Randomized, double-blind, crossover, placebo-controlled clinical trial to evaluate the effects of chicken hot water extract on insulin secretion.

OBJECTIVE: Essence of chicken (EOC), a hot water extract of chicken, is widely consumed in Southeast Asia as a beverage. EOC has an inhibitory effect on the elevation of blood glucose levels and a secretagogue effect on insulin. However, the mechanism by which EOC promotes insulin secretion is unknown. We aimed to verify the postprandial hyperglycemic inhibitory effect and the insulin secretory effect of EOC in healthy adults under appropriate placebo settings. In addition, we aimed to understand the mechanism underlying the insulin secretory effect of EOC. PATIENTS AND METHODS: Thirty-four healthy Japanese adults were fed 68 mL of EOC or control food, followed by 200 g of cooked rice. Blood glucose and plasma insulin levels were measured at 30, 45, 60, 90, and 120 min after the participants ate cooked rice. The trial had a randomized, double-blind, crossover, placebo-controlled design. RESULTS: The ingestion of EOC induced an increase in the maximum blood concentration (Cmax) of insulin and shortened the time required to reach the maximum blood concentration following rice consumption. Ingestion of the test beverage resulted in a significantly higher insulinogenic index than that obtained after ingestion of the control beverage. No side effects were observed in this study. Mechanistic experiments revealed that EOC stimulated significant (p < 0.05) secretion of GLP-1 from NCI-H716 human intestinal L cells at 0.1, 1, and 10 mg/mL. CONCLUSIONS: Consuming EOC when eating rice supports pancreatic function. Daily consumption of EOC could elevate the early-phase insulin response; therefore, it could prevent diabetes in Asians with low insulin secretion.

[Analysis of the long-term prognosis for conjunctival malignant melanomas in Japan].

PURPOSE: To investigate the long-term prognosis for primary conjunctival malignant melanomas in Japan. MATERIALS & METHODS: We conducted a survey of 61 cases which had been reported in a 38-year period (1959 to 1996). We gathered information regarding the survival of patients, the post-operative follow-up period, the causes of death, and recurrences. Answers were obtained segarding 51 cases (84%). Detailed progress was identified in 23 of these cases. The survival rates were calculated using the Kaplan-Meier method. RESULTS: The survival rates were 95.1% after 1 year, 72.9% after 3 years, and 53.4% after 5 years. These values are relatively low compared with those reported in Europe and the United States.

Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines.

We measured the insulin-stimulated amount of Akt1, Akt2, and Akt3 enzymatic activities in four breast cancer cell lines and three prostate cancer cell lines. In the estrogen receptor-deficient breast cancer cells and the androgen-insensitive prostate cells, the amount of Akt3 enzymatic activity was approximately 20-60-fold higher than in the cells that were estrogen- or androgen-responsive. In contrast, the levels of Akt1 and -2 were not increased in these cells. The increase in Akt3 enzyme activity correlated with an increase in both Akt3 mRNA and protein. In a prostate cancer cell line lacking the tumor suppressor PTEN (a lipid and protein phosphatase), the basal enzymatic activity of Akt3 was constitutively elevated and represented the major active Akt in these cells. Finally, reverse transcription-PCR was used to examine the Akt3 expression in 27 primary breast carcinomas. The expression levels of Akt3 were significantly higher in the estrogen receptor-negative tumors in comparison to the estrogen receptor-positive tumors. To see if the increase in Akt3 could be due to chromosomal abnormalities, the Akt3 gene was assigned to human chromosome 1q44 by fluorescence in situ hybridization and radiation hybrid cell panel analyses. These results indicate that Akt3 may contribute to the more aggressive clinical phenotype of the estrogen receptor-negative breast cancers and androgen-insensitive prostate carcinomas.

[A case of bilateral continuous central retinal artery occlusion].

BACKGROUND: We report a case of bilateral simultaneous central retinal artery occlusion (CRAO). The time lag was 21 hours. CASE: The patient was a 78-year-old woman. She had hypertension, arteriosclerosis, and bilateral thrombosis of the legs. Both eyes could only distinguish light and dark at the first medical examination. RESULTS: We started steroid and urokinase therapy and used a vasodilator of peripheral vessels. When the patient left the hospital, visual acuity had improved to the ability of counting fingers (0.01 leftward 30 cm on the left). CONCLUSIONS: Both visual field and color vision had improved slightly. We think this bilateral CRAO resulted from thrombosis of both central retinal arteries at almost the same time.

Identification of a human Akt3 (protein kinase B gamma) which contains the regulatory serine phosphorylation site.

The family of protein kinases called Akt, protein kinase B (PKB), or related to A and C kinase (RAC) have been implicated in numerous biological processes including adipocyte and muscle differentiation, glycogen synthesis, glucose uptake, apoptosis and cellular proliferation. There are 3 known isoforms of this enzyme in mammalian cells (1/alpha, 2/beta and 3/gamma). Akt1 and 2 contain a key regulatory serine phosphorylation site in the carboxy-terminal region of the protein. However, the reported sequence of the rat Akt3 protein differed significantly from this in that it lacked 25 amino acids in the C-terminal region, including this key regulatory serine phosphorylation site (Biochem. Biophys. Res. Commun. 216, 526-534). In the present studies we show that the deduced sequence of human Akt3 contains this serine and that it is phosphorylated in response to insulin. These results indicate that human Akt3 is regulated similarly to Akt1 and Akt2.

Requirement of atypical protein kinase clambda for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes.

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCzeta and PKClambda) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKClambda in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKClambda (lambdaKD or lambdaDeltaNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKClambda, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by lambdaKD or lambdaDeltaNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKClambda was approximately 50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKClambda mutant that lacks the pseudosubstrate domain (lambdaDeltaPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of lambdaDeltaPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKClambda. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKClambda pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.

Posttranscriptional control of adipocyte differentiation through activation of phosphoinositide 3-kinase.

Differentiation of adipocytes is an important aspect of energy homeostasis. Although the transcriptional regulation of adipocyte differentiation is relatively well characterized, the subsequent molecular events remain unclear. The activity of phosphoinositide (PI) 3-kinase precipitated with antibodies to phosphotyrosine has now been shown to increase transiently during adipocyte differentiation of 3T3-F442A and of 3T3-L1 cells. PI 3-kinase activity precipitated with antibodies to insulin receptor substrate 1 (IRS1) and association of subunits of PI 3-kinase with IRS1 were also increased at this stage of differentiation, suggesting that IRS1 contributes to PI 3-kinase activation. Inhibition of the activation of PI 3-kinase by expression of dominant negative mutant subunits of the enzyme prevented adipogenesis, as assessed by lipid accumulation and expression of key adipocyte proteins such as GLUT4, adipsin, and aP2, suggesting that PI 3-kinase activation is essential for adipocyte differentiation. However, these mutant proteins did not affect either the expression of the transcription factor PPARgamma at the mRNA or protein level or the increase in the abundance of mRNAs encoding the adipocyte marker proteins. These results demonstrate that adipocyte differentiation is regulated at the posttranscriptional level and that activation of PI 3-kinase is required for this regulation.

Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport.

A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by approximately 75% in CHO cells and approximately 30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.

Changes in the anterior chamber configuration after small-incision cataract surgery with posterior chamber intraocular lens implantation.

PURPOSE: To report quantitative changes in the anterior chamber configuration after small-incision cataract surgery with implantation of a posterior chamber intraocular lens by means of ultrasound biomicroscopy. METHODS: We examined the anterior chamber configuration of 20 eyes of 20 patients before and 3 months after small-incision cataract surgery (phacoemulsification and aspiration plus foldable intraocular lens implantation through a 3.0- to 4.0-mm self-sealing wound) by means of ultrasound biomicroscopy. The following variables were measured: the anterior chamber depth at the center of the cornea, the angle-opening distance 250 microns from the scleral spur (AOD250), the angle-opening distance 500 microns from the scleral spur (AOD500), and the trabecular-iris angle. RESULTS: The anterior chamber depth at the center of the cornea, AOD250, AOD500, and trabecular-iris angle increased significantly after surgery. The preoperative anterior chamber depth at the center of the cornea and trabecular-iris angle were negatively correlated with the differences between the postoperative and preoperative values (P < .01). The preoperative values of all variables examined were negatively correlated with the ratios of the postoperative value to the preoperative value (P < .002). CONCLUSIONS: The present results showed that small-incision cataract surgery significantly deepened the anterior chamber and widened its angle. The more shallow the preoperative anterior chamber was, the greater the postoperative change of the chamber was; and the more narrow the preoperative angle was, the greater the postoperative change of the angle was.

Phosphoinositide 3-kinase is required for insulin-induced but not for growth hormone- or hyperosmolarity-induced glucose uptake in 3T3-L1 adipocytes.

The(1) regulatory mechanism of glucose uptake in 3T3-L1 adipocytes was investigated with the use of recombinant adenovirus vectors encoding various dominant negative proteins. Infection with a virus encoding a mutant regulatory subunit of phosphoinositide (PI) 3-kinase that does not bind the 110-kDa catalytic subunit (delta p85) inhibited the insulin-induced increase in PI 3-kinase activity co-precipitated by antibodies to phosphotyrosine and glucose uptake in a virus dose-dependent manner. Overexpression of a dominant negative RAS mutant in which Asp57 is replaced with tyrosine (RAS57Y) or of a dominant negative SOS mutant that lacks guanine nucleotide exchange activity (delta SOS) abolished the insulin-induced increase in mitogen-activated protein kinase activity, but had no effect on PI 3-kinase activity or glucose uptake. Although GH and hyperosmolarity attributable to 300 mM sorbitol each promoted glucose uptake and translocation of glucose transporter (GLUT)4 to an extent comparable to that of insulin, these stimuli triggered little or no association of PI 3-kinase activity with tyrosine-phosphorylated proteins. Overexpression of delta p85 or treatment of cells with wortmannin, an inhibitor of PI 3-kinase activity, had no effect on glucose uptake or translocation of GLUT4 stimulated by GH or hyperosmolarity. Moreover, overexpression of delta SOS or RAC17N also did not affect the increase in glucose uptake induced by these stimuli. A serine/threonine kinase Akt, a constitutively active mutant of which was previously shown to stimulate glucose uptake, is activated by insulin, GH, and hyperosmolarity to approximately 4-fold, approximately 2.1-fold, and approximately 2.3-fold over basal level, respectively. These results suggest that insulin-induced but neither GH- or hyperosmolarity-induced glucose uptake is PI 3-kinase-dependent, and neither RAS nor RAC is required for glucose uptake induced by these stimuli in 3T3-L1 adipocytes.

Activation of translation initiation factor eIF2B by insulin requires phosphatidyl inositol 3-kinase.

Eukaryotic initiation factor eIF2B mediates a key regulatory step in peptide-chain initiation and is acutely activated by insulin, although, it is not clear how. Inhibitors of phosphatidylinositide 3-kinase blocked activation of eIF2B, although rapamycin, which inhibits the p70 S6 kinase pathway, did not. Furthermore, a dominant negative mutant of PI 3-kinase also prevented activation of eIF2B, while a Sos-mutant, which blocks MAP kinase activation, did not. The data demonstrate that a pathway distinct from MAP and p70 S6 kinases regulates eIF2B. Glycogen synthase kinase-3 (GSK-3) phosphorylates and inactivates eIF2B. In all cases, eIF2B and GSK-3 were regulated reciprocally. Dominant negative PI 3-kinase abolished the insulin-induced inhibition of GSK-3. These data strongly support the hypothesis that insulin activates eIF2B through a signalling pathway involving PI 3-kinase and inhibition of GSK-3.

Interaction of Nck-associated protein 1 with activated GTP-binding protein Rac.

Bacterially expressed glutathione S-transferase fusion proteins containing Rac1 were used to identify binding proteins of this Rho family GTPase present in a bovine brain extract. Five proteins of 85, 110, 125, 140 and 170 kDa were detected, all of which were associated exclusively with guanosine 5'-[gamma-thio]triphosphate-bound Rac1, not with GDP-bound Rac1. The 85 and 110 kDa proteins were identified as the regulatory and catalytic subunits respectively of phosphatidylinositol 3-kinase. Several lines of evidence suggested that the 125 kDa protein is identical with Nck-associated protein 1 (Nap1). The mobilities of the 125 kDa protein and Nap1 on SDS/PAGE were indistinguishable, and the 125 kDa protein was depleted from brain extract by preincubation with the Src homology 3 domain of Nck to which Nap1 binds. Furthermore, antibodies to Nap1 reacted with the 125 kDa protein. Nap1 was co-immunoprecipitated with a constitutively active form of Rac expressed in Chinese hamster ovary cells. The observation that complex formation between activated Rac and PAK, but not that between Rac and Nap1, could be reproduced in vitro with recombinant proteins indicates that the interaction of Nap1 with Rac is indirect. The 140 kDa Rac-binding protein is a potential candidate for a link that connects Nap1 to Rac. The multimolecular complex comprising Rac, Nap1 and probably the 140 kDa protein might mediate some of the biological effects transmitted by the multipotent GTPase.

Multicenter genetic study of retinitis pigmentosa in Japan: I. Genetic heterogeneity in typical retinitis pigmentosa.

A nationwide, multicenter study of typical retinitis pigmentosa (RP) was carried out in collaboration with 18 hospitals throughout Japan to obtain current information for genetic counseling. We analyzed the genetic heterogeneity of RP based on the parental consanguinity of 434 probands registered during a 6-month period in 1990. A gradual decline in the frequency of consanguineous marriage was recognized among the normal parents of RP patients. The relative frequencies of inheritance patterns were estimated as: autosomal recessive, 25.2%; autosomal dominant, 16.9%; X-linked, 1.6%; and simplex, 56.3%. A comparison of these results with previous reports in Japan revealed a decline in the relative frequency of autosomal recessive cases and an increase in simplex cases. This suggests a decrease in the incidence of autosomal recessive retinitis pigmentosa in Japan, as well as the necessity for exhaustive investigations aimed at identifying inheritance patterns for RP patients seeking genetic counseling.

Multicenter genetic study of retinitis pigmentosa in Japan: II. Prevalence of autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of genetically heterogeneous diseases with autosomal recessive (AR), autosomal dominant, and X-linked modes of inheritance. Autosomal recessive retinitis pigmentosa (ARRP) is the most common form in Japan. A genetic analysis was done to determine the prevalence of ARRP indirectly, to provide an estimation of changing trends in the overall prevalence of RP. Data on the frequency of consanguinity and marriage year of normal parents of 59 ARRP patients were obtained from a nationwide multicenter survey of typical retinitis pigmentosa conducted in 1990. The gene frequency of ARRP was 0.01145 (Dahlberg's formula). In 1990, the number of young symptomatic ARRP patients decreased, while the number of patients aged 40 years and older increased. The total number of symptomatic ARRP patients in 1990 was nearly 21% higher than in 1970. Despite a dramatic decline in consanguinity in recent decades in Japan, the number of ARRP patients has increased. This increase is attributed to greater longevity and overall population growth. Our results suggest that the total number of RP patients has not decreased, and may even have increased.

A role for phosphoinositide 3-kinase in bacterial invasion.

Listeria monocytogenes is a bacterial pathogen that invades cultured nonphagocytic cells. Inhibitors and a dominant negative mutation were used to demonstrate that efficient entry requires the phosphoinositide (PI) 3-kinase p85alpha-p110. Infection with L. monocytogenes caused rapid increases in cellular amounts of PI(3, 4)P2 and PI(3,4,5)P3, indicating that invading bacteria stimulated PI 3-kinase activity. This stimulation required the bacterial protein InlB, host cell tyrosine phosphorylation, and association of p85alpha with one or more tyrosine-phosphorylated proteins. This role for PI 3-kinase in bacterial entry may have parallels in some endocytic events.

Phosphatidylinositol 3-kinase-independent signal transduction pathway for platelet-derived growth factor-induced chemotaxis.

Platelet-derived growth factor (PDGF)-BB is a potent chemoattractant for mesenchymal cells. Intracellular signal transduction for PDGF-induced chemotactic response has been reported to be dependent on phosphatidylinositol 3-kinase (PI3K) activation. Here, we report a PI3K-independent pathway operating for PDGF-induced chemotaxis in vascular smooth muscle cells and other cell types. Two different PI3K inhibitors, wortmannin (WT, 1 nM-1 microM) and LY294002 (100 nM-10 microM), did not inhibit PDGF-induced chemotaxis in smooth muscle cells and Swiss 3T3 cells, whereas WT inhibited activity of PI3K that were immunopurified from PDGF-stimulated cells as well as PI3K purified from cells that were stimulated with PDGF in the presence of the same concentrations of WT. Similarly, WT (100 nM) abolished the increase in intracellular phosphatidylinositol 3,4,5-triphosphate after PDGF stimulation. Furthermore, Chinese hamster ovary/Deltap85 cells overexpressing a dominant negative p85 subunit of PI3K showed a chemotactic response comparable to that of parental cells while showing a remarkable decrease in PI3K activity. Rapamycin, a specific inhibitor of pp70 S6 kinase, which is one of the well characterized downstreams of PI3K, did not inhibit PDGF-induced chemotaxis. Both WT and LY294002 inhibited PDGF-induced amino acid uptake and actin-stress fiber reorganization and partly inhibited PDGF-induced glucose incorporation in Swiss 3T3 cells. Our findings indicate that, in vascular smooth muscle cells and other cell types, the signal transduction for PDGF-induced chemotaxis is independent of PI3K activity while the signal transduction for PDGF-induced amino acid uptake, glucose incorporation, and cytoskeletal reorganization is dependent on PI3K.

Ras-independent and wortmannin-sensitive activation of glycogen synthase by insulin in Chinese hamster ovary cells.

Activation of glycogen synthase is one of the major metabolic events triggered by exposure of cells to insulin. The molecular mechanism by which insulin activates glycogen synthase was investigated. The possible role of Ras and mitogen-activated protein kinase cascade was investigated with a stable cell line, CHO-IR-C/S 46, that overexpresses insulin receptors and a catalytically inactive SH-PTP 2 protein phosphatase and in which insulin does not induce the formation of the Ras-GTP complex or the subsequently activation of the mitogen-activated protein kinase cascade. Insulin activated glycogen synthase in this cell line to a similar extent as in parental CHO-IR cells. The importance of heteromeric phosphoinositide (PI) 3-kinase in insulin activation of glycogen synthase was examined in a stable cell line, CHO-IR/delta p85, that overexpresses insulin receptors and a dominant negative mutant (delta p85) of the 85-kDa subunit of PI 3-kinase that lacks the binding site for the catalytic 110-kDa subunit. Insulin-dependent activation of PI-3 kinase and glucose transport, but not the formation of the Ras-GTP complex, are markedly attenuated in this cell line. In CHO-IR/delta p85 cells, insulin activated glycogen synthase to a similar extent as in parental CHO-IR cells. The failure of overproduction of the mutant (delta p85) protein to inhibit insulin activation of glycogen synthase was also confirmed by transient expression in Rat 1 cells with the use of a recombinant vaccinia virus. However, wortmannin abolished insulin activation of glycogen synthase in all cell lines. These data suggest that existence of a Ras-independent and wortmannin-sensitive pathway for activation of glycogen synthase by insulin.

Requirement for phosphoinositide 3-kinase in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and fat cells by inducing the redistribution of a specific glucose transporter, GLUT4, from intracellular vesicles to the cell surface. Phosphoinositide (PI) 3-kinase has been implicated as a key intermediate in insulin-stimulated glucose transport by studies that have examined the effects of wortmannin and LY294002, which are thought to be specific inhibitors of this enzyme. However, the specificity of these compounds for PI 3-kinase has recently been questioned. Epidermal growth factor, which activates mitogen-activated protein kinase in mouse 3T3-L1 adipocytes, has now been shown to have no effect on PI 3-kinase activity or GLUT4 translocation in these cells. Furthermore, microinjection of a dominant negative mutant of the 85-kDa subunit of PI 3-kinase, which lacks a binding site for the catalytic 110-kDa subunit, inhibited GLUT4 translocation induced by insulin in 3T3-L1 adipocytes; microinjection of the wild-type protein had no effect. These observations indicate that PI 3-kinase is necessary for insulin-induced GLUT4 translocation and glucose transport in adipocytes.

Normal activation of p70 S6 kinase by insulin in cells overexpressing dominant negative 85kD subunit of phosphoinositide 3-kinase.

The role of heteromeric phosphoinositide (PI) 3-kinase activity in insulin signal transduction was studied by investigating the effects of (i) overexpression of a dominant negative mutant p85 (delta p85) that lacks the binding site for p110 (delta p85-overexpressing cells) and (ii) inhibition of PI 3-kinase activity by wortmannin (wortmannin-treated cells). The insulin-induced association of PI 3-kinase activity with insulin receptor sustrate-1 (IRS-1) was inhibited in both wortmannin-treated cells and delta p85-overexpressing cells. However, whereas insulin-induced activation of p70 S6 kinase was completely abolished in wortmannin-treated cells, it appeared normal in delta p85-overexpressing cells. These results raise the possibility that a wortmannin-sensitive pathway independent of heteromeric PI 3-kinase is involved in the activation of p70 S6 kinase by insulin.

1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells.

Insulin stimulation drives the formation of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and 1-phosphatidylinositol 3-kinase (PI 3-kinase; ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137), a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits. This interaction takes place via the phosphorylated YMXM motifs of IRS-1 and the Src homology region 2 (SH2) domains of p85. In this study, the stable overexpression in a Chinese hamster ovary (CHO) cell line of a mutant p85 alpha (delta p85) protein, which lacks a binding site for p110, disrupted the complex formation between IRS-1 and the catalytic subunit of PI 3-kinase in intact cells during insulin stimulation. Activation of insulin receptor kinase and the tyrosine phosphorylation of IRS-1 remained unaffected. In this cell line, both insulin-stimulated accumulation of phosphatidylinositol 3,4,5-trisphosphate and the insulin-stimulated glucose uptake due to the translocation of GLUT1 glucose transporters were markedly impaired, whereas neither phorbol 12-myristate 13-acetate-stimulated glucose uptake nor the insulin-stimulated activation of RAS was impaired. These results suggest that PI 3-kinase is required for glucose transport in insulin signaling in CHO cells.