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Biomedical subjects

K Inukai

Publications and source records attributed to K Inukai.

At least 37 records · Page 2Linked to original sources
PubMed1999

p85/p110-type phosphatidylinositol kinase phosphorylates not only the D-3, but also the D-4 position of the inositol ring.

Activation of p85/p110-type phosphatidylinositol (PI) kinase has been implicated in various cellular activities. This PI kinase phosphorylates the D-4 position with a similar or higher efficiency than the D-3 position when trichloroacetic acid-treated cell membrane is used as a substrate, although it phosphorylates almost exclusively the D-3 position of the inositol ring in phosphoinositides when purified PI is used as a substrate. Furthermore, the lipid kinase activities of p110 for both the D-3 and D-4 positions were completely abolished by introducing kinase-dead point mutations in their lipid kinase domains (DeltaKinalpha and DeltaKinbeta, respectively). In addition, both PI 3- and PI 4-kinase activities of p110alpha and p110beta immunoprecipitates were similarly inhibited by either wortmannin or LY294002, specific inhibitors of p110. Insulin induced phosphorylation of not only the D-3 position, but also the D-4 position. Indeed, overexpression of p110 in Sf9 or 3T3-L1 cells induced marked phosphorylation of the D-4 position to a level comparable to or much greater than that of D-3, whereas inhibition of endogenous p85/p110-type PI kinase via overexpression of dominant-negative p85alpha (Deltap85alpha) in 3T3-L1 adipocytes abolished insulin-induced synthesis of both. Thus, p85/p110-type PI kinase phosphorylates the D-4 position of phosphoinositides more efficiently than the D-3 position in vivo, and each of the D-3- or D-4-phosphorylated phosphoinositides may transmit signals downstream.

1-Phosphatidylinositol 4-Kinase↗
PubMed1999

Imidapril, an angiotensin-converting enzyme inhibitor, improves insulin sensitivity by enhancing signal transduction via insulin receptor substrate proteins and improving vascular resistance in the Zucker fatty rat.

Angiotensin-converting enzyme (ACE) inhibitors are antihypertensive agents, that inhibit the conversion of angiotensin I to angiotensin II, resulting in smooth-muscle relaxation and a reduction of vascular resistance. Recently, it has been suggested that ACE inhibitors improve insulin resistance in diabetic patients. To investigate the effect of an ACE inhibitor on insulin sensitivity, insulin signaling, and circulation, imidapril was administered orally or intraduodenally to Zucker fatty rats. Oral administration of imidapril improved insulin sensitivity based on the results of an oral glucose tolerance test (OGTT) and a decrease in urinary glucose secretion. Phosphatidylinositol 3-kinase (PI 3-kinase) activity associated with hepatic insulin receptor substrate-1 (IRS-1) in the insulin-stimulated condition was significantly enhanced 110% without a significant alteration in tyrosine phosphorylation of IRS-1 in the imidapril-treated group. In muscle, IRS-1 tyrosine phosphorylation and PI 3-kinase activity associated with IRS-1 in the insulin-stimulated condition were enhanced 70% and 20%, respectively, in the imidapril-treated group. In contrast, an alteration of the IRS-2 pathway was observed only in liver; a significant insulin-induced increase in the IRS-2-associated PI 3-kinase over the basal level was observed in the imidapril-treated group but not in the control. In addition, treatment with imidapril was shown to significantly reduce blood pressure and increase blood flow in the liver and muscle. These results suggest that the ACE inhibitor imidapril may improve insulin sensitivity not only by acting directly on the insulin signaling pathway but also by increasing blood flow in tissues via normalization of vascular resistance, a major cause of hypertension.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase.

The hallmark of type 2 diabetes, the most common metabolic disorder, is a defect in insulin-stimulated glucose transport in peripheral tissues. Although a role for phosphoinositide-3-kinase (PI3K) activity in insulin-stimulated glucose transport and glucose transporter isoform 4 (Glut4) translocation has been suggested in vitro, its role in vivo and the molecular link between activation of PI3K and translocation has not yet been elucidated. To determine the role of PI3K in glucose homeostasis, we generated mice with a targeted disruption of the gene encoding the p85alpha regulatory subunit of PI3K (Pik3r1; refs 3-5). Pik3r1-/- mice showed increased insulin sensitivity and hypoglycaemia due to increased glucose transport in skeletal muscle and adipocytes. Insulin-stimulated PI3K activity associated with insulin receptor substrates (IRSs) was mediated via full-length p85 alpha in wild-type mice, but via the p50 alpha alternative splicing isoform of the same gene in Pik3r1-/- mice. This isoform switch was associated with an increase in insulin-induced generation of phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P3) in Pik3r1-/- adipocytes and facilitation of Glut4 translocation from the low-density microsome (LDM) fraction to the plasma membrane (PM). This mechanism seems to be responsible for the phenotype of Pik3r1-/- mice, namely increased glucose transport and hypoglycaemia. Our work provides the first direct evidence that PI3K and its regulatory subunit have a role in glucose homeostasis in vivo.

Animals↗
PubMed1999

Enhanced insulin-stimulated activation of phosphatidylinositol 3-kinase in the liver of high-fat-fed rats.

Insulin receptor substrate (IRS)-1 and IRS-2, which mediate phosphatidylinositol (PI) 3-kinase activation, play essential roles in insulin-induced translocation of GLUT4 and in glycogen synthesis. In this study, we investigated the process of PI 3-kinase activation via binding with IRS-1 and -2 in liver, muscle, and fat of high-fat-fed rats, a model of insulin-resistant diabetes. In the liver of high-fat-fed rats, insulin increased the PI 3-kinase regulatory subunit p85alpha and the PI 3-kinase activities associated with IRS-1 3.6- and 2.4-fold, and with IRS-2, 4.7- and 3.0-fold, respectively, compared with those in control rats. The tyrosine phosphorylation levels of IRS-1 and IRS-2 were not significantly altered, however. In contrast with the liver, tyrosine phosphorylation levels and associated PI 3-kinase proteins and activities were decreased in the muscle and adipose tissue of high-fat-fed rats. Thus, high-fat feeding appears to cause insulin resistance in the liver by a mechanism different from the impaired PI 3-kinase activation observed in muscle and adipose tissue. Taking into consideration that hepatic PI 3-kinase activation is severely impaired in obese diabetic models such as Zucker fatty rats, it is possible that the mechanism by which a high-fat diet causes insulin resistance is quite different from that associated with obesity and overeating due to abnormality in the leptin system. This is the first report to show increased PI 3-kinase activation by insulin in an insulin-resistant diabetic animal model. These findings may be important for understanding the mechanism of insulin resistance in human NIDDM, since a high-fat diet is considered to be one of the major factors exacerbating insulin insensitivity in humans.

Adipose Tissue↗
PubMed1999

No correlation of plasma cell 1 overexpression with insulin resistance in diabetic rats and 3T3-L1 adipocytes.

Membrane glycoprotein plasma cell 1 (PC-1) has been shown to be increased in type 2 diabetes and involved in insulin resistance through inhibiting the insulin receptor tyrosine kinase, which was demonstrated using cultured breast cancer cells. However, other reports have shown contradictory results in Chinese hamster ovary cells and in vitro kinase assay. Thus, we considered it necessary to investigate the effect of PC-1 using highly insulin-sensitive cells. Here, we used two of the following approaches: 1) investigating PC-1 expression levels in insulin-responsive tissues in rat models of diabetes and 2) overexpressing PC-1 in 3T3-L1 adipocytes. We found that PC-1 was highly expressed in insulin-responsive tissues, such as liver and adipose tissue, in normal rats. However, high-fat feeding or streptozotocin-induced diabetes did not change its expression levels in liver, adipose tissue, and skeletal muscle. Thus, PC-1 expression levels were not associated with high-fat-diet-induced insulin resistance or hyperglycemia. Although PC-1 was increased in adipose tissue in Zucker fatty rats (protein level, by 50%; mRNA level, by 90%), its expression levels in liver and skeletal muscle, tissues that are more responsible for whole body glucose metabolism than adipose tissue, did not significantly differ from those in normal rats. Next, we overexpressed PC-1 in 3T3-L1 adipocytes using an adenovirus transfection system. PC-1 expression was markedly increased to a level 16-fold greater than that in normal human adipose tissue, which is higher than the previously reported levels in diabetic patients. However, insulin-induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1, activation of phosphatidylinositol 3-kinase, and glucose uptake were not affected by PC-1 overexpression. These results strongly suggest that increased PC-1 expression is not causally related to insulin resistance.

3T3 Cells↗
PubMed1998

Different subcellular distribution and regulation of expression of insulin receptor substrate (IRS)-3 from those of IRS-1 and IRS-2.

Adipocytes contain three major substrate proteins of the insulin receptor, termed IRS-1, IRS-2, and IRS-3. We demonstrated that IRS-1 and IRS-2 are located mainly in the low density microsome (LDM) fraction and are tyrosine phosphorylated in response to insulin stimulation, leading to phosphatidylinositol (PI) 3-kinase activation. In contrast, IRS-3 is located mainly in the plasma membrane (PM) fraction and contributes to PI 3-kinase activation in the PM fraction. The different cellular localizations of IRS proteins may account for the mechanism of insulin resistance induced by a high fat diet, considering that PI 3-kinase activation in the LDM fraction is reportedly essential for the translocation of GLUT4 in adipocytes. High fat feeding in rats increased both protein and mRNA levels of IRS-3 but decreased those of IRS-1 and IRS-2 in epididymal adipocytes. As a result, selective impairment of insulin-induced PI 3-kinase activation was observed in the LDM fraction, whereas PI 3-kinase activation was conserved in the PM fraction. This is the first report showing that different IRS proteins function in different subcellular compartments, which may contribute to determining the insulin sensitivity in adipocytes.

Adipocytes↗
PubMed1998

Multiple isoforms of the regulatory subunit for phosphatidylinositol 3-kinase (PI3-kinase) are expressed in neurons in the rat brain.

Phosphatidylinositol 3-kinase (PI3-kinase) is a heterodimeric enzyme composed of a catalytic subunit of 110 kDa and an adaptor regulatory subunit. We investigated the presence and localization of five isoforms of the regulatory subunits, p55 alpha, p55 gamma, p85 alpha, p85 beta, and p50 alpha, in the rat brain. In situ hybridization histochemistry using isoform-specific cRNA probes revealed that all five isoforms were expressed in the neurons of the brain. Interestingly, most neuronal cells including Purkinje cells in the cerebellum and pyramidal cells in the cerebrum expressed all five isoforms. Immunohistochemical staining also showed the localization of p55 alpha, p55 gamma, p85 alpha, and p50 alpha in the neuronal cells in the brain. Expression of multiple isoforms in neurons suggests that they may play important roles in signal transduction in the brain.

Animals↗
PubMed1998

Repetitive mitochondrial Ca2+ signals synchronize with cytosolic Ca2+ oscillations in the pancreatic beta-cell line, MIN6.

We examined the relationship between cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial matrix Ca2+ concentration ([Ca2+]m) in the pancreatic beta-cell line, MIN6. [Ca2+]c was monitored in a single or a group (30 cells) of fura-2-loaded MIN6 cells, and [Ca2+]m was measured in a group (1 x 10[6] cells) of MIN6 cells stably transfected with aequorin targeted at the mitochondria. Exogenous ATP (0.25 mmol/l) produced a single transient increase in [Ca2+]c whereas 22 mmol/l KCl produced a sustained plateau increase. ATP and KCl evoked transient increases in [Ca2+]m but with distinct time courses of [Ca2+]m decline: the [Ca2+]m increase induced by ATP decreased more rapidly than that induced by KCl. Nitrendipine (3 micromol/l), a blocker of L-type Ca2+ channels, inhibited both [Ca2+]c and [Ca2+]m signals in response to KCl and tolbutamide, but not those to ATP. Peak levels of [Ca2+]m increase (around 2 micromol/ l) exceeded those of [Ca2+]c increase (around 500 nmol/l). A rise in glucose concentration from 3 to 30 mmol/l induced oscillations of [Ca2+]c that overlay the sustained increases in [Ca2+]c in single cells. An oscillatory increase in [Ca2+]m was similarly observed in response to glucose. Addition of 10 mmol/l 2-ketoisocaproic acid at 20 mmol/l glucose further increased the plateau level of [Ca2+]c and the frequency of [Ca2+]c oscillations, which were correlated with a further increase in [Ca2+]m. In response to pulsatile exposure to KCl, [Ca2+]c and [Ca2+]m increased synchronously. These data suggest that an oscillatory increase in [Ca2+]m in beta cells, the signal which is thought to be necessary for continuous stimulation of mitochondrial metabolism, is produced synchronously with the [Ca2+]c oscillations.

Adenosine Triphosphate↗
PubMed1998

Role of JTT-501, a new insulin sensitiser, in restoring impaired GLUT4 translocation in adipocytes of rats fed a high fat diet.

JTT-501 is an insulin-sensitising compound with an isoxazolidinedione rather than a thiazolidionedione structure. Sprague-Dawley rats fed a high fat diet for 2 weeks were used as an animal model of insulin resistance, and JTT-501 was administered for the final week of the diet. An euglycaemic glucose clamp study showed that the glucose infusion rate (GIR) required to maintain euglycaemia was 57% lower in rats fed a high fat diet than in control rats, and that JTT-501 treatment restored the reduction in GIR produced by the high fat diet. To explain the mechanisms underlying the effects of a high fat diet and JTT-501 treatment, epididymal fat pads were excised and used in the analysis of insulin action. The high fat diet caused: (1) a 58% decrease in insulin receptor substrate-1 (IRS-1) content with a 58% decrease in IRS-1 tyrosine phosphorylation; (2) reductions of 56% and 73% respectively in insulin-induced maximal PI 3-kinase activation in anti-phosphotyrosine and anti-IRS-1 antibody immunoprecipitates; (3) a 46% reduction in the glucose transporter protein, GLUT4 content and, consequently, (4) severely impaired insulin-induced GLUT4 translocation to the plasma membrane and glucose uptake in adipocytes. JTT-501 treatment restored appreciably the protein content and tyrosine phosphorylation level of IRS-1. Insulin-stimulated PI 3-kinase activation was also restored in anti-phosphotyrosine and anti-IRS-1 antibody immunoprecipitates. As reflected by these improvements in insulin signalling, JTT-501 treatment improved considerably insulin-induced GLUT4 translocation to the plasma membrane as well as insulin-induced glucose uptake. However, JTT-501 had no effect on the decrease in GLUT4 content produced by the high fat diet. These observations suggest that JTT-501 enhances insulin signalling and may be effective in reducing insulin resistance.

Adipocytes↗
PubMed1998

Early intervention for very-low-birth-weight infants.

To assess the efficacy of early intervention (EI) for very-low-birth-weight (VLBW) infants, we evaluated 62 2 year old children who were enrolled in an EI program and 48 control subjects aged 2 years. We determined the subjects' developmental quotients (DQ) and obtained information about the parents' evaluation of the children from a questionnaire sent to the parents. There was no significant difference in the DQ between the EI group and the control group. However, based on the responses to the questionnaire, subjects in the EI group showed slight, but statistically marginally significant, improvements in behavioral problems, especially a decrease in hyperkinesia, in adjusting to a circadian sleep cycle, and an improvement in language development, as compared with the control group (P < 0.1). Thus, EI for VLBW infants is considered useful to enhance some areas of development.

Child Behavior↗
PubMed1998

Structural study on a sulfated polysaccharide-peptidoglycan complex produced by Arthrobacter sp.

The structure of a sulfated polysaccharide-peptidoglycan complex (SP-PG) produced by Arthrobacter sp. was analyzed by NMR spectroscopy. In addition, oligosaccharide fragments of the SP-PG-L obtained by HF degradation were analyzed by NMR spectroscopy. These findings indicated that the sulfated polysaccharide (SP) contains a repeating unit composed of two galactofuranosides and a glucopyranoside. The main chain of the trisaccharide is [-->6) beta-D-Galf(1-->6)-beta-D-Galf(1-->ln, with beta-D-Glcp linked to one of the Galfs through a (1-->2) linkage. The sulfated positions of the trisaccharide were identified as C-3 and C-5 of the beta-glucosylated Galf residues, and C-2 or C-3 of the other Galf residue.

Arthrobacter↗
PubMed1998

Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats.

To elucidate the mechanism of obesity-related insulin resistance, we investigated the impaired steps in the processes of phosphatidylinositol (PI) 3-kinase activation through binding with insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) in liver and muscle of Zucker fatty rats. The expressions of IRS-1 and IRS-2 were shown to be downregulated in both liver and muscle in fatty rats (hepatic IRS-1, 83%; hepatic IRS-2, 45%; muscle IRS-1, 60%; muscle IRS-2, 78%), resulting in decreased tyrosine phosphorylation in response to insulin stimulation. Despite the decrease in the tyrosine phosphorylation levels of hepatic IRS-1 and IRS-2 being mild to moderate, associated PI 3-kinase activities were dramatically decreased in fatty rats (IRS-1, 14%; IRS-2, 10%), which may suggest alteration in the sites of phosphorylated tyrosine residues of hepatic IRS-1 and IRS-2. In addition, we demonstrated that the expressions of p85alpha and p55alpha regulatory subunits of PI 3-kinase were reduced (p85alpha, 67%; p55alpha, 54%), and that the p50alpha regulatory subunit was markedly upregulated (176%) in the livers of fatty rats without apparent alterations in expressions of the catalytic subunits p110alpha and p110beta. These alterations may reflect the obesity-related insulin resistance commonly observed in human NIDDM.

Albumins↗
PubMed1998

[Psychological evaluation in vertiginous patients].

Four psychological tests, namely the Cornell Medical Index (CMI), the Manifest Anxiety Scale (MAS), the self-rating depression scale (SDS), and the self-rating anxiety scale (SAS) were performed to investigate the role of psychological factors in vertigo, in a study of 111 vertiginous patients and 81 controls. Vertiginous patients comprised the following three groups: 56 vertebrobasilar insufficiency (VBI group), 29 autonomic nervous dysfunction (AND group), and 26 Ménière's disease (MD group). The incidence of abnormal scores by CMI and SAS in the vertiginous patients was significantly higher than that in the controls (p < 0.01). No significant difference between the vertiginous patients and the controls was observed with respect to MAS and SDS scores. The abnormal rates by CMI in the VBI and AND groups were significantly higher than in controls (p < 0.01). Also, the abnormal rates by SAS in the VBI and MD groups were significantly higher than in controls (VBI group: p < 0.01, MD group: p < 0.05). However, no significant differences between the three groups of vertiginous patients and controls were found with respect to MAS and SDS scores. The average score from other psychological tests in the CMI-abnormal group was significantly higher than that in the CMI-normal group (p < 0.01). There were significant correlations between the MAS and SDS, MAS and SAS, and SDS and SAS scores (r = 0.682-0.688, p < 0.01). The matched data rate between the psychological tests was about 70-80%. Considering the results of MAS and SAS, vertigo induces anxiety forwards vertigo, and thereby initiates a vicious cycle. In vertiginous patients, CMI and SAS are most useful for determining whether or not psychological factors play a role in the onset of and persistence of vertigo. But CMI consist of 195 factors, and therefore, is time concerning. On the other hand, SAS has 20 factors and takes only 5 minutes to complete, thus, SAS is more suitable for screening psychological factors.

Adolescent↗
PubMed1997

14-3-3 protein binds to insulin receptor substrate-1, one of the binding sites of which is in the phosphotyrosine binding domain.

Insulin binding to its receptor induces the phosphorylation of cytosolic substrates, insulin receptor substrate (IRS)-1 and IRS-2, which associate with several Src homology-2 domain-containing proteins. To identify unique IRS-1-binding proteins, we screened a human heart cDNA library with 32P-labeled recombinant IRS-1 and obtained two isoforms (epsilon and zeta) of the 14-3-3 protein family. 14-3-3 protein has been shown to associate with IRS-1 in L6 myotubes, HepG2 hepatoma cells, Chinese hamster ovary cells, and bovine brain tissue. IRS-2, a protein structurally similar to IRS-1, was also shown to form a complex with 14-3-3 protein using a baculovirus expression system. The amount of 14-3-3 protein associated with IRS-1 was not affected by insulin stimulation but was increased significantly by treatment with okadaic acid, a potent serine/threonine phosphatase inhibitor. Peptide inhibition experiments using phosphoserine-containing peptides of IRS-1 revealed that IRS-1 contains three putative binding sites for 14-3-3 protein (Ser-270, Ser-374, and Ser-641). Among these three, the motif around Ser-270 is located in the phosphotyrosine binding domain of IRS-1, which is responsible for the interaction with the insulin receptor. Indeed, a truncated mutant of IRS-1 consisting of only the phosphotyrosine binding domain retained the capacity to bind to 14-3-3 protein in vivo. Finally, the effect of 14-3-3 protein binding on the insulin-induced phosphorylation of IRS-1 was investigated. Phosphoamino acid analysis revealed that IRS-1 coimmunoprecipitated with anti-14-3-3 antibody to be weakly phosphorylated after insulin stimulation, on tyrosine as well as serine residues, compared with IRS-1 immunoprecipitated with anti-IRS-1 antibody. Thus, the association with 14-3-3 protein may play a role in the regulation of insulin sensitivity by interrupting the association between the insulin receptor and IRS-1.

14-3-3 Proteins↗
PubMed1997

Insulin receptor substrate (IRS)-2 is dephosphorylated more rapidly than IRS-1 via its association with phosphatidylinositol 3-kinase in skeletal muscle cells.

Insulin receptor substrate (IRS)-2 is structurally and functionally similar to IRS-1. Indeed, stimulation with insulin or insulin-like growth factor I led to the rapid tyrosine phosphorylation of both IRS-1 and IRS-2, which in turn activated phosphatidylinositol (PI) 3-kinase in L6 cells and rat skeletal muscle. However, IRS-2 was rapidly dephosphorylated (3-10 min after the addition of insulin/insulin-like growth factor I), whereas IRS-1 phosphorylation continued for at least 60 min. The time courses of the PI 3-kinase activity associated with IRS-1 and IRS-2 paralleled the tyrosine phosphorylation of these proteins. Preincubation with sodium orthovanadate, an inhibitor of protein tyrosine phosphatase, blocked the rapid dephosphorylation of IRS-2, suggesting the involvement of tyrosine phosphatase. The activation of PI 3-kinase apparently plays an important role in the rapid dephosphorylation of IRS-2, as IRS-2 dephosphorylation was inhibited markedly by suppressing PI 3-kinase activity with wortmannin or overexpression of the dominant negative p85 subunit of PI 3-kinase, which cannot bind the p110 catalytic subunit. In addition, platelet-derived growth factor stimulation prior to insulin stimulation decreased IRS-associated PI 3-kinase and significantly inhibited the dephosphorylation of IRS-2. Taken together, these observations suggest that IRS-2 plays a unique role in mediating the signals from the insulin receptor to downstream molecules and that this effect is more transient than that of IRS-1. Tyrosine phosphatase and IRS-associated PI 3-kinase activity thus contribute to the rapid dephosphorylation of IRS-2.

Androstadienes↗
PubMed1997

p85alpha gene generates three isoforms of regulatory subunit for phosphatidylinositol 3-kinase (PI 3-Kinase), p50alpha, p55alpha, and p85alpha, with different PI 3-kinase activity elevating responses to insulin.

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine-phosphorylated substrates. We isolated a cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using 32P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85alpha, but the SH3 and bcr homology domains of p85alpha were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85alpha gene product. We suggest that this protein be called p50alpha. Northern blotting using a specific DNA probe corresponding to p50alpha revealed 6.0- and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50alpha protein and its associated PI 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50alpha. Taken together, these observations indicate that the p85alpha gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85alpha, p55alpha, and p50alpha), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50alpha forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85alpha and p55alpha associate only with p110 that can be wortmannin-labeled. Furthermore, p50alpha exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50alpha expression in the liver and its marked responsiveness to insulin, p50alpha appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three alpha isoforms has a different function and may have specific roles in various tissues.

Amino Acid Sequence↗