I
David M. Gibson' Indiana University School of Medicine Indianapolis
I
The Biosynthesis of Fatty Acids
Lipids are found in virtually all living systems. Some classes of lipids form part of the fabric of cell structures, whereas others, such as the triglycerides which fill animal fat cells (adipose tissue), are important reserves of metabolic fuel within the organism. Almost every kind of lipid molecule contains esters of long-chain even-numbered fatty acids as part of its structure. In animal cells the most frequently encountered fatty acids are palmitic, stearic, oleic, and linoleic acids (Fig. 1). The long hydrocarbon cham is hydrophobic in nature (nonpolar) and terminates in a single carboxyl group. The double bonds of the unsaturated fatty acids depicted in Figure 1 have the cis configuration, which means that the carbon chain is bent back upon itself a t these points. Since unsaturated bonds are fixed and do not rotate, a fatty acid like linoleic acid which has two double bonds tends to pack differently within complex lipid structures than do the more saturated acids (1). PALMTIC ACID :
STEARlC ACID : *
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H
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H
Y
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2
uc-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OH "
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*
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intermediates in the metabolism of fatty acids: e.g. palmityl coenzyme A (a thioester) and palmityl carnitine (Fig. 2). A fatty acyl-amide linkage is found in the complex lipid sphingomyelin. Fatty acids may also exist in a "free" state, i.e., not attached with other molecules in covalent linkage, but instead adsorbed or bound to protein molecules. A free fatty acid-plasma albumin complex (often abbreviated FFA) is the means by which fatty acids are transported from the adipose tissues of animals through the blood to the liver and other parts of the organism (9, 3). In animals, fatty acids either originate directly from the fats in the diet, or they are completely synthesized from non-fatty foodstuffs, especially carbohydrates. An exception is linoleic acid (Fig. 1). Although this acid is demanded in the economy of the cell, it cannot be synthesized in toto by animal tissues. Thus, like a vitamin, linoleic acid is an essential dietary constituent (4, 5). De nova synthesis offatty acids from glucose must be complementary to the variable exogenous supply of fatty acids. In general the more fat in the diet, the slower is the rate of fatty acid synthesis in the liver (and adipose cells) (6). A fat-free diet, on the other hand, allows the biosynthetic enzymes full range (7, 8). Thus, fatty acids tend to inhibit their own
Y
OLBIC ACID :
LMOLElC ACID :
Figure 1.
Fatty acids combine in ester lmkage with a variety of alcohol groups, such as the fatty acids in phosphatidyl cholime (lecithin), in triglyceride, and in cholesterol ester (Fig. 2). Other esters, although occurring in very low concentration in cells, are nevertheless important
[ Based on a lecture presented as part of the Symposium on Recent Advances in Biological Chemistry, before the Division of Biochemistry and Division of Chemical Education at the 147th Meeting of the American Chemical Society, Philadelphia, Pennsylvania, April, 1964. Investigations conducted at Indiana University have been supported by grants from the American Heart Association (67.G-139); the American Cancer Society (P-178-D); and the Public Health Sewice, National Institutes of Health (HE-04219-06 and GM-K3-18413), and the Heart Research Center Grant H-6308 of the National Heart Institute.
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Journal o f Chemical Educafion
i i
CHOLESTEROLESTER (Hydrogens Omitfed)
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Figure 2.
P A L ~ Y L CARNI-
formation-a
''negative feedback" control circuit.
Fany Acid Oxidation
The chemical steps by which fatty acids are formed in the cell have only recently been elucidated. In this development many lines of evidence first emerged in the study of how fatty acids, as n~etabolicfuels, are oxidized (9-11). Ihoop, a t the beginning of this century, obtained informat,ion which suggested that fatty acids are degraded two carbons a t a time starting a t the carboxyl end of the chain. I t was postulated that dehydrogena tion and oxidation must take place in the region of the beta (the thud) carbon atom of the fatty acid prior to scission of the terminal two carbons (Fig. 3). The two-carbon units broken off in successive cycles were then oxidized completely to carbon dioxide and water.
RECYCLE
COMPLETE OXIDATION TO C Q and H z 0
Figure 3.
The "Beta Oxidation" scheme of Knoop received final confirmation in most particulars in 1953 when the process was resolved into a set of reactions, each of which was catalyzed by a specific enzyme (Fig. 4) (9, 10, 12). These individual enzyme systems which are located in the mitochondria of the cell are normally coupled so tightly together that free intermediates do not accumulate. Coenzyme A (CoA)? plays a key role (13, 14). Before any chemical change can be imposed on the fatty acid it must first be esterified with CoA-a process which requires the investment of one "high-energy" ATPe molecule. All of the subsequent steps involve acyl CoA intermediates. The center of action is between the secmd and third carbons of the fatty acyl m ~ i e t y . ~The final step in each cycle is the thiolytic cleavage of acetyl CoA from the end of the acyl CoA molecule by reduced CoA (CoASH). The saturated acyl CoA then remaining is two carbons shorter than the original fatty acid initiating the cycle. The process is repeated until the long-chain acyl group has been completely converted to acetyl CoA molecules. The two-carbon fragment,
implicit in the hypothesis of Knoop, is the acetyl part of acetyl CoA. Fatty acid oxidation is geared for energy production in the cell, viz. the synthesis of ATP in the electron transport system of the mitochondria (E.T. in Fig. 4). The oxidation of the reduced nucleotide electron carriers FADH2 and DPNH by molecular oxygen in the E.T. provides the energy for ATP synthesis. Even more ATP is obtained in this manner by the complete oxidation of the acetyl moiety of acetyl CoA in the citric acid cycle (Krebs cycle) (15) to which the fatty acid oxidation cycle must be tightly coupled (9, 16). The identification of acetyl CoA as the two-carbon intermediate in fatty acid oxidation was prerequisite to the understanding of fatty acid synthesis since the acetyl group is also the elemental building block in the assembly of the long hydrocarbon chain. Many early experiments had established (a) that acetate, or molecules which could be converted into acetate (e.g., glucose), provide all of the fatty acid carbons 7 - 4 (b) that both carbons of the acetate molecule were incorporat,ed intact; and (c) that the acetate units ordered then~selves in a repeating head-to-tail sequence (21, 24-27). In theory two reductive steps would be required each time an acetate was accepted into the hydrocarbon chain. An examination of the fatty acid oxidation pathway (Fig. 4) revealed that synthesis could be achieved if the direction of metabolic flow were reversed. Experimentally, however, the mitochondria1 enzymes did not synthesize fatty acids completely from acetate or acetyl CoA ($8,$9)-although, with some modification of the scheme, fatty acids could be lengthened by two or more carbons (29). (See later in Fig. 9.) Consequently attention was focused on those in vitro
"he following abbreviations are used: CoA, coenzyme A; CoASH, reduced CoA; ATP, and ADP, adenosine tri- and diphosphate; TPN and TPNH, oxidised and reduced triphosphopyridie nucleotide; DPN and DPNH, oxidized and redneed diphosphopyridine nucleotide; FAD, flmine adenine dinueleotide; FMN, ffavin mononuoleotide; Pi, inorganic orthophosphate; aGP, alpha glycerol phosphate; DNA, deoxyribonucleio acid; RNA, ribonucleic acid; and C-14, carbon isotope 14. a Note in Figure 4 that the 2-unsaturation formed in beta. oxidation is the trans configuration, and that the 3-hydroxy-acyl intermediate is the i. ( f ) configuration. Volume 42, Number 5, Moy 1965
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237
enzyme syst,ems which did catalyze fatty acid formation from acetate alone. Synthesis of Saturated Fatty Acids
As with whole animals C-1Clabeled acetate is efficiently incorporated into fatty acids by whole liver tissue (as well as adipose tissue, maminary gland, and to a lesser extent other tissues); by homogenates of liver; and by the soluble cytoplasm (the "supernatant") derived from the homogenate by removal of the particulate organelles of the cell, including mitochondria, with high speed ~entrifugation.~ The laboratories of Gurirl and of Popjak established that the coenzymes ATP and CoA were needed to effect acetate incorporation and that citrate greatly stimulated this process (30-32). Langdon showed that TPN is necessary for optimal synthesis by the supernatant system (99)-a finding that clearly distinguished t,he synthetic pathway from the oxidative (see Fig. 4). Studies initiat,ed in Green's laboratory confirmed these cofactor requirements in an assay system employing two enzyme fractions purified from the supernatant of liver (54-57). It was clear that ATP and CoA were needed for acetyl CoA formation, and that TPNH alone provided the reducing potential for synthesis of the fatty acid product,. Other findings substantiated the inlpression that the enzymes of fatty acid oxidation were not involved: (a) When acetyl CoA was used as the acetyl donor, ATP was still needed to bring about its incorporation (98, 99). ( b ) Carbon dioxide was required in this system, although no carbon froin COz entered the final fatty acid structure (58, 59). ( c ) One of the purified enzyme fractions contained proteinbound biotin (40). Avidin, a prot,ein from egg white which specifically hinds biotin, inhibited fatty acid synthesis-a direct demonstration that biotin could f u n d o n as a prosthetic group in an enzyme system. (d) The chief fatty acid product was palmitic acid (over 80%) with smaller quant,ities of other saturated fatty acids (41). Unsaturated fatty acids, such as oleic acid, were not formed in the purified supernatant system. It appeared that these cytoplasmic enzymes comprised the principal pathway by which long-chain saturated fatty acids were synthesized de nova from acet,yl CoA, or, in the intact cell, from glucose by way of pyruvate and acet.yl CoA (96). Palmitic acid once formed was then the starting point for synthesis of other fatty acids by the particulate elements of the cell (24, 42). Continued study of the biotin-containing enzyme by Wakil (S7,49) revealed that it catalyzed the formation of malonyl CoA in the presence of ATP, acetyl CoA, and COz (Fig. 5). This enzyme, now designated acetyl CoA carboxylase, is a member of a new and ubiquitous family of COz-fixingandCop-transferringenzymes which contain biotin. In GO2 fixation, ATP energizes the
'The enzymes are found in the "supernatant" prepared by conventional homogenization techniques. Prolonged centrifugation of the liver supernatant in exes. of 100,000 X g. collects the carboxylase m d synthetase enzymes in the precipitate. Evidence has been obtained that heart sarcasomes may catalyse acetyl incarpar&m into fatty acids without the intervention of supernatant ensyrnes. Cf.HUISMANN, W. C., Biochim. Biophys. Acta, 58,417(1962).
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Journal of Chemical Education
creation of a COz-biotin complex on the enzyme. CO? thus activated may then be transferred to an acyl acceptor (&-46). llalonvl CoA is ranidlv" incoroorated into saturated fatty acids by the second enzyme fraction obtamed from liver supernatant (ST), from yeast (47) and other sources. The purified enzyme system is called fatty acid synthetase. Only those carbons of the n~aloriyl group which were orignially derived from acetyl CoA find their way into fatty acids. Two moles of TPXH are required in this system for each mole of malonyl CoA consumed for synthesis. In addition, an acyl CoA must be present to initiate synthesis if pur~fied enzyme is employed (48, 49). This acyl is best served by acetyl CoA itself, although, depending on the source of enzyme, other short chain acyl groups suffice.
n
/"\NU
-0-2-N
I
"C-C" I
WCY
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I I
CH
'(CHZ14
-C
PROTEIN
The raism d'etre for malonyl CoA in fatty acid syw thesis is that it serves more effectively than acetyl CoA as a two-carbon donor. Malonate provides the two "acetate" carbons which are afIixed to the carboxyl end of a saturated acyl group (Fig. 6). At the same time the remaining carbon atom of the malonate appears again as free COz, thereby facilitating the reaction in the direction of synthesis. By means of this condensationdecarboxylation reaction, the primary lengthening of the carbon chain is achieved (@).
From here the steps resemble the reversal of fatty acid oxidation (Fig. 6) (37, 60): (a) reduction of the beta ket,o group with TFNH to a D(-) beta hydroxy acyl derivative; (b) removal of the elements of water from the latter forming a trans alpha-beta unsaturation; and (c) reduction of the unsaturated bond with TPNH to give the saturated acyl group. (Enzyme-bound flavin adenine niououucleotide, FMN, may be an intermediat,e electron carrier in this reduction.) The new saturated acyl compound then initiates the next cycle of reactions. Seven complete cycles are needed to build up a molecule of palmitic acid from acetyl CoA. Equations representing the stoichiometry of the overall process and the two individual enzyme systems are presented in Figure 7.
Figure 7.
The steps of the synthetase system were depicted in Figure 6 simply as interactions among acyl groups. This was done to avoid the interpretation that CoA esters are obligatory intermediates in these transformations. I t has beeu repeatedly demonstrated that acetyl CoA and malonyl CoA are the primary suhstrates presented to the synthetase enzyme system. However, it is now known t,hat the organic participants in the sequence of reactions leading to synthesis are protein-bound, through sulfhydryl groups, rather than acyl CoA intermediates (50). The synthetase is pictured as a multienzyme complex (50); its molecular weight has been estimated in yeast as 2 X 106. Recently it has been observed that the acyl intermediates are combined with a heat-stable protein of low molecular weight, a polypeptide containing a single sulfhydryl group (51-55). This agent is analogous to CoASH in that it is a carrier for the molecules undergoing change. Acetyl CoA and malonyl CoA must first he converted by transacylase enzymes to the acetyl and malonyl polypeptides before the first steps of condensation and decarboxylation take place. The condensation product, acetoacetyl polypeptide, has been isolated and employed as an intermediate in fatty acid synthesis (52A). The preceding discoveries were initially revealed in bacterial extracts and more recently in plant tissue (62B). There is also reason to believe that the enzyme systems of animals operate in the same manner. Acetoacetate bound to enzyme has been identified in synthetase prepared from avian liver (54) and from yeast (60). The subsequent steps of synthesis (Fig. 6) are probably carried out as acyl polypeptide intermediates (51-55). The final long-chain saturated fatty acyl product is removed from the enzyme complex and ultimately
appears as acyl CoA. The latter ordinarily does not accumulat,e in the cell but is picked up by various acyl acceptors, such as alpha glycerol phosphate, in the formation of the glycerophosphatidss and glycerides (Fig. 2); or the acyl CoA may be processed further by other enzyme systems located in the particulate fractions of the cell, e.g., the acyl carbon chain lengthened or an unsaturation iutroduced (see later). Removal of the fatty acid product insures the continuat,ion of its synthesis. Acetyl CoA, which is t,he source of all carbons in fatty acids, owes its origin in turu to glucose by way of pyruvate. The steps of glycolysis by which pyruvate is generated and the process of pahilitate synthesis are carried out in the soluble cytoplasn~.~The production of acetyl CoA from pyruvate, however, by the enzyule pyruvic dehydrogenase is confined to the mitochondria. These organelles, since they are encased by sets of lipoprotein membranes, do not ordinarily permit facile transport of nucleotides such as DPN and CoA across the boundary. It is not clear therefore if acetyl groups are released by the mitochondrion as acetyl CoA or in some other form. Recent evidence (55, 66) permits the hypothesis that acetyl CoA, arising from pyruvate oxidation, is converted first to acetyl carnitine (55, 56) (Fig. 8). Acetyl carnitine enters into the cytoplasm where it is reconverted to acetyl CoA. Free carnitine is then recycled back to allow further acetyl transport. Although not proved, this model has found couvincing support in the study of long-chain fatty acyl transport into the mitochondrion, the site of fatty acid oxidation (57). In another model (58), intramitochondrial acet,yl CoA is couverted to citrate. Citrate is transported to the cytoplasm to be met by t,he cit,rate cleavage enzyme (59) which regenerates the acetyl CoA (see Fig. 11). This current area of research is clearly important in understanding the control of glucose utilization for fatty acid synthesis.
FATTY A C t E -TRICLYCERIDES
Figure 8.
Formation of Unsaturated Fatty Acids
Palmitic acid is the principal saturated fatty acid produced by the fatty acid syuthetase system. In animals other fatty acids (except linoleic acid) are probably derived directly from palmitic acid by lengthening the carbon chain and by the introduction of cis unsaturations (4?2, 60). Fatty acids may be lengtheued by two or four carbons Volume 42, Number 5, May 1965
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239
Control of Fatly Acid Synthesis
apparently by the same enzymes that catalyze fatty acid oxidation in the mitochondrial membranes (29, 37, 61, 61A). One step that is not found among the oxidation enzymes is the terminal enoyl reductase which requires TPNH (Fig. 9). I n this sequence note that the acetyl group is added directly onto an acyl CoA, and that no ATP is required (except for fatty acid activation). Both DPNH and TPNH are necessary. Fatty acyl CoA molecules with one or more cis uusaturations are lengthened in the same manner. In order to introduce a cis unsaturation into the fatty acid chain, particle-bound enzymes are called into play. The formation of oleic acid was &st discovered with an enzyme preparation from yeast. Following up the observation that yeast required oleic acid if grown in the absence of oxygen (62), Bloomfield and Bloch (63) found that &-A 9-unsaturated fatty acids could be synthesized from saturated long-chain fatty acids (as the acyl CoA) in yeast extracts only in the presence of TPNH and 02. The process, called "oxidative desaturation," has now been demonstrated in the endoplasmic recticulum ("microsome fraction") of animal liver cells (64, 64A). Additional unsaturations are probably introduced into monounsaturated fatty acids in the same way. I n animals new cis unsaturations are inserted three carbons over toward the carboxyl end of the carbon chain (4.9, 60, 64A) (Fig. 10). The formation of polyunsaturated fatty acids is accompanied by chain lengthening as well (42, 60). Thus (the CoA ester of) oleic acid (18 carbons, one unsaturation) is converted in three steps to an eicosatrienoic acid (20 carbons, three unsaturations); and linoleic acid (18 carbons, two unsaturations) into another 'Lessential" fatty acid, arachidonic acid (20 carbons, four unsaturs, tions) (Fig. 10). Although series of polyunsaturated fatty acids can be synthesized in toto from the carbons of glucose (strikingly evident in linoleic acid-deficient animals) (65), these acids cannot completely replace the essential linoleic and arachidonic acids in the economy of the cell.
Fatty acids synthesized de nouo, or acquired from exogenous sources, have several fates in store. They may be oxidized for energy in liver, heart, skeletal muscle, and other tissues; or they may be incorporated into complex lipid structures (Fig. 2). The latter are of two types: (a) First are the "storage" lipids or triglycerides of adipose cells and of liver. Both tissues can convert glucose into triglyceride. However, only the liver releases triglyceride into the blood stream where it is carried as part of the plasma lipoprotein (66, 67, 67A). The adipose cells by contrast release "free fatty acids" (bound to plasma albumin) after hydrolysis of the stored triglycerides (2, 3). (b) The second category of complex lipid includes those which with protein comprise cellular structures such as the mitochondria1 membranes, the endoplasmic reticulum, and the nuclear membrane. (Part of the structure of the plasma lipoproteins is also included in this group.) In a large measure the physical characteristics of the lipoprotein membrane systems depend upon the properties of the contained lipids. The latter in turn reflect the nature of the fatty acid moieties that are part of their structure (68, 69). Thus in linoleic acid deficiency the mitochondria of liver are swollen (70) and their metabolism is altered (5, 71). It seems reasonable to conclude that these changes are secondary to a relative paucity of linoleic and arachidonic acid (or to a relative overloading with more saturated acids) in the lipid constituents of the mitochondrial membranes. By the same token the fatty acid composition of the several types of. plasma lipoproteins is subject to fluctuation and may influence the manner in which the contained lipids are equilibrated with the various tissues (72, 73). I t is therefore of considerable interest to investigators to ascertain which factors control the rate of fatty acid synthesis versus oxidation; to follow the flux of the formed fatty acids into triglycerides or into the structural lipids; and finally to know what regulates the quantity of the several types of fatty acids that are found in the many lipid compartments of the cell. Once the mechanism of an enzyme catalyzed process is reasonably well understood, one can then inquire how the svstem is inteaated into the economv of the intact cell, i.e., how the enzyme machinery is controlled. This
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- S ~ A
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240 / Journal o f Chemical Educofion
W
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Figure 9.
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R - C = C -C-SCOA
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Figure 10.
H
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C - CH - C - HC - C -HO H ~ .
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is clearly important in regard to biosynthetic systems since the amount of a substance being created must not be too great or too small for the demands of cellular function at any particular moment. Synthesis of fatty acids must nicely balance their removal from or consumption in a tissue if a steady state concentration is to be maintained. The rate of synthesis of a cellular constituent, such as a fatty acid, depends almost entirely upon how much "active" enzyme is available for catalysis of its formation. In general, the total tissue or cellular capacity for catalysis by an enzyme is related to (a) the amount of the particular enzyme protein in a cell, and (b) the degree of inhibition or stimulation by certain compounds that are specifically bound by this enzyme (74, 75). In a multieozyme system a "limiting" enzyme is often singled out as the focus for the controlling influences. Although the cellular concentration of any enzyme protein is likewise a balance between its rate of synthesis and degradation, protein formation appears to be the more readily controlled variable. Enhanced synthesis of a protein is initiated when a certain region of the nuclear DNA (the specific "structural" gene) is made free to preside over the formation of a complementary strand of messenger RNA (the template immediately required for elaboration of the enzyme). The nature of the DNA-directed signal has not been clearly defined in animal cells. I t is pictured as a molecule or a set of molecules that arise in the course of metabolism inside the cell. Agents entering from the outside (such as hormones) are not categorically excluded. The second mode of control is due to direct inhibitory or stimulatory influences on existing enzyme protein. This action is mediated by metabolites which are not themselves related in structure to the substrate of the enzyme (the "allosteric effect") (74, 76). In binding, the allosteric metabolite induces a slight modification in the three dimensional structure of the protein (a couformational change) which in the case of an enzyme amplifies or dampens its capacity for catalysis. Certain enzymes are composed of dissociable protein subunits, the complex comprising the quaternary structure. The degree or geometry of association can determine the magnitude of the specific enzyme activity. It has been suggested that an allostericpromoted conformational change in one or more of the enzyme subunits will dictate the potentiality for this association (74, 75). The capacity for synthesis of fatty acids in liver varies under different physiological conditions. In starvation and diabetes, liver cells are virtually unable to incorporate acetate into fatty acids (80, 76). It has recently been shown that the amount of active fatty acidaynthesizing enzymes which can be isolated from the liver of these animals is only 10-20% of normal (77, 78). In diabetes and starvation, the organism depends upon the oxidation of endogenous stores of fatty acids for a supply of energy (especially the fatty acids esterified in the adipose tissue). Thus net synthesis of fats is a strategically useless function in these metabolic states. The observed diminished concentration of the active enzymes catalyzing saturated fatty acid formation (acetyl CoA carboxylase and fatty acid synthetase) can be explained both on the basis
of an allosteric type of inhibition and diminished enzyme synthesis. Many laboratories have now found that long-chain fatty acids or their CoA esters are inhibitory to acetyl CoA carboxylase (and possibly to fatty acid synthetase as well) (6, 79-98). The products formed in this system tend to depress their own manufacture by "allosteric" inhibition thereby serving to maintain a constant rate of fatty acid production in the cell. Extracellular control of these enzymes is effected by the free fatty acids arriving from the adipose tissue (or from absorbed lipids in the fed animal). Citrate and isocitrate are potent activators of the carboxylase enzyme, and one or the other has been routinely employed in any assay involving this enzyme (SO, 55). It has recently been discovered that the carboxylase is stimulated because three protein subunits of the enzyme are brought and held together in the presence of citrate to form a complete, competent enzyme (94). Other work in progress indicates that fatty acids may inhibit the carboxylase enzyme by preventing the citrateinduced aggregation phenomenon ( R.91. ,--,In contrast to starvation, animals that are well-fed or, better, animals that are fed a fat-free diet after a period of starvation (7), show an enormous increase in the capacity for fatty acid synthesis. This is expressed by an increase in the amount of active enzyme that can be isolated from liver (8, 95, 96) and other tissues. I n vivo saturated and monounsaturated fatty acids accumulate (primarily in the triglyceride lipid fraction of liver) while the relative quantity of linoleic and arachidonic fatty acids falls. This sudden and marked disruption in fatty acid composition is secondary to the enhanced capacity for synthesis of the saturated and monounsaturated fatty acids (96). In a matter of hours after initiating fat-free refeeding in rats, all subfractions of the liver cell show the same altered fatty acid pattern. Plasma triglycerides may originate from liver triglycerides (66). Studies in humans indicate that the composition of plasma triglycerides is similarly affected in subjects maintained on a fat-free diet (75). Under these conditions of nutrition glucose fills the metabolic stream, providing carbon for net fatty acid and triglyceride formation. Release of the hormone insulin from the pancreas is enhanced when glucose is available. Insulin facilitates glucose utilization by tissues and dampens the flow of free fatty acids from the adipose tissue. In addition, glucose gives rise to the glycerol backbone of the triglycerides, in the form of alpha glycerol phosphate which is an avid fatty acyl acceptor (97, 98). All of these factors act to diminish the tissue concentration of free fatty acids and their CoA esters (98, 951, even though total fatty acids (esterifiedin triglycerides andglycerophospbatides) are increasing. Evidence has been obtained that there is enhanced synthesis of the enzymes catalyzing fatty acid formation during fat-free feeding (99). Pnromycin and actinomycin D,antibiotics whichblock proteinsynthesis, promptly stop the rapid increase in acetyl CoA carboxylase and fatty acid synthetase activity seen after refeeding a starved animal. Likewise the restoration of enzyme activity seen after injection of insulin in the Volume 42, Number
5, Moy 1965
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241
alloxan diabetic animal is blocked by act,inon~ycin D (QQA). The activity of quite a number of the enzymes which are involved in the conversion of glucose into triglyceride are depressed during starvation and diabetes and hecome elevated after refeeding or insulin injection, respectively: (a) glucose-6-phosphate dehydrogenase (7, Q6), an enzyme system which generates TPNH (another "TPNH enzyme," malic enzyme, also responds in the same direction) (100, 101); (b) the enzyme system of liver endoplasmic reticulum which is concerned with desaturation of stearyl CoA to oleyl CoA (96, 102, 103); the citric acid cleavage enzyme (104, 105, 106); and the enzyme catalyzing glncose phosphorylation (glucokinase) in liver (107, 108). It seems prohable that the increased activity of this group of enzymes and the fatty acid-synthesizing enzymes is the result of adaptive synthesis of new enzyme protein. What is especially interesting is the prospect t,hat these enzymes (which are functionally related in the synthesis of triglyceride from glucose) are produced in tandem, i.e., the adaptive phenomenon is not confined to a single key "limiting" enzyme. The stimulus for new enzyme formation here is obscure hut a t present seems to be related to improved glucose ~tilizat~ion (76), or independently to insulin itself (QQA). Another facet in the control of fatty acid biosynthesis has emerged in a study of linoleic acid deficiency in mice (109). I t was found that the liver enzymes cat& lyzing fatty acid synthesis are elevated in very early linoleic acid deficiency. The dietary deprivation of the essential fatty acid for several days is associated with the accumulation of those acids that have their origin in glucose, viz. palmitic, oleic, and palmitoleic, while the relative concentrations of linoleic and arachidonic acids fall to less than 1%. Supplement,ing the GLUCOSE
T
P
N
H GLUCOSE
- 6 - PHOSPHATE
U GLYCEROL PHOSPHATE
PYRUVATE
\
deficient diet with methyl linoleate reverses this trend, bringing the capacity for synthesis back to nearly normal levels. The role, direct or indirect, of linoleic acid in the control of fatty acid synthesis is not understood. Possibly a lipoprotein bearing linoleic acid or arachidonic acid in its structure is needed to complete the normal control mechanism. In Figure 11 are summarized several aspects of the control of triglyceride synthesis from glucose,a process in which fatty acid formation is an integral part. The enzymic steps numbered 1 through 6 are those which appear to respond by adapt,ive enzyme formation: (1)glucokinase, (2) glucose-6-phosphate dehydrogenase, (3) citrate cleavage enzyme, (4) acetyl CoA carboxylase, ( 5 ) fatty acid synthetase, and (6) the "oxidative desaturation" system. On the right side of the figure is indicated the stimulatory action of citrate on acetyl CoA carhoxylase by a positive arrow, while the "allosteric" inhibition of the same enzyme by long-chain fatty acyl CoA is represented by a negative arrow. The key role of alpha glycerol phosphate as a fat,ty acyl acceptor is noted. I t is produced by reduction of dihydroxy acetone phosphate, an intermediate in glycolysis. Literature Cited (1) SALEM, L., Canad. J . Biochem. Physiol., 40, 1287 (1962). (2) FREDERICICSON, D. S., AND GORDON, R. S., JR., Physiol. Reu., 33,585(1959). (3) VAUGHAN, M., J. Lipid Research, 2,293 (1961). (4) BURR,G. O., Federation Proe., 1, 224 (1942). (5) AAES~ORGENSEN, E., PhysiOl. Reu., 41, 1 (1961). (61 HILL,R., ET AL.,J. B i d . Chem., 233, 30.5 (1958). (7) TEPPERMAN, H.hI., AND TEPPERM.\N, J., Diabetes, 7, 478
- ..-.. .
11459) \ ,
( 8 ) HUBBARD, D. D., ET AL., Bioehem. Biophys. Research
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