THE JOURNAL cm B~LOGICAL CHEMISTRY Vol. 247, No. 6, Issue of March 25, pp. 178Gl800, 1972 Printed in U.S.A. Metabolism of Free Fatty Acids in Isolated Liver Cells FACTORS AFFECTING THE PARTITION BETWEEN ESTERIFICATION AND OXIDATION* (Received for publication, August 30, 1971) JOSEPH A. ONTKO~ From the Oklahoma Medical Research Foundation and the Department of Biochemistry, University of Oklahoma School of Medicine, Oklahoma City, Oklahoma 73104 SUMMARY The structural and metabolic integrity of isolated rat liver cells was verified by the high percentage of trypan blue ex- clusion, a low degree of lactate dehydrogenase release into the medium, a constant rate of gluconeogenesis from L( +)- lactate, and increased oxygen consumption following the addition of 2,4-dinitrophenol. Pahnitic acid, incubated in an albumin-bound form with isolated liver cells, was esterified to form phospholipids, tri- glycerides, diglycerides, and cholesterol esters and was oxidized to CO2 and ketone bodies. In liver cells from fed rats, the major portion of pahnitate was esterified, an inter- mediate quantity was oxidized to ketone bodies, and a smaller amount was oxidized to COz. The partition of pahnitic acid between esterification and ketogenesis was inversed by fast- ing, whereas oxidation to CO2 and the total rate of pahnitate utilization were unaltered. Greater esterification of [‘“Cl- palmitate in cells from fed rats was not a result of carbon recycling via chain elongation or de novo synthesis. Liver cells from fasted rats derived more energy from fatty acid oxidation than cells from fed rats. The results indicate that citric acid cycle flux and endogenous lipolysis were un- affected by fasting. These observations signify that altered partition of free fatty acids between the pathways of oxida- tion and esterification in the liver is a major causative factor in the increased ketogenesis in the fasting state. An increase in [1-14C]pahnitate concentration augmented pahnitate uptake, ketogenesis, and esterification, whereas W02 production was only slightly affected. The estimated citric acid cycle flux and the acetoacetate to P-hydroxybuty- rate ratio were diminished. Increased ketogenesis in re- sponse to sequential elevation of the pahnitate concentration could not be accounted for by diminished citric acid cycle flux and therefore resulted from increased /3 oxidation. Ke- tone body specific activity approached a constant value at ij of 3 to 4. Results indicate intracellular mixing of free fatty acids derived from endogenous lipolysis and from the medium, prior to /3 oxidation. Phospholipid was the pre- dominant esterification product at low concentrations of * This investigation was supported by United States Public Health Service Research Grants HE-12806 and HE-13302. $ Recipient of United States Public Health Service Research Career Develonment Award I-KE-HE-32.143 from the National Heart Institute. added palmitate, but, as phospholipid formation approached saturation, a sigmoid increase in diglyceride and triglyceride formation occurred. Fructose and glycerol each decreased ketogenesis from added pahnitate. Fructose, glycerol, and, to a lesser extent, glucose increased pahnitate esterification in liver cells iso- lated from fasting rats. This effect was characterized by increased conversion of added pahnitate to diglycerides, triglycerides, and phospholipids and decreased conversion to cholesterol esters. These substrates did not alter the rate of fatty acid uptake. Fructose, glycerol, and, to a lesser extent, glucose elevated 14C02 production from [I-14C]pal- mitate. At higher fructose concentrations the elevated 14C02 production was reversed. Results suggest that substrates which enter glycolysis beyond fructose 1,6-diphosphate decrease ketogenesis by competition with fatty acid oxidation and enhance esterifica- tion by the resulting increased availability of long chain free fatty acids and by a separate preferential stimulation of glycerolipid formation. Results indicate that decreased availability of non-fatty acid substrates and, thereby, de- creased competitive oxidation of these substrates is a par- ticipating causative factor in the increased oxidation, and the decreased esterification, of long chain fatty acids in the liver in the fasting state. Free fatty acids in the plasma are rapidly removed from the circulation (1, 2), and the liver plays a dominant role in this process (3, 4). Long chain free fatty acids entering liver cells undergo metabolism via two major pathways (a) oxidation to carbon dioxide and ketone bodies and (b) esterification to form triglycerides, phospholipids, and other fatty acid esters. Little is known about the mechanisms which regulate the par- tition of long chain fatty acids between the pathways of oxida- tion and esterification. Likewise, the factors which control the partition of acetyl CoA, derived from hepatic fatty acid oxida- tion, between the citric acid cycle and ketogenesis are uncertain. In the present study factors affecting the intracellular disposi- tion of free fatty acids were investigated in isolated liver cells. These cells, now obtainable with intact plasma membranes in high yield from the liver (5), possess the metabolic activities of 1788 by guest on March 15, 2020 http://www.jbc.org/ Downloaded from
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THE JOURNAL cm B~LOGICAL CHEMISTRY Vol. 247, No. 6, Issue of March 25, pp. 178Gl800, 1972
Printed in U.S.A.
Metabolism of Free Fatty Acids in Isolated Liver Cells
FACTORS AFFECTING THE PARTITION BETWEEN ESTERIFICATION AND OXIDATION*
(Received for publication, August 30, 1971)
JOSEPH A. ONTKO~
From the Oklahoma Medical Research Foundation and the Department of Biochemistry, University of Oklahoma School of Medicine, Oklahoma City, Oklahoma 73104
SUMMARY
The structural and metabolic integrity of isolated rat liver cells was verified by the high percentage of trypan blue ex- clusion, a low degree of lactate dehydrogenase release into the medium, a constant rate of gluconeogenesis from L( +)-
lactate, and increased oxygen consumption following the addition of 2,4-dinitrophenol.
Pahnitic acid, incubated in an albumin-bound form with isolated liver cells, was esterified to form phospholipids, tri- glycerides, diglycerides, and cholesterol esters and was oxidized to CO2 and ketone bodies. In liver cells from fed rats, the major portion of pahnitate was esterified, an inter- mediate quantity was oxidized to ketone bodies, and a smaller amount was oxidized to COz. The partition of pahnitic acid between esterification and ketogenesis was inversed by fast- ing, whereas oxidation to CO2 and the total rate of pahnitate utilization were unaltered. Greater esterification of [‘“Cl- palmitate in cells from fed rats was not a result of carbon recycling via chain elongation or de novo synthesis. Liver cells from fasted rats derived more energy from fatty acid oxidation than cells from fed rats. The results indicate that citric acid cycle flux and endogenous lipolysis were un- affected by fasting. These observations signify that altered partition of free fatty acids between the pathways of oxida- tion and esterification in the liver is a major causative factor in the increased ketogenesis in the fasting state.
An increase in [1-14C]pahnitate concentration augmented pahnitate uptake, ketogenesis, and esterification, whereas W02 production was only slightly affected. The estimated citric acid cycle flux and the acetoacetate to P-hydroxybuty- rate ratio were diminished. Increased ketogenesis in re- sponse to sequential elevation of the pahnitate concentration could not be accounted for by diminished citric acid cycle flux and therefore resulted from increased /3 oxidation. Ke- tone body specific activity approached a constant value at ij of 3 to 4. Results indicate intracellular mixing of free fatty acids derived from endogenous lipolysis and from the medium, prior to /3 oxidation. Phospholipid was the pre- dominant esterification product at low concentrations of
* This investigation was supported by United States Public Health Service Research Grants HE-12806 and HE-13302.
$ Recipient of United States Public Health Service Research Career Develonment Award I-KE-HE-32.143 from the National Heart Institute.
added palmitate, but, as phospholipid formation approached saturation, a sigmoid increase in diglyceride and triglyceride formation occurred.
Fructose and glycerol each decreased ketogenesis from added pahnitate. Fructose, glycerol, and, to a lesser extent, glucose increased pahnitate esterification in liver cells iso- lated from fasting rats. This effect was characterized by increased conversion of added pahnitate to diglycerides, triglycerides, and phospholipids and decreased conversion to cholesterol esters. These substrates did not alter the rate of fatty acid uptake. Fructose, glycerol, and, to a lesser extent, glucose elevated 14C02 production from [I-14C]pal- mitate. At higher fructose concentrations the elevated 14C02 production was reversed.
Results suggest that substrates which enter glycolysis beyond fructose 1,6-diphosphate decrease ketogenesis by competition with fatty acid oxidation and enhance esterifica- tion by the resulting increased availability of long chain free fatty acids and by a separate preferential stimulation of glycerolipid formation. Results indicate that decreased availability of non-fatty acid substrates and, thereby, de- creased competitive oxidation of these substrates is a par- ticipating causative factor in the increased oxidation, and the decreased esterification, of long chain fatty acids in the liver in the fasting state.
Free fatty acids in the plasma are rapidly removed from the circulation (1, 2), and the liver plays a dominant role in this process (3, 4). Long chain free fatty acids entering liver cells undergo metabolism via two major pathways (a) oxidation to carbon dioxide and ketone bodies and (b) esterification to form triglycerides, phospholipids, and other fatty acid esters.
Little is known about the mechanisms which regulate the par- tition of long chain fatty acids between the pathways of oxida- tion and esterification. Likewise, the factors which control the partition of acetyl CoA, derived from hepatic fatty acid oxida- tion, between the citric acid cycle and ketogenesis are uncertain.
In the present study factors affecting the intracellular disposi- tion of free fatty acids were investigated in isolated liver cells. These cells, now obtainable with intact plasma membranes in high yield from the liver (5), possess the metabolic activities of
the intact tissue and hare therefore provided a very useful sys- In some experiments the same pair of flasks was used for both tem to study the regulation of hepatic fatty acid metabolism. lipid and ketone hod\- analyses.
EXPERIMENTAL PROCEDURES
Aninzals-Male Holtzman rats (200 t,o 300 g) were given water and Rockland rat diet ad l&turn. Livers were removed between 9 and 11 a.m. for cell isolation. In esperiments with fasting rats food was removed between 9 and 11 a.m., and the livers were removed 24 hours later. The anesthetic used was Nembutal.
Liver Cell Isolation Procedure-Liver cells were isolated using the procedure of Berry and Friend (5). The perfusion medium was calcium-free Hanks solution (6). When cells from fed rats were isolated, the perfusion medium contained 1 mg per ml of glucose, whereas the glucose was omitted when cells from fasting rats were isolated. Fifty milligrams of collagenase (Worthing- ton, type CLS) and 100 mg of hyaluronidase (Sigma, type I) were added to a recirculating perfusion volume of 60 to 70 ml. The additional period of perfusion with EDTA (5) was not found necessary and therefore was omitted. After the perfusion and subsequent incubation with shaking at 80 to 90 oscillations per min at 37” for 12 mm, the suspension was poured through lOO- mesh silk sieve cloth (E. H. Sargent) int,o cold centrifuge tubes in an ice bath. Twenty milliliters of cold perfusion medium (previously gassed with 957; O2 and 5%) CO2 but without added enzymes) was then poured on the liver residue on the sieve cloth to wash isolated cells through the cloth. Further manipulation of the liver fragments on the cloth to dislodge isolated cells was avoided since such treatment was observed to cause cell damage. The cells were then centrifuged at 35 x g for 2 min at O-4”. The supernatant was removed and the cells were suspended in cold perfusion medium (previously gassed with 957; 02 and 5% COZ but without added enzymes). after centrifugation at 35 x
g for 2 min, the supernatant was removed and the cells were gently dispersed in cold suspension medium. The suspension medium was Hanks solution without calcium, bicarbonate, or glucose and contained 10 rnM sodium phosphate buffer, pH 7.4. The cells were then recentrifuged at 35 x g for 2 min. After removing the supernatant, the cells were again gently dispersed in cold suspension medium. Cell suspensions were always ex- amined microscopically for trypan blue exclusion. A high per- centage (85 to 957(,) of unstained cells was routinely observed. The dry weight of each suspension was also determined by dry- ing to a constant weight over PZOS in a vacuum desiccator.
Incubation-The isolated liver cells were incubated at 37” in a Dubnoff shaker at 80 to 90 oscillations per min in 25.ml Erlen- meyer flasks with center wells. Each center well contained 0.2 ml of 10yc sodium hydroxide and a folded filter paper. Unless otherwise indicated each flask contained 1 ml of isolated liver cells and 1 ml of 3% albumin-O.75 mM [l-r4C]palmitic acid dis- solved in the suspension medium described above. Crystalline bovine serum albumin (Pentex) was used after treatment with Darco M according to the procedure of Chen (7) to remove free fatty acids. When the concentration of palmitate (Figs. 4 to 6) or albumin (Fig. 7) was varied, the albumin-palmitate and the fatty acid-free albumin solutions were always dissolved in sus- pension medium. All incubations were in duplicate. Two flasks were incubated to determine 14C02 and [curbozyl-14C]ace- toacetate, two flasks for the lipid analyses and two other flasks for the measurement of ketone bodies and other metabolites.
Carbon Dioxide and Acetoacetate Radioactivity--TO, and [carboxyl-**C]acetoacetate were measured in duplicate by trap ping COZ in the center well as previously described (8) except that the period of incubation after aniline citrate addition was 120 min. Folded papers from the center wells were added to count- ing vials containing 1.0 ml of water. Twenty milliliters of the Cab-0-Sil-containing liquid scintillation mixture previously de- scribed (9) was then added. The acetoacetate carboxyl radio- activity was multiplied by 2 to obtain total acetoacetate radio- activity since the carbonyl and carboxyl carbons are equally labeled under these conditions (10). Since acetoacetate and P-hydroxybutyrate are in isotopic equilibrium, P-hydrosybutyr- ate radioactivity was calculated from quantities of acetoacetate and P-hydroxybutyrate found to be present by spectrophoto- metric analysis as described below.
Analyses-At the end of incubation, the isolated liver cells were immediately mixed with 1 volume of ice-cold 30% perchloric acid and after centrifugation the supernatant was adjusted to pH 7 to 8 by adding 0.67 volumes of 20%, KOH followed by small increments of saturated KHCOB. After centrifugation the supernatants were analyzed for acetoacet,ate (11)) P-hydroxy- butyrate (la), and, in certain experiments, for lactate (13) and glucose (14).
Lipid Radioactivity-M, the end of incubation 1 volume of the incubation mixture was added to 20 volumes of chloroform- methanol (2: 1). These extracts were always prepared in dupli- cate. After standing at room temperature for at least 2 hours, the extracts were filtered and then washed according to the pro- cedure of Folch et al. (15). Aliquots of the lipid extracts were evaporated to dryness under nitrogen in counting vials and counted in the scintillation mixture (above) to determine total lipid radioactivity. Other aliquots were evaporated to dryness in a st’ream of nitrogen, dissolved in a small amount of petroleum ether (boiling range 30-60”) and applied to thin layer chroma- tography plates spread with Silica Gel G containing Ultraphor (16) for lipid separation in a solvent system of hexane-diethyl ether-glacial acetic acid (80: 20: 1). Lipids were visualized with ultraviolet light, and the lipid fractions were scraped into vials and counted in the liquid scintillation mixture (above).
Long Chain Fatty Acid Carboxyl Radioactivity-Free fatty acids, phospholipids, and triglycerides were separated by thin layer chromatography as described above and eluted by repeated es- traction with chloroform-methanol (2: 1). Aliquots were evali- orated under nitrogen in 25ml Erlenmeyer flasks containing center wells and decarboxylation was accomplished as described by Brady et al. (17). Center well contents were assayed for radioactivity as described above. This method was validated by obtaining 92 to 95Z recovery of the radioactivity of [l-‘“Cl- palmitic acid and 5.7 to 6.0(;; recovery of the radioactivity from [U-14C]palmitic acid.
RESULTS
Metabolic and Structural Integrity of the Isolated Liver Cells- The initial objective was to determine whether isolated liver cells are a valid system for the study of liver metabolism. Glu- coneogenesis is a critical test for metabolic integrity (18). Berry and Friend (5) reported active gluconeogenesis in liver cells iso- lated by their procedure. Their report did not include sequential measurement of gluconeogenesis in the same cell population as
1790 Regulation of Fatty Acid Metabolism in Isolated Liver Cells Vol. 247, No. 6
a function of time. Gluconeogenesis and lactate utilization were also measured in the present study to characterize further the quality of the isolated cells. Liver cells isolated from rats fasted 24 hours produced 1.1 to 1.9 pmoles of glucose per g dry wt per min when incubated in 10 m&f L( +)-lactate at 37” for 3 hours at cell concentrations of 25 to 30 mg dry wt per ml as de- scribed in Table I. An additional 10 pmoles of lactate was added per ml at 90 min to prevent its complete depletion. Peri- odic measurements of glucose throughout the a-hour incubation established that after a brief lag phase the rate of gluconeogenesis from L(+)-lactate was constant, indicative of complete main- tenance of this multireaction process throughout this period. Longer periods of incubation were not tested.
dium therefore could be accounted for by leakage from the cells which had damaged plasma membranes as indicated by their up- take of trypan blue.
If the isolated liver cells remain structurally and functionally intact during incubation, they should retain soluble cytoplasmic enzymes. The leakage of lactate dehydrogenase from the iso- lated liver cells during 30 min of incubation at 37” therefore was t)ested. The enzyme activity in the cells and medium after in- cubation was assayed after separation of the cells by centrifuga- tion at 100 x g for 5 min. The lactate dehydrogenase activity in the cells was measured after freezing and thawing the cell pellet in fresh medium. Following incubation, 85 to 90% of the initial enzyme activity remained in the cells and only 10 to 15% of the activity was found in the extracellular medium. Like- wise, 10 to 150/,, of the cells were stained by trypan blue prior to incubation. The lactate dehydrogenase released into the me-
Comparative Metabolism of Albumin-bound Palmitic Acid by
Liver Cells Isolated from Fed and Fasted Rats-Validation of this system for the study of fatty acid metabolism in the liver re- quires demonstration that the liver cells remove extracellular long chain free fatty acids from the albumin-bound form, that the cells esterify the fatty acids to diglycerides, triglycerides, phospholipids, and cholesterol esters and oxidize the fatty acids to carbon dioxide and ketone bodies. Furthermore, demon- strated intrahepatic control over these processes resulting from altered conditions in vivo should be operative in the isolated cell system. Therefore, isolated liver cells from fed and fasted rats were incubated with albumin-bound [I-14C]palmitic acid. Rapid uptake and metabolism of the added palmitate ensued. Oxidation and esterification products were measured (Table I). The major pathway of palmitate conversion in the liver cells from fed rats was esterification. In these cells an intermediate quantity of added palmitate was oxidized to ketone bodies and a lesser amount to COZ during the 30-min incubation period.
TABLE II
Ketogenesis by isolated liver cells
Isolated liver cells (1.0 ml) were incubated with 1.0 ml of 397, albumin-O.75 rnnl [l-W]palmitate for 30 min as described in Table I.
TABLE I
Partition of free palmitate between pathways of esterijication and Fed (9)O
oxidation in isolatecl liver cells
One milliliter of isolated liver cells in suspension medium was incubated for 30 min at 37” with 1.0 ml of 3% albumin-O.75 mM [1-W]palmitate in suspension medium. The bovine serum al- bumin was treated to remove free fatty acids (see “Experimental Procedures”) prior to preparation of the albumin-[l-Wlpalmitate. At the end of the incubation period 23 to 587, of the added [l-W- palmitate was recovered unused in these experiments. Therefore, 42 to 777, of the added fatty acid entered various metabolic path- ways during this period. An average of 85% of the total added palmitate radioactivity was recovered in COZ, ketone bodies, fatty acid esters, plus unused palmitate at the end of the incuba-
Acetoacetate after 30 min, nmoles....................... 532 f 68 1192 f 111 <O.OOl
@-Hydroxybutyrate after 30 min, nmoles....................... 277 f 38 504 f 33 <O.OOl
Total ketone bodies after 30 min, nmoles....................... 808 f 1041696 f 138 <O.OOl
Total ketone bodies at zero time, nmoles....................... 128 f 14 284 f 38 <O.OOl
Total ketone bodies produced, nmoles/30 min. 680 f 93 1412 f 116 <O.OOl
Dry wt, mg/ml cell suspension. 29.5 f 3.718.5 f 1.6 <0.02 Total ketone bodies produced,
nmoles/mg dry wt/30 min.... 23.1 f 1.478.6 f 5.1 <O.OOl Final acetoacetate to p-hydroxy-
butyrate ratioc..............1.98 f .122.35 f .14 <O.l Specific activity of ketone bod-
ies, y. of specific activity of addedpalmitated ..__.._..... 65.6 f 5.556.3 f 4.9 <0.5
tion period.
Fed (lO)a
Total conversion of palmi- tate to fatty acid esters CO%, and ketone bodies (nmoles/mg dry wt/30 min).....................
hIg dry wt/ml cell suspen- sion”
Fatty acid esters, To of total conversion
Carbon dioxide, y. of total conversion
Ketone bodies, ‘% of total conversion. . . .
-.
t
-
16.3 f 1.1
24.6 zt 3.9
65.9 f 2.2
6.7 f 0.8
27.5 i 1.7
- Fasted (14)b
16.1 f 0.9
20.2 f 1.7
30.1 f 1.1
6.6 f 0.4
63.3 LIZ 1.1
P
>0.5
>O.l
<O.OOl
>0.5
<O.OOl
a Mean f S.E.M. in liver cells isolated from 10 different fed rats.
* Mean f S.E.M. in liver cells isolated from 14 different fasted rats.
c Each flask contained 1.0 ml of cell suspension.
Fasted (13)6 P
Q Mean f S.E.M. in liver cells isolated from nine different fed rats.
b Mean f S.E.M. in liver cells isolated from 13 different fasted rats.
c The total amounts of acetoacetate and p-hydroxybutyrate found at the end of incubation are shown. These values were used to calculate the final acetoacetate to P-hydroxybutyrate ra- tio, indicative of the mitochondrial oxidation-reduction state at the end of the incubation period. Net acetoacetate and p-hy- droxybutyrate produced during the incubation were not used for this calculation since the ratio gradually increases during the incubation period. The average observed increase in this ratio in 30 min was 11%.
d Dpm per pg of carbon in total ketone bodies f dpm per pg of carbon in added palmitate X 100.
Similar results were obtained for the esterification of albumin- bound [l-14C]palmit.ate and its oxidation to COZ in liver slices (19). A marked increase in oxidative activity and a concomitant decrease in esterifying activity was observed after fasting (Table I), in agreement with previous observations (4, 20-24). Al- though fasting induced marked changes in the partition of free fat,ty acids between pathways of esterification and oxidation, the total rate of utilization of the added free fatty acid was un- affected (Table I).
Ketone body production by the isolated liver cells is shown in Table II. Liver cells isolated from fasted rats contained more ketone bodies prior to incubation and produced more ketone bodies than cells from fed rats. Xcetoacetate was the major ketone body produced. The acetoacetate to fi-hydroxybutyrate ratio at the end of the incubation was greater than 1 in each of the cell preparations summarized in Table II. The final specific activity of the ketone bodies indicates t’hat about 60% of the ketone bodies produced in cells from both fed and fasted rats was derived from the added [V4C]palmitate, at this initial pal- mit,ate concentration, and the remainder was derived from en- dogenous sources.
Rate of Free Fatty Acid Utilization in Isolated Liver Cells- [lJ4C]Palmitic acid present in the medium in an albumin-bound form was progressively removed by isolated liver cells (Fig. I), and concurrently radioactive lipid esters (Fig. 1) and oxidation products (Fig. 2) were formed. Concomitant with the complete removal of albumin-bound [1-14C]palmitate, the formation of
I I I I I
I I I I I 1 2 3 4
-HOURS HOURS
FIG. 1. Rate of utilization and esterification of albumin-bound FIG. 3. Rate of ketone body formation and acetoacetate to
[1-WJpalmitate by isolated liver cells. Cells from a fasted rat p-hydroxybutyrate ratio during the incubation of isolated liver
(19.5 mg dry wt per flask) were incubated with albumin-[l-W]- cells. These data were obtained in the same experiment as de-
palmitate (762,950 dpm) as described in Table I. Duplicate values scribed in Fig. 1. Duplicate values are indicated by the range at
are indicated by the range at each point. O--O, ‘%-free fatty each point. O-0, total ketone bodies; O-0, acetoacetate acid; O---O, W-esterified fatty acid. to o-hydroxybutyrate ratio.
t-
1 I I I 1
I I I I I 1 2 3 4
HOURS
FIG. 2. Rate of formation of WO2 and W-ketone bodies from albumin-bound [I-Wlpalmitate by isolated liver cells. These data were obtained in the same experiment as described in Fig. 1. Duplicate values are indicated by the range at each point. The specific activit,ies of the ketone bodies at 30, 60, 120, and 240 min were 3540, 2320, 1590, and 1290 dpm per pg of carbon. o--o * 14C-total ketone bodies; O--O, 14C02.
1792 Regulation of Fatty Acid Metabolism in Isolated Liver Cells Vol. 247, No. 6
radioactive lipid esters (Fig. 1) and ketone bodies (Fig. 2) ceased. However, WO2 production continued (Fig. 2). Total ketone body production continued at a diminished rate after the added albumin-bound palmitate was depleted and the acetoacetate to P-hydroxybutyrate ratio progressively increased (Fig. 3), as also observed in the rat liver perfused without added fatty acids (24).
E$ects of Palmitate Concentration on Palmitate Utilization by Isolated Liver Cells-The effects of increased palmitate concen- tration on the free fatty acid uptake and metabolism, under conditions of constant albumin cont’ent, are shown in Figs. 4 to 6. The palmitate was added at a constant specific activity. The molar ratio of palmitate to albumin (P) at the beginning of the incubation is shown in the upper abscissa scale. These apparent v values are calculated from the total concentrations of palmitate and albumin present. These are within 170 of the true ? values (ratio of moles of palmitate bound per mole of albumin) since over 99% of the palmitate is bound to albumin under these conditions (25). The P values at each palmitate concentration shown (Figs. 4 to 6) were 0.92, 1.83, 3.45, 5.76, and 8.03 respec- t,ively.
Ket,ogenesis nl~d, to a lesser extent, WOS production increased
i7
6 8 I t I I
35
F
30 A 3
x
25 iT D
2 20 0
i2
w
15 E = L
2 10 $
5
PALMITATE, (mMi
FIG. 4. Effect of palmitate concentration on [lJ4C]palmitate oxidation to CO2 and ketone bodies and on the mitochondrial oxi- dation-reduction potential in isolated liver cells. The liver cells from a fasted rat (22.0 mg dry a-t per flask) were incubated as described in Table I for 30 min. The palmitate concentration was varied. The initial palmitate concentrations tested in this ex- periment, shown by the points in this figure, were 0.21, 0.42, 0.79, 1.32, and 1.84 rn~. The albumin concentration and the [V%]pal- mitate specific activity (830,280 dpm per &mole) remained con- stant. All analyses were in duplicate. The average values are shown. A---A, total ketone bodies, micromoles; O-0, IaC- total ketone bodies, disintegrations per min; n---a, ‘4CO2; O--O, acetoacetate to p-hydroxybutyrate ratio.
as the palmitate concentration increased (Fig. 4). An increase in ketogenesis in response to a sequential increase in fatty acid concentration was also observed in liver homogenates (8) and the perfused liver (26). W02 production became near maxi- mum at a lower palmitate concentration than ketone body pro- duction (Fig. 4). The specific activity of the added palmitate was 4324 dpm per pg of carbon. At the initial palmitate con- centrations of 0.21, 0.42, 0.79, 1.32, and 1.84 mM, the specific activity of the ketone bodies was 2095, 3104, 3617, 3645, and 3780 dpm per pg of carbon. At the palmitate concentrations of 0.79 mM and above, the specific activity of the ketone bodies be- came nearly constant at 850/, of the added palmitate specific activity. As ketogenesis approached a maximum rate (Fig. 4), the production of ketone bodies from extracellular and intra- cellular sources approached a constant proportion. Calculations on the absolute quantity of ketone bodies produced from added palmitate versus endogenous sources showed that at concentra- tions of 0, 0.21, 0.42, 0.79, 1.32, and 1.84 mM added palmitate,
endogenous ketogenesis was 28.8, 24.7, 17.4, 12.8, 13.6, and 11.2 pg of carbon, whereas ketogenesis from the added [l-14C]palmi- tate was 0, 23.3, 44.3, 65.7, 73.0, and 78.0 pg of carbon. The
absolute quantity of ketone bodies derived from endogenous
sources decreased as the extracellular supply of palmitate in- creased.
6-
2 4 6 8 I I I I I I I I
8
6
PALMITATE, imW
FIG. 5. Effect of palmitate concentration on uptake, oxidation, and esterification of [1-l%]palmitate by isolated liver cells. These data were obtained in the same experiment as described in Fig. 4. Palmitate uptake equals total [1-K!]palmitate added minus W- free fatty acid recovered at the end of the 30.min incubation period. Total oxidation products equals 14C02 plus ‘G-total ketone bodies. All analyses were in duplicate. The average values are shown. a---n, palmitate uptake; O---O, %-total oxidation products; O---O, ‘Gesterified fatty acid.
Palmitate uptake also increased as the palmitate concentra- tion was elevated (Fig. 5), as also observed in the perfused liver (26) and in ascites tumor cells (27). Osidative metabolism was the major pathway of palmitate conversion throughout the palmitate concentration range tested (Fig. 5). Total oxidative conversions approached a maximum at a 8 of 3 to 4. Although inflections in palmitate uptake and esterification occurred at this point, these processes continued to increase in a linear fashion as the v increased above 4.
Palmitate esterification was a sigmoid function of palmitate concentration (Fig. 5). Higher concentrations of palmitate were not, tested in this experiment because the maximum solubility of palmitate in the incubation medium was reached at a ij slightly above 8. Analysis of the incorporation of radioactive palmitic acid into various lipid fractions is shown in Fig. 6. At low concentrations of free fatty acid, phospholipid synthesis was more active than triglyceride formation. However, as the free fatty acid concentration increased, phospholipid synthesis be- came saturated, whereas triglyceride synthesis markedly in- creased and became the major lipid ester produced. Triglyc- eride formation increased linearly above a ii of 3. The conversion of [1-Wlpalmitate into the diglyceride fraction also markedly increased as the substrate concentration was elevated. Sigmoid curves of t,riglyceride and diglyceride formation as a function of the free fatty acid substrate concentration were observed (Fig.
r- .8
6 :
8-
6-
-~ v
2 4 6 8 I I I I I I I I
PALMITATE. (mMn)
FIN. 6. Effect of palmitate concentration on incorporation of [I-‘4Clpalmitate into triglycerides, phospholipids, diglycerides, and cholesterol esters in isolated liver cells. These data were ob- tained in the same experiment as described in Fig. 4. All analyses were in duplicate. The average values are shown. O--O, [l%]triglycerides; O---O, [Wlphospholipids; A---A, [‘Qdi- glycerides; V---V, [l*C]cholesterol esters.
The effects of albumin concentration, under conditions of constant fatty acid concentration, are shown in Fig. 7. An in- crease in albumin concentration decreased ketogenesis and lipid ester formation, whereas WOs production was only slightly af- fected.
Evaluation of Fatty Acid Carbon Recycling Via p Oxidation and Resynthesis and Via /3 Oxidation and Chain Elongation-The ob- served carbon 14 incorporation (Table I) indicates that the major path of fatty acid carbon in the liver cells from fed rats was esterification and that this process was significantly decreased by fasting. In addition to direct esterification of the [l-14C]pal- mitate, however, two other possible pathways could be responsi- ble for the higher incorporation of radioactive carbon into the fatty acid esters in liver cells from fed rats. Palmitate carbon recycling via chain elongation and de nowo fatty acid synthesis could each elevate the radioactivity in the fatty acid esters. These processes would result in the incorporation of radioactivity into the methylene carbons of the long chain fatty acids (Fig. 8). Evidence for the formation of [l-14C]acetyl CoA by the action of citrate lyase on citrate produced from oxalacetate and [l-‘“Cl- aectyl CoA by citrate synthase was provided by Srere and
4
?
7 2
x
h 2 m
I
L
I I I I I I
1’ - I I I I I I
1.6 3.2 4.8
ALBUMIN, (%I
Fro. 7. Effect of albumin concentration on the utilization of [l-‘4Clpalmitate by isolated liver cells. Each flask contained 0.5 ml of isolated liver cells (27.3 mg dry wt), 0.5 ml of 3y0 al-
bumin-1.5 mM [lJ%]palmitate (804,460 dpm), and the total albu- min concentration was varied by additions of fatty acid-free albumin dissolved in suspension medium. The final volume in each flask was adjusted to 2.0 ml with suspension medium. The liver cells were isolated from a rat fasted 24 hours. Each flask was incubated at 37” for 30 min. Each point shown is the average of duplicate values. The albumin concentrations tested in this ex- periment, shown by the points in this figure, were 0.79%, 1.58%, 3.16%, and 4.74y0. The P values at these different concentrations of albumin were 3.45, 1.72, 0.86, and 0.57, respectively. O--O, W-total ketone bodies; 0-0, W-free fatty acid remaining at the end of the incubation; A---A, r4C-esterified fatty acid; A--A, wo*.
1794 Regulation of Fatty Acid Metabolism in Isolated Liver Cells Vol. 247, No. 6
(I-- 14C1 FFA Esterlflcatmn [“‘Cl TG W,l PL
KetOgHX?SlS
oxl:~o”~ i 7gatlon ) ;::I )
1’%1 KEM-[I- “Cl ACETYL CoA
-ALACe)
de now synthesis
t
CITRATE+ .L1-‘4C1 ACETYL CoA
Citric acid Cycle
‘TO2 ‘TO?
FIG. 8. Pathways of l-14C-free fatty acid utilization in the liver including conceivable routes of recycling of radioactive carbon by oxidation and subsequent fatty acid chain elongation and de novo fatty acid synthesis. KB, ketone bodies; TG, tri- glycerides; PL, phospholipids.
TABLE III
Effects of ghcose, fructose, and glycerol on jatty acid utilization in isolated liver cells
Liver cells were isolated from rats fasted for 24 hours in these six experiments. Each milliliter of isolated liver cells was incu- bated for 30 min with 1.0 ml of 3% albumin-O.75 mrvr [I-W]palmi- tate as described in Table I. The control flasks contained no other substrates. Glucose (4 mg), fructose (3 mg), and glycerol (10 Mmoles) were added to other flasks as indicated. The dry weights of these six preparations of isolated liver cells were 14.6, 13.0, 14.7, 9.8, 11.3, and 24.5 mg per ml (1.0 ml per flask).
I Quantity of added [l-Wlpalmitate converted
Conversion product
Esterified fatty acids
Carbon dioxide
Ketone bodies
Control
moles”
63.3 64.8 62.8 70.2 53.1 43.7
21.9 11.6
9.7 10.7 18.7 16.0
123.5 138.5 108.5 100.5 145.3 122.7
70 change
29.3 33.5
35.5
0.8 7.3
8.9
-7.7 1.8
-6.1 -
Fructose (8.3 mna)
70 change
71.9 60.1
37.0
-50.2 -16.2
-26.1
-23.7 -25.7
-36.2
-
70 change
90.5
84.9
176.2
31.7
75.7
20.4
-61.7
-41.7
-52.5
0 Nanomoles of added [1-W]palmitate converted per 10 mg dry wt per 30 min.
Bhaduri (28). To determine the extent of carbon recycling via chain elongation and de novo synthesis, the esterified fatty acids were isolated and decarboxylated under conditions in which only the carboxyl carbon is converted to carbon dioxide (see “Experi-
TABLE IV
Effect of glucose, fructose, and glycerol on ketogenesis
These data were obtained in the same experiments as described in Table II
Total ketone bodies produced
Specific activity of ketone bodies
-
Control
nmoles~
585 938
1075 1071 1440
772 dlwa?
of carbonb
4590
3257 2111 2017 5512 6516
GlUCOSe (11.1 InM)
70 change
-5.7 -9.9
-11.4
-2.1 12.9
8.6
Fructose (8.3 mnr)
yx;’
70 change 70 chesge
-9.8 -64.1 -10.1
-52.0 -27.2
-62.2
-15.4 6.i -1T.4
21.5 -12.3
25.6
- a Total ketone body production in nanomoles per 10 mg dry
wt per 30 min. b The specific activity of the added [1-W]palmitate in these
six experiments was 5,815, 5,179, 5,391, 5,322, 10,707, and 10,145 dpm per pg of carbon.
mental Procedures”). The precursor [l-W]palmitate was like- wise decarboxylated, and, by this procedure, 91.5 to 94.97; of the radioactivity in this precursor was recovered in the carbosyl carbon. After incubation of liver cells from fed and fasted rats with [l-r4C]palmitate, as described in Table I, 87.9 to 91.4y0 (fed) and 88.4 to 95.6% (fasted) of the radioactivity in the tri- glyceride and phospholipid fatty acids were recovered in the carboxyl carbon. Chain elongation of [1-W]palmitate and de novo fatty acid synthesis from [lJ4C]acetyl Cob derived from [lJ4C]palmitate were therefore insignificant. Results were similar whether the cells were incubated in flasks with or without center wells containing paper saturated with 10% NaOH and thus with or without the removal of COZ, a requisite for fatty acid synthesis.
Effectsof Glucose, Fructose, and Glycerol on Palmitate Utilization in Isolated Liver Cells-To gain more insight on the factors which determine the partition of free fatty acids between the esterification and oxidative pathways in liver, the influence of certain substrates on these processes was investigated. Glucose, fructose, and glycerol each exerted specific and reproducible ef- fects on the utilization of free fatty acids by isolated liver cells (Table III). All three substrates increased the esterification of added palmitic acid. Glycerol exerted the strongest influence on this process. Fructose also markedly increased esterification, whereas the effect of glucose was minimal. Glycerol and glucose enhanced and fructose decreased oxidation of the added palmitic acid to COZ. The effect of glucose was slight. Glycerol and fructose both inhibited oxidation of the added free fatty acid to ketone bodies, whereas glucose did not affect this conversion. Glycerol was more inhibitory than fructose. Neither glucose, fructose, nor glycerol significantly affected the rate of removal of free fatty acids from the medium.
The effects of glucose, fructose, and glycerol on the production of ketone bodies were also measured. Glucose (11.1 mM), fruc-
FIG. 9. Effects of glucose on the conversion of [l-14C]palmitate to COT, ketone bodies, and esterified fatty acids by isolated liver cells. Cells from a fasted rat (17.3 mg dry wt per flask) were incubated with albumin-[l-14C]palmitate (1,332,400 dpm) as described in Table I for 15 min. The specific activities of the ketone bodies produced in the presence of 0, 2.78, 5.56, 11.11, and 16.67 mM glucose were 5560,5760,5690, 6070, and 6090 dpm per pg of carbon. Duplicate analyses are indicated by the range at each point. n---A, 14C02; O--O, 1%.esterified fatty acids; O--O, K-total ketone bodies.
tose (8.3 mM), and glycerol (5 mM) decreased ketogenesis an average of 9vc, IS%, and 59’$$, respectively (Table IV). The specific activity of the ketone bodies was moderately decreased by fructose but not uniformly altered by glucose or glycerol (see also Figs. 9 to 11). Some fructose carbon may have been con- verted to ketone bodies, although these data do not eliminate other possible causes of the dilution of the ketone body specific activity by fructose.
Determination of radioactivity in individual lipid fractions showed that the increased esterification of the [1-14C]palmitate induced by glucose, fructose, and glycerol was characterized by increased conversion of palmitate into phospholipids, diglycer- ides, and triglycerides, whereas, conversely, the incorporation of [lJ4C]palmitate into the cholesterol ester fraction decreased (Table V).
lS$ects of Glucose, Fructose, and Glycerol Concentration on Pal- mitate Conversions in Isolated Liver Cells-Glucose at a level of 5.6 mM increased the esterification of [l-Wlpalmitate but did not affect ‘4CO2 or 14C-ketone body formation (Fig. 9). Glucose, at 11.1 mu and above, elevated 14CO2 production, and an effect of glucose on the formation of 14C-ketone bodies was only ob- served at a glucose concentration of 16.7 mu. The net produc- tion of total ketone bodies at each glucose level in this series from 0 to 16.7 mM was 1050, 1040, 1060, 950, and 850 nmoles. The specific activity of the ketone bodies was unaltered.
Fructose was markedly more active than glucose in influencing the utilization of free fatty acids by isolated liver cells (Fig. 10). At a level of 1.4 mu, fructose increased esterification and de-
Effects of glucose, fructose, and glycerol on esteri$cation of [I-W]palmitate by isolated livel cells
These data were obtained in the same experiments as described in Table III
Phospholipids
Diglycerides
Triglycerides
Cholesterol esters
nmoleP
21.5 28.0 26.3 24.6 27.1 21.9
10.1 5.8 6.4
11.7 2.8 2.6
29.8 28.8 28.2 29.6 20.4 18.0
2.0 2.3 1.8 4.2 2.9 1.3
-
Glucose (11.1 IrIM)
% change
18.7 31.3
22.8
35.7 24.1
111.1
41.9 40.2
41.2
-15.1 -6.3
-10.0
gbJ;y m
70 chaage
45.6 62.3
106.9
51.4 174.5
154.5
59.7
-16.8 -26.4
-24.1
70 change
63.6
75.5
153.9
114.2
190.5
734.2
112.5
77.0
160.5
-35.3
-34.4
-38.3
(1 Nanomoles of added [lJ%]palmitate converted per 10 mg dry wt per 30 min.
creased 14C-ketone body formation. The effects of fructose on these conversions became maximal at a fructose concentration of 2.8 rnM. The net production of total ketone bodies at each fructose level from 0 to 8.3 mM was 1560, 1390, 1260, 1310, and 1230 nmoles. The specific activity of the ketone bodies was de- creased about 15% at all concentrations of fructose tested. Fructose (1.4 mM and 2.8 mM) increased 14C02 production. However, this effect was completely reversed at a higher fructose level (5.6 mM), and fructose became inhibitory at a concentration of 8.3 mM. The same results were obtained upon repetition of this experiment, thereby demonstrating the highly reproducible nature of these effects. In two other experiments fructose, at a concentration of 2.8 mM, elevated 14C02 production 30% and 39%, whereas at a level of 8.3 mM, fructose repeatedly decreased W02 production (Table III). Higher levels of fructose have not been tested.
At a level of 0.4 mu, glycerol increased esterification of [l-‘“Cl- palmitate and decreased its conversion to ketone bodies (Fig. 11). The net production of total ketone bodies at each glycerol level in this experiment from 0 to 5 mM glycerol was 1460, 1150, 850, 740, and 660 nmoles with no change in ketone body specific activities. An effect on WO2 production became significant at 1 mM glycerol, and, at this concentration, the increased esterifi- cation induced by glycerol reached a maximum. The greatest
1796 Regulation of Fatty Acid Metabolism in Isolated Liver Cells Vol. 247, No. 6
2 4 6 8
FRUCTOSE, (mMi
FIG. 10. Effects of fructose on the conversion of [I-Wlpalmitate to Cog, ketone bodies, and esterified fatty acids by isolated liver cells. Cells from a fasted rat (38.3 mg dry wt per flask) were incubated with albumin-[1-Wlpalmitate (1,591,600 dpm) as described in Table I for 15 min. The specific activities of the ketone bodies produced in the presence of 0, 1.39, 2.78, 5.56, and 8.33 mM fructose were 7470, 6400, 6270, 6120, and 6250 dpm per pg of carbon. Duplicate analyses are indicated by the range at each point. Only the upper or lower half of the range is shown where two curves overlap. A--A, WO2; O--O, 1%-esterified fatty acids; O-0, 14C-total ketone bodies.
enhancement of 14COs production and depression of 14C-ketone body formation was reached at 2.5 mM glycerol. Since the quantity of [lJ4C]palmitate converted to ketone bodies was an order of magnitude greater than that oxidized to completion to 14C02, glycerol exerted a marked net depression on total fatty acid oxidation.
Decarboxylation of the fatty acids in the phospholipids and
triglycerides formed by isolated liver cells from [I-Wlpalmitate in the presence of glucose, fructose, and glycerol clearly showed that recycling of fatty acid carbon by /3 oxidation and subsequent chain elongation or de novo fatty acid synthesis was insignificant. Increased conversion of [I-Wlpalmitate into the lipid esters in the presence of these substrates (Tables III and V) was therefore the result of enhanced direct esterification of the added [l-‘“Cl- palmitate (Fig. 8).
In other experiments the endogenous oxygen consumption of liver cells isolated from fasted rats, measured polarographically using a Clark oxygen electrode at cell concentrations of 6 to 9 mg dry wt per ml of suspension medium, was increased an aver- age of 32y0 immediately following the addition of 2,4-dinitro- phenol at a final concentration of 12.5 pM, indicating that oxida- tion is coupled to phosphorylation in the isolated hepatic cells. In two separate experiments, the effect of 2,4-dinitrophenol on
GLYCEROL, imM)
FIG. 11. Effects of glycerol on the conversion of [l-14C]palmitate to COZ, ketone bodies, total oxidation products, and esterified fatty acids by isolated liver cells. Cells from a fasted rat (38.7 mg dry wt per flask) were incubated with albumin-[l-l%]palmitate (1,474,600 dpm) as described in Table I for 15 min. The specific activities of ketone bodies at 0,0.4, 1.0,2.5, and 5 mM glycerol were 8520, 8370, 8970, 8090, and 8590 dpm per rg of carbon. Each point shown is the average of duplicate values. The ranges were no greater than those shown in Figs. 9 and 10. O-O, 14C02; A----A, ‘4C-esterified fatty acids; O-O, 14C-total oxidation products ; A---A, W-total ketone bodies.
the conversion of [I-Wlpalmitic acid to WO2 in liver cells from
fasted rats was measured. The initial concentration of [l-r4C]- palmitate was 140 PM and albumin was omitted since it binds dinitrophenol. The cells were incubated for 30 min at 37”, and, in the presence of 12.5 pM 2,4-dinitrophenol, r4C02 production from [l-14C]palmitate was stimulated 92yG and 115%. There- fore, adenine nucleotides are involved in governing the activit,y
of the citric acid cycle in the intact hepatocytes.
DISCUSSION
The rapid uptake and active metabolism of free fatty acids by intact liver cells (Table I, Figs. 1 to 3) isolated and incubated as described demonstrates that the pathways of free fatty acid utilization in intact liver are operative in the isolated cells. The reproducible nature of the system provides quantitative defini- tion of these activities. Retention by these cells of alterations in the partition of free fatty acids between the oxidative and esterifying pathways, as induced by fasting (Tables I and II), further validates the use of this system to study the regulation of hepatic fatty acid metabolism. In liver cells isolated by perfusion with EDTA and mechanical treatment, the synthesis of triglycerides from free fatty acids was inactive (29). The ob- served constant rate of gluconeogenesis (see “Results”) provides strong evidence for structural and metabolic integrity of the iso- lated cells under these incubation conditions, since discontinuity
of plasma membranes would have caused leakage and dilution of the cytosol and, thereby, loss of gluconeogenic activity. Main- tenance of cell integrity during incubation was further verified by the low degree of leakage of lactate dehydrogenase (see “Results”).
The partition of added palmitate between the oxidative and esterification pathways was inversed by fasting, while the ketone body specific activity was not appreciably affected (Tables I and II). I f ketone bodies and COZ are derived from the same acetyl CoA pool, or from two different pools in isotopic equilibrium, the specific activities of the CO2 produced by cells isolated from fed and fasted rats should also be similar. Under these conditions, since the CO2 radioactivity in cells from fed and fasted rats was the same (6.7yc of 16.3 nmoles in the fed and 6.6% of 16.1 nmoles in the fasted (Table I)), the citric acid cycle flux in the liver cells was unaltered by fasting. Although this remains tentative pending definitive information on the mitochondrial acetyl CoA precursor pools for the citric acid cycle and ketogenesis, elevated ketogenesis in fasting cannot be accounted for by decreased ac- tivity of the citric acid cycle, since the quantity of palmitate oxidized to COZ was much smaller than the increase in ketogene- sis. These experiments provide further evidence that increased ketogenesis in fasting is not the result of depressed citric acid cycle activity and that increased ketogenesis can occur without such a depression (30-32).
Liver cells from fed rats oxidized an average of 1.09 and 4.48 nmoles of added palmitate to CO2 and ketone bodies, respectively, per mg dry wt per 30 min (calculated from data in Table I). The cells from fasted rats oxidized an average of 1.06 and 10.19 nmoles of added palmitate to COZ and ketone bodies per mg dry wt per 30 min. Based on the known energy yield of these reactions, hepatic cells from rats fasted 24 hours obtained 65% more energy from the added palmitate than cells from well nourished animals. In contrast, other results have indicated that livers from fed and fasted rats oxidized added palmitate in such a way that the total energy derived from the fatty acid was constant (23). The present results are consistent with the concept of caloric homeo- stasis (1). In the fasting animal, as carbohydrate oxidation de- creases, energy derived from fatty acid oxidation increases and fatty acids become the major energy source. This alteration may be even more prominent in the liver in which glucose oxida- tion is markedly depressed in the fasting state, as gluconeogenesis hecomes operative, and fatty acids become the predominant fuel.
Esterification and ketogenesis from added [l-14C]palmitate terminated with removal of [lJ4C]palmitate, whereas ‘4cO~ pro- duction continued (Figs. 1 and 2). Only 14,000 dpm were lost from the fatty acid ester pool after complete depletion of [l-14C] palmitate. Oxidative utilization would have produced about 3,500 dpm of 14COp. According to this estimate, less than 10% of the 14C02 produced after depletion of added [l-14C]palmitate was derived from carbon recycling through the fatty acid ester pool. The continued 14C02 production is apparently derived almost entirely from oxidation of citric acid cycle intermediates and other compounds formed from [1-14C]acety1 CoA during the /3 oxidation of exogenous [I-‘%]palmitate, in agreement with accumulation of water-soluble radioactive organic acids in liver cells during palmitate oxidation (33).
Increased concentration of palmitate in the medium increased ketogenesis and esterification and decreased the acetoacetate to P-hpdroxybutyrate ratio, whereas 14C02 formation was not markedly affected (Figs. 4 to 6). The citric acid cycle rates at
different palmitate concentrations, calculated from the specific activities of ketone bodies formed and r4C02 produced, were 7.5, 5.8, 5.0, 5.3, and 5.6 pg of carbon per 30 min, at initial palmitate concentrations of 0.21, 0.42, 0.79, 1.32, and 1.84 mM. Relative to the rate of CO2 production at 0.21 mM palmitate, the citric acid cycle flux was depressed 23 to 33yc at, the higher palmitate con- centrations. This provides evidence, in addition to that sum- marized by Wieland (34), that in a given metabolic state in- creased fatty acid oxidation depresses the citric acid cycle in liver. At these same palmitate concentrations, ketogenesis pro- ceeded at rates of 48.0, 61.8, 78.5, 86.6, and 89.3 pg of carbon per 30 min. The depression in citric acid cycle flux was slight, relative to the increased carbon flow to ketone bodies, and ac- counts for only 5 to 12% of the increased ketogenesis in response to the increased fatty acid concentration. Therefore, this in- creased ketogenesis is almost exclusively a result of increased /3 oxidation. Although the mechanism of citric acid cycle de- pression is not certain, the fatty acid concentration markedly affected the mitochondrial oxidation-reduction potential as reflected by the altered acetoacetate to fi-hydroxybutyrate ratio (Fig. 4). The diminished citric acid cycle flux may be a conse- quence of the more reduced potential since the oxalacetate to malate ratio should likewise be altered in a similar manner (35). The mild depression in citric acid cycle flux could therefore result from a lower oxalacetate concentration (34-36). It remains possible that other contributory inhibitory effects are operative (37).
The specific activities of the ketone bodies produced in liver cells w-ere not significantly affected by fasting, despite the in- creased magnitude of ketone body formation in the fasting state (Table II). Therefore, fasting increased ketogenesis from endog- enous and exogenous fatty acids to a similar extent. Evidence that endogenous hepatic ketogenesis is derived almost exclusively from fatty acids was previously reported (38). The concentra- tions of free fatty acids in plasma and liver are similar (19). Spector, Steinberg, and Tanaka (27) have presented evidence for an intracellular free fatty acid pool derived from the medium but not exchangeable with medium-free fatty acids. In addition, free fatty acids are released into the cytoplasm by action of phospholipases on membrane phospholipids and lipases on cyto- plasmic triglyceride droplets. In the present experiments, as the extracellular palmitate concentration increased, the quantity of ketone bodies produced from endogenous sources in the liver cells decreased, consonant with intracellular mixing of endogenous free fatty acids derived from lipolysis and exogenous free fatty acids from the medium. It is therefore suggested that free fatty acids derived from endogenous and exogenous sources form a common cytoplasmic pool from which free fatty acids partition between the oxidative and esterification pathways by diffusion to sites of activation and utilization. Thereby these two pathways may be considered competitive. Inhibition or stimulation of either process should influence the other process by alteration of substrate availability, provided both pathways are below maxi- mum velocity.
On this common pool basis the unaltered ketone body specific activity after fasting (Table II) indicates that endogenous lipoly- sis was not appreciably affected by fasting and did not contribute materially to the increased ketogenesis. It is well known that fasting elevates the plasma free fatty acid level (1). Increased plasma free fatty acids enhance ket,ogenesis to some extent even in well nourished rats (39). The present experiments signify that
1798 Regulation of Fatty Acid Metabolism in Isolated Liver Cells Vol. 247, No. 6
increased ketogenesis in the fasting state develops as a conse- quence of two alterations, namely altered partition of long chain fatty acid carbon between the pathways of oxidation and esteri- fication (as shown in Table I and as indicated by other observa- tions (4, 20-24)) and, secondly, an increased level of long chain fatty acids (Fig. 4). The data show (Tables I and II, Fig. 4) that both altered partition and elevated substrate concentration exert marked effects. These results indicate that the former is quantitatively more important. The increment of increased ketogenesis derived from elevated substrate concentration is almost exclusively a result of increased 0 oxidation since, as cal- culated above, only about S70 of this increment can be attributed to inhibition of the citric acid cycle.
As a function of substrate concentration palmitate conversions to ketone bodies and phospholipids were hyperbolic and ap- proached maximum at v of 5, whereas esterification of palmitate into triglycerides was sigmoid and did not reach maximum even at P of 8 (Fig. 6). When p oxidation and phospholipid formation reach near maximum rates, additional free fatty acids enter the pathway of triglyceride synthesis. The mechanism which provides preferential esterification of fatty acids into phospho- lipids, when the free fatty acid supply is low, while restricting entry into triglycerides is not clear. Possibilities include low acyl CoA concentration resulting in a low rate of terminal acyl- ation in triglyceride formation, different binding affinities of the triglyceride and phospholipid-synthesizing enzymes for diglycer- ide, and greater activity of the phospholipid-synthesizing system below maximum velocity owing to higher enzyme concentration or other factors.
As the rates of oxidation and phospholipid formation began to approach maximum at a ii value of about 4, the slope of the curve representing palmitate uptake diminished (Fig. 5). I f limited uptake restricted oxidation and phospholipid formation, it would also have restricted triglyceride formation, which in contrast markedly increased. Therefore, the altered fatty acid uptake appears to be a result of, and not the cause of, approach to maxi- mum utilization of palmitate via oxidation and phospholipid for- mation.
Dietary carbohydrates cause endogenous hyperglyceridemia (40-42). This effect occurs whether the initial plasma triglyc- eride concentration is normal or elevated (41-43). This nor- mally operative response has been attributed to increased plasma triglyceride production (44). Sucrose is more effective than glucose or starch in this regard (45, 46), a difference attributed to the fructose moiety of sucrose (47-49). The liver is the major source of plasma triglycerides derived from endogenous sources (see Reference 3). The present study provides a likely explana- tion for the normally occurring carbohydrate-induced hyperglyc- eridemia and for the greater effectiveness of fructose than glucose in its induction. Fructose was more active than glucose in elevating palmitate esterification in liver cells (Figs. 9 and 10). Although fatty acid synthesis in the liver after carbohydrate intake is operative, most of the fructose carbon incorporated into liver triglycerides is found in glyceride glycerol and relatively little in the fatty acids (50). Similarly, glucose is a poor precur- sor of long chain fatty acids in the liver (50, 51). Results of the present study, together with cited observations, therefore impli- cate increased long chain fatty acid esterification as a more important causative factor than increased hepatic fatty acid syn- thesis in the normally observed hyperglyceridemia following high carbohydrate intake.
The mechanisms by which glucose, fructose, and glycerol influence palmitate utilization in the liver are suggested by cor- relation of the present results with related observations. Fruc- tose and glycerol were far more potent than glucose in decreasing ketogenesis and increasing esterification (Figs. 9 to 11). Dif- ferent concentrations of fructose and glycerol had nearly equal and opposite effects on these two major pathways of free fatty acid utilization. Since oxidation of palmitate to ket,one bodies far exceeded oxidation to COZ, fructose and glycerol clearly in- hibited entry of palmitate into the /3 oxidation pathway. Fruc- tose undergoes rapid metabolism in the liver (50, 52). Lossow et al. (53) observed that fructose decreased the formation of 14C02 and [14C]acetoacetate bv liver slices obtained from rats pre- viously injected with [lJ4C]tripalmitin, supporting the view that carbohydrate utilization restricts the catabolism of long chain fatty acids in this tissue. The rapid catabolism of fructose in the liver may sufficiently compete with fl oxidation to account largely for the decrease in long chain fatty acid oxidation ob- served in the present study. This interpretation is similar to that previously proposed for the antiketogenic effect of fruct,ose in the perfused liver (54).
It has been suggested that the antiketogenic effect of glycerol is due to increased esterification of long chain fatty acids resulting from an elevated glycero-3-phosphate concentration (54). How- ever, Williamson et al. (55) observed that glycerol is also anti- ketogenic in hyperthyroid rat’s in which the hepatic glycero-3- phosphate level is not elevated, providing evidence against a primary effect of glycerol on esterification mediated by glycero-3- phosphate. Since glycerol, like fructose, undergoes rapid ca- tabolism (54, 56), glycerol might also restrict fatty acid oxidation by providing competing oxidizable substrates. The loa,er rate of glucose catabolism in liver is consistent with the lack of an antiketogenic effect of glucose in vitro, except at high concentra- tion (57, Fig. 9). Regarding the mechanism by which anti- ketogenic substrates inhibit the oxidation of long chain fatty acids, whether or not these substrates affect the catabolism of short chain fatty acids is presently inconclusive since the rate- limiting steps and the enzymes involved in long and short chain fatty acid oxidation may differ.
Fructose elevates the glycero-3-phosphate concentration (52, 56, 58) as does glycerol (54-56). Glucose also undergoes con- version to glycero-3-phosphate. The specific st,imulator)- effect of these substrates on glycerolipid formation (Table v) is prob- ably mediated by their conversion to glycero-a-phosphate (57), although this remains indefinite pending conclusive information on the turnover of this intermediate.
Glucose, fructose, and glycerol all increased 14C02 production (Figs. 9 to 11). The effect of glucose was slight. The fructose- induced increase in 14COs production was reversed by higher fructose concentration. The decreased ketone body specific activity indicates that this reversal was caused, at least in part, by isotopic dilution of citric acid cycle intermediates by entry, via pyruvate, of nonradioactive fructose carbon into the cycle, in agreement with the conversion of some fructose carbon to ketone bodies (59). Relative to the small effects of fructose and glycerol on the ketone body specific activity, the observed increase in 14C02 formation in the presence of these substrates (Figs. 10 and 11) reflects increased citric acid cycle flux. Energy yield cal- culations on the results (Figs. 10 and 11) indicate that although fructose and glycerol increased the energy derived from palmitate oxidation via the citric acid cycle, the total energy obtained from
palmitate oxidation was considerably decreased by these sub- strates. Fructose and glycerol initially undergo phosphorylation by ATP, and the ATP concentration in liver is decreased (52,56). Increased phosphorylation of adenine nucleotides inhibits par- tially purified citrate synthase (60, 61) and isocitrate dehydroge- nase (62, 63) and increases the degree of reduction of the mito- chondrial respiratory chain (64-66). It is therefore suggested that the increased citric acid cycle activity in the presence of fructose and glycerol in intact liver cells occurred in part in re- sponse to decreased ATP concentration. The fact that dinitro- phenol also increased r4C02 formation from [lJ4C]palmitate by the liver cells supports this concept. The energy-dependent control of the citric acid cycle has been summarized (37). An effect of decreased adenine nucleotide phosphorylation on the activity of the citric acid cycle, in response to fructose and glyc- erol, could be exerted by a direct effect on one or more citric acid cycle enzymes or by alteration of the mitochondrial NADH: NAD+ ratio or both. Factors other than adenine nucleotides are probably also involved. Notably, increased palmitate oxidation in response to increased palmitate concentration in- creased the mitochondrial oxidation-reduction potential (Fig. 4) and inhibited the citric acid cycle as discussed above. The marked effects of fructose and glycerol on citric acid cycle ac- tivity are therefore probably mediated by the cooperative effects of (a) depletion of ,4TP by initial phosphorylation of these sub- strates and (b) decreased fatty acid oxidation.
In view of the paramount importance of energy production in the maintenance of cell function and the dependence of liver cells on fatty acids for energy in the fasting state, the following events are proposed to account for the influence of glucose, fructose, and glycerol on the partition of long chain free fatty acids between the pathways of oxidation and esterification. It is suggested that the major effect of fructose and glycerol is inhibition of fi oxidation by competitive oxidation which, in con- sequence, promotes esterification by increasing the availability of free fatty acids. All three substrates stimulate esterification by a separate and specific, but lesser, effect on glycerolipid forma- tion, probably mediated by enhanced glycero-a-phosphate for- mation. The more marked effects of fructose and glycerol, than glucose, on esterification are apparently a combined result of this specific effect and, more prominently, the increased availability of long chain fatty acids consequent to restricted fi oxidation. The inhibitory effect of these substrates on fl oxidation may be enhanced by withdrawal of free fatty acids via the specific stimu- lation of glycerolipid formation.
The mechanism of the altered partition of long chain free fatty acid utilization between the pathways of oxidation and esterifica- tion as a result of fasting is suggested by the observed effects of fructose and glycerol on these conversions. Predominant flow of fatty acids into the oxidative pathway in liver cells from fasting rats was immediately restricted in vitro by the addition of fructose and glycerol (Figs. 10 and II), which enter glycolysis beyond fructose 1 , 6-diphosphate and undergo rapid conversion to glucose, pyruvate, and lactate. The immediate antiketo- genie effect of these substrates suggests that an altered concen- tration of enzymes which catalyze p oxidation and ketogenesis is not the basis for the different rates of ketogenesis in liver cells from fed and fasted rats. Evidence for direct inhibitory effects of fructose and glycerol or their metabolites on the activity of these enzymes is lacking. Further, activities of the enzymes which catalyze the conversion of acetyl CoA to acetoacetate in
liver are not appreciably affected by fasting (67). The present results support the suggestion of Krebs et al. (24) that the availa- bility of non-fat substrates may be a determining factor in the rate of hepatic ketogenesis. In addition, insulin may be one of the regulatory factors involved (68). Lack of long chain fatty acid carbon recycling, via oxidation and resynthesis or chain elongation, and the similar citric acid cycle activities in liver cells from fed and fasted rats indicate that hepatic underutilization of acetyl CoA is not involved in the mild ketosis of fasting. There- fore, a deficiency of energy-yielding non-lipid substrates in the liver during fasting, and thereby a decrease in competing oxi- dative processes, may be the dominant factor which enhances hepatic fatty acid oxidation and in consequence decreases esteri- fication in this metabolic state.
Acknozoledgment-The technical assistance of Mrs. Bonnie S. Hunt and Mr. Arthur 0. Searcy is gratefully acknowledged.
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