done on four normals and five animals where BD was added to the diet. No significant differences for normal (T = 1.32), and diabetic (T = 0.75) were noted. The levels of significance for five diabetic insulin-treated and five diabetic insulin-treated where BD was added to the diet were P < 0.025 (T = 2.93).
Synthesis of Organic Mitochondria The synthesis control, diabetic
Acids
from
Pyruvate and Bicarbonate
by Rat Liver
of intermediates for gluconeogenesis by mitochondria from and diabetic-insulin treated rats fed BD are presented in
Table 3. The incorporation of radioactive bicarbonate into organic acids was increased in the presence of glutamate in controls (64% ), diabetic (64%), and diabetic insulin-treated rats (55 % ) . In animals fed BD, there was an increase in the presence of glutamate in l’COV fixation in controls (25 % ) , diabetic (42% ) , and diabetic insulin-fed rats (52% ) . The increase in 14COz fixation in the presence of glutamate can be accounted for by the incorporation of radioactive bicarbonate into aspartate. Similar increases in 14COz incorporation were observed in the presence of caprylate. The estimation of pyruvate carboxylase activity, based on the radioactive 14C02 fixed, represent minimal values because they are calculated on the basis of specific radioactivity of added bicarbonate and do not take into account the dilution of radioactive bicarbonate by metabolically produced nonradioactive carbon dioxide and the loss of l’COI: from intermediates formed in the tricarboxylic acid cycle during metabolism. A more
METABOLIC
CONTROL
157
OF ENZYMES
0.10
s >
0.40
5
0
k
% P
PYRUVATE
B O-30
030
w 3 L 5
a 020
020
e
a
1 z 2i
iz d z
2
5 0.10
0.10
0 7 7 2
0 CONTROL D)ABETIC DIABETIC
0 CONTROL
DIABETIC
WITH INSULIN
PEP
3 PGA
DlASETlC CONTROL WITH INSULIN
DIABETIC
DIABETIC WITH INSULIN
MALATE
Fig. 6.-Liver glucogenic metabolite concentrations. Analyses were performed on four normals, three diabetic, five diabetic insulin-treated and five normals, three diabetic, and five diabetic insulin-treated rats where BD was added to the diet. Levels of significance were for pyruvate in normal P < 0.005 (T = 4.52), in diabetic ns. (T = 1.24), and diabetic insulin-treated ns. (T = 1.39); for PEP in normal P < 0.05 (T = 2.61), in diabetic n.s. (T = 2.20); and diabetic insulin-treated n.s. (T = 0.72); for 2 PGA, in normal P < 0.001 (T = 10.24), in diabetic P < 0.025 (T = 4.39), and diabetic insulin-treated, P < 0.05 (T = 2.68); for 3 PGA in normal P < 0.005 (T = 5.01), in diabetic n.s. (T = 1.52) and diabetic insulin-treated P < 0.01 (T = 3.62); for malate in normal P < 0.05 (T = 2.47), in diabetic n.s. (T = 1.78), and in diabetic insulin-treated n.s. (T = 1.03) reliable estimation of pyruvate carboxylase activity (Fig. 9) was obtained by summing all of the measured metabolites shown in Table 3, which were synthe-
sized through pyruvate carboxylation. The pyruvate carboxylase activity was increased in normal (27% ), diabetic (44% ), and diabetic insulin-treated rats (18%) fed BD. Effect of BD on Glucose Production and Metabolite Concentration in Perfused Livers The rate of glucose production from lactate, and in the presence of added 20 mM BD are summarized in Fig. 10. Twenty-one mg. per cent glucose was produced per 30 minutes from lactate. When livers were perfused with 20 mM BD there was no glucose production. Addition of 20 mM lactate after 30 minutes of perfusion with BD resulted in 8 mg. per cent of glucose produced per 30 minutes. This represents a 62 per cent inhibition in the production of glucose by BD. Further addition of 10 mM fructose led to rapid glucose production, 74 mg. per cent/30 min. This indicates that perfusion of liver with BD does not alter glucose production above the triosephosphate step, and that liver is fully functional with respect to glucose production. Results in Figs. 11 and 12 show the effect of BD on liver gluconeogenic intermediates 20
158
MEHLMAN,
TOBIN AND JOHNSTON
3.4 3.2 3.0 2-B
q
W,thc,ut
i%
With B D
B D
2.6
Fig. 7.-Liver lactate concentrations. Analyses were performed on four normal, three diabetic, and five diabetic insulin-treated rats, and on five normal, three diabetic, and five insulintreated rats where BD was added to the diet. The levels of significance for normals were P < 0.005 (T = 5.43), for diabetic n.s. (T = 0.32) and for diabetic insulin-treated rats n.s. (T = 1.56). 00 06 0 .4 0
2 0 CONTROL
DIABETIC
DIABETIC WITH ,NS”L,N
LIVER
minutes after the addition of substrate (lactate). There was a 60 per cent increase in the liver lactate concentrations (Fig. 12) in the presence of BD and a 73 per cent decrease in pyruvate levels (Fig. 11). The ratio of lactate to pyruvate in liver perfused with BD increased from 13.7 to 8 1.O (Fig. 12). The concentrations of aspartate and malate were greatly increased in the BDperfused liver (Fig. 11). A very large decrease in PEP. 2 PGA was observed in livers perfused with BD. DISCUSSION
Feeding a BD-containing diet to alloxan diabetic rats resulted in an improvement in survival and in general appearance (Fig. 13). In previous studies it has been demonstrated that the addition of BD to diets leads to modifications of tissue lipid content and gluconeogenesis.llJ” The regulation of metabolic pathways is markedly affected by substrates, metabolites, cofactors, concentrations, and distributions of enzymes between compartments. Since enzyme activities and metabolite concentration patterns are exquisitely responsive to both metabolic and dietary state of the animals 313,28,31it may be possible by studying the activities of these enzymes
METABOLIC
CONTROL
m
32
159
OF ENZYMES
With B.D.
Fig. I.-Liver lactateto-pyruvate ratio. Analysis was performed on four normal, three diabetic, and five diabetic insulin-treated rats and on five normal, three diabetic, and five diabetic insulin-treated rats where BD was added to the diet. The levels of significance for normal were n.s. (T = 0.54); for diabetic, P < 0.05 (T = 2.78); and for diabetic insulin-treated, P < 0.01 (T = 3.55).
IT5 5
26
f w 24 l3 Is 20 t zi t- ‘6 : =j 12 k 0
-
6-
5 4 -
CONTROL
DIABETIC
DIABETIC WITH INSULIN
and metabolite patterns to elucidate the mechanism by which BD exerts its effect on gluconeogenesis. The enzymatic activity of PEPcK and malic enzyme are highly responsive to changes in dietary carbohydrate and lipid, as well as to hormones and diabetes,13 and can therefore be utilized as important indicators of the metabolic state of animals. In the adipose tissue (Fig. 4), malic enzyme activity was considerably higher than in liver. Similar patterns in enzyme activity were observed in adipose tissue of diabetic and diabetic insulin-treated rats. In the insulin-treated diabetic rats malic enzyme activity was greater than in adipose tissue of normal rats. The response of enzyme activities in adipose tissue further confirms previous reports32 that adipose tissue is highly responsive to metabolic and hormonal alterations, as is evident from Fig. 1, where adipose tissue weight of these animals is presented. The decrease in adipose tissue weight in diabetic rats is consistent with the requirement for utilization of additional lipid for energy. The smaller decrease in the adipose tissue weight of diabetic BD-fed rats may be due to a reduced requirement of lipid for energy, because this requirement may be met by utilization of dietary BD. The changes in liver tissue content of gluconeogenic metabolites are shown in Fig. 6. In animals fed the diet containing BD the ratio of lactate to pyruvate was increased (Fig. 7). The increase in the lactate to pyruvate ratio represents an increase in the cytoplasmic NADH/NAD+ ratio.“3 There were minimal changes in PEP and 2 PGA levels; however, 3 PGA levels were lower in animals fed BD. The decrease in the 3 PGA level is consistent with the findings that there was an increase in the lactate to pyruvate ratios in BD-fed animals. Pyruvate plays a key role in carbohydrate metabolism, because it can be
160
MEHLMAN,
TOBIN AND JOHNSON
Table 3.-Formation of Tricarboxylic Cycle Acids from Pyruvate, Bicarbonate, Glutamate and Caprylate by Rat Liver Mitochondria From Alioxan and Alloxan Diabetic, Insulin-treated Rats fed 1,3 Butanediol Diet*
_.__~__.
~.
Metabolite Dietary Treatment
30% fat
Condition Addition
of Animals to System
Changes
Pyruvate used
pmoles1minJmg.N
mMolar Control none 6.6 glutamate 0.66 caprylate
0.95 1.16 0.96
0.36 0.59 0.60
0.23 0.19 0.29
0.15 0.28 0.33
0.37 -
Diobeiic none 6.6 glutamate 0.66 caprylate
0.63 0.85 0.75
0.25 0.41 0.40
0.11 0.10 0.16
0.16 0.29 0.25
0.22 -
6.6 glutamate 0.66 caprylate
0.79 0.88 0.83
0.33 0.5 I 0.45
0.18 0.13 0.20
0.19 0.28 0.25
0.27
Control none 6.6 glutamate
0.90 0.92
0.40 0.50
0.21 0.15
0.27 0.33
0.25
Diabetic none 6.6 glutamate 0.66 caprylate
0.80 0.85 0.77
0.31 0.44 0.41
0.17 0.12 0.19
0.22 0.34 0.27
0.24 -
0.85 0.95 0.83
0.31 0.47 0.44
0.17 0.13 0.19
0.23 0.35 0.32
0.26 -
Diabetic with lnsulir~ none
30% fat, 18.3%
I,3 butanediol
-
Diabetic with ltuulin none
6.6 glutamate 0.66 caprylate
.-
* Keactlon mixture for all experiments contained 4 mM ATP, 10 mM MgSO,, 6.6 potassium phosphate, and 6.6 mM triethanolamine buffers. pH 7.4, 6.6 mM pyruvate 13.3 mM KHlWO,, and 5 mg. of fatty acid free albumin in each incubation flask, 6.6 L-glutamate and 0.66 mM caprylate were added as indicated. Mitochondria from 0.5 liver suspended in 0.5 ml. isotonic sucrose were added to each incubation. Incubation was 10 min. at 37’ C for all experiments.
mM and mM Gm. time
carboxylated and used for glucose synthesis or oxidized and used for energy production. The mitochondrial significance of pyruvate metabolism in both kidney and liver for providing precursors of gluconeogenesis has been studied in great detail.‘@33J-40 The changes in the pyruvate carboxylase activity measured in intact mitochondria under these conditions may not represent true activity (Fig. 9) because it is possible to stimulate pyruvate carboxylase activity in mitochondria by altering concentrations and types of substrates, as shown in Table 4. In the presence of 16.3 mM glutamate the 14C0.,_ incorporation in the presence of added glutamate can be accounted for primarily by increased aspartate synthe-
METABOLIC
CONTROL
OF ENZYMES
161
Fig. 9.-Liver mitochondrial pyruvate carboxylase activity.
CONTROL
OIABETIC
DIABETIC WITH INSULIN
sis.2Z Addition of 13.2 mM a-ketoglutarate resulted in 156 percent increase in 14COZ incorporation. This increase in ‘“CO2 incorporation is due not to an increase in pyruvate carboxylation but to a dilution of the radioactive oxalacetate formed from pyruvate carboxylation with the large pool of unlabeled malate formed from a-ketoglutarate. When both a-ketoglutarate and glutamate were used together, a 210 percent increase in 14C02 incorporation was observed. These were still not maximal conditions because cu-ketoglutarate and glutamate are competitive, as evidenced by the decreased levels of aspartate found; also, oxidation of a-ketoglutarate may generate NADH, which can decrease the conversion of malate to oxalacetate and hence to aspartate. Waglet” using broken mitochondria, reported a significant increase in pyruvate carboxylase activity in diabetic rats and a decrease in diabetic insulin-treated rats. However, it must be pointed out that in this study malic enzyme activity and PEPcK activity responded in the expected manner and magnitude in diabetic and diabetic insulin-treated rats. The mitochondrial pyruvate metabolism (Table 3) shows that mitochondria under all experimental conditions examined responded almost identically both in the presence of fatty acids and glutamate. Addition of 20 mM BD to perfused livers resulted in a strong inhibition of glucose production. The rate of glucose synthesis from lactate was decreased by 62 per cent (Fig. 10) in the presence of BD. Addition of 10 mM fructose led to a rapid rate of glucose synthesis, demonstrating that glucose production was not inhibited at the steps above GAP and DHAP. The metabolite concentrations in liver (Fig. 11) perfused with lactate, in the presence of BD, showed a striking change in the levels of gluconeogenic inter-
162
MEHLMAN,
TOBIN
AND JOHNSON
80
60
40
20
LACTATE (20mMOLES) 1 +A8*
._.Hi t
40 20
/
I
FRUCTOSE llOmMOLES)
1,3 BUTANE0 IOL (20mMOLESl
/
0 20
40 PERFUSION
Fig. lO.-Synthesis
PY RUVATE
60
80
TIME,
MINUTES
100
120
of blood glucose from lactate in perfused liver.
ASPARTATE
Fig. 11 .-Metabolite
MALATE
concentrations
PEP
2 PGA
from perfused livers.
3 PGA
METABOLIC
CONTROL
163
OF ENZYMES
Without B.O. With B.D.
a
0 LACTATE
Fig. 12.-Concentration with BD.
RATIO LACTATEf PYRUVATE
of lactate and ratio of lactate to pyruvate in liver perfused
The pyruvate levels were greatly decreased and the levels of malate and aspartate were increased. The 3 carbon phosphorylated intermediates were greatly decreased. These results suggested that BD inhibits gluconeogenesis in perfused liver by blocking the conversion of oxalacetate to phosphoenolpyruvate. The large increase in the lactate to pyruvate ratio from 13.7 to 81 is consistent with an inhibition of gluconeogenesis at this step. The pathway for the catabolism of BD has not been elucidated; however, the following observations have been made: The metabolism of BD in the cytosol leads to an increased production of NADH, as is evident from the increase in the ratio of lactate to pyruvate in perfused livers (Fig. 10). BD is not metabolized by isolated liver or kidney mitochondria. Liver high-speed supernatant fractions rapidly reduce NAD+ to NADH in the presence of BD. Intact animals rapidly oxidize BD-14C to 14C02. Administration of BD orally for two weeks (Table 5) or one hour (Table 6) resulted in a large increase in blood ketone bodies and in the p-hydroxybutyrate to acetoacetate ratio. On the basis of these observations, a pathway for the metabolism of BD is suggested in Fig. 13. The rapid utilization and metabolism by mammalian cells of short-chain carbohydrates can be achieved in the absence of insulin. Studies of this nature offer interesting model systems for the elucidation of metabolic disturbances, mediates.
164
MEHLMAN,
TOBIN AND JOHNSON
Fig. 13.-Alloxan diabetic rats fed dietary BD. Larger animal (left) received 18 per cent BD as replacement of carbohydrate.
Gig - CH - CH2- CH 20H I OH k CH3-CH-CH2CHO
NAD + NADH+H+ +H20
OH
NAD + NADH +H+ ‘b CH3 - CH - CH 2COOH OH Fig.
14sProposed butanediol.
scheme
for metabolism
of 1,3 ‘k
NAD + NADH + H +
CH3 - C - CH 2COOH II 0 CoA ATP CH3 - C - CH2COSCoA II 0 CoA 2CH3-C-SCoA II 0 I Cop + Hz0
METABOLIC
CONTROL
165
OF ENZYMES
Table 4.-!U1uulation of 14C02 Incorporation into Organic Acids in the Presence of CL-Ketoglutarate and Glutarate by Rat Kidney Mitochondria*
mM None 6.6 cu-ketoglutarate 16.3 glutamate 6.6 cu-ketoglutarate and 16.6 glutamate
5.7 4.5 5.3
1.1 6.5 1.5
4.2
6.1
pmoles/mg.N/lO 0.7 1.4 0.8
min.
1.3
1.42
1.0 2.6 1.6
0.38
3.1
* The reaction mixture contained 4 mM ATP, 10 mM magnesium sulfate, 6.6-mM potassium phosphate, and 6.6-mM potassium triethanolamine buffers pH 7.4, 20 mM KHrJCO, and 6.6 mM potassium pyruvate. Final volume was 3 ml., containing a total of 1.2 to 1.7 ml. of isotonic sucrose. Mitochondria from 0.5 Gm. of kidney suspended in 0.5 ml. of isotonic sucrose were added to each incubation and contained 1.0 mg. of nitrogen. (From Mehlman, M. A.: J. Biol. Chem. 243:3289, 1968.) Table S-Effect
of Addition of 20% 1,3 Butanediol of Rats for 14 Days*
to Drinking Water
Experimental Parameters
Examined
Blood glucose mg. per cent Acetoacetate ~moles/mi. /3-Hydroxybutyrate floles/ml. Total ketone bodies cmoles/ml. Ratio. /SHydroxybutyrate Acetoacetate Pyruvate ~moles/ml.
Without
BD (5)
109 0.026 0.094 0.130
+- 4 t 0.003 ? 0.015 ? 0.025
2.85
-c 0.60
0.166 c 0.026
* The animals were allowed to drink water containing killed 1 hr. after they drank water with BD. Table &-Effect
With
102 0.170 0.951 1.121
r + 2 +
6.12
rt 0.45
BD (5)
9 0.011 0.113 0.107
n.s. p < 0.001 p < 0.001 p < 0.001 p < 0.01
0.136 ? 0.027 BD for 2 hr./day.
ns. Animals
were
of 1,3 ButanedioI BD on BIood Ketone Bodies and Blood GIucose Levels Experimental
Parameters
Examined
Blood glucose mg. per cent Acetoacetate, &moles/ml. ,&hydroxybutyrate eoles/ml. Total ketone bodies Ratio /3-hydroxybutyrate acetoacetate
Corm&
yj;hout
112 0.024 0.063 0.087
c 6 +- 0.014 2 0.019 +- 0.032
109 0.052 0.359 0.411
I!I 8 + 0.009 & 0.017 f 0.029
N.S. p < 0.05 p < 0.001 .D <- 0.001
3.07
& 1.31
7.48
? 2.41
p < 0.05
* 3 ml. of 20% BD solution were given orally through killed 1 hr. after administration of BD.
ExperBirn~n;;:
stomach
with
tube and animals were
such as diabetes, under conditions of insulin lack. These compounds can provide a rapidly available energy source for the organisms. Further studies are now in progress concerning enzymes and intermediates involved in the pathway of BD metabolism.
166
MEHLMAN,
TOBIN AND JOHNSON
ACKNOWLEDGMENTS The authors thank Dr. H. A. Lardy for support and help with all aspects of this work, Mr. R. Hanson for perfusion experiments, Mr. Carl Mackerer and Mr. Henry Hahn for help with experiments, and Mr. Lawrence Stem and Mrs. Lucy Riddo for preparing the illustrations.
REFERENCES 1. Friedmann, B., Goodman, E. H., Jr., and Weinhouse, S.: Effects of insulin and fatty acids on gluconeogenesis in the rat. J. Biol. Chem. 242:3620, 1967. 2. Williamson, J. R., Browning, E. T., and Scholz, R.: Control mechanism of gluconeogenesis and ketogenesis. I. Effect of oleate on gluconeogenesis in perfused rat liver. J. Biol. Chem. 244~4607, 1969. 3. Williamson, J. R., Browning, E. T., Scholz, R., Kreisberg, R. A., and Fritz, I. B.: Inhibition of fatty acid stimulation of gluconeogenesis by ( + )-decanoylcarnitine in perfused rat liver. Diabetes 17:194, 1968. 4. Teufel, H., Menahan, L. A., Shipp, J. C., Boning, S., and Wieland, 0.: Effect of oIeic acid on the oxidation and gluconeogenesis from pyruvate-l-r% in the perfused rat liver. Europ. J. Biochem. 2: 182, 1967. 5. Williamson, .I. R., Kreisberg, R. A. and Felts, P. W.: Mechanism for stimulation of gluconeogenesis by fatty acids in the perfused rat liver. Proc. Nat. Acad. Sci. U.S.A. 56: 247, 1966. 6. Herrera, M. G., Kamm, D., Ruderman, N., and Cahill, G. F., Jr.: Non-hormonal factors in the corm01 of gluconeogenesis. Advances Enzym. Regulat. 4:225, 1966. 7. Struck, E., Ashmore, J., and Wieland, 0.: Kurze Mitteilung Stimulierung der Gluconeogenese durch Langkerrige Fettsauren und Glucagon. Biochem. Z. 343: 107, 1965. 8. Krebs, H. A., Hems, R., and Gascoyne, T.: Renal gluconeogenesis. IV. Gluconeogenesis from substrate combinations. Acta Biol. Med. German. 11:607, 1963. 9. Krebs, H. A., Speake, R. N., and Hems, R.: Acceleration of renal gluconeogenesis by ketone bodies and fatty acids. Biochem. J. 94:712, 1965. 10. Stoewsand, G. S., Dymsza, H. A., Mehlman, M. A., and Therriault, D. G.: Influence of 1,3 butanediol on tissue lipids of cold exposed rats. J. Nutr. 83:464, 1966. 11. Mehlman, M. A., Therriault, D. G., Porter, W., Stoewsand, G. S., and Dymsza,
H. A.: Distribution of lipids in rats fed 1,3 butanediol. J. Nutr. 80:215, 1966. 12. Stoewsand, G. S., Dymsza, H. A.. Swift, S. M., Mehlman, M. A., and Therriault, D. G.: Effect of feeding polyhydric alcohols on tissue lipids and the resistance of rats to extreme cold. .I. Nutr. 89:414, 1966. 13. Young, J. W., Shrago, E., and Lardy, H. A.: Metabolic control of enzymes involved in lipogenesis and gluconeogenesis. Biochem. 3 : 1687, 1964. 14. Ochoa, S.: Crystalline condensing enzyme from pig heart. In Colowick, S. P., and Kaplan, N. 0. (Eds.): Methods in Enzymology. I. New York, Academic, 1955, p. 685. 15. Ochoa, S.: A “malic” enzyme from pigeon liver and wheat germ. In Colowick. S. P., and Kaplan, N. 0. (Eds.) : Methods in Enzymology. I. New York, Academic. 1955, p. 739. 16. Ray, P. D., Foster, D. O., and Lardy, H. A.: Paths of carbon in gluconeogenesis and lipogenesis. N. Inhibition by L-tryptophan of hepatic gluconeogenesis at the level of phosphoenolpyruvate formation. J. Biol. Chem. 241:3904, 1966. 17. Bergmeyer, H. V. (Ed.): Methods of Enzymatic Analysis. New York, Academic, 1963. 18. Schneiderer, W. C.: Intracellular distribution of enzymes. III. The oxidation of octanoic acid by rat liver fractions. J. Biol. Chem. 176:289, 1948. 19. Johnson, D. and Lardy, H. A. Isolation of liver of kidney mitochondria. In Estabrook, R. W.. and Pullman, M. E., (Eds.): Methods in Enzymology, Vol. 10. New York, Academic, 1967, p. 94. 20. Walter, P., Petkaw, V., and Lardy, H. A.: Paths of carbon in gluconeogenesis and lipogenesis. III, The role and regulation of mitochondrial processes involved in supplying precursors of phosphoenolpyruvate. J. Biol. Chem. 241:2523, 1966. 21. Mehlman, M. A., Walter, P. and Lardy, H. A.: Paths of carbon in glu-
METABOLIC
CONTROL
OF ENZYMES
coneogenesis and lipogenesis. VII. The synthesis of precursors for gluconeogenesis from pyruvate and bicarbonate by rat kidney mitochondria. J. Biol. Chem. 242:4594, 1967. 22. Mehlman, M. A.: The fate of isotopic carbon in kidney mitochondria synthesizing precursors for glucose from pyruvate and bicarbonate. J. Biol. Chem. 243:3289, 1968. 23. Somberg, E. W., and Mehlman, M. Regulation of gluconeogenesis and A.: lipogenesis: The regulation of mitochondrial pyruvate metabolism in guinea-pig liver synthesizing precursors for gluconeogenesis. Biochem. J. 112:435, 1969. 24. Gorwall, A. G., Bardawill, C. J., and Daub, M. M.: Determination of serum protein by means of the biuret reaction. J. Biol. Chem. 117:751, 1949. 25. Layne, E.: Spectrophotometric and turbidimetric methods for measuring proteins. 111 Methods in Enzymology, Vol. 3. New York, Academic, 1957, p. 447. 26. Veneziale, C. M., Walter, P., Kneer, N. and Lardy, H. A.: Influence of L-tryptophan and its metabolites on gluconeogenesis in the isolated perfused liver. Biochemistry 6:2129, 1967. 27. Mehlman, M. A., Tobin, R. B. and Johnston, J. B.: Influence of dietary 1,3 butanediol on metabolites and enzymes involved in gluconeogenesis and lipogenesis. J. Nun. 100:1341, 1970. 28. Vaughan, D. A. and Winders, R. L.: Effects of diet on HMP dehydrogenases and malic (.TPN) dehydrogenase in the rat. Amer. J. Physiol. 206:1081, 1964. 29. Weber, G.: Behavior and regulation of enzymes synthesis in liver and in hepatomas of different growth rates. Advances Enzym. Regulat., 1:321, 1963. 30. Williamson, J. R., Scholz, R. and Browning, E. T.: II. Interactions between fatty acids oxidation and the citric acid cycle in perfused rat liver. J. Biol. Chem. 244: 4617, 1969. 31. Rognstad, R. and Katz, J.: Gluconeogenesis in the kidney cortex. Effects of Dmalate and amino-oxyacetate. Biochem. J. 116:483, 1970. 32. Therriault, D. G. and Mehlman, M.
167 A.: Effect of prior diet on lipid mobilization in rats during starvation or cold exposure. J. Nutr. 98:25, 1969. 33. Williamson, D. H., Lund, P., and Krebs, H. A.: The redox state of free nicotinamide adenine dinucleotide in the cytoof rat liver. and mitochondria plasm Biochem. J. 103:514, 1967. 34. Henning, H. V., Stumpf, B., Ohly, B., and Seubert, W.: On the mechanism of gluconeogenesis and its regulation. III. The gluconeogenic capacity and the activities of pyruvate carboxylase and PEP carboxylase of rat kidney and rat liver after cortisol treatment and starvation. Biochem. Z. 344:274, 1966. 35. Wagle, S. R., and Ashmore, J.: Studies on carbon dioxide fixation in normal and alloxan-diabetic animals. Biochim. Biophys. Acta 74~564, 1963. 36. Haynes, R. C., Jr.: The fixation of carbon dioxide by rat liver mitochondria and its relation to gluconeogenesis. J. Biol. Chem. 240:4103, 1965. 37. Walter, P., and Strucki, J. W.: Regulation of pyruvate carboxylase in rat liver mitochondria by adenine nucleotides and short chain fatty acids. Europ. J. Biochem. 12:508, 1970. 38. Lardy, H. A., Petkau, V., and Walter, P.: Paths of carbon in gluconeogenesis and lipogenesis: The role of mitochondria in supplying precursors of phosphoenolpyruvate. Proc. Nat. Acad. Sci. U.S.A. 53:1410, 1965. 39. Mehlman, M. A., and Walter, P.: Regulation of gluconeogenesis and lipogenesis inhibition of pyruvate carboxylation in rat kidney mitochondria by malonate and malonyl CoA. Arch. Biochem. Biophys. 127: 590, 1968. 40. Mehlman, M. A.: Inhibition of pyruvate carboxylation by fluorocitrate in rat kidney mitochondria. J. Biol. Chem. 243: 1919, 1968. 41. Haslam, R. J., and Krebs, H. A.: Substrate competition in the respiration of animal tissues: The metabolic interactions of pyruvate and a-oxoglutarate in rat liver homogenates. Biochem. J. 86:432, 1963.
B. JOHNSTON
genesis were examined. The concentration of metabolites in liver was also examined and all changes were found to be minimal. OraI administration of BD greatly increased blood ketone levels and the ratio of p-hydroxybutyrate to acetoacetate. Normal animals fed BD had significantly lower blood glucose levels. Liver perfused with BD also showed decreased glucose production from lactate. Analysis of metabolites from livers perfused with BD showed a large increase in the lactate to pyruvate ratios (from 13.1 to 81.0). Malate and aspartate were increased, whereas pyruvate, PEP, 2 PGA, and 3 PGA were decreased. It is concluded that BD exerts its hypoglycemic effect at the conversion of malate to oxalacetate and hence to PEP. (Metabolism 20: No. 2, February, 149-167, 1971)
INABILITY OF ANIMALS to utilize dietary carbohydrates, mainly glucose, under conditions of insulin deficiency, such as diabetes, leads to enhanced utilization of lipids as the primary energy source and increased synthesis of endogenous carbohydrates. 2 Fatty acids are known to stimulate glucose production from different substrates in perfused livers and tissue slices.“-” Previous studies showed that when part of the dietary carbohydrate was replaced by 1,3-butanediol (BD) as a dietary energy source for the rat, changes in tissue lipid composition were observed.lO-l2 Because in diabetes there is an impairment in the utilization of glucose due to the lack of insulin,
T
HE
From the Department of Biochemistry, University of Nebraska Medical Center, and V.A. Hospital, Omaha, Nebr., and from the Institute for Enzyme Research, University of Wisconsin, Madison, Wis. Supported by NIH and Celanese Corp. grants. MYRON A. MEHLMAN, PH.D.: Associate Professor of Biochemistry, University of Nebraska College of Medicine, Omaha, Nebr. RICHARD B. TOBIN, M.D.: Professor of Medicine and Physiology, University of Nebraska College of Medicine: Associate Chief of Staff and Associate Chief, Medical Service, V.A. Hospital, Omaha, Nebr. JAMES B. JOHNSON, PH.D.: Institute for Enzyzme Research, University of Wisconsin, Madison, Wis. METABOLISM, VOL. 20, No.
2 (FEBRUARY), 1971
149
150
MEHLMAN, TOBIN AND JOHNSTON Table l.-Composition
of Diets Basal
Casein Glucose monohydrate Dextrin Sucrose Lard Corn oil Cellulose Minerals* Vitamins? Choline chloride (50% ) 1,3 Butanediol (BD)
-___
Per Cent
22.0 13.0 13.0 13.0 22.5 7.5 3.4 4.0 I.0 0.4
~~~
.~ ~~~~.._ 1.3 Butanediol
22.0 4.1 4.1 4.0 22.5 7.5 13.4 4.0 1.0 0.4 18.0
* Mineral mix contained (in Gm./Kg. of mix) CaCO,. 4.295; KH,PO,. 343.1; NaC< 250.6; MgSO,, 7 Ha0 99.8; CaHPO,.2H,O; ferric citrate, 6.223; CuSO,, 1,558: MnSO,eH,O, 1.209; ZnCl,, 0.200; KI. 0.005; (NH,),MoO,,.4H,O, 0.0025; and Na,SEO,. 0.015. t Vitamin mix contained (in I.U./Kg. diet) vitamin A, 5,000; vitamin D, 500: Dl-a-tocopheryl acetate, 100; and (in mg./Kg.). Menadione, 5: thiamine HCl, 10: riboflavin, 20; niacin, 50; ascorbic acid, 200; pyridoxine, HCl; p-aminobenzoic acid 100; biotin 0.5: Ca pantothenate, 50; folic acid, 2; inositol 200; and vitamin B,, 0.05.
and the utilization of BD does not require insulin, part of the dietary carbohydrate was therefore replaced by BD. The purpose of the present study is to report results of investigation on feeding a BD-containing diet to normal, diabetic, and diabetic insulin-treated rats, concerning enzyme activities involved in gluconeogenesis and lipogenesis, tissue metabolite levels, mitochondrial pyruvate metabolism, perfused liver metabolite levels, and synthesis. MATERIALS AND METHODS Male albino rats of the Sprague-Dawley strain weighing between 170 and 210 Gm. were housed in individual cages and kept at 25 + 2’ C. Alloxan diabetes was produced by injecting intraperitoneally 120 mg. of alloxan per Kg. body weight into rats previously fasted for two days. Rats were fed Purina chow diet for two weeks before use. During this period about 50 percent of the animals died. Ten diabetic rats were given 4 units of insulin daily by intraperitoneal injection and were maintained on their respective diets for the duration of this experiment. A total of 30 animals ( 10 normal and 20 alloxan diabetic) were divided into six groups of five rats each and fed two diets: (1) 30 percent fat, and (2) 30 percent fat with 18 percent 1,3-butanediol (BD), substituted isocalorically for carbohydrate. The composition of these diets is shown in Table 1. Group 1 (normal). Group 2 (alloxan diabetic 1.and Group 3 (alloxan diabetic insulin-treated) were fed the 30 percent fat diet. Group 4 (normal), Group 5 (alloxan diabetic), and Group 6 (alloxan diabetic insulin-treated) were fed the 30 percent fat diet containing 18 percent BD. The animals were supplied with water and their respective diets ad libitum for two weeks. Food consumption and body weights were measured daily. The animals were killed by cervical dislocation after a 12-hour fast.
Liver and Adipose
Tissue Enzyme
Analysis
A portion of liver or total epididymal fat pads was suspended in 9 volumes of cold 0.25M sucrose, homogenized and centrifuged for 1 hour at 105,000 X g. The supernatant fractions were used for enzyme assays. Phosphoenolpyruvate carboxykinase (PEPcK) was analyzed by the procedures described by Young, Shrago, and Lardy. IX Malic enzyme was assayed by procedures described by Ochoa.l4sis
METABOLIC
CONTROL
Table 2.-Body
Weight and Food Consumption in Normal, Diabetic, and Diabetic Insulin-treated Rats Fed 1,3 Butanediol in Diet C0atKll Without With BD (5)* BD (5)
Parameters Examined Initial Final Food Food
151
OF ENZYMES
body weight, Gm. body weight, Gm. consumed, Gm. efficiencyt
195 2 14t 261&20 209538 31.5
189? 11 229?21 146k-12 35.0
Diabetic With BD (5) OF% 175+-11 168rt20 142+22 -
193c15 189r20 119?19 -
Diabetic with Insulin With BD (5) :FG? 199?8 281?15 213~26 38.4
204+ 12 255216 189220 37.0 -
* Number of animals in parentheses. t Mean + SE. $ As Gm. gain/Gm. food intake x 100.
Tissue Preparation and Metabolite Analysis Part of the liver was removed and quick-frozen in a tissue press that had been precooled in liquid nitrogen. The metabolites were extracted with 6 percent HCIO,, centrifuged, neutralized with 5 percent K&O, to remove perchlorate as previously described, and the clear supematants were frozen and stored at - loo C until analysis. Analysis for metabolites was performed individually on neutralized extracts by enzymatic procedures described in Bergmeyer.
Isolation of Mitochondria
and Analysis for Tricarboxylic
Cycle Acids
Liver samples were pooled from 5 animals in each group, except in Group B diabetic rats fed the 30 percent fat diet, where three animals were used. All experiments were carried out with intact washed liver mitochondria isolated according to the methods of Schneider18 as modified by Johnson and Lardy.1” The methods for studying pyruvate metabolism, processing of samples, 14COs fixation and analysis of tricarboxylic acid cycle intermediates have been previously described.so-2s
Protein and Blood Glucose Determination The mitochondrial nitrogen content was determined by the biuret procedure of Gornall et al.24 The protein on the 105,000 X g. supernatant fraction was analyzed by the biuret procedures described by Layne.25 The blood glucose was analyzed by the glucose oxidase procedure (Calbiochem) .
Liver Perfusion The techniques used in perfusing the liver with lactate, for preparation of extracts and assays of metabolites and glucose levels were described in detail by Veneziale et al.26 The NaH14C03 was obtained from Tracerlab and was diluted with unlabeled KHCO, to give a final concentration of 0.4 pc./pmole. The specific radioactivity of the solution used was determined as previously described .ss The enzymes were purchased from Boehringer Mannheim Corporation. All other reagents were of the highest purity commercially obtainable. RESULTS
Food Consumption, Levels
Body Weight, Adipose
Tissue Weight, and Blood Glucose
The average body weight, food consumption and food efficiency of normal, diabetic, and diabetic insulin-treated rats fed diets containing BD are presented in Table 2. The results show that normal and diabetic insulin-treated rats fed BD consumed less food and had lower body weight gain. These results are
MEHLMAN.
152
CONTROL
DIABETIC
TOBIN
AND JOHNSTON
DIABETICWITH INSULIN
Fig. l.-Adipose tissue weight. Analyses were performed on four normak and five animals where BD was added to the diet. The levels of significance were P < 0.025 (T = 3.14). The levels of significance were n.s. (T = 1.32). No significant differences for adipose tissue weight in diabetic animals (T = 1.33) were noted. Analyses were performed on five diabetic insulin-treated and five diabetic insulin-treated where BD was added to diet. The levels of significance were P < 0.025 (T = 3.17).
in agreement with those in previous publications,l”-l2 which showed that addition of BD to diets leads to a decrease in body weight gain and food consumption. The diabetic rats fed BD also consumed less food and continued to lose weight. The food efficiency was 10 per cent higher in animals fed BD and approximately 20 per cent higher in diabetic insulin-treated rats fed both diets. The adipose tissue weight was decreased in normal rats fed BD and was considerably lower in diabetic rats (Fig. 1). In the diabetic insulin-treated rats the adipose tissue weight was higher than in normal rats and there was no difference between BD-fed rats. In rats without BD there was a 61 per cent decrease in adipose tissue weight of diabetic animals and a 10.3 per cent increase in the adipose tissue weight of the diabetic insulin-treated rats, above normal levels. In rats fed BD the decrease in the adipose tissue weight in diabetic rats was not significantly different from the normal rats. A significant increase above normal in the diabetic insulin-treated rats fed BD was observed. There is a large difference in the adipose tissue weight loss between the diabetic fed and the normal on the BD diet. The diabetic rats fed the BD diet lost considerably less adipose tissue lipid than normal rats. Since adipose tissue is highly re-
METABOLIC
CONTROL
153
OF ENZYMES
250
200
ae P sl 8 3 u
150
8 $
100
50 CONTROL
DIABETIC
DIABETIC WITH ,lNSULlN
Fig. 2.-Blood glucose concentration. Analysis was done on four normals and five animals where BD was added to the diet. The levels of significance were P < 0.01 (T = 3.98). No significant differences for diabetic (T = 2.34) and diabetic insulintreated rats (T = 0.27) where BD was added to the diet were noted. Blood glucose levels for diabetic were 264 i- 19 mg. per cent and for diabetic where BD was added to the diet were 218 + 6 mg. per cent.
sponsive to changes in the physiological condition of the animal, the lipids from adipose tissue are utilized as energy. It is probably of physiological importance that there was less loss of lipids from diabetic BD fed rats as compared with the animals without BD in their diet, since diabetic rats are dependent on fatty acids as their primary energy source. Results presented in Fig. 2 show the average blood glucose levels of normal, diabetic, and diabetic insulin-treated rats fed BD. The blood glucose levels were significantly lower in normal rats fed BD. All other changes in blood glucose levels resulting from BD ingestion were minimal. Phosphoenolpyruvate
Carboxykinase
and Malic Enzyme
Activities
The activities of enzymes are greatly influenced by both dietary and physiological conditionslz*l” and can therefore be used as indicators of metabolic
154
MEHLMAN,
TOBIN
jg
W It hod
m
With B.D.
AND JOHNSTON
B.0
30 CONTROL
DIABETIC
DIABETIC WITH INSULIN
Fig. 3.-Liver phosphoenolpyruvate carboxykinase activity. Analysis was done on four normals and five animals where BD was added to the diet. The level of significance were P < 0.001 (T = 7.43). No significant differences for diabetic (T = 0.23) and diabetic insulin-treated rats (T = 1.30) where BD was added to the diet were noted.
processes. Results in Fig. 3 show that there was a large increase in liver PEPcK activity in diabetic rats and that in the diabetic insulin-treated rats PEPcK activity was suppressed. The normal rats fed the diet containing BD had an increase of 43 per cent in PEPcK activity. There was no significant difference in PEPcK activity of diabetic rats or in the diabetic insulin-treated rats with and without BD. Malic enzyme activity in the adipose tissue was greatly decreased in diabetic rats and increased over normal level in diabetic insulin-treated rats (Fig. 4). No difference in the enzyme activity were observed in rats fed BD. The activity of malic enzyme in liver is shown in Fig. 5. There was a small decrease in malic enzyme activity in normal rats fed BD. In diabetic rats there was no difference between the BD fed rats and a 37 per cent decrease in diabetic insulin-treated rats fed BD. Malic enzyme activity was greatly suppressed in diabetic rats and was increased in diabetic insulin-treated rats to slightly below control values. In adipose tissue the malic enzyme activity was considerably higher than in liver.
METABOLIC
CONTROL
155
OF ENZYMES
q
Without
T T
T
CONTROL
DIABETIC
B.D
mWithB.D.
DIABETIC WITH INSULIN
Fig. 4 .-Adipose tissue malic enzyme activity. Analysis was done on four normals and five animals where BD was added to the diet. No significant difference for normal (T = 0.47), diabetic (T = O.?l), and/or diabetic insulin-treated rats where BD was added to the diet were noted,
Tissue Metabolite
Concentrations
The concentrations of hepatic metabolites in the pathway of gluconeogenesis in normal, diabetic, and diabetic insulin-treated rats fed BD are shown in Fig. 6. The pyruvate concentrations were decreased in controls (30%) and in diabetic and diabetic insulin-treated rats fed BD; however, all changes were minimal. The concentrations of 3 PGA were decreased in control, diabetic, and diabetic insulin-treated rats fed BD; however, these changes were not significant. The 2 PGA levels were increased in diabetic and diabetic insulintreated rats fed BD. The malate levels were significantly lower only in normal rats fed BD. The concentrations of lactate in liver of normal, diabetic, and diabetic insulin-treated rats fed BD are presented in Fig. 7. The liver lactate concentrations of rats fed BD were decreased in controls by 40 per cent. No other changes were significant. The lactate to pyruvate ratios in liver (Fig. 8) were increased in control ( 12% ) , diabetic (29% ) , and diabetic insulin-treated rats (55%) fed BD.
156
MEHLMAN,
TOBIN AND JOHNSTON
32
m
Without
5
With B.D.
B.D.
0
CONTROL
DIABETIC
DIABETIC WITH INSULIN
Fig. S-Liver malic enzyme activity. Analysis was done on four normals and five animals where BD was added to the diet. No significant differences for normal (T = 1.32), and diabetic (T = 0.75) were noted. The levels of significance for five diabetic insulin-treated and five diabetic insulin-treated where BD was added to the diet were P < 0.025 (T = 2.93).
Synthesis of Organic Mitochondria The synthesis control, diabetic
Acids
from
Pyruvate and Bicarbonate
by Rat Liver
of intermediates for gluconeogenesis by mitochondria from and diabetic-insulin treated rats fed BD are presented in
Table 3. The incorporation of radioactive bicarbonate into organic acids was increased in the presence of glutamate in controls (64% ), diabetic (64%), and diabetic insulin-treated rats (55 % ) . In animals fed BD, there was an increase in the presence of glutamate in l’COV fixation in controls (25 % ) , diabetic (42% ) , and diabetic insulin-fed rats (52% ) . The increase in 14COz fixation in the presence of glutamate can be accounted for by the incorporation of radioactive bicarbonate into aspartate. Similar increases in 14COz incorporation were observed in the presence of caprylate. The estimation of pyruvate carboxylase activity, based on the radioactive 14C02 fixed, represent minimal values because they are calculated on the basis of specific radioactivity of added bicarbonate and do not take into account the dilution of radioactive bicarbonate by metabolically produced nonradioactive carbon dioxide and the loss of l’COI: from intermediates formed in the tricarboxylic acid cycle during metabolism. A more
METABOLIC
CONTROL
157
OF ENZYMES
0.10
s >
0.40
5
0
k
% P
PYRUVATE
B O-30
030
w 3 L 5
a 020
020
e
a
1 z 2i
iz d z
2
5 0.10
0.10
0 7 7 2
0 CONTROL D)ABETIC DIABETIC
0 CONTROL
DIABETIC
WITH INSULIN
PEP
3 PGA
DlASETlC CONTROL WITH INSULIN
DIABETIC
DIABETIC WITH INSULIN
MALATE
Fig. 6.-Liver glucogenic metabolite concentrations. Analyses were performed on four normals, three diabetic, five diabetic insulin-treated and five normals, three diabetic, and five diabetic insulin-treated rats where BD was added to the diet. Levels of significance were for pyruvate in normal P < 0.005 (T = 4.52), in diabetic ns. (T = 1.24), and diabetic insulin-treated ns. (T = 1.39); for PEP in normal P < 0.05 (T = 2.61), in diabetic n.s. (T = 2.20); and diabetic insulin-treated n.s. (T = 0.72); for 2 PGA, in normal P < 0.001 (T = 10.24), in diabetic P < 0.025 (T = 4.39), and diabetic insulin-treated, P < 0.05 (T = 2.68); for 3 PGA in normal P < 0.005 (T = 5.01), in diabetic n.s. (T = 1.52) and diabetic insulin-treated P < 0.01 (T = 3.62); for malate in normal P < 0.05 (T = 2.47), in diabetic n.s. (T = 1.78), and in diabetic insulin-treated n.s. (T = 1.03) reliable estimation of pyruvate carboxylase activity (Fig. 9) was obtained by summing all of the measured metabolites shown in Table 3, which were synthe-
sized through pyruvate carboxylation. The pyruvate carboxylase activity was increased in normal (27% ), diabetic (44% ), and diabetic insulin-treated rats (18%) fed BD. Effect of BD on Glucose Production and Metabolite Concentration in Perfused Livers The rate of glucose production from lactate, and in the presence of added 20 mM BD are summarized in Fig. 10. Twenty-one mg. per cent glucose was produced per 30 minutes from lactate. When livers were perfused with 20 mM BD there was no glucose production. Addition of 20 mM lactate after 30 minutes of perfusion with BD resulted in 8 mg. per cent of glucose produced per 30 minutes. This represents a 62 per cent inhibition in the production of glucose by BD. Further addition of 10 mM fructose led to rapid glucose production, 74 mg. per cent/30 min. This indicates that perfusion of liver with BD does not alter glucose production above the triosephosphate step, and that liver is fully functional with respect to glucose production. Results in Figs. 11 and 12 show the effect of BD on liver gluconeogenic intermediates 20
158
MEHLMAN,
TOBIN AND JOHNSTON
3.4 3.2 3.0 2-B
q
W,thc,ut
i%
With B D
B D
2.6
Fig. 7.-Liver lactate concentrations. Analyses were performed on four normal, three diabetic, and five diabetic insulin-treated rats, and on five normal, three diabetic, and five insulintreated rats where BD was added to the diet. The levels of significance for normals were P < 0.005 (T = 5.43), for diabetic n.s. (T = 0.32) and for diabetic insulin-treated rats n.s. (T = 1.56). 00 06 0 .4 0
2 0 CONTROL
DIABETIC
DIABETIC WITH ,NS”L,N
LIVER
minutes after the addition of substrate (lactate). There was a 60 per cent increase in the liver lactate concentrations (Fig. 12) in the presence of BD and a 73 per cent decrease in pyruvate levels (Fig. 11). The ratio of lactate to pyruvate in liver perfused with BD increased from 13.7 to 8 1.O (Fig. 12). The concentrations of aspartate and malate were greatly increased in the BDperfused liver (Fig. 11). A very large decrease in PEP. 2 PGA was observed in livers perfused with BD. DISCUSSION
Feeding a BD-containing diet to alloxan diabetic rats resulted in an improvement in survival and in general appearance (Fig. 13). In previous studies it has been demonstrated that the addition of BD to diets leads to modifications of tissue lipid content and gluconeogenesis.llJ” The regulation of metabolic pathways is markedly affected by substrates, metabolites, cofactors, concentrations, and distributions of enzymes between compartments. Since enzyme activities and metabolite concentration patterns are exquisitely responsive to both metabolic and dietary state of the animals 313,28,31it may be possible by studying the activities of these enzymes
METABOLIC
CONTROL
m
32
159
OF ENZYMES
With B.D.
Fig. I.-Liver lactateto-pyruvate ratio. Analysis was performed on four normal, three diabetic, and five diabetic insulin-treated rats and on five normal, three diabetic, and five diabetic insulin-treated rats where BD was added to the diet. The levels of significance for normal were n.s. (T = 0.54); for diabetic, P < 0.05 (T = 2.78); and for diabetic insulin-treated, P < 0.01 (T = 3.55).
IT5 5
26
f w 24 l3 Is 20 t zi t- ‘6 : =j 12 k 0
-
6-
5 4 -
CONTROL
DIABETIC
DIABETIC WITH INSULIN
and metabolite patterns to elucidate the mechanism by which BD exerts its effect on gluconeogenesis. The enzymatic activity of PEPcK and malic enzyme are highly responsive to changes in dietary carbohydrate and lipid, as well as to hormones and diabetes,13 and can therefore be utilized as important indicators of the metabolic state of animals. In the adipose tissue (Fig. 4), malic enzyme activity was considerably higher than in liver. Similar patterns in enzyme activity were observed in adipose tissue of diabetic and diabetic insulin-treated rats. In the insulin-treated diabetic rats malic enzyme activity was greater than in adipose tissue of normal rats. The response of enzyme activities in adipose tissue further confirms previous reports32 that adipose tissue is highly responsive to metabolic and hormonal alterations, as is evident from Fig. 1, where adipose tissue weight of these animals is presented. The decrease in adipose tissue weight in diabetic rats is consistent with the requirement for utilization of additional lipid for energy. The smaller decrease in the adipose tissue weight of diabetic BD-fed rats may be due to a reduced requirement of lipid for energy, because this requirement may be met by utilization of dietary BD. The changes in liver tissue content of gluconeogenic metabolites are shown in Fig. 6. In animals fed the diet containing BD the ratio of lactate to pyruvate was increased (Fig. 7). The increase in the lactate to pyruvate ratio represents an increase in the cytoplasmic NADH/NAD+ ratio.“3 There were minimal changes in PEP and 2 PGA levels; however, 3 PGA levels were lower in animals fed BD. The decrease in the 3 PGA level is consistent with the findings that there was an increase in the lactate to pyruvate ratios in BD-fed animals. Pyruvate plays a key role in carbohydrate metabolism, because it can be
160
MEHLMAN,
TOBIN AND JOHNSON
Table 3.-Formation of Tricarboxylic Cycle Acids from Pyruvate, Bicarbonate, Glutamate and Caprylate by Rat Liver Mitochondria From Alioxan and Alloxan Diabetic, Insulin-treated Rats fed 1,3 Butanediol Diet*
_.__~__.
~.
Metabolite Dietary Treatment
30% fat
Condition Addition
of Animals to System
Changes
Pyruvate used
pmoles1minJmg.N
mMolar Control none 6.6 glutamate 0.66 caprylate
0.95 1.16 0.96
0.36 0.59 0.60
0.23 0.19 0.29
0.15 0.28 0.33
0.37 -
Diobeiic none 6.6 glutamate 0.66 caprylate
0.63 0.85 0.75
0.25 0.41 0.40
0.11 0.10 0.16
0.16 0.29 0.25
0.22 -
6.6 glutamate 0.66 caprylate
0.79 0.88 0.83
0.33 0.5 I 0.45
0.18 0.13 0.20
0.19 0.28 0.25
0.27
Control none 6.6 glutamate
0.90 0.92
0.40 0.50
0.21 0.15
0.27 0.33
0.25
Diabetic none 6.6 glutamate 0.66 caprylate
0.80 0.85 0.77
0.31 0.44 0.41
0.17 0.12 0.19
0.22 0.34 0.27
0.24 -
0.85 0.95 0.83
0.31 0.47 0.44
0.17 0.13 0.19
0.23 0.35 0.32
0.26 -
Diabetic with lnsulir~ none
30% fat, 18.3%
I,3 butanediol
-
Diabetic with ltuulin none
6.6 glutamate 0.66 caprylate
.-
* Keactlon mixture for all experiments contained 4 mM ATP, 10 mM MgSO,, 6.6 potassium phosphate, and 6.6 mM triethanolamine buffers. pH 7.4, 6.6 mM pyruvate 13.3 mM KHlWO,, and 5 mg. of fatty acid free albumin in each incubation flask, 6.6 L-glutamate and 0.66 mM caprylate were added as indicated. Mitochondria from 0.5 liver suspended in 0.5 ml. isotonic sucrose were added to each incubation. Incubation was 10 min. at 37’ C for all experiments.
mM and mM Gm. time
carboxylated and used for glucose synthesis or oxidized and used for energy production. The mitochondrial significance of pyruvate metabolism in both kidney and liver for providing precursors of gluconeogenesis has been studied in great detail.‘@33J-40 The changes in the pyruvate carboxylase activity measured in intact mitochondria under these conditions may not represent true activity (Fig. 9) because it is possible to stimulate pyruvate carboxylase activity in mitochondria by altering concentrations and types of substrates, as shown in Table 4. In the presence of 16.3 mM glutamate the 14C0.,_ incorporation in the presence of added glutamate can be accounted for primarily by increased aspartate synthe-
METABOLIC
CONTROL
OF ENZYMES
161
Fig. 9.-Liver mitochondrial pyruvate carboxylase activity.
CONTROL
OIABETIC
DIABETIC WITH INSULIN
sis.2Z Addition of 13.2 mM a-ketoglutarate resulted in 156 percent increase in 14COZ incorporation. This increase in ‘“CO2 incorporation is due not to an increase in pyruvate carboxylation but to a dilution of the radioactive oxalacetate formed from pyruvate carboxylation with the large pool of unlabeled malate formed from a-ketoglutarate. When both a-ketoglutarate and glutamate were used together, a 210 percent increase in 14C02 incorporation was observed. These were still not maximal conditions because cu-ketoglutarate and glutamate are competitive, as evidenced by the decreased levels of aspartate found; also, oxidation of a-ketoglutarate may generate NADH, which can decrease the conversion of malate to oxalacetate and hence to aspartate. Waglet” using broken mitochondria, reported a significant increase in pyruvate carboxylase activity in diabetic rats and a decrease in diabetic insulin-treated rats. However, it must be pointed out that in this study malic enzyme activity and PEPcK activity responded in the expected manner and magnitude in diabetic and diabetic insulin-treated rats. The mitochondrial pyruvate metabolism (Table 3) shows that mitochondria under all experimental conditions examined responded almost identically both in the presence of fatty acids and glutamate. Addition of 20 mM BD to perfused livers resulted in a strong inhibition of glucose production. The rate of glucose synthesis from lactate was decreased by 62 per cent (Fig. 10) in the presence of BD. Addition of 10 mM fructose led to a rapid rate of glucose synthesis, demonstrating that glucose production was not inhibited at the steps above GAP and DHAP. The metabolite concentrations in liver (Fig. 11) perfused with lactate, in the presence of BD, showed a striking change in the levels of gluconeogenic inter-
162
MEHLMAN,
TOBIN
AND JOHNSON
80
60
40
20
LACTATE (20mMOLES) 1 +A8*
._.Hi t
40 20
/
I
FRUCTOSE llOmMOLES)
1,3 BUTANE0 IOL (20mMOLESl
/
0 20
40 PERFUSION
Fig. lO.-Synthesis
PY RUVATE
60
80
TIME,
MINUTES
100
120
of blood glucose from lactate in perfused liver.
ASPARTATE
Fig. 11 .-Metabolite
MALATE
concentrations
PEP
2 PGA
from perfused livers.
3 PGA
METABOLIC
CONTROL
163
OF ENZYMES
Without B.O. With B.D.
a
0 LACTATE
Fig. 12.-Concentration with BD.
RATIO LACTATEf PYRUVATE
of lactate and ratio of lactate to pyruvate in liver perfused
The pyruvate levels were greatly decreased and the levels of malate and aspartate were increased. The 3 carbon phosphorylated intermediates were greatly decreased. These results suggested that BD inhibits gluconeogenesis in perfused liver by blocking the conversion of oxalacetate to phosphoenolpyruvate. The large increase in the lactate to pyruvate ratio from 13.7 to 81 is consistent with an inhibition of gluconeogenesis at this step. The pathway for the catabolism of BD has not been elucidated; however, the following observations have been made: The metabolism of BD in the cytosol leads to an increased production of NADH, as is evident from the increase in the ratio of lactate to pyruvate in perfused livers (Fig. 10). BD is not metabolized by isolated liver or kidney mitochondria. Liver high-speed supernatant fractions rapidly reduce NAD+ to NADH in the presence of BD. Intact animals rapidly oxidize BD-14C to 14C02. Administration of BD orally for two weeks (Table 5) or one hour (Table 6) resulted in a large increase in blood ketone bodies and in the p-hydroxybutyrate to acetoacetate ratio. On the basis of these observations, a pathway for the metabolism of BD is suggested in Fig. 13. The rapid utilization and metabolism by mammalian cells of short-chain carbohydrates can be achieved in the absence of insulin. Studies of this nature offer interesting model systems for the elucidation of metabolic disturbances, mediates.
164
MEHLMAN,
TOBIN AND JOHNSON
Fig. 13.-Alloxan diabetic rats fed dietary BD. Larger animal (left) received 18 per cent BD as replacement of carbohydrate.
Gig - CH - CH2- CH 20H I OH k CH3-CH-CH2CHO
NAD + NADH+H+ +H20
OH
NAD + NADH +H+ ‘b CH3 - CH - CH 2COOH OH Fig.
14sProposed butanediol.
scheme
for metabolism
of 1,3 ‘k
NAD + NADH + H +
CH3 - C - CH 2COOH II 0 CoA ATP CH3 - C - CH2COSCoA II 0 CoA 2CH3-C-SCoA II 0 I Cop + Hz0
METABOLIC
CONTROL
165
OF ENZYMES
Table 4.-!U1uulation of 14C02 Incorporation into Organic Acids in the Presence of CL-Ketoglutarate and Glutarate by Rat Kidney Mitochondria*
mM None 6.6 cu-ketoglutarate 16.3 glutamate 6.6 cu-ketoglutarate and 16.6 glutamate
5.7 4.5 5.3
1.1 6.5 1.5
4.2
6.1
pmoles/mg.N/lO 0.7 1.4 0.8
min.
1.3
1.42
1.0 2.6 1.6
0.38
3.1
* The reaction mixture contained 4 mM ATP, 10 mM magnesium sulfate, 6.6-mM potassium phosphate, and 6.6-mM potassium triethanolamine buffers pH 7.4, 20 mM KHrJCO, and 6.6 mM potassium pyruvate. Final volume was 3 ml., containing a total of 1.2 to 1.7 ml. of isotonic sucrose. Mitochondria from 0.5 Gm. of kidney suspended in 0.5 ml. of isotonic sucrose were added to each incubation and contained 1.0 mg. of nitrogen. (From Mehlman, M. A.: J. Biol. Chem. 243:3289, 1968.) Table S-Effect
of Addition of 20% 1,3 Butanediol of Rats for 14 Days*
to Drinking Water
Experimental Parameters
Examined
Blood glucose mg. per cent Acetoacetate ~moles/mi. /3-Hydroxybutyrate floles/ml. Total ketone bodies cmoles/ml. Ratio. /SHydroxybutyrate Acetoacetate Pyruvate ~moles/ml.
Without
BD (5)
109 0.026 0.094 0.130
+- 4 t 0.003 ? 0.015 ? 0.025
2.85
-c 0.60
0.166 c 0.026
* The animals were allowed to drink water containing killed 1 hr. after they drank water with BD. Table &-Effect
With
102 0.170 0.951 1.121
r + 2 +
6.12
rt 0.45
BD (5)
9 0.011 0.113 0.107
n.s. p < 0.001 p < 0.001 p < 0.001 p < 0.01
0.136 ? 0.027 BD for 2 hr./day.
ns. Animals
were
of 1,3 ButanedioI BD on BIood Ketone Bodies and Blood GIucose Levels Experimental
Parameters
Examined
Blood glucose mg. per cent Acetoacetate, &moles/ml. ,&hydroxybutyrate eoles/ml. Total ketone bodies Ratio /3-hydroxybutyrate acetoacetate
Corm&
yj;hout
112 0.024 0.063 0.087
c 6 +- 0.014 2 0.019 +- 0.032
109 0.052 0.359 0.411
I!I 8 + 0.009 & 0.017 f 0.029
N.S. p < 0.05 p < 0.001 .D <- 0.001
3.07
& 1.31
7.48
? 2.41
p < 0.05
* 3 ml. of 20% BD solution were given orally through killed 1 hr. after administration of BD.
ExperBirn~n;;:
stomach
with
tube and animals were
such as diabetes, under conditions of insulin lack. These compounds can provide a rapidly available energy source for the organisms. Further studies are now in progress concerning enzymes and intermediates involved in the pathway of BD metabolism.
166
MEHLMAN,
TOBIN AND JOHNSON
ACKNOWLEDGMENTS The authors thank Dr. H. A. Lardy for support and help with all aspects of this work, Mr. R. Hanson for perfusion experiments, Mr. Carl Mackerer and Mr. Henry Hahn for help with experiments, and Mr. Lawrence Stem and Mrs. Lucy Riddo for preparing the illustrations.
REFERENCES 1. Friedmann, B., Goodman, E. H., Jr., and Weinhouse, S.: Effects of insulin and fatty acids on gluconeogenesis in the rat. J. Biol. Chem. 242:3620, 1967. 2. Williamson, J. R., Browning, E. T., and Scholz, R.: Control mechanism of gluconeogenesis and ketogenesis. I. Effect of oleate on gluconeogenesis in perfused rat liver. J. Biol. Chem. 244~4607, 1969. 3. Williamson, J. R., Browning, E. T., Scholz, R., Kreisberg, R. A., and Fritz, I. B.: Inhibition of fatty acid stimulation of gluconeogenesis by ( + )-decanoylcarnitine in perfused rat liver. Diabetes 17:194, 1968. 4. Teufel, H., Menahan, L. A., Shipp, J. C., Boning, S., and Wieland, 0.: Effect of oIeic acid on the oxidation and gluconeogenesis from pyruvate-l-r% in the perfused rat liver. Europ. J. Biochem. 2: 182, 1967. 5. Williamson, .I. R., Kreisberg, R. A. and Felts, P. W.: Mechanism for stimulation of gluconeogenesis by fatty acids in the perfused rat liver. Proc. Nat. Acad. Sci. U.S.A. 56: 247, 1966. 6. Herrera, M. G., Kamm, D., Ruderman, N., and Cahill, G. F., Jr.: Non-hormonal factors in the corm01 of gluconeogenesis. Advances Enzym. Regulat. 4:225, 1966. 7. Struck, E., Ashmore, J., and Wieland, 0.: Kurze Mitteilung Stimulierung der Gluconeogenese durch Langkerrige Fettsauren und Glucagon. Biochem. Z. 343: 107, 1965. 8. Krebs, H. A., Hems, R., and Gascoyne, T.: Renal gluconeogenesis. IV. Gluconeogenesis from substrate combinations. Acta Biol. Med. German. 11:607, 1963. 9. Krebs, H. A., Speake, R. N., and Hems, R.: Acceleration of renal gluconeogenesis by ketone bodies and fatty acids. Biochem. J. 94:712, 1965. 10. Stoewsand, G. S., Dymsza, H. A., Mehlman, M. A., and Therriault, D. G.: Influence of 1,3 butanediol on tissue lipids of cold exposed rats. J. Nutr. 83:464, 1966. 11. Mehlman, M. A., Therriault, D. G., Porter, W., Stoewsand, G. S., and Dymsza,
H. A.: Distribution of lipids in rats fed 1,3 butanediol. J. Nutr. 80:215, 1966. 12. Stoewsand, G. S., Dymsza, H. A.. Swift, S. M., Mehlman, M. A., and Therriault, D. G.: Effect of feeding polyhydric alcohols on tissue lipids and the resistance of rats to extreme cold. .I. Nutr. 89:414, 1966. 13. Young, J. W., Shrago, E., and Lardy, H. A.: Metabolic control of enzymes involved in lipogenesis and gluconeogenesis. Biochem. 3 : 1687, 1964. 14. Ochoa, S.: Crystalline condensing enzyme from pig heart. In Colowick, S. P., and Kaplan, N. 0. (Eds.): Methods in Enzymology. I. New York, Academic, 1955, p. 685. 15. Ochoa, S.: A “malic” enzyme from pigeon liver and wheat germ. In Colowick. S. P., and Kaplan, N. 0. (Eds.) : Methods in Enzymology. I. New York, Academic. 1955, p. 739. 16. Ray, P. D., Foster, D. O., and Lardy, H. A.: Paths of carbon in gluconeogenesis and lipogenesis. N. Inhibition by L-tryptophan of hepatic gluconeogenesis at the level of phosphoenolpyruvate formation. J. Biol. Chem. 241:3904, 1966. 17. Bergmeyer, H. V. (Ed.): Methods of Enzymatic Analysis. New York, Academic, 1963. 18. Schneiderer, W. C.: Intracellular distribution of enzymes. III. The oxidation of octanoic acid by rat liver fractions. J. Biol. Chem. 176:289, 1948. 19. Johnson, D. and Lardy, H. A. Isolation of liver of kidney mitochondria. In Estabrook, R. W.. and Pullman, M. E., (Eds.): Methods in Enzymology, Vol. 10. New York, Academic, 1967, p. 94. 20. Walter, P., Petkaw, V., and Lardy, H. A.: Paths of carbon in gluconeogenesis and lipogenesis. III, The role and regulation of mitochondrial processes involved in supplying precursors of phosphoenolpyruvate. J. Biol. Chem. 241:2523, 1966. 21. Mehlman, M. A., Walter, P. and Lardy, H. A.: Paths of carbon in glu-
METABOLIC
CONTROL
OF ENZYMES
coneogenesis and lipogenesis. VII. The synthesis of precursors for gluconeogenesis from pyruvate and bicarbonate by rat kidney mitochondria. J. Biol. Chem. 242:4594, 1967. 22. Mehlman, M. A.: The fate of isotopic carbon in kidney mitochondria synthesizing precursors for glucose from pyruvate and bicarbonate. J. Biol. Chem. 243:3289, 1968. 23. Somberg, E. W., and Mehlman, M. Regulation of gluconeogenesis and A.: lipogenesis: The regulation of mitochondrial pyruvate metabolism in guinea-pig liver synthesizing precursors for gluconeogenesis. Biochem. J. 112:435, 1969. 24. Gorwall, A. G., Bardawill, C. J., and Daub, M. M.: Determination of serum protein by means of the biuret reaction. J. Biol. Chem. 117:751, 1949. 25. Layne, E.: Spectrophotometric and turbidimetric methods for measuring proteins. 111 Methods in Enzymology, Vol. 3. New York, Academic, 1957, p. 447. 26. Veneziale, C. M., Walter, P., Kneer, N. and Lardy, H. A.: Influence of L-tryptophan and its metabolites on gluconeogenesis in the isolated perfused liver. Biochemistry 6:2129, 1967. 27. Mehlman, M. A., Tobin, R. B. and Johnston, J. B.: Influence of dietary 1,3 butanediol on metabolites and enzymes involved in gluconeogenesis and lipogenesis. J. Nun. 100:1341, 1970. 28. Vaughan, D. A. and Winders, R. L.: Effects of diet on HMP dehydrogenases and malic (.TPN) dehydrogenase in the rat. Amer. J. Physiol. 206:1081, 1964. 29. Weber, G.: Behavior and regulation of enzymes synthesis in liver and in hepatomas of different growth rates. Advances Enzym. Regulat., 1:321, 1963. 30. Williamson, J. R., Scholz, R. and Browning, E. T.: II. Interactions between fatty acids oxidation and the citric acid cycle in perfused rat liver. J. Biol. Chem. 244: 4617, 1969. 31. Rognstad, R. and Katz, J.: Gluconeogenesis in the kidney cortex. Effects of Dmalate and amino-oxyacetate. Biochem. J. 116:483, 1970. 32. Therriault, D. G. and Mehlman, M.
167 A.: Effect of prior diet on lipid mobilization in rats during starvation or cold exposure. J. Nutr. 98:25, 1969. 33. Williamson, D. H., Lund, P., and Krebs, H. A.: The redox state of free nicotinamide adenine dinucleotide in the cytoof rat liver. and mitochondria plasm Biochem. J. 103:514, 1967. 34. Henning, H. V., Stumpf, B., Ohly, B., and Seubert, W.: On the mechanism of gluconeogenesis and its regulation. III. The gluconeogenic capacity and the activities of pyruvate carboxylase and PEP carboxylase of rat kidney and rat liver after cortisol treatment and starvation. Biochem. Z. 344:274, 1966. 35. Wagle, S. R., and Ashmore, J.: Studies on carbon dioxide fixation in normal and alloxan-diabetic animals. Biochim. Biophys. Acta 74~564, 1963. 36. Haynes, R. C., Jr.: The fixation of carbon dioxide by rat liver mitochondria and its relation to gluconeogenesis. J. Biol. Chem. 240:4103, 1965. 37. Walter, P., and Strucki, J. W.: Regulation of pyruvate carboxylase in rat liver mitochondria by adenine nucleotides and short chain fatty acids. Europ. J. Biochem. 12:508, 1970. 38. Lardy, H. A., Petkau, V., and Walter, P.: Paths of carbon in gluconeogenesis and lipogenesis: The role of mitochondria in supplying precursors of phosphoenolpyruvate. Proc. Nat. Acad. Sci. U.S.A. 53:1410, 1965. 39. Mehlman, M. A., and Walter, P.: Regulation of gluconeogenesis and lipogenesis inhibition of pyruvate carboxylation in rat kidney mitochondria by malonate and malonyl CoA. Arch. Biochem. Biophys. 127: 590, 1968. 40. Mehlman, M. A.: Inhibition of pyruvate carboxylation by fluorocitrate in rat kidney mitochondria. J. Biol. Chem. 243: 1919, 1968. 41. Haslam, R. J., and Krebs, H. A.: Substrate competition in the respiration of animal tissues: The metabolic interactions of pyruvate and a-oxoglutarate in rat liver homogenates. Biochem. J. 86:432, 1963.