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The chemical mediators of insulin action: Possible targets for postreceptor defects
The Chemical Mediators of Insulin Action: Possible Targets for Postreceptor Defects
LEONARD JARETT, M.D. FREDERICK L. KIECHLE, M.D., Ph.D. JANICE C. PARKER, Ph.D. S. LANCE MACAULAY, Ph.D. Philadelphia,
Pennsylvania
From the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia. Requests for reprints should be addressed to Dr. Leonard Jarett, Hospital of the University of Pennsylvania, 3400 Spruce Street, Box 671, Philadelphia, Pennsylvania 19104.
An insulin-sensitive subcellular system was developed from rat adipocytes consisting of plasma membranes and mitochondrfa. Direct addition of insulin, concanavalin A or anti-insulin receptor antibody to this system resulted in the production of a mediator substance from the plasma membrane that caused dephosphorylation of the alpha subunit of pyruvate dehydrogenase in the mitochondria with concomitant activation of the enzyme. The mediator activated pyruvate dehydrogenase by activating the pyruvate dehydrogenase phosphatase and not by inhibiting the pyruvate dehydrogenase kinase. This was similar to the mechanism by which insulin causes activation of the enzyme in the intact cell. The insulin-sensitive mediator material from the adipocyte plasma membrane was acid-stable with a molecular weight of 1,000 to 1,500. Our laboratory has shown that the mediator that activates pyruvate dehydrogenase was present in intact adipocytes, hepatoma ceils, and M-9 lymphocytes. insulin altered the amount or activity of the mediator consistent with the effect of the hormone on the cell. Other laboratories have shown similar effects on skeletal muscle and liver. We have shown the mediator to mimic insulin action on the low Km cyclic adenosine monophosphate (AMP) phosphodiesterase and the (calcium++-magnesium++)-adenosine triphosphatase (Ca++-Mg++)-ATPase of adipocyte plasma membranes in addition to pyruvate dehydrogenase. Other laboratories have shown the mediator to activate glycogen synthase. A body of direct and indirect evidence exists that demonstrates that more than one mediator exists. The chemical nature of the mediator is unknown but probably represents a new family of intracellular mediators of hormone action. These mediators may have clinical relevance in postreceptor defects of obesity and type II diabetes (noninsulin-dependent diabetes mellitus). Insulin is essential for life, and, like other hormones, is a circulating intercellular messenger that coordinates the anabolic functions of diverse tissues, including liver, fat, and muscle, throughout the body. A ‘second messenger,” or chemical mediator, that alters cell function by regulation of enzyme activity is released inside the cell following the binding of most hormones to their specific receptors on the outer surface of a cell. Despite detailed knowledge regarding the insulinreceptor interaction, knowledge of the postreceptor (intracellular) events is minimal, and identification of the actual chemical mediator, or second messenger, for insulin action has remained elusive. Our approach to this problem was to study the direct effects of insulin on subcellular preparations of plasma membranes from rat ad-
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SYMPOSIUM-JARETT
ipocytes. These studies can be performed only if the components and organization of the membrane necessary for insulin to generate a signal have been retained. This report provides the background information on the discovery of the mediator material in an adipocyte subcellular system. It also furnishes evidence that the material is generated from the plasma membrane of the adipocyte, documents the existence of the mediator material in various cell types, lists the insulinsensitive enzymes on which the chemical mediator has been demonstrated to act, and reviews the data supporting the existence of more than one mediator. The possible chemical nature of the mediator or mediators and the mechanism by which they mediate insulin action is discussed. Also, the problems in generation or function of these mediators that may be related to various disease states and associated with postreceptor abnormalities, including both insulin resistance and hypersensitivity, are described. SUBCELLULAR SYSTEM STUDIES Jarett and Smith [l] reported on a highly enriched plasma membrane preparation from rat adipocy-tes, to which the addition of physiologic concentrations of insulin or of concanavalin A rapidly increased the hydrolysis of adenosine triphosphate (ATP) as determined by the release of inorganic phosphate. The addition of physiologic concentrations of insulin to this same preparation of adipocyte plasma membranes was used by Seals et al. [2-51 to generate a material that activated pyruvate dehydrogenase, and it was postulated that the material was a mediator of insulin action. Findings in the initial studies showed that the direct addition of insulin to this adipocyte subcellular system altered the labeling of the components by (T-~*P) ATP [2]. Subsequent electrophoretic analysis revealed that the decreased phosphorylation of two high molecular weight phosphoproteins accounted for the direct effect of insulin on autophosphorylation. One phosphoprotein (molecular weight 120,000) was shown to be of plasma-membrane origin, and the other (molecular weight 42,000) was shown to be of mitochondrial origin [3,4]. The mitochondrial phosphoprotein was identified as the alpha subunit of pyruvate dehydrogenase, and the ability of insulin to decrease its phosphorylation was dependent on the presence of plasma membranes [3]. These effects of insulin on the phosphorylation of the alpha subunit were consistent with the mechanism proposed for the activation of the pyruvate dehydrogenase complex in mitochondria by insulin [6]. These electrophoretic studies suggested that some form of the insulin second messenger was being generated from the plasma membrane to alter the phosphorylation of the mitochondrial protein. The direct addition of insulin to this subcellular system resulted in stimulation of pyruvate dehydrogenase activity [ 51. Not only did the addition of insulin to this subcellular system
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stimulate pyruvate dehydrogenase activity, but also concanavalin A and anti-insulin receptor antibody activated the enzyme. These effects were observed only when the plasma membranes were present. These findings suggested that a chemical mediator was being generated from the plasma membrane and that this messenger was not a piece of the insulin molecule itself. At the time that these studies were performed, Larner et al. [7] isolated a low molecular weight (1,000 to 1,500), acid-stable chemical mediator generated by insulin treatment of skeletal muscle, as determined by the ability of mediator to activate glycogen synthase in vitro. Jarett and Seals [8] demonstrated that this same low molecular weight material isolated from insulintreated muscle stimulated pyruvate dehydrogenase activity in adipocyte mitochondria analogous to the stimulation seen when insulin was added to the plasma membrane-mitochondrial mixture. These findings supported the proposal that this material was an intracellular mediator of insulin action. The mediator released from the plasma membrane of the adipocyte has been partially purified and further characterized by Kiechle et al. [9]. Spontaneous partial release of the mediator as assessed in the pyruvate dehydrogenase assay can occur without hormonal stimulation. Repeated centrifugation followed by washing with TRIS buffer depleted the mediator from the plasma membranes. The supernatant material from plasma membranes prepared in phosphate buffer and treated with insulin stimulated pyruvate dehydrogenase activity more than did supernatant material from untreated membranes. This finding confirmed the report of Seals and Czech [lo]. This supernatant material was stable at pH 7, and increasing concentrations produced a linear increase in pyruvate dehydrogenase activity. Gel filtration of the supernatant material on Sephadex G-25 or G-15 columns revealed one fraction in the molecular weight range of 1,000 to 1,500 which stimulated pyruvate dehydrogenase. This same low molecular weight fraction from supernatant of insulintreated membranes contained a greater quantity or activity of the chemical mediator than did control samples. Thus, this low molecular weight material released from the plasma membrane appears to represent the chemical mediator whose existence was suggested in earlier subcellular system studies. INTACT TISSUE STUDIES To establish a substance as an intracellular mediator of insulin action, the substance must be shown to have a fairly ubiquitous distribution among various cell types, and its concentration must be altered by insulin in a manner consistent with the known effect of insulin on that cell type. The ability of insulin to alter the amount or activity of the mediator in tested cell types is indicated in Table I. This low molecular weight acid-stable
DIABETES SYMPOSIUM-JARETT
material was first identified by Larner et al. [7] in an intact tissue, namely muscle. Later, we showed that treatment of the rat adipocyte with insulin increased the amount or activity of an acid-stable, low molecular weight material, as estimated by the ability of the material to stimulate the activity of pyruvate dehydrogenase
Thus, the insulin-sensitive, low molecular weight material fulfills the role of a true second messenger or mediator by being found in each of the cells tested and by responding in an appropriate manner to insulin treatment of the cells.
[Ill.
INSULIN-SENSITIVE
The IM-9 cultured lymphocyte, a human cell line, was chosen by our group as a control cell to study [ 121. Despite extensive investigations on the binding of insulin to its receptor on the IM-9 lymphocyte, there have been no reports of a biologic response of this cell to the action of insulin. A low molecular weight material was extracted from the IM-9 lymphocyte, which stimulated pyruvate dehydrogenase activity. In contrast to the findings in the adipocyte and skeletal muscle, insulin treatment of IM-9 lymphocytes significantly reduced the amount or activity of the material in the Sephadex C-25 fraction that stimulated pyruvate dehydrogenase activity. This report extended the presence of the insulin mediator to human tissue. Another insulin-sensitive tissue that was tested was liver. Our laboratory studied the mediator released spontaneously from highly enriched plasma membranes prepared from rat liver in TRIS buffer [ 131. Again, the mediator was identified, using pyruvate dehydrogenase stimulation as the bioassay, in the same low molecular weight range fraction from Sephadex chromatography. Recently, Saltiel et al. [ 141 have demonstrated the ability of insulin to stimulate the release of the mediator from liver plasma membranes in a manner analogous to the effect of insulin on adipocyte plasma membranes. To obtain large quantities of material for purification, our laboratory studied the insulin-sensitive H4-II-E-C3’ hepatoma cells proposed by Hoffman et al. [ 151 as a model for studying insulin responsiveness. It was found that insulin produced a dose-dependent increase in the amount of mediator [ 161. The absolute quantity of mediator obtained was striking, and dilutions of the cellular extracts of up to 1500 were required before the material failed to activate pyruvate dehydrogenase.
TABLE I
Cell Types Tested for the Presence of the Chemical Mediator for Insulin Action and the Response to Insulin Treatment
Cells Tested for the Presence of a Mediator for Insulin Action
Effect of Insulin Treatment on the Amount or Activity of Mediator
Skeletal muscle Adipocytes Adipocyte plasma membranes M-9 lymphocytes Liver plasma membranes H4-II-E-C3’ hepatoma cells
Increased increased Increased Decreased Increased Increased
ENZYME STUDIES
The role of the acid-stable, low molecular weight, insulin-sensitive mediator as a second messenger for insulin would be further documented by demonstrating that this material modulates different enzyme systems in a manner analogous to that observed after insulin treatment of intact cells. Table II lists the enzyme systems that have been tested. These enzyme systems were chosen primarily because they modulate insulinsensitive intracellular processes. Larner et al. [ 71 have shown that the insulin-sensitive mediator from skeletal muscle inhibited the activity of cyclic AMP-dependent protein kinase and stimulated the activity of phosphoprotein phosphatase when glycogen synthase was used as a substrate. The changes in the activity of these two enzymes would result in activation of glycogen synthase as seen in studies of insulin-treated intact muscle. The mediator did not affect several cyclic AMP-independent protein kinases that were tested. Studies with the subcellular system have shown that the putative mediator of insulin action stimulated the activity of pyruvate dehydrogenase. The mediator from all of the cell systems described above stimulated the same enzyme. The mechanism by which pyruvate dehydrogenase activity is regulated has been identified [6]. The activity of the enzyme complex is altered by a cyclic AMP-independent protein kinase and a phosphoprotein phosphatase. Phosphorylation of the alpha subunit inactivates the enzyme complex, whereas dephosphorylation of the alpha subunit activates it. The mediator of insulin action could alter pyruvate hydrogenase activity through changes in phosphorylation of the alpha subunit by increasing phosphatase activity or by decreasing kinase activity, or by both. The earlier studies were all carried out in the presence of ATP [2-51, so it was not possible to determine the enzyme pathway utilized. A series of studies were performed in the presence and absence of ATP; of sodium fluoride, a known inhibitor of the pyruvate dehydrogenase phosphatase; and of dichloroacetic acid, an inhibitor of the kinase. These studies utilized the subcellular system [ 171, the active fraction from adipocytes [ 111, muscle [8] and hepatoma cells [ 161, and the supernatant and active fractions from the supernatant of adipocyte plasma membrane [ 91 and liver membranes [ 131. The results of all these studies were identical and indicated that the increase in pyruvate dehydrogenase activity was attributable to activation of the pyruvate dehydrogenase phosphatase and not to any alteration of the January
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TABLE II
Enzyme Systems Tested for Response to the Insulin-Sensitive Chemical Mediator and to Phospholipid
Mediator
Enzyme Cyclic AMP-dependent protein kinase Glycogen synthase phosphoprotein phosphatase Cyclic AMP-independent protein kinase Pyruvate dehydrogenase Cyclic AMP-independent protein kinase Phosphoprotein phosphatase
Decreased Increased No effect Increased No effect Increased
Low Km cyclic AMP phosphodiesterase
Increased
High Km cyclic AMP phosphodiesterase (Ca++-Mg++)-ATPase
No effect Increased
NOTE: AMP = adenosine monophosphate; (CA++-Mg++)-ATPase PS = phosphatidylserine; DPI = phosphatidylinositol-4’-phosphate;
dehydrogenase kinase, which is a cyclic AMP-independent kinase. Insulin treatment of hepatocytes [ 181 or adipocytes [ 19,201 increased the activity of the low Km cyclic AMP phosphodiesterase present in the microsomal fraction. The enzyme system, as it exists in the microsomal membrane fraction of the adipocyte [20], was assayed in the presence of the mediator from several sources. The addition of the active fraction from isolated adipocytes, or adipocyte plasma membranes [21] or from hepatoma cells [ 161 to the microsomal fraction resulted in greater activity of the low Km cyclic AMP phosphodiesterase compared to controls. This increase in activity was concentration dependent. The active fraction from insulin-treated hepatoma cells [ 161 or adipocytes [ 2 l] increased low Km cyclic AMP phosphodiesterase activity to a greater degree than did the material from control hepatoma cells or adipocytes. The mediator from insulin-treated adipocytes or adipocyte plasma membranes had no effect on the high Km cyclic AMP phosphodiesterase. This finding is consistent with the insensitivity of the enzyme to insulin in intact cellular systems [ 18,201. The next enzyme system tested was the insulinsensitive high affinity Ca++-stimulated Mg++dependent ATPase[(Ca++-Mg++)-ATPase] found in the plasma membrane of rat adipocytes, which appears to serve as an enzymatic basis for a calmodulin-sensitive plasma membrane Ca++ transport system [22,23]. Treatment of adipocytes or the adipocyte plasma membrane with physiologic concentrations of insulin up to 100 PUlml decreased enzyme activity and decreased the phosphorylation of a phosphoprotein (molecular weight 110,000). This phosphoprotein has been identified as (Ca++-Mg++)-ATPase [ 24,251. The chemical mediator in the supernatant from the adipocyte plasma membrane was found to stimulate the activity of (Ca++Mg++)-ATPase fourfold and more than doubled the pyruvate
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Effecton Activityof Enzyme Systemsot Phospholipid Not tested Not tested Not tested Increased (PS) Decreased (DPI) Not tested Increased (PS) Decreased (DPI) Increased (PS, PG. lyso PC) Decreased (DPI) No effect (PS, DPI) Not tested
= calcium-stimulated magnesium-dependent adenosine triphosphatase; PG = phosphatidylglycerol; lyso PC = lysophosphatidylcholine.
Ca++ transport [26]. Fractions prepared by Sephadex G-25 chromatography of the supernatant were tested for their ability to alter pyruvate dehydrogenase activity, (Ca++-Mg++)-ATPase activity, and Ca++ transport. Only the low molecular weight fraction, which increased the pyruvate dehydrogenase activity, increased the (Ca++-Mg++)-ATPase activity and calcium transport. Thus, the mediator did alter the enzyme and transport systems, but in a manner opposite to the effect observed with physiologic concentrations of insulin. Recently, H. A. Pershadsingh and J. M. McDonald found that the addition of greater than 300 PUlml of insulin to plasma membranes increased (Ca++-Mg++)-ATPase activity (unpublished observations). These studies demonstrated that the mediator of insulin action controls the activity of glycogen synthase and pyruvate dehydrogenase by altering their state of phosphorylation in a manner similar to that observed in insulin-treated intact cells. The (Ca++-Mg++)-ATPase has been shown to be regulated by phosphorylation or calmodulin, or both [ 271. Thus the mediator could work through either mechanism or other yet to be defined mechanisms. EVIDENCE
OF MORE THAN ONE MEDIATOR
Several different lines of evidence suggest that more than one mediator is generated by the interaction of insulin with the plasma membrane. First, biphasic responses of test systems to increasing concentrations of insulin and other ligands have been reported. That is to say, low concentrations of insulin caused a response that was reversed at higher concentrations. Turakulov et al. [28] first reported that the insulin treatment of rats resulted in an increase in a low molecular weight material in the cytosol of liver, which produced a biphasic response in calcium (Ca++) uptake by mitochondria. Larner et al. [7] have shown that the Sephadex G-25 active fraction from muscle produced a biphasic re-
DIABETES SYMPOSIUM-JARETT
sponse with respect to the activity of the glycogen synthase phosphatase. Studies by Seals and Jarett [5] have demonstrated that the direct addition of insulin, concanavalin A or anti-insulin receptor antibody to the adipocyte subcellular system all produced a biphasic dose-response curve for activation of pyruvate dehydrogenase. Seals and Czech [29] have shown that incubation of adipocyte plasma membranes with increasing insulin concentrations, or their incubation with insulin for increasing periods of time, yielded supernatant material that produced a biphasic stimulation of pyruvate dehydrogenase activity. Saltiel et al. [ 141 have found the same response with liver plasma membranes. One possible explanation of these data could be the existence of two mediators, one having a high affinity for certain enzyme systems and the other a lower affinity for these same systems. When the mediator with a lower affinity for these enzymatic systems is at high concentrations, it may interfere with the action of the first mediator. This second mediator might possess a higher affinity for other enzyme systems, thus reversing the role of the two mediators. More direct proof of the existence of two mediators has been provided by Chen et al. [30]. These investigators separated the active Sephadex G-25 fraction from skeletal muscle into two separate fractions. One fraction stimulated the activity of the cyclic AMP-dependent protein kinase and inhibited that of glycogen synthase phosphoprotein phosphatase. The other fraction retained the ability to inhibit the kinase activity and to increase that of phosphoprotein phosphatase. The biphasic response disappeared with this separation. Some of the data presented heretofore provide further indirect support for the concept of two messengers. The lesser stimulation of pyruvate dehydrogenase activity by the active Sephadex G-25 fraction from insulin-treated IM-9 lymphocytes as compared to the stimulation of this enzyme activity by the extract from control cells could result from insulin generation of the mediator, which blocks the phosphatase without altering the production of the stimulator of the phosphatase. The effects of the mediator isolated from adipocyte plasma membranes on the activity of (Ca++-M$++)-ATPase suggest that at high concentrations of the mediator, the enzyme was activated. Recent studies with the hepatoma cells have shown that increasing concentrations of insulin up to 5 mu/ml did not produce a biphasic effect. One possible explanation for this finding is that only one type of mediator is produced in this cell line in response to the action of insulin [ 161. CHEMICAL NATURE OF THE MEDIATOR The methods used for purification of the mediator of insulin action depend on the composition of the medi-
ators. The active material has been detected in enriched plasma membrane fractions from both adipocytes and hepatocytes. Therefore, the mediator may be a representative of one of the following three primary constituents of plasma membranes: phospholipid, protein, or glycoprotein. Purification of the mediator by standard methods has been difficult. At present, investigations in our laboratory suggest that it is not a simple peptide or glycopeptide. Efforts to identify the chemical nature of the mediator by our laboratory have included attempts to destroy the material by enzymes or to bind the material to columns with specific affinities. The biologic activity of the material can be monitored before and after such experimentation. Incubation of the active material for one hour with nonspecific or specific proteolytic enzymes bound to Sepharose resulted in partial or no inactivation of the factor. Therefore, peptide bonds do not appear to be a structural element essential for the activity of the mediator. This conclusion was supported by the fact that dansylation of the active material had no effect on its activity. The role of sugar or aminosugar residues was determined by chromatography with lectins bound to Sepharose. We were unable to bind the mediator to any lectin column; therefore, we believe it is unlikely that common oligosaccharides are a structural component of the mediator which are essential for activity. Our inability to document a peptide or glycopeptide component led us to consider phospholipids or proteolipids as possible chemical mediators of insulin action. The importance of phospholipids in biologic regulation is becoming more widely recognized, as is indicated by the ever increasing number of published studies on the subject. A literature search has revealed that there is very limited information on the turnover of phospholipids in response to the action of insulin [31-331, but the effects of phospholipase C or A [32,34,35] that mimic the effects of insulin indicated to Rodbell et al. [36] that insulin may cause a change in the disposition of phospholipids within the fat cell membrane. The recent studies that describe a phospholipiddependent, calcium-dependent protein kinase [37-411 suggest a direct relationship between the action of phospholipids and protein phosphorylation [42]. Phospholipids have also been shown to alter the activity of several membrane-bound enzymes directly [43]. Among these are Ca++-ATPase, (Ca++Mg++)-ATPase, pyruvate oxidase, high Km cyclic AMP phosphodiesterase, adenylate cyclase, and glucose6-phosphatase. The above data led us to investigate the possible role of phospholipids as chemical mediators of insulin action. Chloroform-methanol extraction followed by thin layer chromatography of the supernatant from adipocyte plasma membranes and the active Sephadex G-25 fraction from insulin-treated and control hepatoma cells
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DIABETES SYMF’OSIUM4AREll
have shown the presence of 10 phospholipids that were associated with plasma membrane. Dispersions of these and other phospholipids were added individually to pyruvate dehydrogenase and low Km cyclic AMP phosphodiesterase assays. No phospholipid that we tested altered the activity of the high Km cyclic AMP phosphodiesterase from plasma membranes or microsomes. Phosphatidylserine stimulated and phosphatidylinositol-4’-phosphate inhibited pyruvate dehydrogenase activity in a dose-dependent manner from 1 @I to 800 PM, with or without ATP. Eight other phospholipids had no effect, or only slightly inhibited pyruvate dehydrogenase activity. The stimulation of the activity of pyruvate dehydrogenase by phosphatidylserine was reversed by phosphatidylinositol-4’-phosphate. The inhibition of pyruvate dehydrogenase activity by phosphatidylinositol-4’-phosphate was abolished by increasing the Ca++ concentration in the assay. Sodium fluoride, a known pyruvate dehydrogenase phosphatase inhibitor that blocks the ability of the mediator to stimulate pyruvate dehydrogenase, inhibited the activation of the enzyme by phosphatidylserine. The activity of low Km cyclic AMP phosphodiesterase from adipocyte microsomes was stimulated above basal activity by three of these 10 phospholipids as follows: phosphatidylglycerol stimulated this enzyme activity more than did phosphatidylserine, and phosphatidylserine stimulated the enzyme more than lysophosphatidylcholine. The same three phospholipids activated the adipocyte plasma membrane enzyme, but the order of activation was changed: lysophosphatidylcholine activated it more than phosphatidylserine, and phosphatidylserine activated it more than phosphatidylglycerol. Kinetic studies indicated that these three phospholipids had no effect on the apparent Km for cyclic AMP for either enzyme. However, the apparent V, of both enzymes was increased in the same order as the stimulation of enzyme activity. Phosphatidylinositol4’-phosphate inhibited the activity of low Km cyclic AMP
phosphodiesterase in a dose-dependent manner. Kinetic studies with the microsomal low Km cyclic AMP phosphodiesterase have shown that phosphatidylinositol_4’-phosphate did not alter the apparent V, but increased the apparent Km. The marked similarities between the effect of phosphatidylserine and the mediator of insulin action on insulin-sensitive enzymes and the counterregulatory role of phosphatidylinositol-4’phosphate suggest that phosphatidylserine, phosphatidylinositol_4’-phosphate, or other related substances may represent the molecules involved in mediating the intracellular effects of insulin. More extensive studies are needed to delineate the role of phospholipids or proteolipids in insulin action. SUMMARY
AND CLINICAL SIGNIFICANCE
These studies would suggest that the mediators of insulin action may represent a new family of intracellular messengers, possibly involving various phospholipids or proteolipids. To date the data concern only some of the short-term effects of insulin. The mechanisms by which these potential mediators alter regulatory enzymes seem to be varied and complex, including control of phosphorylation; control of affinity for calcium (&I++) or calmodulin, 01 both: control of fluidity of membranes; or direct allosteric effects. These exciting new observations open numerous avenues of future investigation on the molecular mechanism of insulin actions. These studies have potential clinical significance as well as basic interests. The postreceptor defects of insulin resistance in obesity and type II diabetes (noninsulin-dependent diabetes mellltus) may result in part from abnormal release or metabolism of the mediators. Likewise, reactive hypoglycemia may result in part from excess production of the mediators. Oral hypoglycemic agents may carry out their extra-islet effects directly by stimulating release of the mediators or indirectly by making the membranes more sensitive to insulin stimulation. Positive identification of the insulin mediator will permit the testing of these theories.
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stimulation in adipocytes of the plasma membrane Ca*+transport [Ca*+ + Mg*+]-ATPase system and mitochondrial pyruvate dehydrogenase by a supernatant factor from isolated plasma membrane. Biochem Biophys Res Commun 1981; 100: 857-864. Vincenzi FF, Hinds TR: Calmodulin and plasma membrane calcium transport. In: Cheung WY, ed. Calcium and cell function, vol I. New York: Academic Press, 1980: 128186. Turakulov IaKh, Gainutdinov MKh, Lavina II, et al.: [insulin dependent cytoplasmic regulation of Ca++ ion transport in liver mitochondria. Reo Acad Sci USSR 1977: 234: 1471-1474.1 (Russ) ’ Seals JR, Czech MP: Characterization of a pyruvate dehydrogenase activator released by adipocyte plasma membranes in response to insulin. J Biol Chem 1981; 256: 2894-2899. Chen K, Galasko G, Huang L, Kellogg J, Larner J: Studies on the insulin mediator. II. Separation of two antagonistic biologically active materials from fraction II. Diabetes 1980; 29: 659-681. Garcia-Sainz JA, Fain JN: Effect of insulin, catecholamines and calcium ions on phospholipid metabolism in isolated white fat-cells. Biochem J 1980; 186: 781-789. Rodbell M: Metabolism of isolated fat cells. II. The similar effects of phospholipase C (Clostriiium perfringens (Y toxin) and of insulin on glucose and amino acid metabolism. J Biol Chem 1966; 241: 130-739. De Torrontegui G, Berthet J: The action of insulin on the incorporation of [32P]phosphate in the phospholipids of rat adipose tissue. Biochem Biophys Acta 1966; 116: 477481. Rodbell M, Jones AB: Metabolism of isolated fat cells. Ill. The similar inhibitory action of phospholipase C (Clostridium perfringens (Y toxin) and of insulin on lipolysis stimulated by lipolytic hormones and theophylline. J Biol Chem 1966; 241: 140-142. Blecher M: Effects of insulin and phospholipase A on glucose transport across the plasma membrane of free adipose cells. Biochem Biophys Acta 1967; 137: 557-571. Rodbell M, Jones AB, Chiappe de Cingolani GE, Birnbaumer L: The actions of insulin and catabolic hormones on the plasma membrane of the fat cells. Recent Prog Horm Res 1968; 24: 215-254. Kaibuchi K, Takai Y, Nishizuka Y: Cooperative roles of various membrane phospholipids in the activation of calciumactivated, phospholipiddependent protein kinase. J Biol Chem 1981; 256: 7146-7149. Wrenn RW, Katoh N, Wise BC, Kuo JF: Stimulation by phosphatidylserine and calmodulin of calcium-dependent phosphorylation of endogenous proteins from cerebral cortex. J Biol Chem 1980; 255: 12042-12048. Kuo JF, Andersson RGG, Wise BC, et al.: Calciumdependent protein kinase; widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calmodulin, and trifluoperazine. Proc Natl Acad Sci USA 1980; 77: 7039-7043. Katoh N, Wrenn RW. Wise BC, Shoji M, Kuo JF: Substrate proteins for calmodulin-sensitive and phospholipkt-sensitive Ca*+dependent protein kinases in heart, and inhibition of their phosphorylation by palmitoylcarnitine. Proc Natl Acad Sci USA 1981; 78: 4813-4817. Kishimoto A, Takai H, Mori T, Kikkawa U, Nishizuka Y: Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. J Biol Chem 1980; 255: 2273-2276. Michell B: A cellular control cascade of lipid degradation and protein phosphorylation. Trends Biochem Sci 1981; 6: 7. Fourcans B, Jain MK: Role of phospholipids in transport and enzymic reactions. Adv Lipid Res 1974; 12: 147-226.
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37
LEONARD JARETT, M.D. FREDERICK L. KIECHLE, M.D., Ph.D. JANICE C. PARKER, Ph.D. S. LANCE MACAULAY, Ph.D. Philadelphia,
Pennsylvania
From the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia. Requests for reprints should be addressed to Dr. Leonard Jarett, Hospital of the University of Pennsylvania, 3400 Spruce Street, Box 671, Philadelphia, Pennsylvania 19104.
An insulin-sensitive subcellular system was developed from rat adipocytes consisting of plasma membranes and mitochondrfa. Direct addition of insulin, concanavalin A or anti-insulin receptor antibody to this system resulted in the production of a mediator substance from the plasma membrane that caused dephosphorylation of the alpha subunit of pyruvate dehydrogenase in the mitochondria with concomitant activation of the enzyme. The mediator activated pyruvate dehydrogenase by activating the pyruvate dehydrogenase phosphatase and not by inhibiting the pyruvate dehydrogenase kinase. This was similar to the mechanism by which insulin causes activation of the enzyme in the intact cell. The insulin-sensitive mediator material from the adipocyte plasma membrane was acid-stable with a molecular weight of 1,000 to 1,500. Our laboratory has shown that the mediator that activates pyruvate dehydrogenase was present in intact adipocytes, hepatoma ceils, and M-9 lymphocytes. insulin altered the amount or activity of the mediator consistent with the effect of the hormone on the cell. Other laboratories have shown similar effects on skeletal muscle and liver. We have shown the mediator to mimic insulin action on the low Km cyclic adenosine monophosphate (AMP) phosphodiesterase and the (calcium++-magnesium++)-adenosine triphosphatase (Ca++-Mg++)-ATPase of adipocyte plasma membranes in addition to pyruvate dehydrogenase. Other laboratories have shown the mediator to activate glycogen synthase. A body of direct and indirect evidence exists that demonstrates that more than one mediator exists. The chemical nature of the mediator is unknown but probably represents a new family of intracellular mediators of hormone action. These mediators may have clinical relevance in postreceptor defects of obesity and type II diabetes (noninsulin-dependent diabetes mellitus). Insulin is essential for life, and, like other hormones, is a circulating intercellular messenger that coordinates the anabolic functions of diverse tissues, including liver, fat, and muscle, throughout the body. A ‘second messenger,” or chemical mediator, that alters cell function by regulation of enzyme activity is released inside the cell following the binding of most hormones to their specific receptors on the outer surface of a cell. Despite detailed knowledge regarding the insulinreceptor interaction, knowledge of the postreceptor (intracellular) events is minimal, and identification of the actual chemical mediator, or second messenger, for insulin action has remained elusive. Our approach to this problem was to study the direct effects of insulin on subcellular preparations of plasma membranes from rat ad-
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DIABETES
SYMPOSIUM-JARETT
ipocytes. These studies can be performed only if the components and organization of the membrane necessary for insulin to generate a signal have been retained. This report provides the background information on the discovery of the mediator material in an adipocyte subcellular system. It also furnishes evidence that the material is generated from the plasma membrane of the adipocyte, documents the existence of the mediator material in various cell types, lists the insulinsensitive enzymes on which the chemical mediator has been demonstrated to act, and reviews the data supporting the existence of more than one mediator. The possible chemical nature of the mediator or mediators and the mechanism by which they mediate insulin action is discussed. Also, the problems in generation or function of these mediators that may be related to various disease states and associated with postreceptor abnormalities, including both insulin resistance and hypersensitivity, are described. SUBCELLULAR SYSTEM STUDIES Jarett and Smith [l] reported on a highly enriched plasma membrane preparation from rat adipocy-tes, to which the addition of physiologic concentrations of insulin or of concanavalin A rapidly increased the hydrolysis of adenosine triphosphate (ATP) as determined by the release of inorganic phosphate. The addition of physiologic concentrations of insulin to this same preparation of adipocyte plasma membranes was used by Seals et al. [2-51 to generate a material that activated pyruvate dehydrogenase, and it was postulated that the material was a mediator of insulin action. Findings in the initial studies showed that the direct addition of insulin to this adipocyte subcellular system altered the labeling of the components by (T-~*P) ATP [2]. Subsequent electrophoretic analysis revealed that the decreased phosphorylation of two high molecular weight phosphoproteins accounted for the direct effect of insulin on autophosphorylation. One phosphoprotein (molecular weight 120,000) was shown to be of plasma-membrane origin, and the other (molecular weight 42,000) was shown to be of mitochondrial origin [3,4]. The mitochondrial phosphoprotein was identified as the alpha subunit of pyruvate dehydrogenase, and the ability of insulin to decrease its phosphorylation was dependent on the presence of plasma membranes [3]. These effects of insulin on the phosphorylation of the alpha subunit were consistent with the mechanism proposed for the activation of the pyruvate dehydrogenase complex in mitochondria by insulin [6]. These electrophoretic studies suggested that some form of the insulin second messenger was being generated from the plasma membrane to alter the phosphorylation of the mitochondrial protein. The direct addition of insulin to this subcellular system resulted in stimulation of pyruvate dehydrogenase activity [ 51. Not only did the addition of insulin to this subcellular system
32
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stimulate pyruvate dehydrogenase activity, but also concanavalin A and anti-insulin receptor antibody activated the enzyme. These effects were observed only when the plasma membranes were present. These findings suggested that a chemical mediator was being generated from the plasma membrane and that this messenger was not a piece of the insulin molecule itself. At the time that these studies were performed, Larner et al. [7] isolated a low molecular weight (1,000 to 1,500), acid-stable chemical mediator generated by insulin treatment of skeletal muscle, as determined by the ability of mediator to activate glycogen synthase in vitro. Jarett and Seals [8] demonstrated that this same low molecular weight material isolated from insulintreated muscle stimulated pyruvate dehydrogenase activity in adipocyte mitochondria analogous to the stimulation seen when insulin was added to the plasma membrane-mitochondrial mixture. These findings supported the proposal that this material was an intracellular mediator of insulin action. The mediator released from the plasma membrane of the adipocyte has been partially purified and further characterized by Kiechle et al. [9]. Spontaneous partial release of the mediator as assessed in the pyruvate dehydrogenase assay can occur without hormonal stimulation. Repeated centrifugation followed by washing with TRIS buffer depleted the mediator from the plasma membranes. The supernatant material from plasma membranes prepared in phosphate buffer and treated with insulin stimulated pyruvate dehydrogenase activity more than did supernatant material from untreated membranes. This finding confirmed the report of Seals and Czech [lo]. This supernatant material was stable at pH 7, and increasing concentrations produced a linear increase in pyruvate dehydrogenase activity. Gel filtration of the supernatant material on Sephadex G-25 or G-15 columns revealed one fraction in the molecular weight range of 1,000 to 1,500 which stimulated pyruvate dehydrogenase. This same low molecular weight fraction from supernatant of insulintreated membranes contained a greater quantity or activity of the chemical mediator than did control samples. Thus, this low molecular weight material released from the plasma membrane appears to represent the chemical mediator whose existence was suggested in earlier subcellular system studies. INTACT TISSUE STUDIES To establish a substance as an intracellular mediator of insulin action, the substance must be shown to have a fairly ubiquitous distribution among various cell types, and its concentration must be altered by insulin in a manner consistent with the known effect of insulin on that cell type. The ability of insulin to alter the amount or activity of the mediator in tested cell types is indicated in Table I. This low molecular weight acid-stable
DIABETES SYMPOSIUM-JARETT
material was first identified by Larner et al. [7] in an intact tissue, namely muscle. Later, we showed that treatment of the rat adipocyte with insulin increased the amount or activity of an acid-stable, low molecular weight material, as estimated by the ability of the material to stimulate the activity of pyruvate dehydrogenase
Thus, the insulin-sensitive, low molecular weight material fulfills the role of a true second messenger or mediator by being found in each of the cells tested and by responding in an appropriate manner to insulin treatment of the cells.
[Ill.
INSULIN-SENSITIVE
The IM-9 cultured lymphocyte, a human cell line, was chosen by our group as a control cell to study [ 121. Despite extensive investigations on the binding of insulin to its receptor on the IM-9 lymphocyte, there have been no reports of a biologic response of this cell to the action of insulin. A low molecular weight material was extracted from the IM-9 lymphocyte, which stimulated pyruvate dehydrogenase activity. In contrast to the findings in the adipocyte and skeletal muscle, insulin treatment of IM-9 lymphocytes significantly reduced the amount or activity of the material in the Sephadex C-25 fraction that stimulated pyruvate dehydrogenase activity. This report extended the presence of the insulin mediator to human tissue. Another insulin-sensitive tissue that was tested was liver. Our laboratory studied the mediator released spontaneously from highly enriched plasma membranes prepared from rat liver in TRIS buffer [ 131. Again, the mediator was identified, using pyruvate dehydrogenase stimulation as the bioassay, in the same low molecular weight range fraction from Sephadex chromatography. Recently, Saltiel et al. [ 141 have demonstrated the ability of insulin to stimulate the release of the mediator from liver plasma membranes in a manner analogous to the effect of insulin on adipocyte plasma membranes. To obtain large quantities of material for purification, our laboratory studied the insulin-sensitive H4-II-E-C3’ hepatoma cells proposed by Hoffman et al. [ 151 as a model for studying insulin responsiveness. It was found that insulin produced a dose-dependent increase in the amount of mediator [ 161. The absolute quantity of mediator obtained was striking, and dilutions of the cellular extracts of up to 1500 were required before the material failed to activate pyruvate dehydrogenase.
TABLE I
Cell Types Tested for the Presence of the Chemical Mediator for Insulin Action and the Response to Insulin Treatment
Cells Tested for the Presence of a Mediator for Insulin Action
Effect of Insulin Treatment on the Amount or Activity of Mediator
Skeletal muscle Adipocytes Adipocyte plasma membranes M-9 lymphocytes Liver plasma membranes H4-II-E-C3’ hepatoma cells
Increased increased Increased Decreased Increased Increased
ENZYME STUDIES
The role of the acid-stable, low molecular weight, insulin-sensitive mediator as a second messenger for insulin would be further documented by demonstrating that this material modulates different enzyme systems in a manner analogous to that observed after insulin treatment of intact cells. Table II lists the enzyme systems that have been tested. These enzyme systems were chosen primarily because they modulate insulinsensitive intracellular processes. Larner et al. [ 71 have shown that the insulin-sensitive mediator from skeletal muscle inhibited the activity of cyclic AMP-dependent protein kinase and stimulated the activity of phosphoprotein phosphatase when glycogen synthase was used as a substrate. The changes in the activity of these two enzymes would result in activation of glycogen synthase as seen in studies of insulin-treated intact muscle. The mediator did not affect several cyclic AMP-independent protein kinases that were tested. Studies with the subcellular system have shown that the putative mediator of insulin action stimulated the activity of pyruvate dehydrogenase. The mediator from all of the cell systems described above stimulated the same enzyme. The mechanism by which pyruvate dehydrogenase activity is regulated has been identified [6]. The activity of the enzyme complex is altered by a cyclic AMP-independent protein kinase and a phosphoprotein phosphatase. Phosphorylation of the alpha subunit inactivates the enzyme complex, whereas dephosphorylation of the alpha subunit activates it. The mediator of insulin action could alter pyruvate hydrogenase activity through changes in phosphorylation of the alpha subunit by increasing phosphatase activity or by decreasing kinase activity, or by both. The earlier studies were all carried out in the presence of ATP [2-51, so it was not possible to determine the enzyme pathway utilized. A series of studies were performed in the presence and absence of ATP; of sodium fluoride, a known inhibitor of the pyruvate dehydrogenase phosphatase; and of dichloroacetic acid, an inhibitor of the kinase. These studies utilized the subcellular system [ 171, the active fraction from adipocytes [ 111, muscle [8] and hepatoma cells [ 161, and the supernatant and active fractions from the supernatant of adipocyte plasma membrane [ 91 and liver membranes [ 131. The results of all these studies were identical and indicated that the increase in pyruvate dehydrogenase activity was attributable to activation of the pyruvate dehydrogenase phosphatase and not to any alteration of the January
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DIABETES SYMPOSIUM-JARETT
TABLE II
Enzyme Systems Tested for Response to the Insulin-Sensitive Chemical Mediator and to Phospholipid
Mediator
Enzyme Cyclic AMP-dependent protein kinase Glycogen synthase phosphoprotein phosphatase Cyclic AMP-independent protein kinase Pyruvate dehydrogenase Cyclic AMP-independent protein kinase Phosphoprotein phosphatase
Decreased Increased No effect Increased No effect Increased
Low Km cyclic AMP phosphodiesterase
Increased
High Km cyclic AMP phosphodiesterase (Ca++-Mg++)-ATPase
No effect Increased
NOTE: AMP = adenosine monophosphate; (CA++-Mg++)-ATPase PS = phosphatidylserine; DPI = phosphatidylinositol-4’-phosphate;
dehydrogenase kinase, which is a cyclic AMP-independent kinase. Insulin treatment of hepatocytes [ 181 or adipocytes [ 19,201 increased the activity of the low Km cyclic AMP phosphodiesterase present in the microsomal fraction. The enzyme system, as it exists in the microsomal membrane fraction of the adipocyte [20], was assayed in the presence of the mediator from several sources. The addition of the active fraction from isolated adipocytes, or adipocyte plasma membranes [21] or from hepatoma cells [ 161 to the microsomal fraction resulted in greater activity of the low Km cyclic AMP phosphodiesterase compared to controls. This increase in activity was concentration dependent. The active fraction from insulin-treated hepatoma cells [ 161 or adipocytes [ 2 l] increased low Km cyclic AMP phosphodiesterase activity to a greater degree than did the material from control hepatoma cells or adipocytes. The mediator from insulin-treated adipocytes or adipocyte plasma membranes had no effect on the high Km cyclic AMP phosphodiesterase. This finding is consistent with the insensitivity of the enzyme to insulin in intact cellular systems [ 18,201. The next enzyme system tested was the insulinsensitive high affinity Ca++-stimulated Mg++dependent ATPase[(Ca++-Mg++)-ATPase] found in the plasma membrane of rat adipocytes, which appears to serve as an enzymatic basis for a calmodulin-sensitive plasma membrane Ca++ transport system [22,23]. Treatment of adipocytes or the adipocyte plasma membrane with physiologic concentrations of insulin up to 100 PUlml decreased enzyme activity and decreased the phosphorylation of a phosphoprotein (molecular weight 110,000). This phosphoprotein has been identified as (Ca++-Mg++)-ATPase [ 24,251. The chemical mediator in the supernatant from the adipocyte plasma membrane was found to stimulate the activity of (Ca++Mg++)-ATPase fourfold and more than doubled the pyruvate
34
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Effecton Activityof Enzyme Systemsot Phospholipid Not tested Not tested Not tested Increased (PS) Decreased (DPI) Not tested Increased (PS) Decreased (DPI) Increased (PS, PG. lyso PC) Decreased (DPI) No effect (PS, DPI) Not tested
= calcium-stimulated magnesium-dependent adenosine triphosphatase; PG = phosphatidylglycerol; lyso PC = lysophosphatidylcholine.
Ca++ transport [26]. Fractions prepared by Sephadex G-25 chromatography of the supernatant were tested for their ability to alter pyruvate dehydrogenase activity, (Ca++-Mg++)-ATPase activity, and Ca++ transport. Only the low molecular weight fraction, which increased the pyruvate dehydrogenase activity, increased the (Ca++-Mg++)-ATPase activity and calcium transport. Thus, the mediator did alter the enzyme and transport systems, but in a manner opposite to the effect observed with physiologic concentrations of insulin. Recently, H. A. Pershadsingh and J. M. McDonald found that the addition of greater than 300 PUlml of insulin to plasma membranes increased (Ca++-Mg++)-ATPase activity (unpublished observations). These studies demonstrated that the mediator of insulin action controls the activity of glycogen synthase and pyruvate dehydrogenase by altering their state of phosphorylation in a manner similar to that observed in insulin-treated intact cells. The (Ca++-Mg++)-ATPase has been shown to be regulated by phosphorylation or calmodulin, or both [ 271. Thus the mediator could work through either mechanism or other yet to be defined mechanisms. EVIDENCE
OF MORE THAN ONE MEDIATOR
Several different lines of evidence suggest that more than one mediator is generated by the interaction of insulin with the plasma membrane. First, biphasic responses of test systems to increasing concentrations of insulin and other ligands have been reported. That is to say, low concentrations of insulin caused a response that was reversed at higher concentrations. Turakulov et al. [28] first reported that the insulin treatment of rats resulted in an increase in a low molecular weight material in the cytosol of liver, which produced a biphasic response in calcium (Ca++) uptake by mitochondria. Larner et al. [7] have shown that the Sephadex G-25 active fraction from muscle produced a biphasic re-
DIABETES SYMPOSIUM-JARETT
sponse with respect to the activity of the glycogen synthase phosphatase. Studies by Seals and Jarett [5] have demonstrated that the direct addition of insulin, concanavalin A or anti-insulin receptor antibody to the adipocyte subcellular system all produced a biphasic dose-response curve for activation of pyruvate dehydrogenase. Seals and Czech [29] have shown that incubation of adipocyte plasma membranes with increasing insulin concentrations, or their incubation with insulin for increasing periods of time, yielded supernatant material that produced a biphasic stimulation of pyruvate dehydrogenase activity. Saltiel et al. [ 141 have found the same response with liver plasma membranes. One possible explanation of these data could be the existence of two mediators, one having a high affinity for certain enzyme systems and the other a lower affinity for these same systems. When the mediator with a lower affinity for these enzymatic systems is at high concentrations, it may interfere with the action of the first mediator. This second mediator might possess a higher affinity for other enzyme systems, thus reversing the role of the two mediators. More direct proof of the existence of two mediators has been provided by Chen et al. [30]. These investigators separated the active Sephadex G-25 fraction from skeletal muscle into two separate fractions. One fraction stimulated the activity of the cyclic AMP-dependent protein kinase and inhibited that of glycogen synthase phosphoprotein phosphatase. The other fraction retained the ability to inhibit the kinase activity and to increase that of phosphoprotein phosphatase. The biphasic response disappeared with this separation. Some of the data presented heretofore provide further indirect support for the concept of two messengers. The lesser stimulation of pyruvate dehydrogenase activity by the active Sephadex G-25 fraction from insulin-treated IM-9 lymphocytes as compared to the stimulation of this enzyme activity by the extract from control cells could result from insulin generation of the mediator, which blocks the phosphatase without altering the production of the stimulator of the phosphatase. The effects of the mediator isolated from adipocyte plasma membranes on the activity of (Ca++-M$++)-ATPase suggest that at high concentrations of the mediator, the enzyme was activated. Recent studies with the hepatoma cells have shown that increasing concentrations of insulin up to 5 mu/ml did not produce a biphasic effect. One possible explanation for this finding is that only one type of mediator is produced in this cell line in response to the action of insulin [ 161. CHEMICAL NATURE OF THE MEDIATOR The methods used for purification of the mediator of insulin action depend on the composition of the medi-
ators. The active material has been detected in enriched plasma membrane fractions from both adipocytes and hepatocytes. Therefore, the mediator may be a representative of one of the following three primary constituents of plasma membranes: phospholipid, protein, or glycoprotein. Purification of the mediator by standard methods has been difficult. At present, investigations in our laboratory suggest that it is not a simple peptide or glycopeptide. Efforts to identify the chemical nature of the mediator by our laboratory have included attempts to destroy the material by enzymes or to bind the material to columns with specific affinities. The biologic activity of the material can be monitored before and after such experimentation. Incubation of the active material for one hour with nonspecific or specific proteolytic enzymes bound to Sepharose resulted in partial or no inactivation of the factor. Therefore, peptide bonds do not appear to be a structural element essential for the activity of the mediator. This conclusion was supported by the fact that dansylation of the active material had no effect on its activity. The role of sugar or aminosugar residues was determined by chromatography with lectins bound to Sepharose. We were unable to bind the mediator to any lectin column; therefore, we believe it is unlikely that common oligosaccharides are a structural component of the mediator which are essential for activity. Our inability to document a peptide or glycopeptide component led us to consider phospholipids or proteolipids as possible chemical mediators of insulin action. The importance of phospholipids in biologic regulation is becoming more widely recognized, as is indicated by the ever increasing number of published studies on the subject. A literature search has revealed that there is very limited information on the turnover of phospholipids in response to the action of insulin [31-331, but the effects of phospholipase C or A [32,34,35] that mimic the effects of insulin indicated to Rodbell et al. [36] that insulin may cause a change in the disposition of phospholipids within the fat cell membrane. The recent studies that describe a phospholipiddependent, calcium-dependent protein kinase [37-411 suggest a direct relationship between the action of phospholipids and protein phosphorylation [42]. Phospholipids have also been shown to alter the activity of several membrane-bound enzymes directly [43]. Among these are Ca++-ATPase, (Ca++Mg++)-ATPase, pyruvate oxidase, high Km cyclic AMP phosphodiesterase, adenylate cyclase, and glucose6-phosphatase. The above data led us to investigate the possible role of phospholipids as chemical mediators of insulin action. Chloroform-methanol extraction followed by thin layer chromatography of the supernatant from adipocyte plasma membranes and the active Sephadex G-25 fraction from insulin-treated and control hepatoma cells
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35
DIABETES SYMF’OSIUM4AREll
have shown the presence of 10 phospholipids that were associated with plasma membrane. Dispersions of these and other phospholipids were added individually to pyruvate dehydrogenase and low Km cyclic AMP phosphodiesterase assays. No phospholipid that we tested altered the activity of the high Km cyclic AMP phosphodiesterase from plasma membranes or microsomes. Phosphatidylserine stimulated and phosphatidylinositol-4’-phosphate inhibited pyruvate dehydrogenase activity in a dose-dependent manner from 1 @I to 800 PM, with or without ATP. Eight other phospholipids had no effect, or only slightly inhibited pyruvate dehydrogenase activity. The stimulation of the activity of pyruvate dehydrogenase by phosphatidylserine was reversed by phosphatidylinositol-4’-phosphate. The inhibition of pyruvate dehydrogenase activity by phosphatidylinositol-4’-phosphate was abolished by increasing the Ca++ concentration in the assay. Sodium fluoride, a known pyruvate dehydrogenase phosphatase inhibitor that blocks the ability of the mediator to stimulate pyruvate dehydrogenase, inhibited the activation of the enzyme by phosphatidylserine. The activity of low Km cyclic AMP phosphodiesterase from adipocyte microsomes was stimulated above basal activity by three of these 10 phospholipids as follows: phosphatidylglycerol stimulated this enzyme activity more than did phosphatidylserine, and phosphatidylserine stimulated the enzyme more than lysophosphatidylcholine. The same three phospholipids activated the adipocyte plasma membrane enzyme, but the order of activation was changed: lysophosphatidylcholine activated it more than phosphatidylserine, and phosphatidylserine activated it more than phosphatidylglycerol. Kinetic studies indicated that these three phospholipids had no effect on the apparent Km for cyclic AMP for either enzyme. However, the apparent V, of both enzymes was increased in the same order as the stimulation of enzyme activity. Phosphatidylinositol4’-phosphate inhibited the activity of low Km cyclic AMP
phosphodiesterase in a dose-dependent manner. Kinetic studies with the microsomal low Km cyclic AMP phosphodiesterase have shown that phosphatidylinositol_4’-phosphate did not alter the apparent V, but increased the apparent Km. The marked similarities between the effect of phosphatidylserine and the mediator of insulin action on insulin-sensitive enzymes and the counterregulatory role of phosphatidylinositol-4’phosphate suggest that phosphatidylserine, phosphatidylinositol_4’-phosphate, or other related substances may represent the molecules involved in mediating the intracellular effects of insulin. More extensive studies are needed to delineate the role of phospholipids or proteolipids in insulin action. SUMMARY
AND CLINICAL SIGNIFICANCE
These studies would suggest that the mediators of insulin action may represent a new family of intracellular messengers, possibly involving various phospholipids or proteolipids. To date the data concern only some of the short-term effects of insulin. The mechanisms by which these potential mediators alter regulatory enzymes seem to be varied and complex, including control of phosphorylation; control of affinity for calcium (&I++) or calmodulin, 01 both: control of fluidity of membranes; or direct allosteric effects. These exciting new observations open numerous avenues of future investigation on the molecular mechanism of insulin actions. These studies have potential clinical significance as well as basic interests. The postreceptor defects of insulin resistance in obesity and type II diabetes (noninsulin-dependent diabetes mellltus) may result in part from abnormal release or metabolism of the mediators. Likewise, reactive hypoglycemia may result in part from excess production of the mediators. Oral hypoglycemic agents may carry out their extra-islet effects directly by stimulating release of the mediators or indirectly by making the membranes more sensitive to insulin stimulation. Positive identification of the insulin mediator will permit the testing of these theories.
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Jarett L, Smith RM: The stimulation of adipocyte plasma membrane magnesium ion-stimulated adenosine triphosphatase by insulin and concanavalin A. J Biol Chem 1974; 249: 5195-5199. Seals JR, McDonald JM. Jarett L: Direct effect of insulin on the labeling of isolated plasma membranes by [ y3*P] ATP Biochem Biophys Res Commun 1978; 83: 1365-1372. Seals JR, McDonald JM, Jarett L: Insulin effect on protein phosphorylation of plasma membranes and mitochondria in a subcellular system from rat adipocytes. I. kfentification of insulin-sensitive phosphoproteins. J Biol Chem 1979; 254: 699 l-6996. Seals JR, McDonald JM, Jarett L: Insulin effect on protein phosphorylation of plasma membranes and mitochondria in a subcellular system from rat adipocytes. II. Character-
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