Perspectives in Diabetes Some Thoughts on the Mechanism of Action of Insulin JOHN H. EXTON

Proposed mechanisms by which insulin exerts its effects are discussed. Evidence for a role for the tyrosine kinase activity of the insulin receptor and of a phosphorylation/dephosphorylation cascade is presented. The possible roles of phospholipid breakdown, diacylglycerol, and protein kinase C are discussed. The hypothesis that insulin elicits the hydrolysis of a glycosyl phosphatidylinositol to form a mediator of certain of its actions is considered in detail. The evidence that a G protein is involved in insulin action is analyzed. Diabetes 40:521-26,1991

Editor's note: Every now and then welcome humor grins out from under the mounds of paper found on editors' desks. It did so recently in correspondence from a renowned Southern humorist.

December 11, 1990 R. Paul Robertson, MD Editor, Diabetes Dear Paul, By this hand, I do commit the enclosed work that thou didst ask this humble scribe to render forth upon this near Christmastide. Verily, I hath laboured mightily on this task, wearing thin many quills, draining many wells of ink, and pestering many purveyors of parchment. At times, it has been a veritable pestilence, forcFrom the Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, and Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee. Address correspondence and reprint requests to John H. Exton, MD, PhD, Howard Hughes Medical Institute, Research Laboratories, Vanderbilt University School of Medicine, Rudolph Light Hall, Room 802, Nashville, TN 372320295. Received for publication 17 December 1990 and accepted in revised form 11 January 1991.

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ing me to broach butts of marmsy or seek the counsel of soothsayers, wizards, and other learned gentlemen. I have heroically curbed the urge to jest and pun in a valiant effort to give the work an air of lofty scholarship melded with understanding of unfathomable depths. Attempts to keep miscreant lawyers and disgruntled scribblers, at bay have been made by combining exquisite judgment and impeccable balance with mellifluous phrasing. Prithily, my liege, I yield unto thee this work of mere man that e'en the very gods would stumble to give birth to. Your humble servant, John H. Exton, MD, PhD December 28, 1990 John H. Exton, MD, PhD Dear Liegeman, I am in receipt of your humbly proffered yet prodigious paradigm of potential phospholipid-provided perturbations in insulin action. It languished not even a fortnight on mine desk despite its untimely arrival near Winter Solstice, which greatly limits reading hours due to scant solar lumination in this bleak Northern clime. I deem your opus intriguing and thank thee for it. Despite thy consultations with various wizards and soothsayers, thy verbiage is not unduly homogenized. Howe're, I understand not your use of "marmsy;" hereabouts we inbibe malmsey. Perhaps you could send me a draft of thine mysterious Southern concoction. More important, mislead not thyself by thinking mellifluous phrases will hold at bay the lawyers! To quote mine neighbouring Bard, "We must first kill all the lawyers and only then take our rest!" Doubtless thou will feel pleasure in learning that thine words on parchment carry such persuasive force and clarity of thought that I will send them forthwith to ye olde publisher without burdening thou with inspection

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by anonymous authorities whose judgment can sometimes be delivered on the bias. Thank thee! His Majesty Paulibus Rex Editor

January 7, 1991 R. Paul Robertson, MD My Liege, Truly thou art a scholar and poet of the first water, even though thou nightly doth quaff a tankard or two of malmsey—not to be confused with marmsy, a crude liquor that Southern peasantry doth swill upon, inflaming their senses and provoking much debauchery and unseemliness. I have heard tell that genteel Northern folk, upon passing marmsy through alembics and other various purifying devices doth make malmsey, but that this fluid hath the same effects upon their bodies as the cruder form. I thank thee that thou have vouchsafed to send mine screed to the presses with such dispatch. May Master Caxton and his fellow rollers do their inky work so that these words be broadcast to all and sundry. A pox on all lawyers and others that feast on man's suffering and frailty! Would that I could drown them all in a butt of malmsey! I remain, Your humble servant, John H. Exton, MD, PhD P.S. Some scholars and lesser wits that have looked upon the above-mentioned screed and the letter that journeyed with it northward, have opined that the letter and that written in return by thine own hand doth bring them greater pleasure than the screed! These gentlefolk doth humbly suggest that these letters and others that they may engender be also dispatched to Master Caxton that they may be inked and rolled also.

January 1 1 , 1991 John E. Exton, MD, PhD Dear Liegeman, As you like it. Sincerely, R. Paul Robertson, MD Editor

MEMBRANE-ASSOCIATED SIGNALING SYSTEMS

There are several general mechanisms by which a signal from a surface receptor is passed to the interior of a cell. The most widespread involves guanine nucleotide-binding proteins (G proteins) that are activated by interaction of agonists with receptors and act on various effector proteins. A mechanism used by many growth factors involves activation of an intrinsic receptor-protein tyrosine kinase, and evidence

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is emerging that some signaling could involve receptor-associated protein tyrosine phosphatases. Another mechanism used by certain neurotransmitters involves regulation of an ion channel that is part of the receptor complex. Recently, a new system has been discovered that is used by atrial natriuretic and certain other peptides and involves guanylate cyclase as an intrinsic component of the receptor. There are also many agonists that interact with plasma membrane receptors but for which the signaling mechanisms are unknown. In general, a receptor or receptor subtype is not directly coupled to two different types of signaling mechanism, e.g., to both tyrosine kinase and G proteins. However, a receptor may couple to more than one G protein, and a G protein may interact with more than one effector. Furthermore, some effectors, e.g., phosphoinositide phospholipase C, can be controlled by both G proteins and tyrosine phosphorylation, although different isozymes are involved. Certain phospholipase isozymes and G proteins can also be phosphorylated by protein kinase C (PKC) or cAMP-dependent protein kinase, and certain cell proteins can be multiply phosphorylated by these and other kinases. These interactions provide many opportunities for cross talk between the different signaling systems. PROTEIN PHOSPHORYLATION/DEPHOSPHORYLATIONSIGNALING SYSTEM FOR INSULIN

There is no dispute concerning the structure of the insulin receptor and of the location of its insulin-binding and tyrosine kinase domains (1-3). The tyrosine kinase activity of the 0subunit has been rigorously explored, and most studies indicate that this activity is essential for insulin action. Thus, deletions and truncations that result in loss of insulin-stimulated kinase activity or natural or induced mutations in the ATP-binding site cause loss of insulin effects on hexose transport and other processes (1-5). Likewise, inhibition of kinase activity by monoclonal antibodies or other inhibitors impairs insulin effects in several cell types (4-7). Although there is strong evidence that kinase activity is essential for insulin action, some studies with mutated or chimeric receptors indicate that certain other changes in the cytoplasmic tail of the p-subunit can interfere with signal transmission (2,3,5). A major question is the identity of the immediate cellular targets of the insulin-stimulated tyrosine kinase. One substrate is the receptor itself (6-8). This raises the possibility that it is a conformational or other change induced in the receptor by autophosphorylation rather than the increased tyrosine kinase activity per se that is involved in generating or modulating some insulin effects. This possibility is supported by the finding that mutations of the autophosphorylation sites alter insulin responses (6,7). The postulated conformational change induced by autophosphorylation could either control access of certain substrates to the tyrosine kinase or permit the cytoplasmic tail to interact noncovalently with proteins involved in the signal transduction. If the latter change were induced by some monoclonal antibodies to the insulin receptor with little or no activation of the tyrosine kinase, this could explain why these antibodies stimulate glucose and amino acid transport (8,9). The insulin receptor phosphorylates many substrates on

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tyrosine in vitro, and many substrates have been identified in intact cells, especially in the presence of phosphatase inhibitors. These cellular substrates include many proteins (e.g., 15,000, 60,000, 120,000, 185,000, or 240,000 Mr) of unknown function (4,5), phosphatidylinositol 3-kinase (10), and a 42,000-/Wr kinase that phosphorylates microtubuleassociated protein 2 (MAP2) (11). There are also many proteins that are phosphorylated on serine and/or threonine in response to insulin in intact cells. Examples are the receptor itself, ATP citrate lyase, acetyl-CoA carboxylase, S6 ribosomal protein (5,12,13), an isozyme of cAMP phosphodiesterase (14), Raf-1 protein kinase (15,16), and some proteins of unknown function (5,13). The functional significance of these phosphorylations is unclear except in the case of cAMP phosphodiesterase, which is activated. The simplest explanation of the above findings is that stimulation of the receptor tyrosine kinase and/or a resultant change in the receptor lead to the phosphorylation and activation of one or more protein serine/threonine kinases. In this regard, insulin treatment of several cell lines results in the phosphorylation and/or stable activation of various serine/threonine kinases assayed in vitro, e.g., MAP2 kinase, casein kinase II, protease-activated kinase II, two forms of S6 kinase, and Raf-1 kinase (4,5,11-13,15-18). However, none of these has been shown to be phosphorylated by the insulin-receptor tyrosine kinase in vitro, although MAP2 kinase is phosphorylated on tyrosine and threonine in intact cells, and dephosphorylation of either residue(s) results in inactivation (11,18). MAP2 kinase can phosphorylate and activate the 85,000-/Wr form of S6 kinase from Xenopus and may thus participate in a phosphorylation cascade initiated by insulin (8), but MAP2 kinase does not phosphorylate or activate the 70,000-/Wr form of S6 kinase that predominates in mammalian cells (17), indicating that another kinase is involved in its activation by insulin. Potential targets of phosphorylation are the insulin-sensitive forms of the glucose transporter, but there is no evidence that these proteins are phosphorylated in response to insulin. Obviously, a reason why no serine/threonine kinase has been found to be directly phosphorylated by the receptor tyrosine kinase is that its interaction with receptor may be noncovalent. It is well known that insulin promotes the dephosphorylation of several proteins (5,13). In some cases, this is attributable to a decrease in cAMP, whereas in others, it is independent of cAMP. There is much indirect evidence that insulin stimulates protein phosphatase activity (5,13), and there have been reports that it activates a type 1 phosphatase in several tissues (19). Recently, evidence has been presented that an insulin-stimulated protein kinase activates type 1 phosphatase in skeletal muscle by phosphorylating its G subunit on a specific serine (19). This results in activation of glycogen synthase and inactivation of phosphorylase kinase and can thus explain the effects of insulin on glycogen metabolism. However, based on the identities of other proteins dephosphorylated in response to insulin, other phosphatases, e.g., type 2A, are probably involved. The mechanisms by which these phosphatases are stimulated are unknown. Potential complications arising from the simultaneous activation of protein kinases and phosphatases could be obviated if these enzymes exhibited high substrate specificity

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or if they and/or their substrates were highly localized in the cell. Because insulin controls plasma membrane transport processes, cytosolic and mitochondrial enzyme activities, and nuclear transcriptional events, any mechanism of action must affect activities in all of these compartments. A phosphorylation/dephosphorylation mechanism operating in the cytosol could affect the phosphorylation state of proteins located in the cytosol and on the cytoplasmic surfaces of organelles. It could also result in the formation of molecules that enter organelles and alter the activity of proteins within them, or it could alter the translocation of such molecules into the organelles. PHOSPHOLIPID-SIGNALING SYSTEM FOR INSULIN

In contrast to the mounting evidence that insulin initiates a phosphorylation/dephosphorylation cascade, data supporting a role for phospholipid or glycophospholipid hydrolysis in insulin action remain fragmentary and controversial. Thus, some laboratories report that, in some cell types, insulin rapidly elevates diacylglycerol (DAG) measured chemically or isotopically, whereas other groups see no changes in cells that are major targets of the hormone (20-24). There have also been reports that insulin activates PKC (20,22,25,26), although this has been disputed (27,28), and the evidence that there is an associated translocation is inconsistent. Addition of active phorbol esters to many cells induces some effects similar to those caused by insulin but also some that are the opposite (13,20,27), and the patterns of protein phosphorylation are different (27,29,30). Furthermore, some insulin effects persist in cells depleted of PKC (27,29,30). Although, it is possible that an isozyme of PKC is involved in some actions of insulin, the data can also be explained if this kinase acted on some of the same substrates as the insulin-stimulated protein kinase(s). Because insulin produces very different effects from Ca2+-mobilizing agonists, if insulin acts in part by generating DAG, this must be chemically different from that generated by these agonists or must be in a different cellular compartment. However, there have been no chemical analyses of the fatty acid composition and molecular species of the DAG in any cell type treated with insulin and no studies of its intracellular location. Despite the inconsistency in the reported effects of insulin on DAG, which is surprising because of the large increases reported by one group (21,22), possible sources of this lipid may be considered. In many cells, insulin stimulates de novo synthesis of DAG, but this is usually glucose dependent. Other ways in which insulin could elevate DAG are by hydrolysis of phospholipids, glycophospholipids, or triacylglycerol; inhibition of the synthesis of triacylglycerol, phosphatidylcholine, or phosphatidylethanolamine; increased phosphatidate phosphohydrolase activity; and reduced DAG kinase or DAG lipase activity. Of these possibilities, the only ones proposed are de novo synthesis and the hydrolysis of a phospholipid or glycophospholipid. If insulin generates DAG through hydrolysis of a phospholipid, this should be identifiable by its characteristic fatty acid composition or molecular-species profile or its selective labeling by certain fatty acids. Labeling of the head group of the phospholipid, and isotopic or chemical measurement of the released head group should identify it more specifically.

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Some early reports indicated that insulin stimulated the hydrolysis of inositol phospholipids in adipose tissue (e.g., 31). However, efforts to confirm these findings have been unsuccessful, and it is generally accepted that insulin does not increase inositol 1,4,5-trisphosphate (IP3) or elevate cytosolic Ca2+ (23,32). On the other hand, there is evidence that insulin stimulates the synthesis of phosphoinositides and other phospholipids in adipose tissue and BC3H1 cells (21,23,33), due in part to increased de novo synthesis of phosphatidic acid (21,23,34). There is a single report that insulin stimulates the release of [3H]phosphocholine from BC3H1 cells previously labeled with [3H]choline (35), but the implication that phosphatidylcholine hydrolysis is stimulated needs further support. GLYCOSYL PHOSPHATIDYLINOSITOL (GPI)-SIGNALING SYSTEM FOR INSULIN

The hypothesis that insulin signaling involves the release of an inositol phosphate glycan (IPG) from a GPI (20,36) arose out of earlier reports from the laboratories of Jarett (37) and Lamer (38) of insulin-dependent second messengers that influenced the activities of several enzymes. Despite its initial attractiveness, most features of the system remain unclear and controversial. In any cell signaling system, the chemical nature of the components must be defined before the system can be understood. This applies particularly to postulated second messenger molecules, which must be synthesized ultimately and shown to reproduce part or all of an agonist's action(s). It has been proposed that the insulin-sensitive GPI is similar in structure to the GPIs that anchor many cell surface proteins, e.g., trypanosome-variant surface glycoprotein, 5'-nucleotidase, alkaline phosphatase, and acetylcholinesterase (20,36,39). The structure of the GPI anchor for the variant surface glycoprotein is defined, and there is extensive knowledge of the composition of the other anchors (39,40), but the structures of the insulin-sensitive GPI and its IPG product remain elusive. Isotopic labeling studies of the insulin-sensitive GPI have provided some areas of agreement, but there are also some differences, perhaps arising from heterogeneity. Most reports indicate the presence of glucosamine and myo-inositol (20,36,39), although there is evidence for c/i/ro-inositol in rat liver and H35 hepatoma cells (41). Most studies also report the incorporation of labeled myristic acid into a DAG moiety (20,36,39), whereas an alkylacylglycerol structure containing mainly palmitic acid has been proposed for the hepatoma cell GPI (42). The presence of phosphate and galactose has been reported (42) but not that of ethanolamine or mannose, which are present in all GPI anchors (39,40). However, it is not always demonstrated that the material being analyzed is insulin sensitive. The pitfalls in the area are illustrated by the finding that a material synthesized by liver microsomes and reported to be GPI is actually a mixture of lysophospholipids and does not contain hexosamine or ethanolamine (43). With respect to the insulin-sensitive IPG, even less is certain. Surprisingly, no data on the chemical composition of the active material released from plasma membranes by insulin action have been published. Instead, investigators

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have mainly examined fractions produced by bacterial phosphoinositide phospholipases that mimic the actions of insulin on certain enzymes (20,36,39). Although the chromatographic and electrophoretic properties of these compounds have been stated to be identical to those of the material(s) produced by insulin (20,36,44), published data supporting this crucial contention are very limited and not conclusive (45). In most cases, the IPG fractions used for chemical and biological studies have undergone minimal or no purification after preparation by the action of bacterial phospholipases on GPI fractions isolated from cell or membrane lipid extracts by sequential thin-layer chromatography. In accordance with the GPI data, most IPG fractions can be labeled with glucosamine and either myo-inositol or c/i/ro-inositol (20,36). The extent of labeling with phosphate or galactose is uncertain. Because of the absence of chemical or other assays, the concentrations of IPG and GPI are unknown in any tissue. In accord with their function, the GPI anchors for cell surface proteins project from the surface of the cell (39,40). However, the orientation of the insulin-sensitive GPI is uncertain (20,36), and a surface projection seems a priori inappropriate for a molecule that is presumed to be cleaved to an intracellular messenger. Either it has the opposite membrane orientation or some mechanism exists for the cellular uptake of the IPG messenger. The idea that IPG can be taken up by cells is supported by reports that adding IPG preparations of undefined purity to intact cells modifies the activities of certain intracellular enzymes (20,46). However, hexose transport is not stimulated, and many insulin-sensitive enzymes are not affected (46). Furthermore, some enzymes that are altered are not generally recognized as insulin targets. Antibodies to the trypanosome-variant surface glycoprotein have been reported to block the action of insulin on pyruvate dehydrogenase but not hexose transport or glycogen synthase in BC3H1 cells (47), but the mechanism of the inhibition is uncertain. Second messengers for hormones or neurotransmitters usually have primary cellular targets to which they bind with high affinity, e.g., the cyclic nucleotide protein kinases, PKC, calmodulin, and the IP3 receptor. These proteins undergo specific changes and amplify the message by interacting with many proteins or by utilizing other mechanisms. The published effects of IPG indicate that it interacts with diverse enzymes in vitro and does not have a primary target (20,36,46). If these observations are correct, then an important amplification step is missing from this signaling system. It also means that all the target enzymes must have binding sites for IPG that are capable of influencing the diverse activities of the enzymes. If insulin action is mediated in part through the hydrolysis of a unique GPI or phospholipid (20,36), there must be a mechanism by which the relevant phospholipase is activated. This could involve tyrosine phosphorylation or a G protein. There is much evidence that the 7 r isozyme of phosphoinositide, phospholipase C, is phosphorylated and activated by the tyrosine kinases of the epidermal growth factor and platelet-derived growth factor receptors, but efforts to show similar phosphorylation by the insulin receptor have been unsuccessful (48). A partial purification of a GPI-

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specific phospholipase C from liver membranes has been achieved (49), but it is not known if this is insulin responsive or can be phosphorylated by the insulin receptor. POSSIBLE ROLE FOR G PROTEINS

The idea that insulin action is partly mediated by a G protein has resulted in many studies. It has been found that high concentrations of guanine nucleotides (0.1-1 mM) inhibit insulin binding to plasma membranes from some but not all cells (50-52) and that insulin alters GTP7S binding to membranes from BC3HI cells but not from liver or adipocytes (50,52). It has also been shown that pertussis toxin alters insulin binding and inhibits some but not all insulin effects in several cell types (52-55), but it is unclear that these effects are the direct result of ADP ribosylation of a G protein. Interaction between the insulin receptor and a G protein(s) is suggested by the observations that GTP7S inhibits insulininduced tyrosine phosphorylation of several proteins in L6 muscle cells and adipocyte plasma membranes (50,51) and that insulin treatment of liver plasma membranes inhibits pertussis toxin-catalyzed ADP ribosylation of Gj (55), but again these effects may be indirect. An earlier report that insulin inhibits cholera toxin-dependent ADP ribosylation of a 25,000-Mr protein, termed Gins, in liver membranes (56) has not been reproduced (57), and the 25,000-Mr protein may have been the cholera toxin A subunit (57). There have also been studies showing that the insulin receptor from human placenta or rat liver can phosphorylate the a-subunits of Gi, Go, and G, (58-60), but there is no evidence that these proteins are phosphorylated in intact cells (55). Insulin-stimulated tyrosine phosphorylation of a G protein would represent a novel means of cellular signal transduction. However, such a phosphorylation should alter the interaction of the G protein with known insulin-responsive effectors. Currently, few, if any, of the accepted enzyme targets of insulin are known to be regulated by G proteins. SUMMARY

The major theme of this Perspective is to delineate experiments to confirm or refute key aspects of proposed mechanisms of insulin action. In an area where there is little general agreement, it seems that the tyrosine kinase activity of the receptor is essential for insulin action and that multiple signaling mechanisms are involved. Because it is not known at what steps the pathways diverge, the various proposed mechanisms are not mutually exclusive. Whatever particular mechanism one espouses—e.g.,aphosphorylation/dephosphorylation cascade initiated by the insulin-receptor tyrosine kinase; the hydrolysis of a specific glycosyl phospholipid or phospholipid to yield DAG, IPG, or other messengers; or a resultant or independent activation of a putative PKC isozyme—there are serious deficiencies in knowledge. Major uncertainties with the phosphorylation/dephosphorylation mechanism are its linkage to the insulin-receptor tyrosine kinase and the mechanisms involved in dephosphorylation. Problems with the proposed PKC mechanism are uncertainties about the extent to which it operates, if at all, and about the isozyme involved arid its mechanism of activation. Difficulties with the GPI hypothesis relate to a serious lack of knowledge about its chemical structure and that of its IPG

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product, its location in the plasma membrane, the mechanism by which insulin promotes its cleavage, and the mechanism of action of IPG on its target enzymes. In addition, the possible involvement of G proteins lacks any direct support. Surprisingly, methods and approaches exist to explore many of these deficiencies, especially the rigorous chemical identification of postulated signaling components, but many investigators have been deterred by the slow progress in the area and the lack of unanimity on many points. I hope that the next few years will refute the jocular characterization of insulin as a hormone without a mechanism of action! ACKNOWLEDGMENTS

I thank Judy Childs for typing seemingly interminable versions of this paper and the innumerable colleages who made helpful comments during its preparation and/or supplied reprints and preprints.

REFERENCES 1. Rosen OM: Banting Lecture 1989: structure and function of insulin receptors. Diabetes 38:1508-11, 1989 2. Wente SR, Rosen OM: Insulin-receptor approaches to studying protein kinase domain. Diabetes Care 13:280-87, 1990 3. Olefsky JM: The insulin receptor: a multifunctional protein. Diabetes 39:1009-16, 1990 4. Kasuga M, Izumi T, Tobe K, Shiba T, Momomura K, Tashiro-Hashimoto Y, Kadowaki T: Substrates for insulin-receptor kinase. Diabetes Care 13:317-26, 1990 5. Avruch J, Tornqvist HE, Gunsalus JR, Yurkow EJ, Kyriakis JM, Price DJ: Insulin regulation of protein phosphorylation. In Handbook of Experimental Pharmacology. Vol 92. Cuatrecasas P, Jacobs S, Eds., Berlin, Springer. Verlag, 1990, p. 313-66 6. Shechter Y, Yaish P, Chorev M, Gilon C, Braun S, Levitzki A: Inhibition of insulin-dependent lipogenesis and anti-lipolysis by protein tyrosine kinase inhibitors. EMBO J 8:1671-76, 1989 7. Saperstein R, Vicario PP, Strout HV, Brady E, Slater EE, Greenlee WJ, Ondeyka DL, Patchett AA, Hangauer DG: Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells. Biochemistry 28:5694-701, 1989 8. Hawley DM, Maddux BA, Patel RG, Wong K-Y, Mamula PW, Firestone GL, Brunetti A, Verspohl E, Goldfine ID: Insulin receptor monoclonal antibodies that mimic insulin action without activating tyrosine kinase. J Biol Chem 264:2438-44,1990 9. Soos MA, O'Brien RM, Brindle NPJ, Stigter JM, Okamoto AK, Whittaker J, Siddle K: Monoclonal antibodies to the insulin receptor mimic metabolic effects of insulin but do not stimulate receptor autophosphorylation in transfected NIH 3T3 fibroblasts. Proc Natl Acad Sci USA 86:5217-21, 1989 10. Endemann G, Yonezawa K, Roth RA: Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receptor tyrosine kinase. J Biol Chem 265:396-400, 1990 11. Ray LB, Sturgill TW: Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo. Proc Natl Acad Sci USA 85:3753-57, 1988 12. Czech MP, Klarlund JK, Yagaloff KA, Bradford AP, Lewis RE: Insulin receptor signaling: activation of multiple serine kinases. J Biol Chem 263:11017-20, 1988 13. Denton RM: Early events in insulin actions. In Advances in Cyclic Nucleotide and Protein Phosphorylation Research. Vol 20. Greengard P, Robison GA, Eds. New York, Raven, 1986, p. 293-341 14. Degerman E, Smith CJ, Tornqvist H, Vasta V, Belfrage P, Manganiello VC: Evidence that insulin and isoprenaline activate the cGMP-inhibited low Km cAMP phosphodiesterase in rat fat cells by phosphorylation. Proc Natl Acad Sci USA 87:533-37, 1990 15. Blackshear PJ, Haupt DM, App H, Rapp UR: Insulin activates the Raf-1 protein kinase. J Biol Chem 265:12131-34, 1990 16. Kovacina KS, Yonezawa K, Brantigan DL, Tonks NK, Rapp UR, Roth RA: Insulin activates the kinase activity of the Raf-1 proto-oncogene by increasing its serine phosphorylation. J Biol Chem 265:12115-18, 1990 17. Price DJ, Gunsalus JR, Avruch J: Insulin activates a 70-kDa S6 kinase through serine/threonine-specific phosphorylation of the enzyme polypeptide. Proc Natl Acad Sci USA 87:7944-48, 1990 18. Anderson NG, Mailer JL, Tonks NK, Sturgill TW: Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature (Lond) 343:651-53, 1990

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MECHANISM OF INSULIN ACTION 19. Dent P, Lavoinne A, Nakielny S, Caudwell FB, Watt P, Cohen P: The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle. Nature (Lond) 348:302-308, 1990 20. Saltiel AR, Osterman DG, Darnell JC, Chan BL, Sorbara-Cazan LR: The role of glycosylphosphoinositides in signal transduction. Recent Prog Horm Res 45:353-82, 1989 21. Farese RV, Cooper DR, Konda TS, Nair G, Standaert ML, Davis JS, Pollet RJ: Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes. Biochem J 256:175-84, 1988 22. Cooper DR, Hernandez H, Kuo JY, Farese RV: Insulin increases the synthesis of phospholipid and diacylglycerol and protein kinase C activity in rat hepatocytes. Arch Biochem Biophys 276:486-94, 1990 23. Augert G, Exton JH: Insulin and oxytocin effects on phosphoinositide metabolism in adipocytes. J Biol Chem 263:3600-609, 1988 24. Turinsky J, Bayly BP, O'Sullivan DM: 1,2-Diacylglycerol and ceramide levels in rat skeletal muscle and liver in vivo: studies with insulin, exercise, muscle denervation, and vasopressin. J Biol Chem 265:7933-38, 1990 25. Cooper DR, Konda TS, Standaert ML, Davis JS, Pollet RJ, Farese RV: Insulin increases membrane and cytosolic protein kinase C activity in BC3H-1 myocytes. J Biol Chem 262:3633-39, 1987 26. Egan JJ, Saltis J, Wek SA, Simpson IA, Londos C: Insulin, oxytocin, and vasopressin stimulate protein kinase C activity in adipocyte plasma membranes. Proc Natl Acad Sci USA 87:1052-56, 1990 27. Spach DH, Nemenoff RA, Blackshear PJ: Protein phosphorylation and protein kinase activities in BC3H-1 myocytes. J Biol Chem 261:1275053, 1986 28. Klip A, Ramlal T: Protein kinase C is not required for insulin stimulation of hexose uptake in muscle cells in culture. Biochem J 242:131 -36,1987 29. Blackshear PJ, Nemenoff RA, Hovis JG, Halsey DL, Stumpo DJ, Huang J-K: Insulin action in normal and protein kinase C-deficient rat hepatoma cells: effects on protein phosphorylation, protein kinase activities, and ornithine decarboxylase activities and messenger ribonucleic acid levels. Mol End 1:44-52, 1987 30. Blackshear PJ, Witters LA, Girard PR, Kuo JF, Quamo SN: Growth factorstimulated protein phosphorylation in 3T3-L1 cells. J Biol Chem 260:13304-15, 1985 31. Farese RV, Kuo JY, Babischkin JS, Davis JS: Insulin provokes a transient activation of phospholipase C in the rat epididymal fat pad. J Biol Chem 261:8589-92, 1986 32. Klip A, Ramlal T: Cytoplasmic Ca 2+ during differentiation of 3T3-L1 adipocytes: effect of insulin and relation to glucose transport. J Biol Chem 262:9141-46, 1987 33. Pennington SR, Martin BR: Insulin-stimulated phosphoinositide metabolism in isolated fat cells. J Biol Chem 260:11039-45, 1985 34. Vila MC, Milligan G, Standaert ML, Farese RV: Insulin activates glycerol3-phosphate acyltransferase (de novo phosphatidic acid synthesis) through a phospholipid-derived mediator: apparent involvement of Gia and activation of a phospholipase C. Biochemistry 29:8735-40, 1990 35. Nair GP, Standaert ML, Pollet RJ, Cooper DR, Farese RV: Effects of insulin and phorbol esters on diacylglycerol generation and synthesis and hydrolysis of phosphatidylcholine in BC3H-1 myocytes. Biochem Biophys Res Commun 154:1345-49, 1988 36. Saltiel AR: Second messengers of insulin action. Diabetes Care 13:24456,1990 37. Jarett L, Kiechle FL: Intracellular mediators of insulin action. Vitam Horm 41:51-78, 1984 38. Larner J: Banting Lecture 1987: insulin-signaling mechanisms: lessons from the Old Testament of glycogen metabolism and the New Testament of molecular biology. Diabetes 37:262-75, 1988 39. Low MG, Saltiel AR: Structural and functional roles of glycosyl-phosphatidylinositol in membranes. Science 239:268-75, 1988 40. Doering TL, Masterson WJ, Hart GW, Englund PT: Biosynthesis of glycosyl phosphatidylinositol membrane anchors. J Biol Chem 265:611-14, 1990

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41. Mato JM, Kelly KL, Abler A, Jarett L, Corkey BE, Cashel JA, Zopf D: Partial structure of an insulin-sensitive glycophospholipid. Biochem Biophys Res Commun 146:764-70, 1987 42. Mato JM, Kelly KL, Abler A, Jarett L: Identification of a novel insulinsensitive glycophospholipid from H35 hepatoma cells. J Biol Chem 262:2131-37, 1987 43. Thakkar JK, Raju MS, Kennington AS, Foil B, Caro JF: [3H]myoinositol incorporation into phospholipids in liver microsomes from humans with and without type II diabetes. J Biol Chem 265:5475-81, 1990 44. Saltiel AR, Cuatrecasas P: Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycolipid. Proc Natl Acad Sci USA 83:5793-97, 1986 45. Saltiel AR, Fox JA, Sherline P, Cuatrecasas P: Insulin-stimulated hydrolysis of a novel glycolipid generates modulation of cAMP phosphodiesterase. Science 233:967-72, 1986 46. Witters LA, Watts TD, Gould GW, Lienhard GE, Gibbs EM: Regulation of protein phosphorylation by insulin and an insulinomimetic oligosaccharide in 3T3-L1 adipocytes and fao hepatoma cells. Biochem Biophys Res Commun 153:992-98, 1988 47. Romero G, Gamez G, Huang LC, Lilley K, Luttrell L: Anti-inositolglycan antibodies selectively block some of the actions of insulin in intact BC3H1 cells. Proc Natl Acad Sci USA 87:1476-80, 1990 48. Nishibe S, Wahl Ml, Wedegaertner PB, Kim JJ, Rhee SG, Carpenter G: Selectivity of phospholipase C phosphorylation by the epidermal growth factor receptor, the insulin receptor, and their cytoplasmic domains. Proc Natl Acad Sci USA 87:424-28, 1990 49. Fox JA, Soliz NM, Saltiel AR: Purification of a phosphatidylinositol-glycanspecific phospholipase C from liver plasma membranes: a possible target of insulin action. Proc Natl Acad Sci USA 84:2663-67, 1987 50. Burdett E, Mills GB, Klip A: Effect of GTP^S on insulin binding and tyrosine phosphorylation in liver membranes and L6 muscle cells. Am J Physiol 258:C99-108, 1990 51. Davis HW, McDonald JM: Insulin receptor function is inhibited by guanosine 5'-[>-thio]triphosphate (GTP[S]). Biochem J 270:401-407, 1990 52. Luttrell L, Kilgour E, Larner J, Romero G: A pertussis toxin-sensitive Gprotein mediates some aspects of insulin action in BC3H-1 murine myocytes. J Biol Chem 265:16873-79, 1990 53. Luttrell LM, Hewlett EL, Romero G, Rogol AD: Pertussis toxin treatment attenuates some effects of insulin in BC3H-1 murine myocytes. J Biol Chem 263:6134-41, 1988 54. Ciaraldi TP, Maisel A: Role of guanine nucleotide regulatory proteins in insulin stimulation of glucose transport in rat adipocytes. Biochem J 264:389-96, 1989 55. Rothenberg PL, Kahn CR: Insulin inhibits pertussis toxin-catalyzed ADPribosylation of G-proteins: evidence for a novel interaction between insulin receptors and G-proteins. J Biol Chem 263:15546-52, 1988 56. Heyworth CM, Whetton AD, Wong S, Martin BR, Houslay MD: Insulin inhibits the cholera-toxin-catalyzed ribosylation of a M r 25,000 protein in rat liver plasma membranes. Biochem J 228:593-603, 1985 57. Lynch CJ, Blackmore PF, Johnson EH, Wange RL, Krone PK, Exton JH: Guanine nucleotide binding regulatory proteins and adenylate cyclase in livers of streptozotocin- and BB/Wor-diabetic rats. J Clin Invest 83:2050-62, 1989 58. Zick Y, Sagi-Eisenberg R, Pines M, Gierschik P, Spiegel AM: Multisite phosphorylation of the a subunit of transducin by the insulin receptor kinase and protein kinase C. Proc Natl Acad Sci USA 83:9294-97, 1986 59. O'Brien RM, Houslay MD, Milligan G, Siddle K: The insulin receptor tyrosyl kinase phosphorylates holomeric forms of the guanine nucleotide regulatory proteins G, and Go. FEBS Lett 212:281-88, 1987 60. Krupinski J, Rajaram R, Lakonishok M, Benovic JL, Cerione RA: Insulindependent phosphorylation of GTP-binding proteins in phospholipid vesicles. J Biol Chem 263:12333-41, 1988

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