Alterations in Insulin Receptor and Substrate Phosphorylation in Hypertensive Rats. C. Ronald Kahn1 and Mario J.A. Saad ABSTRACT. The Insulin Receptor

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1 Alterations in Insulin Receptor and Substrate Phosphorylation in Hypertensive Rats C. Ronald Kahn1 and Mario J.A. Saad CR. Kahn, M.J.A. Saad, Research Division, Joslin Diabetes Center. Department of Medicine, Brigham and Women s Hospital. and Harvard Medical School, Boston, MA (J. Am. Soc. Nephrol. 1992; 3: ) ABSTRACT Insulin stimulates tyrosine phosphorylation of the insulin receptor and of an endogenous substrate of -185 kd (insulin receptor substrate I or IRS-I) in most cell types. Tyrosine phosphorylation of insulin recepbr and of IRS- I have been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the study presented here, the levels and phosphorylation state of the insulin receptor and IRS- I in liver and muscle after insulin stimulation in vivo have been examined in spontaneously hypertensive rats (SHR) by immunoblotting with antipeptide antibodies to insulin receptor and IRS-I and antiphosphotyrosine antibodies. It was found that the levels of insulin receptor and IRS-I protein in liver and muscle are similar in controls (Wistar-Kyoto rats) and SHR. By contrast, there is a decrease in autophosphorylotion in the liver and muscle of SHR and a parallel decrease in phosphorylation of IRS-I. These data indicate that reduced insulin receptor kinase activity and reduced substrate phosphorylation may play an important role in the impaired insulin action in the hypertensive rat. Key Words; Insulin receptor kinase. insulin resistance, insulin receptor substrate, hypertension. spontaneously hypertens/ye rat A lthough the relationship between insulin and cellular metabolism has been under intense study since the discovery of the hormone in , it is only with the recent characterization of the insulin receptor and related down-strain components of the insulin response system that we have come to have I Correspondence to Dr. CR. Kahn, Research Division, Joslin Diabetes Center. One Jostin Place, Boston, MA I /0304-0S69$03.00/0 Journal of the American Society of Nephrology Copyright C 1992 by the American Society of Nephrology even a rudimentary understanding of this relationship on the molecular level. The importance of understanding insulin action has been pointed out by the prevalence of Insulin resistance and by the fact that insulin resistance has an important role in the pathogenesis of many disorders, including obesity, diabetes melhitus, uremia, and hypertension. A broad understanding of the current concepts of the mechanism of insulin action is necessary if we are to illucidate the molecular mechanisms involved in the insulin resistance of hypertension and other pathophysiologic states. INSULIN ACTION The Insulin Receptor The actions of insulin at the cellular level are mitiated by insulin binding to its plasma membrane receptor (1-5). This receptor is present in virtually all mammalian tissues, although the concentration varies from less than 1 00 receptors per cell on circuhating erythrocytes to more than 200,000 receptors per cell on adipocytes and hepatocytes. The insulin receptor is a heterotetrameric glycoprotemn consisting of two a-subunits, each of Mr of 135,000, and two fisubunits, each of Mr of 95,000, linked by disulfide bonds to give a 13-a-a-$ structure (2,5). The a-subunit is entirely extracelluhar and contains the insulinbinding site. The 13-subunit is a transmembrane protein and is responsible for signal transduction. The insulin receptor gene is situated on the short arm of chromosome 19. The structure of the insulin receptor gene was established in and consists of 22 exons and 21 introns (6-8). Two forms of insulin proreceptor are synthesized from this gene, which contained either 1,370 or 1,382 amino acids. The difference in size is accounted for by the a- subunit and depends upon alternate splicing of exon 1 1, which adds or deletes 1 2 amino acids. The relative abundance of these two slice variants is reguhated in a tissue-specific manner (9). The implications for functional differences between the two variants is at an early stage and is controversial but has been suggested to play a role in non-insulin dependent diabetes melhitus (10). The extracellular a-subunit confers high affinity insulin binding to the receptor. Chemical modification and mutagenesis studies suggest that specific domains, particularly the region encoded by exons 2 and 3, are most critical for higand binding (1 1 ). Cys- Journal of the American Society of Nephrology S69

2 Alterations in Insulin Receptor and Substrate Phosphorylation in SHR teine residues in the a-subunit and ectodomain of the a-subunit participate in disulfide bonding between the a-subunit and the fl-subunit. Thus far, only one specific cystemne of the a-subunit (Cys647) has been directly demonstrated to participate in the binding of the a-is subunits (12) and the cysteines involved in a-a binding are uncertain. The transmembrane domain of the (3-subunit is responsible for transducing the signal of insulin binding through the membrane to the cytoplasmic domain. Phosphorylation in Insulin Action A major breakthrough in understanding transmembrane signaling by the receptor came in 1982 with the finding that the a-subunit of the receptor was an insulin-stimulated protein kinase capable of phosphorylating itself and other substrates on tyrosine residues (1 3). Insulin binds to the a-subunit and stimulates tyrosine phosphorylation of the fl-subunit of the insulin receptor. ATP acts as the phosphate donor, and phosphorylation occurs exclusively on tyrosmne residues. Insulin stimulates the kinase primanly by increasing the catalytic velocity ( Vmax) rather than by changing substrate affinity (14). The exact mechanism of the insulin stimulation is unknown, but it appears to involve a release of an inhibitory effect exerted by the a-subunit on the fisubunit function. Autophosphorylation of the insulin receptor occurs through a cascade of intramolecular phosphorylation. As a result, at least five tyrosmnes in the intracellular portion of the fl-subunit are phosphorylated. Three of these tyrosines occur in a cluster at residues 1 158, 1 162, and on the fl-subunit (15).2 When all three of these tyrosines are phosphorylated, the kinase is further activated toward exogenous substrates. This autophosphorylation appears to be important for signal transduction, because kinase activity remains enhanced as long as these residues remain phosphorylated, even if insulin is allowed to dissociate from the receptor ( 1 6). Changing any of these tyrosine residues to phenylalanines by in vitro mutagenesis results in a receptor that is not as fully activated as a kinase and is inefficient in signal transduction (17). In intact cells, the receptor also undergoes serine and threonine phosphorylation (18). This can be stimulated by prolonged insulin treatment, phorbol esters, and camp analogs, and is presumably the result of phosphorylation of the receptor by protein kinase C and/or protein kinase A (19). In contrast to tyrosine phosphorylation, which activates the ki- 2 Residue numbers are based on the +exon I I form of the receptor. The -exon I I form contains the same 1-subunit sequence, but all positions are 12 amino acids closer to the N-terminal origin of the proreceptor. i.e., these tyrosines would have the numbers and 1151, respectively. nase, serine phosphorylation tends to inactivate the kinase (20). This exquisite regulation of insulin receptor kinase activity by multisite phosphorylation provides an important potential mechanism for the regulation of insulin signaling in physiologic and pathologic states. Direct evidence that the tyrosmne kinase activity of the insulin receptor is required for insulin action has come from several directions. The most convincing evidence derives from in vitro mutagenesis experiments in which a lysine residue at position 1030 has been changed to some other amino acid such as alanine. On the basis of analogy to other kinases, this lysine residue is critical in the ATP binding site of kinase (8,9). When such lysine mutants are cxpressed in cells, they bind insulin normally, but do not bind ATP, and thus are totally inactive as kinases and totally ineffective in mediating the insulin stimulation of cellular metabolism (21). This is true for all effects of insulin including both the acute metabohic and more chronic growth-promoting effects of the hormone. Mutagenesis of one or more of the major tyrosine autophosphorylation sites in the cluster 1 158, 1 162, and produces similar but less dramatic effects. Mutation of all tyrosines in this cluster, however, produces a receptor that has a loss of activity similar to that of the ATP binding site mutants (22). Alterations of receptor kinase in insulin-resistant states are also consistent with the importance of normal phosphorylation in normal insulin action (see below). Exactly how the receptor kinase ultimately transmits its signal is still uncertain. Most Investigation has focused on a model involving a phosphorylation cascade, i. e., insulin induces receptor autophosphorylation, which activates the receptor kinase, which in turn phosphorylates one or more cellular substrates (Figure 1, Model 1 ). These substrates could be enzymes (serine kinases or phosphoprotemns phosphatases) or enzyme inhibitors whose activity is changed by this phosphoryhation, and this leads to a cascade of secondary phosphoryhation and dephosphorylation reactions. This phosphoryhation cascade model is supported by the fact that insulin treatment results in phosphorylation and dephosphorylation of several cellular enzymes on serine residues (23,24). The Insulin Receptor Substrate Several primary endogenous substrates for insulin receptor have been uncovered. These are proteins rapidly and directly phosphorylated on tyrosmne residues by the active receptor kinase. The first, and best studied, is a protein that migrates as a broad band between 160 and 190 kd and is termed ppl85 or insulin receptor substrate 1 (IRS-i) (25). In cells transfected with the normal human insulin receptor 570 Volume 3 Supplement I 1992

3 Kahn and Saad Figure 1. Schematic representation of the insulin receptor and early steps in insulin action. The receptor is described in the Text and consist of two (binding) subunits and two f3 (kinase) subunits. Insulin binding stimulates the kinase, resulting in autophosphorylation of the receptor on tyrosine residues. Two models of subsequent steps in the pathway are depicted. Model I represents the phosphorylation cascade; Model 2 represents a noncovalent interaction pathway. Both are described in the Text. cdna, there is marked enhancement of pp 1 85 phosphorylation coincident with an enhancement of insuhin action (26). Phosphorylation of this protein is reduced in cells expressing receptors that have ATP binding site and/or autophosphorylation site mutations, in proportion to the reduction in insulin receptor kinase activation (22). Important evidence of a role for pp 1 85/IRS- 1 phosphorylation in insulin action has come from an unexpected finding after in vitro mutagenesis of the insulin receptor in the juxtamembrane region at or around tyrosine 960. These mutant receptor molecules bind insulin normally and are fully active as kinases in vitro. This is not surprising, because Tyr 960 is not a site of autophosphorylation in the active kinase domain. However, when transfected into cells, these mutant receptors fail to phosphorylate ppi8s. This lack of substrate phosphorylation correlates with a lack of transmission of signals for insulin action on glycogen or DNA synthesis (27). We have recently cloned IRS- 1 at the cdna level from both rat liver and human muscle (28). The rat liver cdna for IRS- 1 codes for a protein of 1,235 amino acids that does not closely resemble any of the proteins or cdna in existing gene data banks. Several features of the predicted protein are striking. First, there is no transmembrane domain, i.e., IRS-i is cytophasmic in localization. There is a sequence consistent with the consensus sequence for nucleotide (ATP or GTP) binding, but there is no clear homology to known protein kinases. On the basis of consensus sequence analysis, there are multiple potential serine, threonine, and tyrosine phosphoryhation sites. Six of the tyrosine phosphorylation sites have the repetitive sequence YMXM where Y is tyrosine, M is methionine, and X is any amino acid. Two additional phosphorylation sites have the related sequence YXXM. The YMXM motif does not occur in the insulin receptor but has been observed in several other tyrosmne kinases. In these, the YMXM domain appears to be involved in the interaction of these proteins noncovalently with the signaling molecules. These signaling molecules are characterized by the presence of domains that bind the YMXM motif termed SH2 (src homology 2). Recently, we have demonstrated an association of the enzyme phophatidyhinositol 3 -kinase (Ptdlns 3 - kinase) with IRS-i after insulin stimulation (28). Ptdlns 3 -kinase consists as a 1 10-kd catalytic subunit and an 85-kd regulatory subunit that contains two SH2 domains (29). The association between Ptdlns 3 -kinase and IRS-i probably occurs through phosphorylated YMXM motifs on IRS- 1 and the SH2 domains on the 85-kd subunit. Insulin also increases total Ptdlns 3 -kinase activity and the cellular concentration of Ptdlns 3-phosphate. Although tyrosmne Journal of the American Society of Nephrology 71

4 Alterations in Insulin Receptor and Substrate Phosphorylation in SHR phosphorylation of the 85-kd subunit of the Ptdlns 3 -kinase has been reported after growth factor stimulation (29), there is no evidence for phosphorylation of an 85-kd band during insulin stimulation. Instead, activation may occur simply because of the binding of the Ptdlns 3 -kinase to IRS-i. Thus, insulin stimulates IRS- 1 phosphorylation and phosphorylated IRS- 1 binds other signal transduction molecules, propagating insulin action. Several lower-molecular-weight endogenous substrates have also been identified. These include hipocortin 2 (30) and a 1 5-kd protein termed protein 442 or AP-2 (31). The latter is an abundant fatty acid-binding protein present in fat cells that has homology to myehin P-2 protein. Whether phosphorylation affects the function of either of these molecules and their roles in insulin action are unknown - Other Components of the Insulin Action Cascade Not all of insulin s actions on cells can be directly related to changes in phosphoryhation or to a phosphorylation cascade. For example, at present, there is no evidence that insulin stimulates phosphorylation or dephosphorylation of the glucose transporter or any proteins at the glucose transport pathway (32). Insulin-stimulated glucose transport appears to result primarily from a transhocation of glucose transported from an intracellular pooh to the plasma membrane (33). What drives this translocation is unknown, but some monoclonal antibodies to the insulin receptor appear to be able to stimulate glucose transport without activating the receptor kinase (34). Furthermore, cells expressing certain mutant receptors defective in autophosphoryhation appear to still mediate some insulin actions such as the stimulation of S6 kinase ( 1 7). These observations have led to a second model of action in which receptor autophosphorylation is viewed as changing the conformation of the receptor fl-subunit, allowing it to interact noncovalently with some other effector system (Figure 1, Model 2). Evidence for a conformational change after insulin binding and autophosphoryhation has come from studies with antibodies to specific domains of the fl-subunit that bind to the receptor only after it undergoes phosphorylation or after insulin binds to the a-subunit (35). Insulin action on cells also appears to activate one or more phosphohipases, including a specific Ptdlns phospholipase C. This activation has been suggested to generate an inositol glycan compound capable of mimicking several of insulin s action on cells (36). Full chemical characterization of this presumed mediator, however, has not yet been achieved. Thus, it has not been possible to test the importance of this pathway in intact cells. This and other effector systems for insulin action may be linked indirectly, I.e., via a G protein or ras-related protein, rather than directly to the receptor (37). Indeed, insulin has been shown to stimulate GTP loading of a protein present in receptor immunoprecipitates (38). Insulin treatment also modifies the ability of the a-subunit of G or 0,, In liver membranes to be ADP ribosylated by pertussus toxin (39). INSULIN RESISTANCE Insulin resistance is defined simply as any situation in which there is a subnormal response to a given concentration of insulin. It is well known that the effects of insulin are plelotropic, but the term insulin resistance usually refers to the actions of insulin on glucose homeostasis. Table 1 summarizes the many causes of insulin resistance. These include a large number of genetic, immune, and regulatory abnormalities in insulin action and range from very rare genetic defects to very common disorders such as obesity, hypertension, and type II diabetes. Because the action of insulin involves many gene products, pathophysiologically, primary defects could be due to a mutation affecting any protein between the receptor and the final insulin-regulated proteins. The insulin receptor locus has received the greatest attention, and to date, over 20 receptor mutations have been identified in patients with insulin resistance (40). Functionally important defects in other genes that are central to insulin action have not yet been described, although certainly, they must occur. Candidate genes include signaling intermediates between the receptor and downstream pathways like IRS- 1, the GLUT4 glucose transporter (the glucose transporter protein that appears to be responsible for the TABLE I. Causes and classification of insulin resistance Genetic Type A syndrome Leprechaunism Lipoatropic diabetes Immunological Anti-insulin antibodies Anti-insulin receptor antibodies Regulatory Obesity (may have a genetic component) Type II diabetes (definitely has a genetic component) Type I diabetes Metabolic (uremia, acidosis, etc) Physiological (puberty, pregnancy) Dysendocrinopathies Hypertension-associated Other Subcutaneous degradation 572 Volume 3 Supplement I 1992

5 Kahn and Saad insulin-stimulated uptake of glucose in muscle and fat), and several enzymes Involved in the intracehlular pathways of glucose metabolism. A point mutation of uncertain physiologic significance has been identified in 1 of 32 patients with non-insulin dependent diabetes melhitus (40). Many physiologic states and circulating factors can adversely affect the action of insulin. The secondary nature of these defects is established by the removal of the factor or state and by the reversal of the defect after cells are removed from the abnormal In vtvo milieu. Glucocorticolds, glucagon, catecholamines, and growth hormone induce insulin resistance in cases of the excess secretion of individual hormones such as occurs in specific endocrinopathies, and the combined effects of these hormones are observed in the setting of infection and stress. Hyperinsulmnemia itself down-regulates insulin receptors and results in postreceptor cellular desensitization (40). Insulin resistance can be demonstrated in patients with insuhinomas and in normal subjects after the infusion of insulin. Several complex physiologic and pathologic states including obesity, non-insulin dependent diabetes mellitus, diabetic ketoacidosis, uremia, cirrhosis, fasting, pregnancy, puberty, and aging are associated with resistance to insulin. Although rare, one of the best-characterized factors that can affect the sensitivity of target cells to insulin are spontaneously occurring antibodies to the insulin receptor (41). Hypertension as an Insulin-Resistant State Substantial evidence from several sources points to the presence of impaired insulin action in patients with essential hypertension. Epidemiologic studies indicate that fasting insulin levels are significantly elevated in hypertension (42) and that blood pressure correlates strongly with plasma insulin concentration (43). Second, hypertensive patients are hyper- Insulinemic after a standard glucose load when compared with control and require infusion of considerably less glucose to maintain euglycemla during hyperinsuhmnemic glucose clamps (44). The significance of the relationship between insulin resistance and hypertension has not been established. It is not known whether hypertension Is a cause or a consequence of insulin resistance or whether these two abnormalities arise coincidentally as a result of some other factor. The potential clinical consequences of insulin resistance can be divided into two broad categories: (1) those that result from deficient insulin action, and (2) those that result from excessive insulin in the circulation, which may act on less-resistant or nonresistant tissues. Impaired glucose tolerance and diabetes are clearly the consequences of deficient insulin action. The notion that compensatory hyperinsulinemia can actually produce excessive insulin action was first suggested by two clinical features common to many patients with severe resistance to the glucose-lowering effects of insulin-the skin Icsion of acanthosis nigricans and ovarian thecal hyperplasla with hyperandrogenism (45). Both of these have been suggested to result from the effects of insulin on these nonclassical target tissues of insulin action. On the basis of the latter hypothesis, several mechanisms have been proposed by which hyperinsulinemia may cause hypertension. First, by virtue of its ability to promote the renal tubular reabsorption of sodium, insulin causes an increase in total-body sodium and extracellular fluid volume so that higher renal (and therefore arterial) perfusion pressures are necessary to maintain sodium balance (46). Second, insulin increases sympathetic nervous system activ- Ity in humans and resistance to this action of insulin on the sympathetic nervous system does not appear to develop in obese subjects (47). The increased sympathetic activity may then contribute to hypertension by Increasing sodium reabsorption, cardiac output, and peripheral vascular resistance (48). Third, insuhin stimulates the activity of the Na/H exchanger, which is linked to Ca2 exchange (46). The mntracellular accumulation of Na and Ca2 is presumed to enhance the sensitivity of vascular smooth muscle to the pressor effects of norepinephrine, angiotensmn II, and volume expansion. Fourth, insulin may cause vascular hypertrophy, thereby increasing peripheral vascular resistance (46). Fifth, physiologic concentrations of insulin decrease the catecholamine-induced production of prostaglandin (PG) 12 (prostacydin) and PGE2, two potent vasodilators, in adipose tissue. Hypermnsulinemia may increase peripheral vascular resistance and blood pressure by inhibiting the stimuhatory effect of adrenergic agonists on the production of PGI2 and PGE2 in adipose tissue (49). Although these mechanisms may explain the link between insulin resistance, hyperinsulmnemia, and hypertension, it is important to emphasize that patients with type A syndrome of insulin resistance, who have severe insulin resistance with insulin 1evels usually 1 0- to 1 00-times normal, do not have hypertension. Whether this reflects a unique feature of the mechanisms of this disorder, desensitization of this action of insulin, or some other mechanism is unknown. MOLECULAR MECHANISM OF INSULIN RESISTANCE IN AN ANIMAL MODEL OF HYPERTENSION (SPONTANEOUSLY HYPERTENSIVE RAT) The spontaneously hypertensive rat (SHR) has been shown to be insulin resistant and hyperinsuhinemic when compared with the control Wistar-Kyoto (WKY) strain (50), although this has not been uni- Journal of the American Society of Nephrology 573

6 Alterations in Insulin Receptor and Substrate Phosphorylation in SHR..,...,,.,., seswb formly observed in all studies (51). In an attempt to evaluate this insulin resistance, Reaven et at, studied insulin-stimulated glucose uptake in isolated adipocytes of the SHR (52). The results showed that the insulin-stimulated glucose uptake is lower in adipocytes isolated from SHR compared with that in WKY rats. Insulin resistance did not seem to be related to a decrease in the number of insulin receptors or in the ability of insulin to stimulate insulin receptor autophosphorylation in vitro or insulin receptor tyrosine kinase against exogenous substrates. Taking advantage of the recent sequenced data, we have prepared antipeptide antibodies to IRS- 1. With these along with antiphosphotyrosmne antibodies and antiinsuhin receptor antibodies, it is now possible to directly access insulin-stimulated tyrosine phosphorylation of both the insulin receptor and its substrate IRS- 1 in the liver and muscle of intact rats. Briefly, the method consists of the administration of insulin via the portal vein followed at 30 by the extraction and homogenization of liver in boiling SDS containing buffer, and 90 s later the homogenization of muscle in Triton X-l00 buffer with phosphatase and protease inhibitors. The proteins from these two tissues can then be analyzed by SDS polyacrylamide gel electrophoresis and immunoblotting with antiphosphotyrosmne antibody and antipeptide antibody against insulin receptor and IRS- 1. In the basal state, a closely spaced doublet band with a molecular mass between 1 10,000 and 1 20,000 was observed in phosphotyrosmne blots of the liver and muscle extracts (Figures 2A and 3A). We have referred to this band as ppl2o: it is the major constitutive phosphotyrosine protein in most tissues. After insulin infusion into the portal vein, a phosphotyrosmne band with a Mr of 95,000, previously identified as the insulin receptor fl-subunit, appeared and became prominently phosphorylated. In addition, after insulin injection, a broad phosphotyrosyl protein migrating between 1 65,000 and 1 85,000 consistent with IRS- 1, the endogenous substrate of the insulin receptor kinase, was also detectable. Similar results were obtamed in muscle. Using this procedure, we studied SHR and WKY control rats of similar body weights (298 ± 6 versus 303 ± 5 g, respectively). Systolic blood pressure was significantly higher in the SHR (201 ± 4 versus i i9 ± 4 mm Hg). As is apparent in Figures 2 and 3, insulin-induced receptor phosphorylation and IRS-i phosphoryhation were decreased in the liver and musdc of SHR as compared with that in the control rats. By direct immunoblotting with antipeptide antibodies, the levels of insulin receptor and IRS- 1 protein were similar in controls and SHR in both tissues (Figures 2B, 2C, 3B, and 3C). These results indicate that the insulin resistance in these animals may be related to a decrease in insulin receptor and IRS- i Insulin Cl) 60 >. 40 -a < 20 0 Anti-Phosphotyrosine WKY + A Antibody SHR + Kd Anti IRS-i Antibody SHR B WKY IR IRS-i IR IRS-i; Protein Phosphorylation Figure 2. (A) Insulin-stimulated tyrosine phosphorylation in intact liver from WKY rats and SHR. Briefly, rats were anesthetized, and the abdominal wall was incised to expose the viscera. Normal saline (lanes I and 3) or io M insulin (lanes 2 and 4) was infused into the portal vein as a bolus injection and, 30 s later, liver was excised and homogenized in extraction buffer A (50 mm HEPES (ph 7.5), 1% SDS, and phosphatase inhibitors) at 100#{176}C for 5 mm; 90 s later, muscle was excised and homogenized in extraction buffer B (50 mm HEPES (ph 7.5), i% Triton-X 100, protease, and phosphatase inhibitors) at 4#{176}C. After centrifugation, aliquots with the same amount of protein were resolved on SDS-6% polyacrylamide gel, transferred to nitrocellulose, detected with antiphosphotyrosine antibodies and ( 25I)protein A, and subjected to autoradiography. (B) IRS-I levels in liver from WKY rats and SHR. Aliquots from the same samples (lanes I and 3) were also resolved on SDS-6% polyacrylamide gels, transferred to nitrocellulose, and detected with anti-irs-i antibody instead of antiphosphotyrosine antibody. (C) Insulin receptor (IR) and IRS-I levels and phosphorylation in liver of SHR. Insulin receptor levels were determined by immunoblotting with an anti-insulin receptor antibody as described in the Text. The values are derived from four to eight separate experiments determined by scanning densitometry and are represented as the mean ± SE. indicates differences from control (WKY) at P< phosphorylation but not to a decrease in the levels of these proteins. As previously described, the autophosphorylation S74 Volume 3 Supplement I 1992

7 Kahn and Saad Anti.Phosphotyrosine Antibody WKY SHR Insulin Kd 205 Anti IRS.1 Antibody SHR WKY #{216}.4 phorylation and kinase activity and fail to mediate any of the biological effects of insulin. Reduction in Kd IRS- i phosphorylatlon with normal protein level is also observed in CHO cells expressing insulin recep- 205 tors with a mutation at tyrosine 960 (27) and tyrosines , , and (22). In these cells, the i i U) < 20 0 A L IR IRS-i1 IR Protein Phosphorylation Figure 3. (A) Insulin-stimulated tyrosine phosphorylation in intact muscle from WKY rats and SHR. The proteins from the muscle were isolated and processed as described in the legend to Figure IA. Extracts from rats injected with normal saline are in lanes I and 3, and those with io M insulin are in lanes 2 and 4. (B) IRS-I levels in muscle from WKV rats and SHR. Aliquots from the same samples (lanes I and 3) were also resolved on SDS-6% polyacrylamide gels, transferred to nitrocellulose, and detected with anti-irs-i antibody instead of antiphosphotyrosine antibody. (C) Insulin receptor(lr) and IRS- I levels and phosphorylation in muscle of SHR. Insulin receptor levels were determined by immunoblotting with an anti-insulin receptor antibody as described in the Text. The values are derived from four to eight separate experiments determined by scanning densitometry and are represented as the mean ± SE. indicates differences from control (WKY) at P< of the insulin receptor and the phosphorylation of IRS- 1 (kinase activity of insulin receptor toward endogenous substrate) appear to be important for signal transduction. Direct evidence that the tyrosmne kinase activity of insulin receptor is required for insuhin action derives from in vitro mutagenesis experiments in which a lysmne residue at position i030 (ATP-binding site) has been changed and from studies of naturally occurring mutants (21,40). When overexpressed in various cell types, these receptors display an absence of insulin-stimulated autophos- ii6 80 B 1 16 absence of biological activity correlates specifically with the inability of the mutant receptor to stimulate 80 the tyrosyl phosphoryhation of IRS- 1. Thus, overexpression of these mutants described above produces a state of cellular insulin resistance and provides a strong argument in favor of the role of insuhin-receptor autophosphorylation/kinase activity in mediating some, if not all, of the biological effects of insulin. The reduced level of phosphorylation of IRS- 1 in liver and muscle in SHR may play a role in the impaired insulin action. As the level of IRS-i protein In liver and muscle was normal, the decrease in IRS- 1 phosphorylation appears to reflect changes in insulin receptor kinase activity and the ability of the receptor to use this protein substrate. Comparison of our results with a previous study (52) that did not find reduced receptor phosphorylation and kinase activity in SHR is complicated by differences in tissue studied. More importantly, in the previous study, the receptors were first extracted from the membrane and partially purified and the in vitro kinase assays were performed with an exogenous phosphoacceptor substrate. Although informative, this latter approach is susceptible to many potential biochemical artifacts resulting from cell homogenization and receptor purification procedures, e.g., proteolysis and/or dephosphorylation of the receptor by contaminating phosphoprotein phosphatases, as well as removal of the receptor from the plasma membrane where interactions with other cellular components may influence receptor activity. The reduced insulin receptor autophosphoryhation and IRS-i phosphorylation in liver and muscle of SHR appear to be regulatory, because we have observed similar alterations in other animal models of insulin resistance associated with hypermnsuhinemia, such as the ob/ob mouse (53). Whether these regulatory defects reflect alterations in receptor serine phosphorylation or some other regulatory event is unknown. Increased activation of protein kinase C (PKC) has been demonstrated in some tissues of SHR (54), and as discussed above, increased PKC activity increases serine phosphoryhation of the insulin receptor and thus tends to inactivate the tyrosine kinase. Also, IRS- 1 appears to be a substrate of PKC but effects of the serine phosphorylation of this protein are unknown. Cell plasma membranes of SHR differ from those of normotensive rats in many ways, including activity of cation flux, calcium binding, phosphoinositide metabolism, and physicochemical properties as Journal of the American Society of Nephrology 575

8 Alterations in Insulin Receptor and Substrate Phosphorylation in SHR,..; r.r xr... :.. i., measured with a variety of inserted probes. These abnormalities are present on different tissues, suggesting that a ubiquitous membrane component may be altered (55). Kato and Takenawa have shown increased phospholipase C on erythrocyte ghosts of SHR (56). Recently, our laboratory demonstrated that treatment of intact cells (IM-9 lymphocytes and NIH- 3T3 fibroblasts) with phosphohipase C reduced insuhin-stimulated receptor and IRS- 1 phosphorylation (57). These effects of phosphohipase C are not observed with insulin receptors purified on wheat germ agglutinin-agarose. suggesting that the phosphohipid environment in the plasma membrane is an important modulator of transmembrane signaling within the insulin receptor heterotetramer and at the level of substrate phosphorylation. Further direct studies of PKC and phosphohipase C activity in the liver and muscle of SHR would be needed to clarify this point. In summary, this study demonstrates that reduced insulin-induced receptor and IRS-i phosphoryhation is present in liver and muscle of SHR. In this setting, altered phosphorylation and/or activity of these proteins may play a role in the production of an insulinresistant state. ACKNOWLEDGMENTS The authors thank Terri-Lyn Bellman for excellent secretarial assistance. This work was supported in part by grants from the NIH OK to CR. Kahn. Joslin s Diabetes and Endocrinology Research Center grant (DK 36836). and the Marilyn M. Simpson Research Program In Diabetes (CR. Kahn). During this work. Dr. Saad held a fellowship from the CNPq. Brazil. REFERENCES 1. Freychet P. Roth J, Neville DM Jr: Insulin receptor in the liver: Specific binding of [125II insulin to the plasma membrane and its relation to insulin bioactivity. Proc Natl Acad Sd USA 1971:68: Cuatrecasas P: Affinity chromatography and purification of the insulin receptor of liver cell membranes. Proc Natl Acad Sci USA 1972:69: Kahn CR: Current concepts of the molecular mechanism of insulin action. Annu Rev Med 1985:36: Massague JP, Pilch PF, Czech MP: Electrophoretic resolution of three major insulin receptor structures with unit stoichiometries. Proc Natl AcadSciUSA 1981:77: Kasuga M, Hedo JA, Yamada KM, Kahn CR: Structure of insulin receptor and its subunits. J Biol Chem 1982:257: Ebina Y, Ellis L, Jarnagin K, et at. : The human insulin receptor cdna: The structural basis for hormone-activated transmembrane signalling. Cell 1985:40: Ullrich A, Bell JR, Chen EY, et at. : Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature (Lond) 1985:313: Seino S, Seino M, Nishi S, Bell GI: Structure of the human insulin receptor gene and characterization of its promoter. Proc Nath Acad Sci USA i989;86:i i Goldstein BJ, Muller-Wieland D, Kahn CR: Variation in insulin receptor mrna expression in human and rodent tissues. Mol Endocrinol 1987: 1: Mosthaf L, Vogt B, Haring HU, Ullrich A: Altered expression of insulin receptor types A and B in the skeletal muscle of non-insulin dependent diabetes melhitus patients. Proc Natl Acad Sci USA 1991:88: Dc Meyts P, Gu JL, Shymko RM, Kaplan BE, Bell GI, Whittaker J: Identification of a higandbinding region of the human Insulin receptor encoded by the second exon of the gene. Mol Endocrinol 1990:4: Cheatham B, Kahn CR: Cysteine 647 in the insulin receptor is required for normal covalent interaction between a- and fl-subunits and si - nal transduction. J Biol Chem i992; Kasuga M, Karlsson FA, Kahn CR: Insulin stimuhates the phosphoryhation of the 95,000-dalton subunit of its own receptor. Science i 982:215: White MF, Haring HU, Kasuga M, Kahn CR. Kinetic properties and sites of autophosphorylation of the partially purified receptor from hepatoma cells. J Biol Chem i984;259: White MF, Shoelson SE, Seutmann H, Kahn CR: A cascade of tyrosmne autophosphorylatlon in the fl-subunit activates the insulin receptor. J Biol Chem 1988:263: Yu KT, Czech MP: Tyrosine phosphoryhation of the insulin receptor fl-subunit activates the receptor-associated tyrosine kinase activity. J Biol Chem 1984:259: Ellis L, Clauser E, Morgan DO, Edery M, Roth RA, Rutter WJ: Replacement of insulin receptor tyrosmne residues i 162 and 1 i63 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell 1 986:45:72 i Kasuga M, Zick Y, Blithe DL, Karlsson FA, Haring HU, Kahn CR: Insulin stimulation of phosphorylation of the fl-subunit of the insulin receptor formation of both phosphoserine and phosphotyrosmne. J Biol Chem 1982:257: 989 i Takayama S, White MF, Lauris V, Kahn CR: Phorbol esters modulate insulin receptor phosphorylation and insulin action in hepatoma cells. Proc Natl Acad Sci USA i 984:81: Takayama 5, White MF, Kahn CR: Phorbol ester induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity. J Biol Chem 1988:263: Ebina Y, Araki E, Taira M, et at.: Replacement of hysine residue in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and receptor kinase activity. Proc Nath Acad Sci USA 1987:84: Wilden PA, Siddle K, Haring E, Backer JM, White MF, Kahn CR: The role of insulin receptor kinase domain autophosphorylation In receptormediated activities: Analysis with insulin and anti-receptor antibodies. J Biol Chem 1992; 267:i37i S76 Volume 3 Supplement I 1992

9 -..#{149},,. #{149}y W... Kahn and Saad 23. Kahn CR: Current concepts of the molecular mechanism of insulin action. Annu Rev Med i 985: Czech MP: The nature and regulation of the insulin receptor. Annu Rev Physiol 1984:47: White MF, Maron R, Kahn CR: Insulin rapidly stimulates tyrosmne phosphoryhation of a Mr 185,000 protein in intact cells. Nature (Lond) 1985:318: White MF, Shoelson SE, Keutmann H, Kahn CR: A cascade of tyrosine autophosphorylation in the fl-subunit activates the insulin receptor. J Biol Chem i 988:263: White MF, Livingston JN, Backer JM, Dull T, Ulirich A, Kahn CR: Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity. Cell 1988:54: Sun XJ, Rothenberg P. Kahn CR, et at. : Structure of the insulin receptor substrate IRS-i defines a unique signal transduction protein. Nature (Lond) 1991:352: Carpenter CL, Cantley LC: Phosphoinositide kinases. Biochemistry 1990:29: Karasik A, Pepinsky RB, Shoelson SE, Kahn CR: Lipocortins 1 and 2 as substrates for the insulin receptor kinase In rat liver. J Biol Chem 1988:263: Bernier M, Laird DM, Lane MD: Insuhin-activated tyrosine phosphorylation of a i 5-kilodalton protein in intact 3T3-Li adipocytes. Proc Natl Acad Sci USA i987;84:i844-i Gibbs EM, Allard WJ, Lienhard GE: The glucose transporter in 3T3-L1 adipocytes is phosphorylated in response to phorbol ester but not in response to insulin. J Biol Chem 1986:261: i Cushman SW, Wardzala LI: Potential mechanisms of insulin action on glucose transport in the isolated rat adipose cell: Apparent translocation of intracellular transport systems to the plasma membrane. J Biol Chem i980:255: Forsayeth JR, Caro JF, Sinha MK, Maddux BA, Goldfine ID: Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity. Proc Nath Acad Sci USA 1987:84: Perlman R, Bottaro DP, White MF, Kahn CR: Conformational changes in the a- and fl-subunits of the insulin receptor identified by antipeptide antibodies. J Biol Chem 1989:269: Saltiel AR, Cuatrecasas P: Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycohipid. Proc Nath Acad Sd USA i986;83: Burgering BMT, Medema RH, Maassen JA, Wetcling MLV, McCormick F, Bos JL: Insulin stimulation of gene expression mediated by p2 1 ras activation. EMBO J 1991:10: Medema RH, Burgering BMT, Bos JL: Insulininduced p2 1 ras activation does not require protein kinase C, but a protein sensitive to phenylarsmne oxide. J Biol Chem 1991:266: i Rothenbcrg P. Kahn CR: Insulin inhibits pertussis-toxin catalyzed ribosylation of G-proteins. J Biol Chem i988;263:15546-i Moller DE, Flier JS: Insulin resistance-mechanisms, syndromes, and implications. N Engl J Med 1991:325: Flier JS, Kahn CR, Roth J: Receptors, antireceptor antibodies and mechanisms of insulin resistance. N Engh J Med 1979:300: Modan M, Halkin H, Almog 5, et at. : Hyperinsulinemia-a link between hypertension, obesity and glucose intolerance. J Clin Invest 1985: 75: Lucas CP, Estigarribia JA, Darga LL, Reaven GM: Insulin and blood pressure in obesity. Hypertension 1985:7: Ferrannini E, Buzzigoli G, Bonadonna R, et at.: Insulin resistance in essential hypertension. N Engl J Med i987;3i7: Flier JS: The metabolic importance of acanthosis nigricans. Arch Dermatol 1 985: i 21: DeFronzo HA, Ferrannini E: Insulin resistance: A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 1991:14: Daly PA, Landsberg L: Hypertension in obesity and NIDDM: Role of insulin and sympathetic nervous system. Diabetes Care ; 14: Rowe JW, Young JB, Minaker KL, Stevens AL, Pallotta J, Landsberg L: Effect of insulin and glucose infusions on sympathetic nervous system activity in normal man. Diabetes 1981:30: Axelrod L: Insulin, prostaglandmns, and the pathogenesis of hypertension. Diabetes 1991: 40: i Mondon CE, Reaven GM: Evidence of abnormahities of insulin metabolism in rats with spontaneous hypertension. Metabolism i988;37: Buchanan TA, Sipos GF, Madrilejo N, Liu Chaplin, Campese VM: Hypertension without peripheral insulin resistance in spontaneously hypertensive rats. Am J Physiol i992:25:e14-ei Reaven GM, Chang H, Hoffman BB, Azhar 5: Resistance to insulin-stimulated glucose uptake in adipocytes isolated from spontaneously hypertensive rats. Diabetes 1 989:38: Saad MJA, Araki E, Miralpeix M, Rothenberg PL, White MF, Kahn CR. Regulation of insulin receptor substrate 1 in liver and muscle of animal models of insulin resistance. J Clin Invest 1992, in press. 54. Makita N, Yasuda H: Alterations of phosphoinositide-specific phosphohipase C and protein kinase C in the myocardium of spontaneously hypertensive rats. Basic Res Cardiol 1990:85: Devynck MA, Pernollet MG, Nunez AM, et at.: Diffuse structural alterations in cell membranes of spontaneously hypertensive rats. Proc Nat! Acad Sd USA 1982:79: Kato H, Takenawa T: Phosphohipase C activation and diacylglycerol kinase inactivation lead to an increase in diacylglycerol content in spontaneoushy hypertensive rat. Biochem Biophys Res Commun 1987:146: Zoppini G, Kahn CR: Effect of phospholipase treatment on insulin receptor signal transduction. Diabetologia 1 992:35: Journal of the American Society of Nephrology 577