Regulation of Intermediary Metabolism, with Special Reference to the Control Mechanisms in Insect Flight Muscle

Regulation of Intermediary Metabolism. with Special Reference to the Control Mechanisms in Insect Flight Muscle BERTRAM SACKTOR Gerontology Research Center National Institute of Child Health and Human Development National Institutes of Health. Baltimore. Maryland. U.S.A. I . Introduction . . . . . . . . . . . . . . . . . . 268 I1. Physiological Properties and Structural Organization of Insect Flight . . . . . . . . . . . . . . . . . . . . Muscle 269 A . Utilization of Oxygen during Flight . . . . . . . . . 269 Supply of Oxygen and Fuel to the Flight Muscle B. . . . . 269 C. Nature of the Substrate Consumed during Flight . . . . 271 D . Properties of the Contractile Proteins . . . . . . . . 271 E . Morphological Organization of Flight Muscle . . . . . . 275 F. Structural-Functional Correlates . . . . . . . . . . 281 111. Regulation of Carbohydrate Metabolism . . . . . . . . . 281 A . Glycogenolysis . . . . . . . . . . . . . . . 283 B. Phosphorylase b Kinase . . . . . . . . . . . . . 295 C. Glycogen Synthetase . . . . . . . . . . . . . . 295 D . Trehalase . . . . . . . . . . . . . . . . . 296 E. Biosynthesis of Trehalose . . . . . . . . . . . . 300 F. Glycolysis . . . . . . . . . . . . . . . . 303 G . Identification of Other Loci of Control of Metabolism . . 310 IV . Regulation of Fat Metabolism . . . . . . . . . . . . . 312 A . Fatty Acid Catabolism . . . . . . . . . . . . . 313 B. The Role of Carnitine . . . . . . . . . . . . . 314 C. Biosynthesis of Fat . . . . . . . . . . . . . . 316 D . Mobilization and Transport of Fat . . . . . . . . . 319 V . Regulation of Mitochondria1 Metabolism . . . . . . . . . 322 A . The Respiratory Chain and Oxidative Phosphorylation . . 323 B. Control of Pyruvate Oxidation . . . . . . . . . . 325 C. Control of Proline Oxidation . . . . . . . . . . . 330 D . Control of a-Glycero-P Oxidation . . . . . . . . . 332 E. The Energy-Dependent Accumulation of CaZ+and Pi . . . 333 F . Interactions of Metabolic Effectors with the Respiratory Chain 3 34 VI . Conclusions . . . . . . . . . . . . . . . . . . . 336 References . . . . . . . . . . . . . . . . . . . . . 338 261

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I . INTRODUCTION

During the rapid transition of a tissue from a “resting” state to one carrying on intense physiological work, the metabolic rate of the tissue increases many-fold. This indicates that the tissue has the full capacity to carry out glycolytic and oxidative reactions at the higher rate, in fact, classical determinations ’of activities of individual enzymes and concentrations of substrates in the tissue may suggest that metabolism should occur at maximal rates at all times. This is not the case, however, because biochemical pathways in living organisms are regulated or controlled. Studies of the regulation of metabolism, especially of systems in vivo, were initiated, therefore, with the following question in mind. What are the mechanisms by which energy-yielding biochemical pathways become activated instantaneously; or, perhaps more to the point, what are the mechanisms for keeping energy reserves from being used until needed? Previously, studies on the regulatory mechanisms of glycolysis and oxidative metabolism, in vivo, were limited to viable yeast or ascites cells or, in vitro, to a wide variety of preparations, ranging from reconstructed systems with purified enzymes to the more physiological isolated perfused heart or brain made anoxic by decapitation. The approach in our laboratory has been to seek regulatory processes in intact animals that are amenable to this kind of experimental inquiry. A unique system to determine the control mechanisms in a living animal during the transition from rest to activity is found in the flying insect. Initiation of flight induces an instantaneous increase in rate of energy transformation which is far in excess of that for any other biological system. In addition, muscular activity of the insect is readily controllable without use of traumatic experimental techniques. Moreover, as will be shown subsequently, kinetic measurements, in vitro, can be used successfully to predict the factors which control activity, in vivo. The energy used by insect muscle in performance of work which, at times, can total more than one million successive wingbeats with rates as great as lOOO/s, is ultimately derived from chemical reactions going on within the muscle. The nature and regulation of these chemical interconversions in the muscle and in other tissues, such as in fat body, that have significant and direct effects on the metabolism of the muscle, are the subjects of this review.

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11. PHYSIOLOGICAL PROPERTIES A N D STRUCTURAL ORGANIZATION

OF INSECT FLIGHT MUSCLE A. UTILIZATION OF OXYGEN DURING FLIGHT

The over-all level of metabolism, or biochemical interconversions, in the working muscle may be estimated from either the respiratory exchange or the depletion of the animals’ depots of fuel. In terms of cal/g of muscle/hr, values as high as 2400 for the bee during prolonged periods of continuous flight have been reported (Weis-Fogh, 1952). The cost of flight in insects can also be measured by comparing the rate of oxygen uptake during flight with that of the same insect at rest. Increases as great as 50-100 times the resting values have been recorded in a variety of insect species (see Sacktor, 1965). For example, Davis and Fraenkel (1940) reported that the resting respiratory rate of the blowfly, Luciliu, is 33-50 pl/g/min and this is increased 30-50 times during flight. Some individuals have oxygen consumptions of about 3000 pl/g/min during flight, thus elevating their resting rates approximately 100-fold. Such large increases in respiration upon initiation of flight are not restricted to Diptera and Hymenoptera, which have the asynchronous or fibrillar type of muscle striation and are characterized by a high frequency of movement of their wings. Essentially identical increases in oxygen uptake between individuals at rest and during flight have been observed in Orthoptera and Lepidoptera, which have the synchronous or close-packed type of muscle morphology and, in general, have relatively slow rates of wingbeat. For instance, in a variety of moth species, Zebe (1954) reported oxygen uptakes of from 7-1 2 pl/g/min at rest. These increased to values of 700-1 660 pl during flight, an increment of over 100-times in some cases. The 50to 100-fold increase in respiratory rate upon initiation of flight indicates that there is a large degree of control of respiration in muscle, in vivo. The mechanism of respiratory control will be discussed in a later section. B. SUPPLY OF OXYGEN AND FUEL TO THE FLIGHT MUSCLE As pointed out, oxygen uptake upon initiation of flight may reach a rate of 3000 pl/g/min. In spite of these enhanced respiratory rates, the observations that Drosophilu (Chadwick, 1953) and locusts (Krogh and Weis-Fogh, 195 1) can maintain flight for hours while accruing no, or only a small, oxygen debt indicate that the metabolic processes are not limited by the availability of oxygen. In insect

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flight muscle, myoglobin and hemoglobin are absent and air is conveyed directly to muscle through an elaborate conduit of tracheae. In most muscles the rich tracheolar system invades the fibers. Electronmicrographs, which will be described in detail later, show tracheoles to be in close opposition with mitochondria in a “mitochondrion-tracheole continuum” (Edwards and Ruska, 1955). The minute distances between tracheoles and oxygen-consuming elements of muscle suggest that diffusion suffices to transport at least part of the extra oxygen utilized. In fact, Weis-Fogh (1964, 1967) calculated that in small insects, including the flies, Drosophilu, Muscu, and Culliphoru, diffusion of respiratory gases is sufficient to account for the entire transport between the spiracles and the end of the tracheoles even at the highest rate of metabolism. In large insects, such as in dragon flies, locusts and wasps, the primary tracheole supply must be strongly ventilated while diffusion is sufficient in the remaining part of the air tubes. The ventilating mechanisms may be of two types: abdominal movements which result in abdominal pumping of air and hemolymph (in Vespu) and movements of the thoracic walls which are caused by the wing movements themselves and which result in thoracic pumping at wing-beat frequency (in Aeshnu). Weis-Fogh (1964) suggested that pumping of air and blood due to shortening of the fibers of the flight muscle is of little importance for the exchange of gases but of major importance for the supply of metabolic fuel. In many insects the flight muscles are so large that diffusion alone is quite inadequate t o transport substrate from the surface t o the interior and that exchange is achieved by a combination of diffusion and muscular pumping. He claimed that because of the inefficiency of the tidal process of moving blood into and out of intramuscular spaces as the muscle relaxes and contracts that the concentration of sugar in the blood must be very high (of the order of 0.5-1%) to provide gradients that can supply substrate to the catabolic enzymes in the interior of the fibers at the rates required for the exceptionally high activity of this tissue. The dependence of the rate of wingbeat on concentration of blood sugar in the blowfly (Clegg and Evans, 1961) is relevant to this view. Although muscular pumping may be important, the organization of the transverse tubular system (the Tsystem) and specific facilitated transport mechanisms may also have significant functions in transporting substrates. These latter concepts will be discussed more fully in later sections.

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C. NATURE OF THE SUBSTRATE CONSUMED DURING FLIGHT

Metabolic energy in working muscle is generated by oxidation of foodstuffs by atmospheric 0 2 ,with concomitant production of C02 . Measurements of the volumes of C 0 2 liberated and of O2 consumed, with calculation of the ratio of these volumes, the RQ, have been of value by virtue of inferences they permit in regard to the kind of substance undergoing oxidation. A compilation and full discussion of the data on the RQ during flight as well as the depletion of the insects’ reserves after flight was made previously (Sacktor, 1965). In summary, with insects having the asynchronous fibrillar type muscle, such as Diptera and Hymenoptera, the RQ is equal to unity and carbohydrates are the main, if not the exclusive, substrate. In those insects having the synchronous type of muscle, including Lepidoptera and Orthoptera, RQ values of 0.73 are found and fats are depleted, even though some species (moths) were gorged with glucose (Zebe, 1954). Locusts, roaches and aphids may use both carbohydrates and fat. Glycogen and trehalose are used during the initial period of flight; however, as flight continues, the RQ decreases and fat becomes the principal fuel and is able to sustain flight for hours. Also, flight muscle homogenates of the cecropia moth oxidize sugars and glycogen, and a moderately active trehalase was reported (Stevenson, 1968; Gussin and Wyatt, 1965). The role of amino acids as substrates for flight was considered earlier (Sacktor, 1961, 1965). The rapid utilization of proline on initiation of flight of the blowfly (Sacktor and Wormser-Shavit, 1966) to prime Krebs cycle activity (Sacktor and Childress, 1967) will be dealt with later. A unique example of the utilization of proline as an energy-furnishing reserve is found in the Tsetse-fly (Bursell, 1963, 1966). D. PROPERTIES OF THE CONTRACTILE PROTEINS

An understanding of the biochemical control mechanisms in insect flight muscle demands a knowledge of many aspects of insect organization, especially the structural and mechanical characteristics of the muscle as well as the physiological and biochemical properties of the contractile elements. These subjects have been the object of much experimental inquiry and a full description of them is clearly beyond the scope of the present discussion. Fortunately, excellent reviews on the properties of the contractile proteins have been published recently, including those by Maruyama (1 9 6 9 , Pringle (1967a, b) and Ruegg (1968). However, since the significances of various metabolic regulatory mechanisms become evident only when

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related to physiological events, a minimum of pertinent and essential features of the contractile processes in insect muscle must be noted. 1. Excitation-Contraction Coupling There are two fundamentally different ways in which the wing-beat frequency of insect flight muscle is determined. In one way, found in synchronous or afibrillar type muscles (Roeder, 1951), wingbeat frequency is determined by the activity of the central nervous system; each cycle of mechanical activity is triggered by one or a short burst of motor nerve impulse. This classical kind of excitation-contraction coupling is found in moths, dragonflies, and locusts and the flight of these insects is characterized by a rate of wingbeat of about 5-30/s. In the other way, found in asynchronous or fibrillar type muscles (Pringle, 1949), contraction is not triggered by nerve impulses and by action potentials in synchrony with the contraction cycle, rather mechanical activity is maintained by a self-oscillatory mechanism and is merely initiated and terminated by motor nerve control. Flies, bees, beetles and most bugs have this kind of flight muscle and these species have rapid rates of wingbeat, over 1OOO/s in some instances. Pringle and collaborators (see Pringle, 1967a) have shown that fibrillar muscle oscillates because of a unique property of the myofibrils themselves, in which there is a delay between changes of length and changes of tension in a mechanically resonant system. ATP is the source of energy and Ca and Mg ions are necessary activators. The threshold concentration for Ca2+ is approximately l o p 7 to lO-'M (Jewel1 and Ruegg, 1966); sensitivity of the muscle preparation to Ca2+depends on the concentration of MgZ+,which has a weak antagonistic effect on the action of Ca2+,and on mechanical conditions. The effect of muscle length is of particular importance, as stretching the fiber seems to be equivalent to raising the concentration of Ca2+.

2. Myofibrillar A TPase A Ca-activated myofibrillar ATPase in asynchronous flight muscle was reported some time ago (Sacktor, 1953a). More recently, vom M) are needed for Brocke (1966) noted that low levels of CaZ+( activation and that the degree of CaZ+ stimulation is less in oscillatory than in non-oscillatory muscles. This latter observation was further explored by Maruyama et al. (1 968), who showed that in synchronous muscle ATPase activity is low in the absence of Ca2+

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and that activity increases greatly over a narrow range of concentration of Ca2+. On the other hand, asynchronous muscle has a greater ATPase in the absence of Ca2+ but shows a much smaller increase over a wide range of concentration. The low Ca*+sensitivity of fibrillar flight muscle can be of marked physiological importance in that the small increment of Castimulation makes possible further increases in ATPase activity during stretch and oscillation (Ruegg and Tregear, 1966; Chaplain, 1967) and that the fibrillar muscle is less sensitive to small changes in concentration of Ca2+ over the physiological range. 3. Relaxing Factor Activity Tsukamoto e t al. (1966) described a granular fraction from locust flight muscle that inhibits myofibrillar ATPase, retards superprecipitation of actomyosin, and, in the presence of ATP, removes Ca2+ from myofibrils because of their strong Ca2+-binding capacity. These observations suggest that the locust granules are capable of acting as a relaxing factor. Indeed, the Ca2+-binding capacity of this insect preparation is comparable to that of rabbit skeletal muscle. Further, Smith (1966) showed, as will be illustrated in the next section, that locusts, having the synchronous-type muscle, have a well-developed sarcoplasmic reticulum, the source of the Ca2+ secluding vesicles. On the other hand, the sarcoplasmic reticulum is drastically reduced in asynchronous muscle (Smith, 1966). Since adequate Ca2+ cannot be released rapidly by the remnants of the reticulum, it is unlikely that the rapid rise and fall of tension occurring during oscillatory activity of fibrillar muscle involves the sarcoplasmic reticulum. In fact, Jewel1 and Ruegg (1966) and Chaplain (1967) demonstrated that glycerinated fibers carry out oscillatory contraction in the presence of ATP, Mg2+, and Ca2+, and that the frequency is independent of changes in concentration of Ca2+.Thus, fluctuations in the level of Ca2+ probably are not involved in the actual mechanism of oscillatory contraction, although Ca2+ is necessary to activate this system. Significantly, actomyosin ATPase is inhibited by excess MgATP and the allosteric inhibition is counteracted by physiological levels of Ca2+ (Chaplain, 1966). The possible role of mitochondria in maintaining Ca2+fluxes must not be overlooked in view of recent experiments by Carafoli e t al. (1969) showing energydependent rapid uptake and release of Ca2+ by mitochondria from blowfly flight muscle.

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E. MORPHOLOGICAL ORGANIZATION O F FLIGHT MUSCLE

The complexities and interactions of the biochemical systems in flight muscle become fully appreciated in light of structuralfunctional studies. The morphological organization of the tissue provides a basis for the biochemical similarities and differences between synchronous and asynchronous muscles, for the distinctive aggregation of enzymes into intracellular compartments, and for the regulatory mechanisms of the various metabolic pathways. Our knowledge of the structure of flight muscle stems largely from the elegant studies of David S . Smith (Smith, 1961a,b, 1963, 1965, 1966a, b). The reader is urged to examine these papers in detail since only the barest essentials consistent with the present discussion will be given here. The general organization of insect flight muscle was described in the pioneering investigations of Von Siebold (1848), Kolliker ( 1 857), and Watanabe and Williams (195 1). Among the distinguishing characteristics of the tissue is the large size of the fibrils, their arrangement into giant fibers, with diameters of up to several hundred microns, that are massed in turn, to fill the greatest part of the insect thorax and the short sarcomeres formed by restriction of the I-band. In addition, elongate columns of closely-packed mitochondria lie between the fibrils, the fibers are invaded by a rich tracheolar system and nuclei are few in number and often located peripherally. All the components of the muscle fiber are enveloped in an exceedingly thin sarcolemma. Structural unity results, in part, from the network of tracheae. Electron microscopic studies have elaborated the general morphological features of the tissue and have pointed out the complex, fine structure of insect flight muscle. A representative of the synchronous or close-packed, type of musclc is described in Fig. 1. This longitudinal section of the dragonfly muscle shows cylindrical fibrils approximately 2.3 p in length and 0.6 p in diameter. The sarcomeres are delineated by the prominent Z-bands. The I-bands are much-reduced. Transverse sections (Figs 2 and 3) indicate that the Fig. 1 . Longitudinal section of a synchronous type flight muscle of the dragonfly, Sympetmm, showing satcomere striations indicated by the prominent Z-bands. The mitochondria are arranged precisely opposite the sarcomeres. Cisternae of the sarcoplasmic reticulum lie in a bead-like fashion between the fibrils and mitochondria. Transversely sectioned profiles of the T-system tubules lie midway between the 2-band and center of the sarcomere. (Unpublished photo, kindly furnished by David S. Smith, similar to Fig. 3 in Smith, 1966b) ~35,000.

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actin filaments lie opposite to and between pairs of myosin filaments (Smith, 1966b). In the dragonfly, the mitochondria are slab-like and very large, and are interposed between the myofibrils and arrayed alongside each fibril precisely opposite the sarcomeres defined by successive Z-bands. In internal structure the mitochondria contain an enormous number of doubly lamellate cristae arranged in whirled and subparallel arrays. The large number of cristae is indicative of the high metabolic activity of these mitochondria. As first described by Smith (1966a), the fibrils of the muscle fibers are invested with two distinct series of membrane-limited components: a longitudinal system of cisternae, the sarcoplasmic reticulum; and a transversely oriented tubular system, the T-system. As seen in Fig. 1, membranes of the sarcoplasmic reticulum lie between the fibrils and the mitochondria in a continuous sheet bearing fenestrations which afford a beaded appearance. In synchronous muscle, the T-system tubules transverse the fiber midway between the Z-line and the middle of the sarcomere, lying in an indentation in the surface of the adjoining mitochondrion (Fig. 1). The two membrane systems enter into intimate association, in a dyad configuration. In the region of the dyad the fenestrations of the sarcoplasmic reticulum are absent; electron opaque material is present in the reticulum cisternae while the interior of the T-system appears to be entirely devoid of structural content. The origin of the T-system is apparent from Fig. 2. In the dragonfly the tubules are seen interposed between cisternae of the sarcoplasmic reticulum and the mitochondria and are derived as open invaginations of the plasma membrane at the surface of the fiber. Tubules opening directly to the interfiber space are in evidence. Figure 3 shows a fiber soaked in ferritin prior to fixation. The electron-dense particles of ferritin are clearly seen in the T-system space, between fibrils as well as between the fibrils and mitochondria, demonstrating the deep infusion of the muscle fiber by the extracellular milieu. The fine structure of flight muscle of the asynchronous or fibrillar type is illustrated in Figs 4 and 5. The cylindrical fibrils are very large, approximately 2 p in diameter and 3 p in length. The I-band is very narrow. As in other fibrillar muscles (Smith, 1961a and 1963; Shafiq, 1963; Ashhurst, 1967; Gregory et al., 1968), the Fig. 2. Transverse section of Sympetmm flight muscle showing origin of the transverse T-system as direct invaginations of the plasma membrane. (Unpublished photo, kindly furnished by David S. Smith, similar to Fig. 6 in Smith, 1966b) ~40,000.

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mitochondria of the blowfly, Phormiu, are ovoid and irregular in shape, up to 4 p in length, and are not precisely aligned with respect to the myofibrillar striations. The extensive sarcoplasmic reticulum that surrounds the fibrils in synchronous muscle is markedly reduced in the asynchronous muscle (Fig. 4). The reticulum is represented only by small flattened vesicles, closely applied to the T-system tubules in a dyad configuration. On the other hand, the tubular plasma membrane system, derived from the circumtracheolar sheaths drawn into the fiber around the invaginated tracheoles, is profusely distributed throughout the fibers and plasma membranes lie in close proximity to the mitochondria (Fig. 5). Ferritin injected into the blowfly in vivo is distributed throughout, and exclusively in, the T-system (Smith and Sacktor, 1970). The numerous and large extracellular spaces between the myofibrils and delineated by the tracheolar plasma membrane system (Fig. 5) have been described erroneously by some recent authors as degenerative mitochondria, lysosomes, and storage vesicles. The blowfly flight muscle mitochondrion, as is true for other mitochondria, has a distinct outer limiting membrane and cristae which are continuous with the inner membrane of the mitochondrion. The cristae are arrayed as parallel plates, 30-35 cristae per micron (Smith, 1963; Gregory et al., 1967). The cristae are fenestrated; the fenestrations of successive cristae are aligned to form cylindrical channels within the mitochondrial matrix (Figs 4 and 5, and Smith, 1963). The membranes of the cristae are about 70 A in thickness, and the intervening spaces, the matrix of the mitochondrion and the intracristal spaces, are each approximately 100 A wide. The osmotically active matrix space was reported to be about 2.5 pl/mg mitochondrial protein (Hansford, 1968). Cristae, derived from osmotically disrupted mitochondria and negatively stained, are displayed as membraneous ribbons flanked on the side by spherical particles, each 80-95 A in diameter (Fig. 6 and Smith, 1966). Each particle is attached to the membrane by a stalk. Smith (1963) calculated that each cubic micron of the mitochondrion contains 200,000 particles, representing 10% of the total mitochondrial volume. As will be discussed subsequently, these particles are the sites of ATP synthesis, at least in mammalian mitochondria (Kagawa and Racker, 1966). Fig. 3. Transverse section of Symperncrn flight muscle soaked in ferritin solution prior to fixation in glutaraldehyde. (From Smith, 1966b,reproduced with kind permission of David S. Smith and J. Cell Biol.) ~105,000.

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F. STRUCTURAL-FUNCTIONAL CORRELATES

The physiological role of the T-system in excitation-contraction coupling was suggested from the observations of Hill (1948, 1949) that the transition from rest to activity following depolarization is so rapid in large fibers as to preclude the possibility that the activation is mediated by diffusion into the sarcoplasm of a substance released inside the surface cell membrane by membrane depolarization. The current view maintains that in muscles with a well-developed sarcoplasmic reticulum and T-system, surface depolarization of the plasma membrane is spread internally along the tubules of the T-system, which is morphologically and physiologically continuous with the external plasma membrane. Excitation is transferred from Tsystem to sarcoplasmic reticulum at the dyad. Ca2+is released from the cisternae of the sarcoplasmic reticulum, which diffuses to the myofibrils and activates the myofibrillar ATPase concomitant with contraction of the muscle. Following contraction, Ca2+ is actively reaccumulated by the sarcoplasmic reticulum and MgATP dissociates the actin myosin filaments, permitting relaxation. The resting potential across the surface plasma membrane and across the junction between the T-system and sarcoplasmic reticulum is then re-established. In fibrillar flight muscle, which has a well developed T-system but a reduced sarcoplasmic reticulum, the situation is less clear. It is possible that while excitation-contraction coupling at the onset of activity may be initiated and subsequently maintained by a mechanism similar to that occurring in other striated fibers, the mechanical activity of the muscle in the oscillatory state is controlled by an intrinsic property of the myofibrillar elements. The marked reduction in sarcoplasmic reticulum seems to rule out the possibility that sarcomere length is controlled by movement of Ca2+into and out of the reticulum. However, other evidence indicates that Ca2+is vitally important, not only in initiating the oscillatory process and in activating the myofibrillar ATPase but, as will be seen later, in participating in the control of the critical energy-furnishing metabolic enzymes in this muscle. 111: REGULATION OF CARBOHYDRATE METABOLISM

The chemical sources of muscular energy in an insect, such as the Fig. 4. Transverse section of asynchronous type flight muscle of the blowfly, Phormiu. Note the large diameter of the fibrils and mitochondria as well as the absence of an extensive sarcoplasmic reticulum around the fibrils. Remnants of the sarcoplasmic reticulum are in juxtaposition with the T-system tubules and appear as dyads. (From Smith and Sacktor, 1970) ~ 2 8 , 0 0 0 .

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blowfly, include glycogen depots in flight muscle and fat body, and trehalose and other sugars in blood and gastrointestinal tract. The depletion of these reserves during exercise and the general description of the glycolytic and oxidative pathways in flight muscle have been discussed previously (Sacktor, 1965). The present review represents a continuation of these findings, but with special emphasis on the biochemical control system in this muscle, which on initiation of flight undergoes a transition from a state of minimal to that of maximal metabolic flux. By measuring coincident and sequential changes in concentrations of carbohydrates, glycolytic and citric acid cycle intermediates, amino acids and nucleotides at the start of flight and during the “steady state” of a continuous flight, the loci of metabolic control have become evident (Sacktor and WormserShavit, 1966; Sacktor and Hurlbut, 1966). A. GLYCOGENOLYSIS

1. Utilization of Glycogen

Figure 7 describes the changes in concentrations of carbohydrates in flight muscle of the blowfly during flight. Changes in concentration are evident 5 s after induction of flight. Although the concentration of glycogen is not measurably decreased during this brief initial period, after about 2 min of flight glycogen in flight muscle does serve as a major energy reserve, supplying approximately 2.5 pmoles (as equivalents of glucose) each minute until it is depleted after about 10 min. This rapid utilization of glycogen in muscle, commencing shortly after flight is induced, indicates control of glycogenolysis. The concentration of glycogen in the fat body does not significantly decrease during the first 5 min of flight. However, the depletion of this depot is large after about 15 min and becomes more marked with flights of longer durations. Thus, during flight, glycogen is mobilized from its two principal storage loci at independent rates; glycogen in muscle being used before the polysaccharide in fat body.

2. Characterization of Flight Muscle Glycogen Until recently, virtually nothing was known of the physical or biochemical properties of glycogen from insects. Since Bueding and Orrell (1964) have shown that the molecular properties of the Fig. 5 . Longitudinal section of Phomia flight muscle including dyads and tracheoles surrounded by invaginated plasma membrane of muscle fiber. (From Smith and Sacktor, 1970) ~ 2 5 , 0 0 0 .

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polysaccharide may vary with the tissue of origin and with the metabolic state of the tissue, a characterization of glycogen from flight muscle is essential to understanding how glycogenolysis is activated. Examination of glycogen in situ (Fig. 8), reveals that it is located mostly in the interfibrillar sarcoplasm with scattered deposits on the myofibrils (Childress et al., 1970).The glycogen is in the form of alpha particles or rosettes which vary in size with diameters of up to 0.25 micron. The individual components comprising the rosettes, the beta particles, are not clearly delineated. However, the polydisperse nature of native glycogen is apparent from electron micrographs of isolated glycogen (Fig. 8(d)). Incubation of the flight muscle tissue with amylase following fixation results in disappearance of the glycogen rosettes, leaving only an amorphous network of electron dense material in this region. Sections of the Fig. 6. A portion of the cristae from disrupted mitochondria of the blowfly, Culliphoru, negatively stained with phosphotungstate. The laterally placed particles (ATPase) are attached by stalks to the axial structure. (From Smith, 1963, reproduced with kind permission of David S . Smith andJ. CellBioZ.) ~550,000. A.1.P.-10*

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Fig. 8. Localization of blowfly flight muscle glycogen in situ. Glycogen rosettes are located between mitochondria (Mi) and myofibrils (M). The area within the square is shown in higher magnification in Fig. 8(b) ~ 5 , 8 2 0 ,bar represents 1 . 0 ~ (b) . Glycogen rosettes (arrows) are prominent. ~ 7 5 , 0 0 0 bar , represents 0 . 2 ~(c) . Section similar to (b) but treated with a-amylase. Only amorphous electrondense material can be seen adjacent to the mitochondria. ~ 7 5 , 0 0 0 ,bar represents 0 . 2 ~ (d) . Isolated rosettes of negatively stained glycogen. ~ 7 6 , 5 9 0bar , represents 0 . 2 ~(From . Childress el al., 1970.)

muscle examined by light microscopy following this treatment are no longer PAS positive. Native glycogen, isolated from blowfly flight muscle by a mild aqueous procedure, is a white amorphous powder containing less than 0.1% protein (Childress et al., 1970). On incubation with phosphorylase b, amylo-1, 6-glucosidase, AMP and Pi, the polysaccharide is degraded completely to glucose-1-P (90.5%) and glucose 9.5%). Sedimentation analysis of the pure glycogen also

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shows that it is polydisperse (Fig. 9), with particles having molecular weights as high as 50-100 million. Enzymatic degradation of the glycogen reveals that the outer chains are quite short, only about 25% of the total glucosyl residues being released by phosphorylase alone. Treatment of native glycogen with hot alkali, typical conditions for the extraction of glycogen from tissues, increases the release of the glucosyl residues to 3176, reflecting the availability of

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additional terminal residues which previously were resistant to enzymatic attack, perhaps because of steric hindrance. Chemical alteration of native glycogen by the harsher alkali treatment is also demonstrated by a marked lowering of the sedimentation coefficient (Fig. 9), indicating a five- to 1 0-fold decrease in molecular weight. The significance of using native glycogen in kinetic studies of glycogenolysis is evident from the data in Fig. 10, showing that flight muscle phosphorylase a has a lower affinity for native flight muscle glycogen than for the same substrate treated with alkali or for preparations of glycogen from other species. The apparent K, values are 0.09 and 0.29 mM for the alkali-treated and native glycogens, respectively, in the presence of saturating levels of Pi and AMP. A much greater difference between K, values for the two substrates is observed at low AMP levels. Values for maximum velocity with the native substrate are approximately 50% of the values obtained with alkali-treated or commercial glycogens. In addition, phosphorylase a

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has differing affinities for Pi depending on which glycogen is present as cosubstrate. In the presence of saturating levels of AMP and glycogen, the apparent Km values for Pi are 4.5 and 9.5 mM, respectively, for alkali-treated and native glycogens. In view of these glycogendependent differences in kinetic properties of the phosphorylases, it is advisable to use native glycogen, extracted from flight muscle tissue, as substrate in experiments on the regulation of glycogenolysis in flight muscle. I

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I

I

I

I

I

KOH glyc -AMP

04

08

12

16

I

I

I

20

24

28

[GLYCOGEN] mM END GROUP Fig. 10. A comparison of the effects of concentration of native and alkaliextracted glycogens on the initial velocity of flight muscle phosphorylase a. The concentration of Pi was 80 mM and of AMP, when present, was 1 mM. (From Childress et al., 1970.)

3. Glycogen Phosphorylase The glycogen phosphorylases, phosphorylase a and b, from flight muscle of the blowfly have been characterized by the recent studies

REGULATION OF INTERMEDIARY METABOLISM

289

of Childress and Sacktor ( 1970). Glycogen phosphorylase catalyses the reaction: Glycogen,

+ Pi * Glycogen, -, + Glucose- 1-P

It has been calculated that the phosphorylases comprise approximately 1.5% of the total muscle protein and have a potential activity of 9.6 pmoles xmin-' x g-' wet wt of thorax, a value more than adequate to account for the rate of glycogenolysis during flight. Studies of sedimentation velocity and electrophoresis on polyacrylamide gel demonstrate a high degree of homogeneity of the purified enzyme preparations. The SZO value for flight muscle phosphorylase b is 7.4. Estimations of the molecular weight of purified phosphorylase b by sedimentation equilibrium, by gel filtration, and by calculation from sedimentation and diffusion coefficients indicate a value of approximately 100,000. Flight muscle phosphorylase a, purified as the a form or converted from b to a with phosphorylase b kinase and ATP just before sedimentation analysis, also has a SZO of 7.4, demonstrating that flight muscle phosphorylase a has the same molecular weight as does the b form of the enzyme. This is in marked contrast to the rabbit muscle enzyme. Other studies on the purified blowfly enzyme show that phosphorylase b is not dissociated in to smaller molecular species by p-chloromercuribenzoate and that pyridoxal phosphate is the prosthetic group. Table I shows an amino acid analysis of flight muscle phosphorylase b. For comparison, data are also presented on the composition of human, rabbit, and frog muscle phosphorylases. Although the overall compositions are similar, striking differences between the flight muscle enzyme and the vertebrate muscle enzymes are found in the contents of half-cystine, arginine, and lysine residues. The kinetic properties of Phormia flight muscle phosphorylase a and b have been examined in vitro and during flight (Childress and Sacktor, 1960,1970). The interactions between cosubstrates, glycogen and Pi with phosphorylase a are shown in Fig. 11. These double reciprocal plots give a series of straight lines which intersect in the fourth quadrant, suggesting a bimolecular, sequential mechanism in which increasing levels of either substrate enhance the binding of the other. The values for apparent K, for Pi range from 47 mM at 0.15 mM glycogen to 7.3 mM at 1.7 mM glycogen; the values for apparent K, for glycogen range from 6.4 mM at 1.6 mM Pi to 0.72 mM at 40.3 mM Pi. In contrast to phosphorylase a, double

290

B. SACKTOR

TABLE I Amino acid

composition

Amino Acid Aspartic acid Threonine Serine Glutamic acid Proline Alanine Me thionine Isoleucine Leucine Tyrosine Valine Glycine Lysine Histidine Arginine Phenvlalanine Try piophane Half-cystine

of

rabbit, human, phosphoelases

frog, and flight

Rabbita

Human"

Frogb

Phormia

100 35 26 99 37 65 22 51 83 37 63 50 54 25 70 41 13 9

100 34 26 94 35 66 23 49 80 35 60 49 50 25 68 42 12 9

100 37 37 92 47 60 22 47 76 38 63 51 46 21 57 38 14 12

100 35 32 95 38 66 24 53 79 40 52 57 66 16 35 32 10 60

muscle

Compositions are expressed in percentiles relative to the values of Aspartate which were set arbitrarily at 100. Therefore, the differences in amino acid compositions of the four enzymes which are found by this method of calculation are not due to differences in molecular weight or absorbence indices. a Data of Appleman et ul. (1963); data of Metzger et ul. (1968). (From Childress and Sacktor, 1970.)

reciprocal plots of velocity vs. substrate concentration for phosphorylase b are non-linear. However, as in the case of phosphorylase a, increasing levels of one substrate enhance the binding of the other. Although not required for activity, low levels of AMP stimulate phosphorylase a two- to three-fold at saturating levels of substrates and ten-fold or higher at very low substrate levels. The apparent K, for AMP is 0.6 pM at saturating levels of Pi and glycogen. Lowering the level of either substrate decreases the affinity of the enzyme for the activator. Moreover, AMP increases the affinity of phosphorylase a for both substrates. The apparent K, for glycogen is decreased by one-third and that for Pi is lowered from 100 mM to 9 mM in the presence of 10.w and high concentrations of AMP, respectively. Unlike the a form of the enzyme, phosphorylase b has an absolute requirement for AMP. Furthermore, levels of AMP 100-fold greater than those which stimulate phosphorylase a are needed to stimulate

29 1

REGULATION OF INTERMEDIARY METABOLISM

I

1

.-c

2.4

I

I

I

I

I

1,

I

aJ

c

E

m

E

1

1

.-=

E

2.0 1.6

1 -0 43 L E

0

1.2

LL

4 -

0.8

c3 v)

% 0.4 E a

0.0

I

0

I

200

I

400

I

600

Fig. 1 1 . Initial velocity of phosphorylase “a” as a function of substrate concentration at several fixed levels of cosubstrate. The AMP concentration is 1.6 mM. The apparent K, for Pi are, respectively, 47.6, 23.7, 16.3, 11.2 and 7.3 mM in order from the lowest to the highest glycogen concentration. The apparent K , for glycogen are, respectively, 6.4, 4.5, 2.6, 1.7 and 0.7 mM in order from the lowest to the highest Pi concentration. (From Childress and Sacktor, 1970.)

the b form of the enzyme. As with phosphorylase a, however, increasing amounts of AMP lower the apparent K, values for Pi and glycogen, and increasing levels of either substrate lowers the affinity of phosphorylase b for AMP. Childress and Sacktor (1970) have found that ATP is a potent inhibitor of phosphorylase b but not of phosphorylase a. The Ki value for ATP is approximately 2 mM. At infinite concentration of AMP there is little change in Vmax, suggesting competitive inhibition with respect to AMP. Consistent with this view is the fact that ATP causes increases in the apparent K, values for both glycogen and Pi. Other nucleoside triphosphates are less potent as inhibitors while

292

B. SACKTOR

various metabolites, including trehalose, glucose-6-P and arginine-P, at concentrations found in flight muscle, are non-inhibitory.

4. Flight Muscle Phosphorylase a and b in Rest and Flight These detailed kinetic data for the flight muscle phosphorylases coupled with knowledge of the concentrations in resting and working muscle of various metabolites that affect phosphorylase activity T A B L E I1 values for flight muscle Comparison of metabolite levelsU a n d a p p a r e n t K, phosphorylases u n d e r in vivo conditions

Metabolite

in vivo in vi vo Level Level a t rest d u r in g flight

mM Glycogen

Pi AMP

ATP

6-9

14 0.2 14

mM

6+O.7Sc 15 0.6

13

Apparent K, valueb b

mM 0.6 10 0.5

2(K;)

Potential activity at simulated conditions o f Rest Flight

a

b

mM

8

1.7

0.001 -

a

a

b

%

%

%

%

3

64

9

65

.__

Metabolite levels, expressed by Sacktor and Wormser-Shavit (1966) and Sacktor and Hurlbut (1966) as pmoles x g-l wet wt of thorax, were doubled with the assumption that muscle represents half the thorax and the remainder of the thorax does not contribute to the metabolite concentration. The apparent K, values were determined from the kinetic data at the metabolite concentrations which exist in vivo under resting conditions. The level of glycogen in vivo decreased steadily during flight to a value of 0.75 mM after 10 min of flight where it remained steady. A glycogen concentration of 1% corresponds to 5.9 mM end groups. Glycogen data are given in mM end groups. (From Childress and Sacktor, 1970.)

(Sacktor and Wormser-Shavit, 1966; Sacktor and Hurlbut, 1966), enable estimation of the state of the phosphorylases, in vivo. The calculated apparent K, values for cosubstrates and activator of phosphorylase a and b are shown in Table 11. The data indicate that the level of glycogen present is sufficient to saturate the enzymes except after 10 min or more of flight when the muscle glycogen reserve is near depletion. The level of Pi under resting conditions is 14 mM, increasing slightly to 15 mM during flight. In this concentration range, which is slightly above the K, value, phosphorylase activity is quite dependent upon changes in the Pi level. The levels of AMP in vivo are 100-fold greater than the K,

REGULATION OF INTERMEDIARY METABOLISM

293

value for phosphorylase a and it seems reasonable to assume that the enzyme is saturated with AMP at all times. This level of AMP is near the K, value for phosphorylase b and thus the activity of the b form of the enzyme is responsive to changes in AMP level from 0.2 mM at rest to 0.6mM during flight. However, the level of ATP, which is strongly inhibitory to phosphorylase b, is very high in this tissue and decreases only slightly, from 14 mM to 13 mM, upon initiation of flight. Table I1 also shows the predicted relative activities of phosphorylase a and b under conditions of rest and flight. The strong inhibitory effect of ATP on phosphorylase b is most critical. In flight, phosphorylase b retains only 9% of its potential activity, while the a form of the enzyme, unaffected by ATP, retains 65%. Even though the changes in metabolite concentrations which occur during the transition from rest to flight cause a three-fold stimulation of phosphorylase b, the maximal rate of the b form is still far too low to account for the rate of glycogenolysis during flight. Based on the specific activity of 9.6 units of activity x g-’ wet wt of thorax, when assayed with native glycogen at 30°C and the fact that the potential activity of phosphorylase is the same whether it exists in the a or the b form, it can be estimated from the kinetic data that at least 50% of the total phosphorylase must be present in the a form in order to account for the rate of glycogen breakdown that occurs during flight (a minimum of 2.5 pmoles glucosyl residues x min-’ x g-’ wet wt of thorax). Using special precautions t o prevent alterations in the relative TABLE 111 Relative amounts of phosphorylases “a” and “b” in vivo at rest and during flight State

% of total phosphorylase in the “a” form

Resting-Unmounted 17.8f 3.6 (17) Mounted-Rested 2 hr 3 4 . 3 f 8.5 (27) Flown 5 s 63.9 12.3 (16) Flown 15 s 69.1 f 13.4 (16) Flown 30 s 72.2 f 12.3 (10) Flown 6 0 s 71.5 f 14.2 (10) Flown 10 min 72.1 13.1 (10) The figures shown represent mean values and their standard deviations. The number of extracts, each consisting of five thoraces, is shown in parentheses. (From Childress and Sacktor,

* *

1970.)

294

B. SACKTOR

proportions of phosphorylase a and b, Childress and Sacktor (1970) have measured the levels of both forms of the enzyme in flight muscle during the transition from rest to activity (Table 111). An increase in the relative amount of phosphorylase a upon initiation of flight is demonstrated. The level of phosphorylase a reaches its maximum of about 70% at 15 s of flight and remains there during a flight of 10 min. The total amount of phosphorylase present (a plus b ) does not change during flight. This level of phosphorylase a is adequate t o account for the observed rate of glycogenolysis during flight. From the kinetic data obtained under in vitro conditions, a 70% level of phosphorylase a can catalyze a rate of glycogenolysis of 4.3 pmoles glucosyl residues x min-' x g-' wet wt of thorax as compared to a minimum rate of 2.5 actually measured (Sacktor and Wormser-Shavit, 1966).

5. Glycogenolysis in Fat Body

As noted previously, glycogen stored in the fat body is used as a major energy depot during flights of long durations (also see review by Sacktor, 1965). In the blowfly, glycogen from the abdomen, presumably from the fat body, is consumed primarily after 10 min of flight and this reserve may support activity for at least an hour of continuous flight (Sacktor and Wormser-Shavit, 1966; Clegg and Evans, 1961). In general, essentially nothing is known of the mechanisms that initiate glycogenolysis in the fat body and knowledge of the biochemical properties of the phosphorylases in this tissue is fragmentary. The phosphorylases in the fat body from various stages of Cynthia and cecropia silkmoths have been partially characterized by Stevenson and Wyatt (1964) and Wiens and Gilbert (1967a). Activation by AMP is found and K, values for glucose-1-P and glycogen are given. It is obvious that, in view of the complicated kinetics of flight muscle phosphorylases, the values reported for fat body phosphorylases are to be interpreted with caution. Nevertheless, it is clear that when fat body glycogen is degraded, glucose does not leave the tissue. Instead, sugars are mobilized into blood as the disaccharide trehalose (Clegg and Evans, 1961). This trehalose, in turn, pools with blood trehalose and is transported to muscle, where it supplies energy for flight. There is reasonable evidence that hormones may enhance glycogenolysis in fat body. Besides changes in phosphorylase activity during development (Wyatt, 1967; Wiens and Gilbert, 1967b), Steele (1961, 1963) and others (McCarthy and Ralph, 1962; Ralph, 1962;

295

REGULATION OF INTERMEDIARY METABOLISM

Bowers and Friedman, 1963) have shown that corpus cardiacum extract stimulates the release of trehalose from isolated fat body and that the chief precursor of the released disaccharide is glycogen. However, the slow response of fat body phosphorylase to the hormone, 2 hr in some cases, makes the role of this hormonal mechanism in flight metabolism debatable. B. PHOSPHORYLASE b KINASE

Childress and Sacktor (1970) have shown that the rapid glycogenolysis induced at the initiation of blowfly flight requires the conversion of phosphorylase b to a. This conversion is catalyzed by the enzyme, phosphorylase b kinase. The mechanisms by which phosphorylase b kinase, itself, becomes activated to transform phosphorylase from the b to the a form, are, therefore, of significance to the regulation of flight muscle metabolism. The kinase is localized with phosphorylase a phosphatase, and phosphorylases a and b in the post-mitochondrial supernatant of the muscle, and can be readily isolated. In some recent experiments with Phormia flight muscle, Hansford and Sacktor (1970b) have found that phosphorylase b kinase is stimulated by Ca2+ at concentrations > with maximal enhancement at about g atoms/litre. These concentrations of Ca2+ are within the physiological range of the cation. An additional increase in phosphorylase b kinase activity is obtained by high concentrations of Pi. Both Pi and Ca2+seemingly function by increasing the Vmax of the enzyme. C. GLYCOGEN SYNTHETASE

Despite the importance of glycogen and its metabolism in flight muscle, little is known of the mechanisms by which the polysaccharide is synthesized. There is evidence that in insect muscle, as in mammalian tissues, the synthesis of the a-l,4-glucosidic linkage in glycogen is a function of UDP-glucose-glycogen transglycosylase (glycogen synthetase): UDP-glucose + Glycogen,

f,

UDP + Glycogen,,

,

The UDP-glucose is generated by UDP-glucose pyrophosphorylase by the reaction: Glucose-1-P + UTP * UDP-glucose + PPi Trivelloni ( 1960) has shown incorporation of 14C-glucose from UDP-glucose into glycogen and release of UDP by extracts of

296

B. SACKTOR

thoracic muscles of locusts. On the other hand, the histochemical findings of Hess and Pearse (1961) are not in full agreement with these data. These authors claim that little glycogen is deposited by the transglycosylase reaction but much glycogen is formed by a reversal of the phosphorylase reaction. However, as pointed out previously (Sacktor, 1 9 6 3 , since the histochemical method is dependent on the visualization of synthesized glycogen, the apparent low activity of the UDP-glucose system may have resulted from the presence of catabolically active phosphorylase which prevents the accumulation of glycogen from UDP-glucose by rapidly breaking down the newly formed polysaccharide. Hess and Pearse (1961) do report low phosphorylase and high UDPglucose-glycogen transglycosylase activities in the leg muscle of the locust, however. The synthesis of glycogen by the glycogen synthetase pathway in fat body of insects is well documented (see Wyatt, 1967). The synthetase is bound t o the particulate glycogen (Murphy and Wyatt, 1965; Vardanis 1967); in fact, with preparations from bee larvae it is not necessary to add glycogen as a primer. Glucose-6-P activates the synthetase, as in mammalian tissues. In cecropia larval fat body (Murphy and Wyatt, 1965), the K, for glucose-6-P is 0.6 mM and the K, for UDP-glucose is 1.6 mM. Glucose-6-P activates without significantly changing the K, for UDPglucose, an effect which resembles that found with the D form of the enzyme in mammalian tissues (Rosell-Perez and Larner, 1964). In the bee (Vardanis, 1967), the K, is slightly less than that in the silkmoth, but glucose-6-P increases approximately 10-fold the affinity of the synthetase for UDP-glucose. Thus, the bee has properties of both the D and I forms of the mammalian enzyme. Vardanis has suggested that the main factor limiting incorporation of glucose into glycogen is the length of outer branch chains in the primer. When the limit is reached, incorporation stops. Addition of glucose-6-P causes incorporation to resume until a higher limit is attained. He reasons that either glucose-6-P changes the specificity of the enzyme for primer outer chains, or that glucose-6-P activates a glucose-6-P dependent form of the enzyme that can utilize longer outer chains as effective primer units. The possible hormonal regulation of glycogen synthesis has been suggested (Van Handel and Lea, 1965; Wyatt, 1967). D. TREHALASE

1. Utilization of Trehalose In addition to glycogen, it is now well established that the

REGULATION OF INTERMEDIARY METABOLISM

297

disaccharide trehalose ( 1-a-D-glucopyranosyl-u-D -glucopyranoside) also supports flight muscle activity. The enzyme trehalase is in flight muscle (Sacktor, 1955). Trehalose, the principal blood sugar in many species of insects (see Wyatt, 1967), is found in muscle, and is reduced in concentration within these loci after flight (Bucher and Klingenberg, 1958; Clegg and Evans, 1961; Sacktor and WormserShavit 1966). A correlation has been observed between the titer of trehalose in the blood and the frequency of wingbeat in the blowfly (Clegg and Evans 1961). Figure 7 describes sequential changes in the concentration of trehalose on initiation of flight and during the steady-state of a continued flight (Sacktor and Wormser-Shavit, 1966). The concentration of disaccharide falls precipitously, approximately 1 pmole x g-I wet wt during the first 5 s, and continues to decrease rapidly for about 30 s. The level decreases progressively but at a lesser rate during the remainder of flight. This rapid initial utilization of trehalose coupled with the relatively slow rate of glycogenolysis on induction of flight suggests that during the first few seconds of flight the catabolism of trehalose probably serves as the major energydonor reaction. It has been pointed out earlier (Sacktor and Wormser-Shavit, 1966), that two kinetically different pools of trehalose are indicated from the discontinuity in the rate at which trehalose is utilized (Fig. 7). One is metabolized at a great rate and becomes exhausted within 30s; the other is depleted gradually during sustained flight. The kinetics of the latter pool resemble closely those of the decrease in the concentration of trehalose in the blood of the thorax during flight. The former pool has been designated as “muscle trehalose”. However, it may represent the trehalose in the extracellular fluids of the tubular system which invaginates the muscle profusely (Fig. 5 ) , and is compartmented from the sugar in the blood. If this interpretation is found to be valid, then all the trehalose is, in fact, extracellular and the enzyme trehalase, which hydrolyzes the disaccharide to its two glucose moieties, functions in the transport of sugar across the cell membrane. The marked decrease in the concentration of trehalose at the onset of exercise is coincident with the rapid and marked increase in the level of glucose. As shown in Fig. 7, the concentration of glucose increases by over 2 pmoles x g-I (wet wt) during the first few seconds of flight. These opposite changes, occurring at a time when there has been considerable enhancement of glycolysis, clearly indicates that the cleavage of trehalose to glucose by trehalase has

298

B. SACKTOR

been facilitated greatly and is controlled. The elevation of the concentration of glucose is transient, however; within 30 s its concentration has returned fo the original level. The changes in the intramuscular concentration of glucose reveal suggestions of overshoots and periodic fluctuations. This indicates that the steady-state levels of metabolites are not reached monotonically but in an oscillatory manner. Such oscillations are indicative of fine adjustments in the regulatory mechanisms of glycolysis in the muscle during exercise.

2. Hydrolysis of Trehalose by Trehalase

The enzyme trehalase hydrolytically splits trehalose into two glucose moieties: Trehalose + H, 0 + 2 Glucose Reports on the activity of trehalase in a host of insect species and various tissues are common and these have been ably tabulated by Wyatt (1967). In general, two different trehalases, specific for trehalose, have been characterized and both may be found in the same insect, silkmoth (Gussin and Wyatt, 1965), roach (Gilby et al., 1967), and blowfly (Sacktor, unpublished). One type is represented by the enzyme from intestine; the other has been described from muscle. The gut trehalase is soluble, has a pH optimum in the range 5.0-5.7 and has a K, mostly less than 1 mM.The enzyme from muscle is largely associated with particles, has a pH optimum less acid and has a K, value indicating less affinity for substrate. The particulate thoracic muscle trehalase from roaches, locusts and cecropia silkmoths can be activated several-fold by various physical and chemical treatments, i.e. freezing and thawing, detergents, phospholipase A, that tend to disrupt lipoprotein structure (Zebe and McShan, 1959; Gussin and Wyatt, 1965; Gilby et al.. 1967; Stevenson, 1968). After activation, the pH optimum of the muscle trehalase remains the same as that of the original particulate preparation but the K, is lowered to approximately one-half. It has been suggested (Gussin and Wyatt, 1965) that this activation phenomenon may be related to the biological regulation of the muscle trehalase but further studies from the same laboratory cast doubt on this hypothesis (Gilby et al., 1967). They point out that in the housefly and two species of blowflies, insects which utilize carbohydrate for flight energy, the muscle trehalase is not activated by freezing and thawing. However, their values of trehalase activity

REGULATION OF INTERMEDIARY METABOLISM

299

in Dipteran muscle preparations are already high. In fact, no further increase should have been expected since they are probably measuring the uncontrolled rate. It can be calculated that for Phormia, at least, the activity of trehalase in the muscle homogenate is sufficient to account for the rate of trehalose utilization by the blowfly on initiation of flight (Sacktor and Wormser-Shavit, 1966). The mechanism of regulation of the membrane-bound trehalase in insect muscle remains unknown despite much experimental probing. An important question is the precise cellular localization of the enzyme and to date the fragmentary observations are seemingly inconsistent. The first report of trehalose catabolism in flight muscle (Sacktor, 1955) has established a requirement for both the cytosol and particulate fractions in the complete oxidation of the disaccharide. Since it is now known that some of the glycolytic enzymes are localized exclusively in the cytosol whereas the respiratory chain is in the mitochondria, and that the complete catabolism of trehalose to C 0 2 and H,O involves both these metabolic pathways, the need for the two fractions is understandable and, therefore, these original observations cannot be used as an argument for any specific localization of trehalase. Approximately 25% of the total trehalase is found in the 100,000 x g for 20 or 30 min supernatant fraction of flight muscle from Phormia blowfly (Hansen, 1966), cecropia silkmoth (Gussin and Wyatt, 1965), and Blaberus roach (Gilby et al., 1967). In both cecropia and Blaberus, the post-mitochondria1 “microsomal” fraction contains about half the total enzyme with relatively high specific activities. Appreciable activity remains with a low-speed fraction that contains myofibrils and nuclei, but mitochondria show very low activity. In contrast, Zebe and McShan (1959) and Hansen (1966) claim that mitochondria are the chief locus of muscle trehalase in the roach Leucophaea and blowfly, respectively. However, cellular fractions, other than the myofibrils of Phormia, apparently have not been tested. Wyatt (1967) correctly points out that “microsomes” is an operational term rather than a specific morphologic‘al entity. By the procedures used in his experiments, the “microsomal” fraction contains sarcoplasmic reticulum, ribosomes, glycogen, and perhaps elements of the plasma membrane. It is clear from the electron micrographs shown in Figs 2 and 5 that the plasma membrane, as indicated by the Tsystem, deeply invaginates the muscle. Thus, this membrane makes intimate contact with other cellular components and, in view of the tendency of lipoprotein membranes to adhere to

300

B. SACKTOR

neighbouring particulates, claims as to the localization of enzymes by cosedimentation alone without appropriate controls with marker enzymes and electron microscopic examination of the pellets may be somewhat rash. In preliminary studies with flight muscle of Phormia (Sacktor and Reed, unpublished), substantial trehalase activity is found cosedimenting with mitochondria. However, electronmicrographs of the preparations frequently show what appears to be a third mitochondrial membrane, which suggests the adherence of an extra mitochondrial component. Other studies with marker enzymes clearly show that trehalase is located on or exterior to the outer mitochondrial membrane. If trehalase is, in fact, part of the plasma membrane, the seemingly discrepant observations of Hansen ( 1966) with blowflies and Gussin and Wyatt (1 965) and Gilby et al. (1967) with silkmoths and roaches, respectively, may be reconciled. In the latter insects, which have the synchronous type muscle with a welldeveloped sarcoplasmic reticulum, fragments of the plasma membrane may adhere to the reticulum and appear in the “microsomal” fraction. On the other hand, in the asynchronous muscle of the blowfly the sarcoplasmic reticulum is markedly reduced while the T-system and mitochondria are well-developed. Disruption of cell structure by homogenization, however mild, may cause the plasma membranes to collapse on t o the mitochondria, and trehalase activity will be evident in isolated mitochondria. Such hypothesis for the cellular localization of trehalase is consistent with the proposed function of the enzyme in transporting the sugar into the cell. This transport process will be activated, in a manner yet to be discovered, upon neuroelectrical stimulation of the muscle, depolarization of the plasma membrane and initiation of contraction. E. BIOSYNTHESIS OF TREHALOSE

Clegg and Evans (1961) have suggested that the intensity of flight in Phormia is determined largely by the interaction of two processes: the rate of trehalose utilization by the flight muscle and the rate of trehalose synthesis. Exhaustion results when trehalose cannot be supplied to the muscle at the necessary rate. While this view may be an oversimplification of the mechanisms regulating flight muscle, it is perfectly clear that the synthesis of trehalose is an important component of the overall process. A number of investigators (Treherne, 1958; Winteringham, 1959; Clegg and Evans, 1961; Wyatt, 1967) have shown that 14C-glucose injected into insects is converted to trehalose. Clegg and Evans (1961) introduced labeled

REGULATION OF INTERMEDIARY METABOLISM

301

glucose into blood of adult Phormia, previously starved for 24 hr, and measured the rate of trehalose formation in uivo. Within 30 s, radioactivity is measurable in trehalose of blood. The percentage of radioactivity appearing in blood trehalose increases very sharply in time, reaching about 50% within 2 min and about 90% at 10 min following the injection. Since over 96% of the total injected radioactivity is recovered as either trehalose or glucose, it is apparent that little glycogen is formed under these experimental conditions. Glucose, fructose and mannose are converted to trehalose with equal rapidity, while incorporation of galactose is appreciably slower. This pattern of conversion is similar to that for the rates of oxidation of these monosaccharides by fly flight muscle homogenates (Sacktor, 1955). Candy and Kilby (1 959) and Clements (1959) have shown that biosynthesis of trehalose takes place rapidly in the locust fat body whereas blood, leg muscle, and gut tissues are largely inactive in this respect. The synthesis in fat body has been collaborated by Clegg and Evans (1961) for the blowfly and the woodroach, and by other workers with a variety of insect species. Clegg and Evans (1961) also confirm that I4C-glucose is not incorporated into trehalose by mid-gut or blood of the blowfly, but a limited quantity of disaccharide is synthesized by flight muscle, although the possible contamination of the muscle by the diffuse fat body in the thorax is not ruled out. However, Trivelloni (1960) and Hines and Smith (1963) have detected trehalose synthesis in locust muscle. Clegg and Evans ( 1961) have demonstrated that the trehalose that is rapidly synthesized by isolated fat body is immediately released to the incubation medium. One may conclude from these data that the primary site of trehalose synthesis is the fat body and that the rate of disaccharide formation and liberation by this tissue is sufficiently rapid for it to be the source of most of the trehalose that accumulates in blood and is utilized in flight. The enzymes catalyzing the conversion of glucose to trehalose in the fat body have been described by Candy and Kilby ( 196 1 ) and are identical with those leading to the formation of the disaccharide in yeast, as previously discovered by Cabib and Leloir (1958). The pathway from glucose to UDP-glucose is common to the synthesis of both trehalose and glycogen. In the biosynthesis of trehalose, UDP-glucose serves as donor of one of the glucose moieties. The other hexose moiety stems from glucose-6-P, which, upon condensation with the nucleotide in a reaction catalyzed by the

302

B. SACKTOR

enzyme, trehalose-6-P synthetase, forms trehalose-6-P: UDPglucose

+ Glucose-6-P -,Trehalose-6-P + UDP

This is followed by the dephosphorylation of trehalose-6-P by a specific phosphatase (Friedman, 1960): Trehalose-6-P + H, 0 + Trehalose + Pi Murphy and Wyatt (1965) have studied the kinetics of the synthesis of trehalose in fat body of cecropia larvae. The K, for UDPglucose is 0.3 mM. Glucose-6-P shows more complex kinetics, a plot of glucose-6-P concentration against velocity yields a sigmoid curve with half-maximal velocity at about 5 mM. High concentrations of glucose-6-P are inhibitory. Mgz+ enhances the binding of glucose-6-P. These allosteric properties (Monod et al., 1963, 1965) are lost when the enzyme is partially purified or mildly maltreated. After such treatment the enzyme has classical Michaelis-Menton kinetics towards glucose-6-P with a K, of 5 mM. Significantly, trehalose-6-P synthetase is strongly inhibited by trehalose. The extent of inhibition varies not only with the trehalose concentration but also with the levels of glucose-6-P and Mgz+.Trehalose decreases the affinity of the enzyme for glucose-6-P and is non-competitive with respect to UDP-glucose. Treatments that cause loss of sigmoid kinetics towards glucose-6-P also reduce the sensitivity of the enzyme to trehalose. This feedback inhibition of trehalose-6-P synthetase by trehalose may be important to the mechanism of regulation of blood trehalose. Murphy and Wyatt (1965) suggest that elevation in glucose (by ingestion or other processes) causes a rise in glucose-6-P, and this will activate both glycogen and trehalose-6-P synthetase, the former reaching saturation (K, = 0.6 mM) before the latter (K, = 5 mM). However, trehalose-6-P synthetase has a greater affinity for UDP-glucose, K, = 0.3 mM as compared to 1.6 mM for glycogen synthetase. This will enable the preferential synthesis of trehalose when UDPglucose levels are low. When trehalose accumulates sufficiently to inhibit trehalose-6-P synthetase, the UDP-glucose level rises and allows increased synthesis of glycogen. This mechanism can provide for rapid production of both trehalose and glycogen when glucose is increased, followed by readjustments to homeostasis in the titer of blood trehalose. The scheme also provides a rational for the increased synthesis of trehalose after starvation (Clegg and Evans, 1961), when the blood trehalose titer is low; and the decreased

REGULATION OF INTERMEDIARY METABOLISM

303

synthesis of disaccharide induced by corpus cardiacum hormone in well-fed flies (Friedman, 1967), when a high level of blood trehalose is expected. On the other hand, Friedman (1968) has suggested an alternative mechanism which may partially explain the action of trehalose on trehalose-6-P synthetase. In addition to inhibiting the synthetase by decreasing the affinity of the enzyme for glucose-6-P (Murphy and Wyatt, 1965), Friedman has found that the rate of GLYCOGEN

G LY C 0 L Y T l C EN0-PRODUCTS

Fig. 12. A simplified schematic representation of the interactions of hexoses, trehalose and glycogen in the regulation of carbohydrate metabolism. A = Hexokinase. Inhibited by glucose-6-P. B = Glucose-6-P phosphatase. Activated by trehalose. C = Phosphoglutomutase. D = UDP-glucose pyrophosphorylase. E. = Glycogen synthetase. Activated by glucose-6-P. F = Glycogen phosphorylase. Inhibited by ATP. G = Trehalose-6-P synthetase. Inhibited by trehalose. H = Trehalose-6-P phosphatase. I = Trehalase. Control mechanism as. yet unknown. J = Glycolysis. Control at phosphofructokinase.

glucose-6-P phosphatase is increased greatly and specifically by trehalose. Thus, the accumulation of trehalose may result in feedback inhibition by removing a necessary substrate for trehalose-6-P synthetase without affecting the synthesis of glycogen. It is evident that hexoses, trehalose and glycogen mutually influence one another and that their cross-reactions are both numerous and complex. A simplified schematic representation of some of these interactions is illustrated in Fig. 12. F. GLYCOLYSIS

I . General Considerations The principal pathway for catabolism of carbohydrate in muscle

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of insects is unquestionably a variant of the classical EmbdenMeyerhof glycolytic scheme. An alternative route, designated by different authors as the ‘tpentose-P pathway”, “hexose monophosphate oxidation shunt”, or the “Warburg-Dickens pathway”, although prominent in some tissues during the life-cycle of the insect and present in muscle (Sacktor, 1965; Chefurka, 1965), probably contributes little to the energetics of the muscle. VogeTl et al. (1959) have reported that the activities of glucose-6-P dehydrogenase in muscle from wing and leg of locusts are only 0.1% of those of enzymes in glycolysis and Agosin et al. (1961) have noted that in thoracic extracts of the bug, Triatoma, 6-phosphogluconate dehydrogenase is even less active than is glucose-6-P dehydrogenase. Additional facets pertaining to the significance of the hexose monophosphate oxidative shunt in muscle as well as a description of the reaction sequences are to be found in previous reviews (Sacktor, 1965, Chefurka, 1965). The early studies of glycolysis in insects and details of the individual enzymatic reactions have also been thoroughly discussed (Sacktor, 1965; Chefurka, 1965) and no attempt will be made here to characterize each step in the pathway. Rather, the overall system and its regulation in muscle will be considered. It is now quite clear that flight muscle of a variety of insect species have a very high activity of a-glycero-P dehydrogenase and an extremely low activity of lactate dehydrogenase. Thus carbohydrates, whether originating as trehalose, glycogen on monosaccharides, yield stoichiometrically pyruvate and a-glycero-P as the products of glycolysis (Fig. 13). Comparative studies by several investigators (see Table I of Sacktor, 1965; Brosemer, 1967; Brosemer and Marquardt, 1966) on a-glycero-P and lactate dehydrogenase activities in different kinds of muscle reveal a reciprocal correlation between these activities. The highest ratio of a-glycero-P to lactate dehydrogenase activities is found in flight muscle of Dipterous and Hymenopterous insects. In this respect, the contrast between flight and leg muscle of a given species, between flight muscle of insects that are active flyers and the same muscle of related species that have lost during evolutionary development the ability to fly, and between some insect and mammalian muscle, is particularly striking. The significance of the formation of pyruvate and a-glycero-P in glycolysis becomes evident in light of the a-glycero-P cycle (Bucher and Klingenberg, 1958; Estabrook and Sacktor, 1958a; Sacktor and Dick, 1962). The cycle consists of two reactions:

REGULATION OF INTERMEDIARY METABOLISM

3 05

NADH(DPNH) + H + + Dihydroxyacetone-P + NAD+(DPN+) + a-glycero-P a-glycero-P + 1 / 2 O2+ Dihydroxyacetone-P + H20 NADH(DPNH) + H + + 1/ 2 O2-+ NAD+(DPN') + H20 Sum :

As shown in Fig. 13, the a-glycero-P cycle provides a mechanism whereby NADH, which is formed extramitochondrially in the glyceraldehyde-3-P dehydrogenase reaction, becomes oxidized. With the virtual absence of both lactate dehydrogenase and a direct mitochondrial oxidation of extramitochondrial NADH, because mitochondria are impermeable to NADH, glycolytically formed NADH is reoxidized, concomitant with the reduction of dihydroxyacetone-P and formation of a-glycero-P, by the extremely active soluble a-glycero-P dehydrogenase of the cystosol. The a-glycero-P is oxidized, in turn, by the mitochondrial a-glycero-P dehydrogenase, a flavoprotein, thereby regenerating additional GLUCOSE

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dihydroxyacetone-P. This dihydroxyacetone-P is then available for further oxidation of extramitochondrial NADH. Accordingly, the cycle is a shuttle system, in which NAD-linked substrates, in reduced and oxidized states, respectively enter and leave the mitochondria. In this way, hydrogen or reducing equivalents from the extramitochondrial pool of NADH pass the cytosol-mitochondria1 barrier and are oxidized by the mitochondria1 respiratory chain. Further, the cyclic process is self-generating in that only a catalytic quantity of dihydroxyacetone-P is needed to oxidize the NADH being continuously formed (Sacktor and Dick, 1962). This suggests that most of the dihydroxyacetone-P that is produced by the aldolase reaction can be isomerized to glyceraldehyde-3-P and that essentially all of the carbon of the carbohydrate metabolized in a prolonged flight is convertible to pyruvate and is available for further oxidation via the Krebs citric acid cycle. Thus the system is remarkably efficient, in that end-products of glycolysis, such as lacate, need not accumulate wastefully as it does in exercising vertebrate muscle. The oxidation of the two end-products of glycolysis, pyruvate and aglycero-P, by flight muscle mitochondria will be examined in a subsequent section. In considering glycolysis in muscle, the usable chemical energy derivable from the reactions is central to the discussion. The net yield of productive chemical energy from glycolysis may be computed from a balance of the moles of ATP consumed and regenerated per mole of carbohydrate degraded to end-products. As illustrated in Figs 12 and 13, when the substrate is glucose, which becomes available to muscle either as the result of the action of trehalase on trehalose or as free hexose, 1 mole of ATP is consumed at each of two kinase steps, the phosphorylations of glucose and of fructose-6-P. Since in anaerobic glycolysis in insect flight muscle the chief end-products are 1 mole each of pyruvate and a-glycero-P from each mole of glycosyl residue, the highenergy phosphate transferred to ADP to generate ATP is different from that typically described for mammalian tissues. The anaerobic formation of a-glycero-P is not concomitant with synthesis of ATP. In the formation of pyruvate, one equivalent of ATP results from the conversion of 1,3-diPglycerate to 3-Pglycerate and another is generated when P-enolpyruvate is transformed to pyruvate. Thus, per mole of glucose glycolyzed, 2 moles of ATP are used and 2 moles are regenerated, with no net gain of ATP. When starting with glycogen as the substrate instead of glucose, the synthesis of glucose-6-P is achieved

307

REGULATION OF INTERMEDIARY METABOLISM

without involvement of ATP. Hence, per mole of glucose residue glycolyzed, only 1 mole of ATP is utilized (the fructose-6-P kinase reaction), and the net gain in ATP is 1 mole. Thus, anaerobic glycolysis in insect muscle is extremely inefficient with respect to conservation of energy in the form of ATP and the need for an essentially completely aerobic metabolism is now fully appreciated. Indeed, the modifications, both anatomical and biochemical, that have evolved in the rapidly flying insect are largely those which provide for an efficient aerobic metabolism.

2.Control of Glycolysis

From concurrent measurements of the concentrations of glycolytic intermediates in flight muscle of the blowfly at rest and after periods of induced flight, the enzymatic reaction regulating glycolysis has been identified (Sacktor and Wormser-Shavit, 1966). On initiation of flight, the concentration of glucose-6-P decreases and that of fructose-6-P remains essentially constant. In contrast, the 4.

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308

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concentration of fructose-l,6diP increases during the first 5 s of the start of contraction (Fig. 14). The level of fructose-l,6-diP in the muscle reaches a peak at 15 s. The concentration of the hexose diphosphate returns to the resting level by the second minute of flight and from 10 to 60 min of the flight is maintained at a value slightly less than that measured initially. The slight decrease in the concentration of the hexose monophosphates coincident with a rapid accumulation of fructose- 1,6diP under conditions of maximal glycolytic flux brought about by initiation of active contraction identifies an additional crossover or control point, i.e. the phosphofructokinase reaction. The mechanism whereby the phosphofructokinase reaction is facilitated in the transition from a slowly metabolizing resting muscle to an intensely active working muscle becomes evident from an examination of changes in concentrations of the adenine nucleotides and Pi in flight muscle at the initiation of flight (Sacktor and Hurlbut, 1966). In earlier in vitro studies with mammalian enzymes, Lardy and Parks (1956) have discovered that, although ATP is a substrate for the reaction, excess ATP is inhibitory to phosphofructokinase. Passonneau and Lowry ( 1962) have found that the inhibition by ATP may be overcome by either ADP, AMP, Pi, 3’,5kyclic AMP, fructose-l,6-diP or, more effectively, by a combination of these activators. Lowry et al. (1964) postulate that whenever formation of ATP does not keep up with use of ATP, however slight, then Pi, ADP, and, particularly, AMP will increase and that this combination enhances phosphofructokinase activity autocatalytically. As shown in Fig. 15, on initiation of flight the concentration of ATP in blowfly flight muscle decreases whereas the levels of Pi, ADP and, especially, AMP increase. These changes, as flight begins, are in complete accord with their theory and extend the hypothesis to a working muscle in vivo. Recent studies by Grasso and Miglioro Natalizi (1968) with roach leg muscle confirm the effects of ATP and other phosphates on the phosphofructokinase in an insect tissue. Thus, their findings are consistent with the previously suggested mechanism for regulation of phosphofructokinase in insects. The pattern of changes in the concentrations of the other glycolytic intermediates in flight muscle during flight does not reveal any other locus of control of glycolysis (Sacktor and WormserShavit, 1966). The concentrations of dihydroxyacetone-P and 3-Pglycerate reflect the changes in the concentration of

REGULATION OF INTERMEDIARY METABOLISM

309

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fructose-l,6diP although neither increases as much as that of the hexose diphosphate. The concentrations of glyceraldehyde3-P, 2-P-glycerate and 2-Penolpyruvate are low initially and do not change significantly during prolonged muscular work (Sacktor and Wormser-Shavit, 1966). A purified hexokinase from honeybee flight muscle is inhibited by its products, glucose-6-P and ADP (Ruiz-Amil, 1962), but the significance of this inhibition in regulation of glycolysis in the working muscle is uncertain. Interestingly, the concentration of glucose-6-P in blowfly flight muscle at rest greatly exceeds the value observed after exercise (Fig. 7) and the ratio of glucose-6-P to fructose-6-P approaches the expected value of 3 : 1 during continuous flight, suggesting that the isomerase reaction is not at equilibrium in the resting muscle. A.1.P.-11

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G . IDENTIFICATION OF OTHER LOCI OF CONTROL OF METABOLISM

Sequential analyses of metabolites in flight muscle during flight have uncovered three additional points of regulation, the oxidations of a-glycero-P, pyruvate and proline. As noted above, aglycero-P and pyruvate are the end-products of glycolysis and they, as well as proline, are metabolized further by mitochondrial oxidations. The evidence on the identification and significance of these control sites will be presented in this section, whereas the mechanisms of the regulation will be discussed later in conjunction with a general exposition of regulation of oxidative activity in mitochondria.

1 .a-Glycero-P Oxidation Flight muscle of Phormia contains an exceptionally high concentration of aglycero-P, about 1.5 pmoles x g- 1 wet wt of thorax, a value 10-100 times those of other phosphorylated glycolytic intermediates (Fig. 14, Sacktor and Wormser-Shavit, 1966) and approximating the apparent K, of the mitochondrial dehydrogenase (Estabrook and Sacktor, 1958a). Significantly, the concentration of a-glycero-P does not change during flight. This is in contrast with the findings of a large accumulation of a-glycero-P during anaerobic glycolysis in insect muscle in vitro (Kubista, 1957; Chefurka, 1958; Heslop et al., 1963). It indicates that glycolysis in insect flight muscle during prolonged and continuous work is completely aerobic, the a-glycero-P produced concomitant with oxidation of glycolytically formed NADH being immediately oxidized by the extraordinarily active mitochondrial a-glycero-P dehydrogenase. Since, as pointed out previously, only a catalytic quantity of the regenerated dihydroxyacetone-P is needed in the a-glycero-P cycle, the excess triosephosphate is metabolized to pyruvate. Thus, these in vivo data demonstrate that essentially two equivalents of pyruvate are formed from each mole of hexose, verifying the concept and function of the a-glycero-P cycle in the intact organism. 2. Pyruvate Oxidation The concentration of pyruvate increases strikingly on initiation of flight (Fig. 14), doubling within the first few seconds and reaching a maximal value in 15 s. Thereafter, this concentration decreases rapidly so that by the second minute a steady-state level only slightly higher than that seen at rest is achieved. Coincident with the onset of active contraction and with the substantial rise in the concentration

REGULATION OF INTERMEDIARY METABOLISM

311

of pyruvate, there is an enormous accumulation of alanine in the flight muscle (Fig. 16, Sacktor and Wormser-Shavit, 1966). About 1 pmole/g (wet wt of thorax) is formed within 5 s. The concentration of alanine continues to increase after 15 s, the time at which the concentration of pyruvate reaches its maximum. A maximal concentration of alanine is measured after 5 min of flight. Polecek and Kubista (1960) have also noted an increased concentration of pyruvate within the muscle of the roach after a flight of 1-3 min and Kirsten et al. (1 963) have found elevated concentrations of pyruvate and alanine after a 20 s flight of the locust. In addition to the formation of alanine after the onset of flight, pyruvate is also converted to acetyl carnitine (Childress et al., 1967). A four-fold increase in acetyl carnitine parallels the four-fold increase in pyruvate. After about 1 min of flight the levels of both acetyl carnitine and pyruvate decrease, with acetyl carnitine attaining a steady-state concentration of about twice that in the muscle at rest. The increase in concentration of pyruvate and accumulations of both alanine and acetyl carnitine in the transition from rest to flight suggest that, on the initiation of flight, pyruvate is not metabolized in the citric acid cycle as fast as it is formed by glycolysis. This indicates that there is a limitation in the oxidation of pyruvate that is relieved shortly after the onset of flight. The nature of this control will be considered subsequently.

3.Pro line Oxidation The large accumulation of alanine with the induction of contraction has prompted a search for potential sources of the amino group (Sacktor and Wormser-Shavit, 1966). As shown in Fig. 16, the increase in alanine approximates the decrease in proline, which is present in resting muscle at a remarkable concentration of over 6 pmoles x g-1 wet wt of thorax. A slight decrease is found in the concentration of glutamate, while no consistent or significant changes are observed in the concentrations of aspartate, free NH;, total amide, glutamine, or asparagine within the muscle on initiation of active contraction or during prolonged flight. The coincident and stoichiometric relationship between the formation of alanine and the utilization of proline suggests that the amino moiety of alanine is derived mostly from the large store of free proline. Bursell (1963) has also reported a decrease in the concentration of proline during short flights of the tsetse fly and has suggested that the amino acid is of significance as an energy reserve in this pest because of the

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blood-sucking habit of the latter as well as its lack of carbohydrate stores. The triggering of the rapid decrease in the concentration of proline in flight muscle of the blowfly on initiation of flight demonstrates the sixth enzymatic site of regulation in the rest-to-flight transition. IV. REGULATION OF FAT METABOLISM

As in the present discussion of carbohydrate metabolism, this examination of the regulation of fat metabolism will not include all the details of the biosynthetic and degradative pathways nor the multitude of data on various aspects of the biochemistry of lipids. These topics have been dealt with in earlier reports by Gilby (1 965), Tietz (1965), and Sacktor (1965) and more recently in the comprehensive review by Gilbert ( 1967a). The present discussion is limited, in part, by the author’s interests and, therefore, will treat topics related to flight muscle metabolism more extensively than subjects not necessarily of less importance.

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313

A. FATTY ACID CATABOLISM

1. Utilization of Fatty Acids As described in a previous section, the fact that some insects deplete their reserves of fat during sustained flight indicates that fat can serve as a metabolic fuel for muscular contraction. In these cases, the importance of lipid should not be underestimated. Lipids are the most concentrated source of energy, yielding per gram over twice as many calories as d o carbohydrates or proteins. However, it should be reemphasized, as pointed out earlier (Sacktor, 1965), that locusts and roaches, species which utilize fats in a sustained flight, will first consume their carbohydrates (Bucher and Klingenberg, 1965 ; Hofmanova et al., 1966). Furthermore, the conclusion that moths exclusively utilize fats for flight (Zebe, 1954; Domroese and Gilbert, 1964) is not indubitable. The RQ of a prolonged flight is not indicative of the metabolic events at the beginning of flight; indeed, the RQ of flying moths fed glucose, although low, is higher than those of individuals not fed (Zebe, 1954). Moreover, the observations that added glucose fails to increase the endogenous rate of 0, uptake in muscle homogenates and that the relative rates of 14C02 produced from labeled glucose and pyruvate are low (Domroese and Gilbert, 1954) may not indicate the full potential of the muscle because there is no indication that the conditions are optimal for these substrates in these experiments. In fact, Stevenson (1968a) has shown that flight muscle homogenates of the Southern armyworm moth, Prodenia, can completely and rapidly oxidize glucose, trehalose and glycogen and that mitochondria isolated from this muscle metabolize pyruvate plus malate at a rate comparable to that measured with mitochondria from flies. The glycogen content of Prodenia is relatively low, but it is sufficient to support flight for about 8 min. On the other hand, the ability of fly mitochondria to oxidize fatty acids is extremely limited (Sacktor, 1955; Childress et al., 1967), although Gregory et al. (1968) claim that in the development of the flight muscle in the pupa of the blowfly, Lucilia, fatty acids are utilized significantly for the synthesis of ATP. Thus, the distinction between insects that supposedly use only fats and those that utilize only carbohydrates is even more equivocal than has been suggested previously (Sacktor, 1965). 2. Oxidation of Fatty Acids in Muscle Based on known values of 0, uptake in locusts during flight, Beenakkers ( 1965) has calculated that the flight muscle will consume

314

B. SACKTOR

fatty acids at a rate of 2 3 mg x g-' wet wt of muscle x hr-'. In vitro studies by Meyer et al. (1960), Domroese and Gilbert (1964) and Bode and Klingenberg (1-964) show the utilization of fatty acids by flight muscle at a rate only a fraction of this calculated in vivo rate. However, with the work of Stevenson (1 966, 1968b) using isolated flight muscle mitochondria from Prodenia, rates of respiration greater than 700 pl O2 x mg-' mitochondrial protein x hr-' are reported, and these values approach the required in vivo rates. The requirement for ATP, MgZ+,CoA and a member of the Krebs cycle (for priming) for maximal rates of oxidation of fatty acid by flight muscle preparations (Meyer et al., 1960; Domroese and Gilbert, 1964), suggests that in insects the activation of the fatty acid to its acyl CoA derivative and, in turn, its catabolism via the poxidation pathway of fatty acids is the same as that established for vertebrate systems. Although the overall process of P-oxidation has yet to be demonstrated in insects, the findings of 0-ketoacylthiolase and P-hydroxyacyl dehydrogenase in locust flight and leg muscle (Zebe, 1960; Beenakkers, 1963a, b) as well as fatty acyl-CoA synthetase in moths (Domroese and Gilbert, 1964; Stevenson, 1968b), strongly support the presence of the entire sequence of reactions in muscle of insects. The enzymes are localized largely in the mitochondria of the flight muscle (Beenakkers, 1963a; Stevenson, 1968b). Successive repetition of the P-oxidation cycle results in the complete degradation of even-numbered fatty acids to acetyl CoA. The acetyl CoA generated by degradation of fatty acids pools with acetyl CoA arising from the oxidative decarboxylation of pyruvate, derived largely from glycolysis. The fate of acetyl CoA, upon its entry into the citric acid cycle, will be considered later. B. THE ROLE OF CARNITINE

In pioneering experiments, Friedman and Fraenkel (1 955) have shown that extracts of mammalian tissues mediate the reversible acyl transfer between CoA and carnitine. The physiological significance of this reaction becomes apparent with the finding of Fritz (1 955) that carnitine stimulates the rate of oxidation of fatty acids. This and other studies have led to the hypothesis (Fritz and Marquis, 1965) that fatty acyl CoA thioesters d o not readily penetrate mitochondrial membranes, whereas fatty acyl carnitine esters do, and that the formation of carnitine esters by acyl transferases effects the translocation of fatty acyl groups to the site of fatty acid oxidation. In accord with this view, Beenakkers (1963b) and Bode and

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315

Klingenberg (1964) have found that added carnitine markedly stimulates the oxidation of fatty acids in locust flight muscle and that fatty acids supplied as acyl carnitine esters are metabolized at even greater rates. The requirement for carnitine for oxidation of fatty acids is strongly correlated with the presence in the muscle of active carnitine-acetyl and -palmityl transferases (Beenakkers and Klingenberg, 1964; Beenakkers et al., 1967). Particularly striking are the differences in transacetylase activity between the flight muscle of two insects, those of the locust, which oxidize fatty acids, and those of the bee, which utilize only carbohydrates in flight. The enzyme is absent from flight muscle of the bee, whereas it is very active in locust flight muscle. Conflicting with this scheme, however, are the surprising observations that flight muscle of two species of moths oxidize palmitate vigorously without added carnitine and that carnitine palmityl transferase cannot be detected in the muscles (Stevenson, 1966, 1968b). Interestingly, the flight muscle of the blowfly, which like the bee is deficient in fatty acid oxidase and has only a negligible capacity to oxidize palmityl carnitine, has a high content of carnitine and a very active acetyl carnitine transferase (Childress et al., 1967). It has been found in the fly, but not in the bee, that carnitine affects carbohydrate utilization, via a role in pyruvate metabolism. The acetyl carnitine transferase in mitochondria from flight muscle of Phormia catalyzes the synthesis of acetyl carnitine from carnitine and acetyl CoA, derived from pyruvate. Formation of acetyl carnitine has been demonstrated both in vitro and in vivo; on initiation of flight its concentration in flight muscle increases four-fold, paralleling the increase in pyruvate (Childress et al., 1967). Approximately 90% of the acetyl carnitine transferase in flight muscle of Phormia is found in the mitochondria (Childress et al., 1967). Exogenous acetyl CoA, in the presence of carnitine, is not oxidized by mitochondria, although acetyl carnitine is oxidized with a QO, of over 300. This indicates that the blowfly mitochondrial inner membrane is not permeable to the thioester and that the mitochondrial carnitine acetyl transferase does not transfer acetyl groups from extramitochondrial acetyl CoA to carnitine and, thus, into the mitochondrial matrix. Instead, the evidence suggests that the mitochondrial enzyme mediates the transfer of acetyl groups out of the mitochondria. On the other hand, the apparent presence of about 10% of the carnitine acetyl transferase activity in the extramitochondrial fraction of the muscle may permit the extramitochondrial

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acetylation of carnitine, with subsequent transport of the acetyl carnitine into the mitochondria. In contrast to the situation in blowfly mitochondria, in both locust (Beenakkers and Henderson, 1967) and moth (Stevenson, 1968b) flight muscle, exogenous acetyl CoA plus carnitine as well as acetyl carnitine, but not acetyl CoA alone, are oxidized at appreciable rates. From this, it has been inferred that there are two pools of acetyl carnitine transferase; one between the outer and inner membrane, the other within the cristae space. The formation of acetyl carnitine in the blowfly at the start of flight raises the question as to its physiological significance. It has been shown, as discussed in a previous section, that during the initial phase of flight pyruvate is generated faster than it is utilized via the Krebs cycle. Childress et al. (1967) have offered several possible ways in which the formation of acetyl carnitine during this critical period can be of advantage t o the blowfly. These possibilities include: (1) lowers the acetyl CoA : CoA ratio, alleviating inhibition of pyruvate decarboxylase; (2) makes free CoA available for the oxidation of a-ketoglutarate t o succinate, providing oxaloacetate for citrate synthesis; (3) increases the energy production from glycolysis from 6 to 12 moles of ATP per mole of glucose; and (4) provides a readily oxidizable substrate (acetyl carnitine) rather than alanine during the period prior t o the maximal activation of pyruvate oxidation. A more comprehensive discussion of these alternative mechanisms has been presented earlier (Childress et al., 1967). C. BIOSYNTHESIS OF FAT

Data on the mechanisms for biosynthesis of fats in insects are exceedingly limited. In general, the existing knowledge tends to suggest that insects synthesize fat by pathways similar to those which operate in microbial and mammalian systems. Acetate, injected into insects, is readily transformed into fats (Robbins et al., 1960; Louloudes et al., 196 1;Sedee, 1961 ; Sridhara and Bhat, 1964; and Bade, 1964). Labeled acetate is incorporated primarily into C I 6 C l 8 fatty acids, both saturated and unsaturated. Strong (1963) has reported the conversion of U-l"C-glucose to fats in aphids; the pattern of fatty acids formed from glucose is not significantly different from that produced with acetate as precursor. Experiments with specifically labeled glucose indicate synthesis of fatty acid via pyruvate, decarboxylation of pyruvate and incorporation of 2-carbon units into lipids (Horie et al., 1968).

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317

Interestingly, Van Handel and Lum (1 96 1) have found that female mosquitoes, but not houseflies nor male mosquitoes, can synthesize fatty acids from glucose. Locust fat body, in vitro, converts the carbon moiety of amino acids into fat (Clements, 1959). For the most part, the fatty acids synthesized from acetate and glucose are found combined as triglycerides and phospholipids (Tietz, 196 1). Clements (1959) and Zebe and Mcshan (1959b) have demonstrated the in vitro biosynthesis of fatty acids using isolated, intact fat body and cell-free homogenates of fat body, respectively. Primarily, saturated long-chain fatty acids are formed, although Bade and Clayton (1963) have shown that stearate is rapidly desaturated to oleate. The cell-free system requires or is stimulated by ATP, Mg2+,glutathione, bicarbonate, malonate, CoA, an intermediate of the Krebs cycle, and NADP (Zebe and McShan, 1959b; Tietz, 1961). Under optimal conditions, the particle-free supernant (20,000 x g for 20 min) is as active as the whole homogenate in incorporating acetate into fatty acids. Preincubation with avidin completely inhibits fatty acid synthesis. The inhibition is reversed by biotin. The stimulatory effect of NADP on production of fatty acids strongly suggests a role for NADPH in reductive synthesis. An active hexose monophosphate pathway, which yields NADPH in the oxidations of glucose-6-P and 6-P-gluconate, has been discovered in fat body of silkworms (Horie et ul., 1968). Moreover, the rate of NADPH oxidation in this tissue is remarkably dependent on both malonyl CoA and acetyl CoA (Horie, 1968). These results, although fragmentary at best, do support the view that insects synthesize fatty acids in a manner similar to those of mammals and microorganisms. A generalized scheme for fatty acid biosynthesis has been described in the recent review of Gilbert (1 967a). Zebe and McShan (1959b) have demonstrated that, although fat body is a major site of fat formation, synthesis of fatty acids can take place in muscle. Glucose is incorporated into fatty acids in muscle at a rate approximately 20% that in fat body. Mitochondria from Drosophilu larvae are also capable of de novo synthesis of fatty acids (Goldin and Keith, 1968). Acetate is incorporated into even-number, saturated and monoenoic, fatty acids from 12-18 carbons in chain length. Antimycin A, an inhibitor of the mitochondrial respiratory chain, lowers the rate of incorporation of acetate but the rate can be partially restored by ATP. Labeled acetate appears in free fatty acids, neutral lipids, as well as phospholipids. It is not known whether this mitochondria1 system for fatty acid A.1.P.-1 1

318

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biosynthesis differs from that found in the soluble fraction of the fat body (Tietz, 1961). The mechanism by which fatty acids are incorporated into glycerides in insects is essentially unknown. Isolated fat body from cecropia, roach and locust incorporate 14C-palmitate into diglyceride and triglyceride (Chino and Gilbert, 1965; Tietz, 1962, 1967). Incorporation into phospholipid is negligible. The total amount of triglyceride is approximately 5 0 times greater than that of the diglyceride, so that most of the label appears in the triglyceride fraction. However, the specific activity of the diglyceride is at least 50 times greater than that of the triglyceride (Tietz, 1967). Preliminary studies from Tietz's laboratory indicate that the biosynthesis of glycerides proceeds by the pathway previously shown for mammalian tissues, namely : a-Glycero-P + 2 Acyl-CoA +. Phosphatidic acid Phosphatidic acid + H20+. DqP-Diglyceride + Pi D-a,P-Diglyceride + Acyl-CoA +.Triglyceride

Tietz (1967) reports that the relative amounts of palmitate incorporated into diglyceride and triglyceride will depend on the relative concentrations of a-glycero-P and diglyceride available as acceptors for palmityl CoA. Using fat body of resting locusts, which have a relatively high level of tissue a-glycero-P, biosynthesis of diglyceride predominates. In fat body from flown locusts, which is depleted of glycogen and the concentration of a-glycero-P is, therefore, low, the incorporation of palmitate into triglyceride will prevail. Addition of glucose to the incubation medium containing fat body from flown locusts will initiate a-glycero-P formation, and the biosynthesis of diglyceride will proceed at a higher rate. There are suggestions that lipid synthesis in fat body is under hormonal regulation. Bodenstein (1953) has reported that in the roach, Periplunetu, removal of the corpora allata results in a quantitative increase in the fat deposited in the fat body. This effect of the corpus allatum has been confirmed in the blowfly (Orr, 1964). Vroman et ul. (1965) have shown that incorporation of acetate into the triglyceride fraction is more than doubled in the allatectomized insects, whereas incorporation into phospholipid is not appreciably affected. Allatectomy also noticeably slows the turnover of both triglyceride and phospholipids and it has been suggested that the corpora allatum hormone increases triglyceride by regulating the mechanisms responsible for the utilization of lipids, i.e. ovarian

REGULATION OF INTERMEDIARY METABOLISM

319

development (also, see Gilbert, 1967b). In the female mosquito, the medial neurosecretory cells restrict the synthesis of glycogen and stimulate triglyceride synthesis (Van Handel and Lea, 1965). Removal of these cells greatly increases the storage capacity for glycogen at the expense of triglyceride storage. However, these authors caution against ascribing the effects directly to a neurosecretory cell hormone since bilateral sectioning of the combined nervus corporis allati and esophagi, which emanate from these cells, has the same metabolic effect as removal of the neurosecretory cells. D. MOBILIZATION AND TRANSPORT OF FAT

It has been calculated that a locust possessing about 180 mg of flight muscle consumes fatty acids at a rate of 4.1 mg x hr-1. The fatty acid content of the muscle is about 3 mg, while the fat body has more than 18 mg (Beenakkers, 1965). Since the locust flies continuously for 7-8 hr and its reserve of carbohydrate can last for only 1-2 hr, it is obvious that during flight fat is mobilized in the fat body and is transported to the flight muscle, presumably by way of the blood. 1. Release of Fatty Acids from Fat Body

I’ietz (1962) has found that when fat body of locusts, previously prelabeled with 14C-palmitate, is incubated in hernolymph, glycerides are released from the tissue into the medium. The effect of blood is specific; little glyceride is released in phosphate-saline, bovine serum, or buffered solutions of albumins. The amount of glyceride released is proportioaal to the amount of hemolymph that is added. Effectiveness of the blood is not affected by dialysis, but is destroyed by heating, and is inhibited by fluoride and cyanide. Confirmatory observations have been reported by Chino and Gilbert (1965) for three additional species. Further, they have noted that the release of glyceride is inhibited by azide and dinitrophenol. Chino and Gilbert (1965) have concluded that in pupal and adult cecropia, as well as in the roach and grasshopper, that the glyceride released by prelabeled fat body is in the form of diglyceride. Little triglyceride is liberated. Free fatty acids are found in the incubation medium; however, this release is not specific for insect hemolymph but is also enhanced by serum albumin. The metabolic inhibitors that block the release of diglyceride stimulate the yield of free fatty acids. The apparent accelerated rate of release of free fatty acids by the inhibitors is attributed t o their interference with the incorporation of

3 20

B. SACKTOR

free fatty acid into neutral lipid. According to Gilbert (1967a), the release of free fatty acids is a passive process which follows a concentration gradient and depends on the level of free fatty acid in the fat body. Recently, the possible presence of two different glyceride pools in fat body, only one of which directly releases fatty acids to the blood, has been suggested (Beenakkers and Gilbert, 1968). As noted previously, when isolated fat body is incubated with IT-palmitate, the fatty acid is incorporated into both di- and triglycerides. The specific activity of the diglyceride in the tissue is 50 times that of triglyceride, although the amount of triglyceride in the fat body is far greater (Chino and Gilbert, 1965; Tietz, 1967). When the prelabeled fat body is transferred to a medium containing hernolymph, the quantity of diglyceride that is released is greater than its concentration in the fat body, although the amount in the tissue does not change. However, its specific activity is markedly reduced. Consequently, the average specific activity of the glycerides in the blood is much greater than the average specific activity of the glycerides in the fat body because the diglyceride with an extremely high specific activity is continuously being released into hemolymph, while the low specific activity triglyceride largely remains in the fat body. When the fat body is prelabeled in vivo and the specific activities of the di- and triglyceride are identical, the specific activity of the diglyceride in the fat body is not reduced during diglyceride release (Tietz, 1967). The general conclusion that diglyceride is specifically released from the fat body of insects (Gilbert, 1967a), may now need revision. Wlodawer and co-workers (1966, 1967) have suggested that, in the larvae of the waxmoth, Galleria, prelabeled fat body releases free fatty acids into the hemolymph and these are subsequently incorporated into triglyceride by an active lipase in the blood. Very little radioactivity is found in diglyceride although the diglyceride represents the largest fraction of the glycerides. The findings of Cook and Eddington ( 1967) with Periplunetu are also inconsistent with the views of Gilbert and Tietz. Analytical as well as isotopic analyses show that free fatty acids and triglyceride are the major lipids released from fat body. Diglyceride appears to be the principal lipid in the hernolymph, however, as shown previously by Chino and Gilbert (1965). The contrast between the data of Cook and Eddington and those of Chino and Gilbert (1965) on the identification of the fatty acid that is released may be due to the

REGULATION OF INTERMEDIARY METABOLISM

321

latter authors’ use of counts rather than analyses, of levels of hemolymph which are well below the optimal for triglyceride release, and of not considering fully the consequences of diffusion. During flight the concentration of diglyceride in the hemolymph of bcusts increases several-fold (Beenakkers, 1965; Tietz, 1967). The levels of triglyceride and phospholipids in the blood do not change. Whether there is a change in the free fatty acid content of the blood of flown locusts is still inconclusive (Beenakkers, 1965 ;Tietz, 1967). The fat body is the source of the increased diglyceride in the blood and it has been suggested that the excess fatty acids are transported to the muscle for utilization in flight. However, until the turnover rates of the hernolymph lipids during the flight are known, it is difficult to assess the relative contributions of the different forms of lipid to flight metabolism.

2. Transport of Fat in Hemolymph

The specific requirement for hemolymph in the liberation of lipids from the fat body suggests the involvement of a blood protein in the release and/or transport mechanism. Indeed, Tietz (1962) has shown that a lipoprotein component of locust blood becomes radioactive after incubation with prelabeled fat body and Chino and Gilbert ( 1955) have identified a diglyceride-hemolymph protein conjugate. In cecropia, four distinct protein bands are resolved electrophoretically, three of which are lipoproteins. Almost all the radioactive glyceride is concentrated with a single fraction (Chino and Gilbert, 1965). The three classes of lipoproteins have been isolated by ultracentrifugal techniques (Thomas and Gilbert, 1968). A high density lipoprotein contains about 75% of the total lipid. Electrophoresis demonstrates that each class is composed of several lipoprotein species. Previously, a diglyceride-bound lipoprotein from Cynthia has been isolated by chromatography and is described as a globulin-like protein (Chino et al., 1967). Acrylamide gel electrophoresis of the isolated lipoprotein reveals two protein bands, indicating also that the fraction obtained is not yet pure. Mayer and Candy (1967) have examined changes in hemolymph lipoprotein during flight of the locust. Electrophoresis of blood from resting locusts shows eight protein components, only two (Group A) of which contain lipid. After a 2 hr flight, the total lipid content (mainly diglyceride) of the hemolymph increases three to four times the resting value. Part of this increase, 70%, is accounted for by an increase in the lipid content of the A group lipoproteins. The

322

B. SACKTOR

remaining 30% is found in a second pair of proteins (Group B), which previously have been devoid of lipid. Group A lipoprotein contains both tri- and diglycerides, whereas Group B lipoprotein contains chiefly diglyceride. Three hours after flight has stopped the hemolymph pattern has returned to the resting state.

3. Uptake of Fat by Muscles The mechanisms by which the glycerides from the lipoproteins in the blood are transported into the muscle are essentially unknown. Lipase activities have been demonstrated in leg and flight muscles (George and Bhakthan, 1960a, b , 196 1). Considerably greater activities of the enzyme are found in flight muscle of locusts and dragonflies than in that of bumblebees. This difference between species seems to be correlated with locusts’ and dragonflies’ dependence on fat as a metabolic fuel during prolonged flights, whereas bumblebees, if like the honeybee, rely exclusively on carbohydrate for the energy for flight. Significantly, the lipase in flight muscle of cecropia hydrolyzes diglyceride at a rate of five times that of triglyceride (Gilbert et al., 1965). The lipase is not Ca*+-activated, As a consequence of lipase activity, free fatty acids and glycerol are formed. The role of cartinine in the transport of fatty acids has been considered previously. Glycerol is glycogenic and follows the pathway of carbohydrate in metabolism. Flight muscles of flies oxidize glycerol at a very slow rate (Sacktor, 1955). Presumably, glycerol is first converted to a-glycero-P at the expense of ATP in a reaction catalyzed by glycerol kinase. The diminutive rate of oxidation of glycerol relative to that of a-glycero-P suggests that the kinase reaction is rate-limiting.

V. REGULATION OF MITOCHONDRIAL METABOLISM

The large increase in rate of oxygen uptake upon initiation of flight indicates that there is an exceptionally high degree of respiratory control in flight muscle, in vivo. Since cellular respiration is attributed almost exclusively to mitochondria (Watanabe and Williams, 195 1 ; Sacktor, 1953b), an examination of mitochondrial metabolism is crucial to the understanding of the regulatory mechanisms at the three mitochondrial loci of control in flight muscle; namely, the oxidations of pyruvate, proline and a-glycero-P.

REGULATION OF INTERMEDIARY METABOLISM

3 23

A. THE RESPIRATORY CHAIN AND OXIDATIVE PHOSPHORY LATION The primary energy-conserving reaction in mitochondria is the formation of ATP coupled to the exergonic passage of protons and electrons from substrate to molecular oxygen via the respiratory chain. This vital process, oxidative phosphorylation, accounts for over 90% of the ATP generated. The components of the respiratory chain (NAD, flavoproteins, quinone, and the cytochomes), their spectral properties, concentrations and kinetic parameters, have been examined in detail in mitochondria of flight muscle of flies (Chance and Sacktor, 1958; Estabrook and Sacktor, 1958b) and locusts (Klingenberg and Bucher, 1959, 196 1 ). These fundamental studies, and some others which are largely confirmatory in nature, have been reviewed in depth (Sacktor, 1965). Since then, recent advances on the respiratory enzymes in insects include the isolation, crystallization and determination of the amino acid composition of cytochrome b from houseflies (Ohnishi, 1966a, b) and the establishment of the primary structure of cytochrome c from flight muscle of Cynthia (Chan and Margoliash, 1966). A general discussion of the current views of the reaction mechanisms of oxidative phosphorylation can be found elsewhere. The status of oxidative phosphorylation in insects has been summarized by Sacktor (1 965) and Harvey and Haskell (1966). Recently it has become clear that the respiratory pigments and phosphorylating enzymes are essential constituents of the mitochondria1 inner membrane (Figs 4 and 5). If analogous to the situation as found in mammalian mitochondria (Kawaga and Racker, 1966), the respiratory chain of insects is an integral part of the cristae and the phosphorylating machinery is localized in the stalked spherical particles, shown in Fig. 6. Although these knobs have not been isolated from insect preparations, a Mg*+- and DNP-activated (Sacktor, 1953a; Sacktor and Cochran, 1957), cold-labile (Mills and Cochran, 1967), oligomycin-sensitive (Hansford and Sacktor, 1970a) ATPase has been described. A fairly typical experiment illustrating classical respiratory control by ADP is shown in Fig. 17. Mitochondria exhibit high rates of respiration in the presence of ADP and Pi (State 3) but respire only slowly in the absence of phosphate acceptor (State 4). The respiratory control ratio, defined as the ratio of the rate of oxygen uptake in the presence of added ADP to the rate of respiration after the added ADP has been completely utilized, is approximately 6 in this experiment (Childress and Sacktor, 1966). The ADP : 0 ratio is

324

B. SACKTOR

defined as the ratio of the pmoles of ADP added t o the patoms of oxygen utilized, induced by the addition of ADP. A value of about 2.7 for the oxidation - of pyruvate approaches the theoretical maximum of 3.0 for this substrate. These ratios are in general agreement with those obtained manometrically by Gregg e t al. (1960) and Van den Bergh and Slater (1962) with fly mitochondria

F l

RESPIRATORY CONTROL DURING PYRUVATE OXIDATION

2.67

5.95

RATES IN MATOMS 0 2 / M I N I

c . 2 #MOLE ADP

Fig. 17. Respiratory control of pyruvate oxidation by blowfly mitochondria. (From Qlildress and Sacktor, 1966.)

and potentiometrically by Klingenberg and Bucher (1 959) with locust mitochondria. Although the stimulation of the rate of pyruvate oxidation by ADP is apparently indicative of tightly coupled mitochondria, respiratory control values with aglycero-P as substrate are relatively small when measured with the same insect preparations. Thus, R.C. ratios ranging from no stimulation to a high of about 3 have been reported for isolated mitochondria (Sacktor, 1954; Sacktor and Cochran, 1958; Gregg et al., 1960;Van den Bergh and Slater 1962; Birt, 1961; Klingenberg and Bucher, 1959; Hansford, 1968), and from 3-5 with the use of teased muscle preparations (Sacktor and Packer, 196 1). As emphasized previously

REGULATION OF INTERMEDIARY METABOLISM

3 25

(Sacktor, 1965), it is most important to point out that these observed stimulations of pyruvate and a-glycero-P oxidation by ADP, although of considerable significance, are much too small to account, alone, for the physiological control of respiration in the insect initiating flight. 9. CONTROL OF PYRUVATE OXIDATION As discussed in a previous section, the increase in concentration of pyruvate and accumulations of alanine and acetyl carnitine in the transition from rest to flight demonstrates that, on initiation of flight, pyruvate is not metabolized in the Krebs cycle as fast as it is formed by glycolysis. This indicates that there is a control on the oxidation of pyruvate that is released shortly after the onset of flight. The mechanisms of this regulation will be considered at this time. 1. Respiratory Substrates and Permeability of Mitochondria

Although historically there has been much dispute as to the relative abilities of fly flight muscle mitochondria to oxidize a-glycero-P, pyruvate and Krebs cycle intermediates (see Sacktor, 1965), it is now clear, as shown in Table IV, that intact mitochondria oxidize, at appreciable rates, only exogenous a-glycero-P, pyruvate, TABLE IV Respiratory activities of mitochondria from blowfly flight muscle Substrate a! -Glycero-P

Pyruvate Acetyl carnitine Proline Citrate a-Ketoglutarate Succinate Fumarate Malate Glutamate Asuarate

QOz 1400 65 5 330 130

10 45 60 35

30

25 10

QO, = rlOz x mg-I mitochondria1 protein x h r l . Data compiled from Sacktor and Childress (1967), Childress e l al. (1967), Childress and Sacktor (1966) and Chance and Sacktor (1958).

326

B. SACKTOR

acetyl carnitine and, to a lesser extent, proline. Other experiments (Van den Bergh and Slater, 1962; Hansford, 1968) show approximately equal rates of oxidation with a-glycero-P and pyruvate. Indeed, in some instances pyruvate is oxidized at twice the rate for a-glycero-P (Hansford, 1968). In contrast, Krebs cycle intermediates, such as citrate, a-ketoglutarate, succinate and malate, the amino acids glutamate and aspartate, and NADH, added to isolated mitochondria are not effective respiratory substrates (Chance and Sacktor, 1958; Van den Bergh and Slater, 1962; Childress and Sacktor, 1966; Childress e t al., 1967; Sacktor and Childress, 1967). An explanation for the low rates of oxidation of these substrates has been suggested by Van den Bergh and Slater (1962), who have discovered the unusual phenomenon that the mitochondria are not readily permeable to these compounds. Subjecting mitochondria to sonic disintegration or freezing-thawing, procedures that damage the mitochondrial membranes, increase the respiratory rates with these substrates many-fold (Van den Bergh and Slater, 1962; Sacktor and Childress, 1967). On the other hand, the oxidations of a-glycero-P and pyruvate are not stimulated by the disruptive techniques, provided cofactors are added back to the reaction. Van den Bergh (1967) can find no evidence in fly flight muscle mitochondria for the presence of specific exchange-diffusion carriers for Krebs cycle substrate anions.

2. The Function of Proline in Pyruvate Oxidation Isolated mitochondria rapidly lose the capacity to oxidize pyruvate (Childress and Sacktor, 1966; Sacktor and Childress, 1967). This loss can be reversed by proline but not by Krebs cycle intermediates nor glutamate (Fig. 18). Since blowfly flight muscle mitochondria are permeable to proline, but not t o other amino acids nor metabolites of the citric acid cycle, these findings suggest that proline enhances the rate of pyruvate metabolism by penetrating the mitochondrial barrier, forming intramitochondrial precursors of oxaloacetate, enabling the synthesis of citrate, and effecting the complete oxidation of pyruvate via the Krebs cycle at a maximal rate. In support of this view, it has been found that proline is metabolized by flight muscle with formation of A'-pyrroline-5carboxylate and glutamate (Bursell, 1963; Brosemer and Veeradhadrappa, 1965; Sacktor and Childress, 1967). Transamination of glutamate with pyruvate gives rise to alanine and a-ketoglutarate. The intramitochondrial a-ketoglutarate is further metabolized to

REGULATION OF INTERMEDIARY METABOLISM

3 27

dicarboxylic acids, forming oxaloacetate and C 0 2 (Sacktor and Childress, 1967; Bursell, 1967). Alternatively, the bicarbonate liberated by this sequence of reactions can be used to provide oxaloacetate by the enzyme pymvate carboxylase (Hansford and Chappell, 1968). The carboxylase has been reported in flight muscle (Lewis and Price, 1956; Pette et al., 1962; Bursell, 1965). RESTORATION OF PVRUVATE OXIDATION I V PROLINE

A-ASSAYED IMMEDIATELV B,C,L D ASSAYED AFTER 4 0 MIN EXPOSURE TO MEDIUM

0 . 9 4 n A T O M 02

\

M W

CVRUVATE IN MEDIUM INITIALLY

1.0 m M PYRUVATE IN MEDIUM INITIALLY

I.

\\

5.0 m M PROLINE IN MEDIUM INITIALLY

5.0 mM PROLINE

10mM GLUTAMAT

.._ .nM PVRUVATE

5.0 n M PROLINE

A R A T E S I N IA A T O M S

Fig. 18. Restoration of pyruvate oxidation by proline. (From Sacktor and Childress, 1967.)

The control on pymvate oxidation at the initiation of flight suggests that, in vivo, flight muscle mitochondria may be deficient in Krebs cycle intermediates and that these are generated from proline. By this mechanism, one of the limitations in the oxidation of pyruvate during flight is relieved.

3. The Requirement for Piand ADP for Activation at the Dehydrogenase Level During a study of kinetic factors controlling Krebs cycle activity in mitochondria from flight muscle of the blowfly, Calliphora, Hansford and Chappell (1968) have discovered that the rate of pyruvate oxidation is markedly influenced by Pi. As shown in Fig. 19, more than 25 mM Pi is required for a maximal State 3 rate. It is

328

B. SACKTOR

unlikely that this requirement has anything to do with respiratory chain phosphorylation as only 2 mMPi is needed for the ADP-stimulated oxidation of a-glycero-P. In contrast, the State 4, or controlled, rate of pymvate oxidation is unaffected by Pi. Thus, in the presence of ADP, the rate of oxygen uptake with pymvate increases by a factor of about 10 as the concentration of Pi is raised from 1.3 to 25 mM.

Concn. of Phosphate mM Fig. 19. The dependence upon Pi concentration of the rate of pyruvate oxidation by blowfly mitochondria. Incubation mixture contained pyruvate, bicarbonate, ATP and an excess of ADP. (From Hansford, 1968.)

This finding implies that the enzyme catalyzing the rate-limiting step of the Krebs cycle requires a very high level of Pi. Likely candidates are the substrate level phosphorylation concomitant with sketoglutarate oxidation and the NAD-linked isocitrate dehydrogenase. Substrate level phosphorylation has been largely ruled out, since at 1.2 mM Pi gramicidin-treated mitochondria oxidize a-ketoglutarate at a rate well in excess of that needed to be

329

REGULATION OF INTERMEDIARY METABOLISM

consistent with the very low rate of pyruvate oxidation at this level of Pi (Hansford, 1968). On the other hand, the NADdependent isocitric dehydrogenase of blowfly ritochondria has an absolute dependence on Pi (Fig. 20). Moreover, the requirement for Pi is very high; at 5 mM only 5% of the maximal activity is expressed, activity is 80%of maximum at 30 mM Pi (Hansford and Chappell, 1968).

. In al

d

0

E 1

E

z

0 bV 3

n

W LY

n

a

z

0

0

20

4Q

60

80

100

mM PHOSPHATE Fig. 20. The effect of Pi on blowfly NAD-isocitric dehydrogenase activity. (From Hansford and Chappell, 1968.)

Further support for the view that isocitrate dehydrogenase is subject to tight regulation and may limit pyruvate oxidation in the Krebs cycle has come from the finding that the enzyme is activated by ADP, as shown in Fig. 2 1. The mechanism of action of ADP is to lower the K, for isocitrate (Hansfordland Chappell, 1968). In the presence of a concentration of isocitrate approximating that found in the mitochondrion, there is a 20-fold increase in enzyme activity on adding ADP. It has also been found that the dehydrogenase is strongly inhibited by ATP (Hansford and Chappell, 1968). Thus,

330

B. SACKTOR

although the stimulation by ADP and inhibition by ATP are dependent on the concentrations of the effectors, the activity of isocitric dehydrogenase is determined by the relative proportions of the two nucleotides in a mixture of a fixed total concentration of adenine nucleotide. A concentration of adenine nucleotide in the mitochondria of about 6 mM has been estimated (Price and Lewis,

0 1 2 3 4 Concn. of ADP mM

Fig. 21. The dependence of isocitric dehydrogenase activity upon the level of ADP. (From Hansford, 1968.)

1959; Hansford, 1968), and, if one assumes that in resting flight muscle most of the adenine nucleotide is ATP (Sacktor and Hurlbut, 1966), then the control of isocitrate dehydrogenase and, in turn, pyruvate oxidation by this mechanism must be quite rigorous. C. CONTROL OF PROLINE OXIDATION

As described in Fig. 16, the level of proline in flight muscle drops abruptly on the initiation of flght. It has been suggested that the mitochondria1 oxidation of proline is facilitated by the rest-to-flight

33 1

REGULATION OF INTERMEDIARY METABOLISM

transition and that this oxidation is crucial in providing the Krebs cycle intermediates necessary for the rapid and complete oxidation of pyruvate (Sacktor and Childress, 1967). In view of this role, the mechanism for the regulation of proline oxidation is important. Hansford and Sacktor (1970a) have found that the oxidation of proline by flight muscle mitochondria from Phormiu is stimulated by

0 12

-

0.10

f

s ' 0.08 N

0 v3

s

0.06

m

=l

w

k 0.04 a 0.02

2

1

3

mM ADP Fig. 22. The effect of ADP on proline oxidation. The uncoupling agent, FCCP, is in the reaction mixture. (From Hansford and Sacktor, 1970a.l

ADP in the presence of uncoupling agents and oligomycin (Fig. 22). The stimulation is enhanced further by high levels of Pi. These findings indicate that the site of action of the nucleotide is proline dehydrogenase, rather than on the respiratory chain. The mode of action of ADP is to lower the apparent K, for proline (Hansford and Sacktor, 1970a). Significantly, the K, in the presence of ADP approximates the concentration of proline found in

332

B. SACKTOR

the muscle, at rest (Sacktor and Wormser-Shavit, 1966). In a manner analogous to that for isocitric dehydrogenase, ATP inhibits proline dehydrogenase. Therefore, the activity of the enzyme is dependent, in part, on the composition of the adenine nucleotide mixture. As the nucleotide in the resting muscle is predominantly ATP, the small changes in ADP and ATP levels which are found when blowflies begin to fly (Sacktor and Hurlbut, 1966) will lead to a considerable enhancement in the rate of proline oxidation. D. CONTROL OF a-GLYCERO-P OXIDATION

For both pyruvate and proline oxidations, the key dehydrogenase is sensitive to a signal of the metabolic state of the muscle. It is also evident that for the third mitochondria1 site of regulation, u-glycero-P oxidation, control is manifested at the dehydrogenase level. Estabrook and Sacktor (1958a) have found that EDTA blocks 0

.-c €

0.7

H 0-6 0

-as 0.5 1

Y

b

3

2 !!

6

0.4 0.3 0.2

rc

0

Q,

0.1

. c .

b

'0

4 8 12 16 20 24 Concn. of D L Glycerol-3-P mM

Fig. 23. The effect of aglycero-P concentration on the rate of oxidation by blowfly flight muscle mitochondria, in the presence and absence of free calcium. Reaction mixture contains FCCP. (From Hansford and Chappell, 1967.)

REGULATION OF INTERMEDIARY METABOLISM

333

a-glycero-P oxidation and that this inhibition is reversed by the divalent cations, Ca2+ and Mg2+, and by additional substrate. The locus of inhibition has been observed to be at the dehydrogenase level. Based on these findings, a hypothesis has been suggested that regulation of a-glycero-P oxidation is achieved by reversal of the inhibited state by either the accumulation of substrate or, more likely, by release of divalent ions coincident with nervous stimulation of the muscle at the initiation of flight. This suggestion has received extensive confirmation through the work of Hansford and Chappell (1967). They have found that the metal ion involved is Ca2+,rather than Mg2+, and activation is maximal at very low levels of Ca2+.The level of free calcium that is required is about 5 x 10-7 g-ions/litre. As shown in Fig. 23, Ca2+has been seen to act by lowering the K, of the a-glycero-P dehydrogenase for its substrate (Hansford and Chappell, 1967). A plot of enzyme activity versus concentration of a-glycero-P reveals allosteric kinetics. At about 2 mM a-glycero-P, which is the physiological level in flight muscle (Sacktor and Wormser-Shavit, 1966), a 10-fold increase in rate is obtained on adding Ca2+. Increasing Ca2+ from 10-8 to 10-6 g-ion/litre causes a progressive increase in the ADPstimulated (State 3) rate, without an increase in the resting (State 4) rate. E. THE ENERGY-DEPENDENT ACCUMULATION OF Ca2+ AND Pi

It has been shown that Pi markedly stimulates the rate of pyruvate and proline oxidations and that Ca2+ enhances the oxidation of a-glycero-P. The need for very high levels of Pi has suggested that blowfly mitochondria may be capable of accumulating Pi against a concentration gradient. Direct support for this has been obtained by Hansford and Chappell (1968), who have found in Calliphora an energydependent uptake of Pi to about 60 mM. The accumulation of Pi is supported by both pyruvate and a-glycero-P and is inhibited and reversed by FCCP. Recently, Carafoli et al. (1969) have demonstrated that Phormia flight muscle mitochondria accumulate Ca2+ to a level as high as 400 nmoles xmg-1 protein (Fig. 24). The uptake requires respiratory energy derivable from either a-glycero-P or pyruvate. Uncoupling agents block and reverse, in part, the accumulation. Ca2+ uptake is not accompanied by ejection of H+. Instead, the simultaneous uptake of the Pi anion is required; inhibition of Pi uptake by mersalyl inhibits Ca2+binding. Acetate or chloride anions do not substitute for the Pi anion.

334

1

B. SACKTOR

4oo.

0 LL a

PYRUVATE PROLINE

300

++ 200 m 0

cn W

0

2

100

c

+ACETATE

0

0

3 6 MINUTES

Fig. 24. 'Ihe accumulation of (3% by blowfly flight muscle mitochondria. (From Carafoli et al., 1969.) F. INTERACTIONS OF METABOLIC EFFECTORS WITH THE RESPIRATORY CHAIN

Changes in the steady state oxidation-reduction level of the components of the respiratory chain in flight muscle mitochondria during the transition from a controlled to an active metabolic state have been examined by Chance and Sacktor (1958), Sacktor and Packer (1961), in flies, and Klingenberg and Bucher (1961), in locusts. In general, it has been seen that the respiratory components are largely oxidized in the presence of oxygen and absence of substrate. They become considerably reduced upon addition of a-glycero-P or pyruvate. For the most part, the extent of reduction is graded along the chain, the percentage decreasing from dehydrogenase to oxygen. (With a-glycero-P, reduction of NAD is due to reverse electron transport.) In most of these experiments a-glycero-P has been the substrate, and it has been found that addition of ADP

335

REGULATION OF INTERMEDIARY METABOLISM

or uncoupling agents cause the respiratory components to become more oxidized as the rate of oxygen uptake increases. When the added ADP has become phosphorylated to ATP, the respiratory rate decreases and the steady state levels of the cytochromes become more reduced. A second addition of ADP again initiates a transition to an increased respiratory rate (see Fig. 17), and decreased levels of reduction of cytochromes. When dissolved oxygen becomes exhausted and respiration ceases, the respiratory pigments go completely reduced.

TI

H

1 minute

Reduct ion

r

o*=0

(A1

glycerolphosphate I

02=0 I

I

I

Ca**and ADP

ADP

ADP

NADH Fig. 25. The effect of the simultaneous addition of ADP and CaB on the steady state levels of the respiratory components. oGlycero-P is the substrate. (From Hansford, 1968.)

Similar measurements of the redox states of cytochrome c and NAD have been made by Hansford (1968, 1969). The classical picture is seen when ADP is added to blowfly mitochondria respiring with a-glycero-P (Fig. 25). Surprisingly, however, during pyruvate oxidation, ADP addition results in an increased reduction even though, as described previously, the rate of respiration increases many-fold. Added Pi also causes a reduction, especially in the presence of oligomycin. These findings strongly support the concept that ADP and Pi activate a rate-limiting step in pyruvate oxidation (isocitrate dehydrogenase) even more than the respiratory chain. Hansford has also found that, when u-glycero-P is the substrate, added Ca*+increases reduction of the respiratory carriers at the same time that respiration is increased. Figure 25 shows an attempt to simuiate flight when both ADP and Ca2+ are added simultaneously,

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as may happen at the initiation of flight. It is evident that both cytochrome c and NAD become more reduced. These observations give strong support to the hypothesis that respiratory control in flight muscle may be due to control at the substrate level as well as to control in the respiratory chain (Chance and Sacktor, 1958; Estabrook and Sacktor, 1958a; Hansford and Sacktor, 1970a). Hansford’s elegant experiment also provides the experimental explanation for Keilin’s (1925) observations that the reduced cytochrome bands appear in the flight muscle, in situ, when the waxmoth starts to flap its wings. VI. CONCLUSIONS

This review has attempted to analyze the metabolic events that are fundamental to the transition of a muscle from a controlled resting state to one so active that energy transformations are taking place at a rate far in excess of that for any other biological process. The approach has been to identify the enzymatic reactions that are facilitated during the transition, to determine the mechanisms of activation at each locus of control, and then to formulate a working hypothesis that will unite the experimental findings into an overall scheme for the metabolic regulation of this tissue. This summarization should be premised with the knowledge that many of the experimental observations have been made with isolated enzymes and subcellular organelles. Thus, the in vivo environment can never be adequately reconstructed. Moreover, knowledge of the metabolite levels permitting simulation of in vivo conditions assumes uniform distribution of these substances and excludes compartmentation. These reservations made, the sites of regulation in fly flight muscle have been identified by measuring coincident and sequential changes in the concentrations of the metabolic intermediates. On the basis of the crossover theorem and initiation of utilization of stored reserves, these loci are: phosphorylase and trehalase, which determine the entrance of carbohydrate into the catabolic pathway; phosphofructokinase, which is rate-limiting for glycolysis; the mitochondria1 oxidations of a-glycero-P and pyruvate, the two end-products of glycolysis; and the oxidation of proline, which presumably provides intermediates for the initiation of the Krebs cycle. It is implicit to this discussion of the mechanisms of activation that, when the muscle is at rest and the rate of metabolism is low,

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the regulatory enzymes are in an inhibited state. Conversely, initiation of contraction sets off a chain of biochemical events that deinhibit or activate the enzymes, allowing for the accelerated rate of metabolism. With this in mind, it is noteworthy that high levels of ATP (or MgATP), a condition likely to be found in the muscle at rest, inhibit actomyosin ATPase, phosphorylase b, phosphofructokinase, isocitrate dehydrogenase and proline dehydrogenase. On the other hand, it is now known that the products of ATP breakdown, ADP, AMP and Pi, which are formed when the muscle contracts, counteract the effects of ATP or activate phosphorylase b, phosphofructokinase, isocitrate dehydrogenase and proline dehydrogenase. Significantly, and perhaps more than fortuitously, the low level of Ca2+,about 5 x 1O-' g-ions/litre, that activates myofibrillar ATPase, is the same concentration that activates phosphorylase b kinase and a-glycero-P dehydrogenase. Using these facts, as well as others cited previously in the review, it is tempting to hypothesize a scheme for the metabolic regulation of blowfly flight muscle. With the arrival of an electrical impulse and depolarization of the sarcolemma, the CaZ+which is sequested in the remnants of the sarcoplasmic reticulum is liberated. The free Ca2+ activates myofibrillar ATPase and phosphorylase b kinase. Phosphorylase b kinase converts the inhibited phosphorylase b to an active phosphorylase a, resulting in glycogenolysis. With the dephosphorylation of ATP by actomyosin and formation of ADP, AMP and Pi, phosphofructokinase becomes activated autocatalytically, enabling glycolysis to proceed at a maximum rate. The decrease in the concentration of extramitochondrial ATP by the myofibrillar ATPase, with the resultant increase in extramitochondrial ADP, also initiates a concentration-dependent exchange with the intramitochondrial adenine nucleotides; ATP leaves and ADP enters the mitochondria. The mitochondria will also accumulate Pi and Ca2+, now available. The decrease in mitochondrial ATP coupled with the increase in mitochondrial ADP, Pi and Ca2+ deinhibits the dehydrogenases. Ca2+, by lowering the K, of the u-glycero-P dehydrogenase, increases the oxidation of u-glycero-P. A 10-fold increase in oxidation is to be expected, at the level of substrate found in the tissue. A shift in the proportion of ATP and ADP initiates proline oxidation, also by reducing the K, of the enzyme for its substrate. With the limitation to pyruvate oxidation relieved by ample Krebs cycle intermediates derived from proline,

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ADP and Pi affects a 20-fold increase in isocitrate dehydrogenase activity, thus facilitating pyruvate oxidation. With controls at the dehydrogenase levels removed, the rates of oxidation of pyruvate and a-glycero-P via the respiratory chain, activated, in turn, several-fold by ADP in the classical manner, become sufficiently rapid to account for the oxygen uptake of the blowfly in flight. Concomitant with this respiration is the synthesis of the ATP for continued contraction. REFERENCES Agosin, M., Scaramelli, N. and Neghme, A. (1961). Intermediary carbohydrate metabolism of Triatoma infestans (Insecta; Hemiptera). I. Glycolytic and pentose phosphate pathway enzymes and the effect of DDT. Comp. Biochem. Physiol. 2, 143-159. Appleman, M. H., Yunis, A. A., Krebs, E. G. and Fischer, E. H. (1963). Comparative studies on glycogen phosphorylase-the amino acid composition of rabbit and human skeletal muscle phosphorylase. J. biol. Chem. 238, 1358-1361. Ashhurst, D. E. (1967). The fibrillar flight muscle of giant water-bugs: an electronmicroscope study. J. Cell Sci. 2,425-444. Bade, M. L. (1964). Biosynthesis of fatty acids in the cockroach, Eurycotis jloridana. J. Insect Physiol. 10,333-34 1. Bade, M. L. and Clayton, R. B. (1963). Cholesterol esters of the cockroach, Eurycotis floridana. Nature, Lond. 197, 77-79. Beenakkers, A. M. Th. (1963a). Enzyme der fettsaure-oxydation in den flugmuskeln von Locusta migratoria wiihrend firer entwicklung. Biochem. Z. 337,436-439. Beenakkers, A. M. Th. (1963b). Fatty acid oxidation in insect muscles. Acta physiol. pharmacol. neerl. 12,332-335. Beenakkers, A. M. Th. (1965). Transport of fatty acids in Locusta migratoria during sustained flight. J. Insect Physiol. 11,879-888. Beenakkers, A. M. Th., DeWaide, J. H., Henderson, P. T. and Lutgerhorst, A. (1967). Fatty acid oxidation and some participating enzymes in animal organs. Comp. Biochem. Physiol. 22, 675-682. Beenakkers, A. M. Th. and Gilbert, L. I. (1968). The fatty acid composition of fat body and haemolymph lipids in Hyalophora cecropia and its relation to lipid release. J. Insect Physiol. 14,481-494. Beenakkers, A. M. Th. and Henderson, P. T. (1967). The localization and function of camitine acetyltransferase in the flight muscles of the locust. Eur. J. Biochem. 1, 187-192. Beenakkers, A. M. Th. and Klingenberg, M. (1964). Carnitine-coenzyme A transacetylase in mitochondria from various organs. Biochim. biophys. Acta 84,205-207. Birt, L. M. (1961). Flight muscle mitochondria of Lucilia cuprina and Musca dornestica. Estimation of the pyridine nucleotide content and of the

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