Side-chain modified vitamin D analogs require activation of both PI 3-K and erk1,2 signal transduction pathways to induce differentiation of human promyelocytic leukemia cells

Synthetic analogs of vitamin D for potential use in differentiation therapy should selectively regulate genes necessary for differentiation without inducing any perturbations in calcium homeostasis. PRI-1906, an analog of vitamin D2, and PRI-2191, an analog of vitamin D3 bind nuclear vitamin D receptor (nVDR) with substantially lower affinity than 1,25-dihydroxyvitamin D3 (1,25-D3), but have higher differentiation-inducing activity as estimated in HL-60 leukemia cellmodel. To examine how their increased differentiation-inducing activity is regulated we tested the hypothesis that membrane-mediated events, unrelated to nVDR, take part in the differentiation in response to PRI-1906 and PRI-2191. The induction of leukemia cell differentiation in response to the analogs of vitamin D was inhibited by LY294002 (phosphatidylinositol 3-kinase inhibitor), PD98059 (inhibitor of MEK1,2, an upstream regulator of extracellular-signal regulated kinase) and rapamycin (p70S6K inhibitor) pointing out that activation of signal transduction pathways unrelated to nVDR is necessary for differentiation. On the other hand, inhibition of cytosolic phospholipase A2 accelerated the differentiation of HL-60 cells induced by either 1,25-D3 or by the vitamin D analogs suggesting possible existence of a feedback loop between extracellular-signal regulated kinases and phospholipase A2.     

The in vitro effect of 1 alpha,25-dihydroxyvitamin D3 on insulin production by neonatal rat islets

The in vitro effect of 1 alpha,25-dihydroxyvitamin D3 on the function of beta cells of the endocrine pancreas was investigated. Neonatal islets maintained in serum-free medium, or medium supplemented with 0.5% fetal bovine serum achieved a 2.5-fold increase in medium insulin levels in response to 10(8) M 1 alpha,25-dihydroxyvitamin D3 (P less than 0.001). The effect of 1,25-dihydroxyvitamin D3 required at least 96 h treatment to become evident and was similar at medium glucose concentrations of 10 and 20 mM. Cell-associated insulin was increased in 1 alpha,25-dihydroxyvitamin D3-treated cultures maintained in 0.5% serum. These data suggest that 1 alpha,25-dihydroxyvitamin D3 may have a direct effect in the beta cell.     

G2/M arrest by 1,25-dihydroxyvitamin D3 in ovarian cancer cells mediated through the induction of GADD45 via an exonic enhancer

1,25-Dihydroxyvitamin D3 suppresses the growth of multiple human cancer cell lines by inhibiting cell cycle progression and inducing cell death. The present study showed that 1,25-dihydroxyvitamin D3 causes cell cycle arrest at the G2/M transition through p53-independent induction of GADD45 in ovarian cancer cells. Detailed analyses have established GADD45 as a primary target gene for 1,25-dihydroxyvitamin D3. A DR3-type vitamin D response element was identified in the fourth exon of GADD45 that forms a complex with the vitamin D receptor.retinoid X receptor heterodimer in electrophoresis mobility shift assays and mediates the dose-dependent induction of luciferase activity by 1,25-dihydroxyvitamin D3 in reporter assays. Chromatin immunoprecipitation assays have shown that the vitamin D receptor is recruited in a ligand-dependent manner to the exonic enhancer but not to the GADD45 promoter regions. In ovarian cancer cells expressing GADD45 antisense cDNA or GADD45-null mouse embryo fibroblasts, 1,25-dihydroxyvitamin D3 failed to induce G2/M arrest. Taken together, these results identify GADD45 as an important mediator for the tumor-suppressing activity of 1,25-dihydroxyvitamin D3 in human ovarian cancer cells.     

pp60Src mediates insulin-stimulated sequestration of the beta(2)-adrenergic receptor: insulin stimulates pp60Src phosphorylation and activation

Insulin stimulates a rapid phosphorylation and sequestration of the beta(2)-adrenergic receptor. Analysis of the signaling downstream of the insulin receptor with enzyme inhibitors revealed roles for both phosphatidylinositol 3-kinase and pp60Src. Inhibition of Src with PP2, like the inhibition of phosphatidylinositol 3-kinase with LY294002 [2-(4-morpholynyl)-8-phenyl-4H-1-benzopyran-4-one], blocked the activation of Src as well as insulin-stimulated sequestration of the beta(2)-adrenergic receptor. Depletion of Src with antisense morpholinos also suppressed insulin-stimulated receptor sequestration. Src is shown to be phosphorylated/activated in response to insulin in human epidermoid carcinoma A431 cells as well as in mouse 3T3-L1 adipocytes and their derivative 3T3-F422A cells, well-known models of insulin signaling. Inhibition of Src with PP2 blocks the ability of insulin to sequester beta(2)-adrenergic receptors and the translocation of the GLUT4 glucose transporters. Insulin stimulates Src to associate with the beta(2)-adrenergic receptor/AKAP250/protein kinase A/protein kinase C signaling complex. We report a novel positioning of Src, mediating signals from insulin to phosphatidylinositol 3-kinase and to beta(2)-adrenergic receptor trafficking.     

Essential role of protein kinase C zeta in the impairment of insulin-induced glucose transport in IRS-2-deficient brown adipocytes

Insulin receptor substrate-2-deficient (IRS-2(-/-)) mice develop type 2 diabetes. We have investigated the molecular mechanisms by which IRS-2(-/-) immortalized brown adipocytes showed an impaired response to insulin in inducing GLUT4 translocation and glucose uptake. IRS-2-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity was blunted in IRS-2(-/-) cells, total PI 3-kinase activity being reduced by 30%. Downstream, activation of protein kinase C (PKC) zeta was abolished in IRS-2(-/-) cells. Reconstitution with retroviral IRS-2 restores IRS-2/PI 3-kinase/PKC zeta signalling, as well as glucose uptake. Wild-type cells expressing a kinase-inactive mutant of PKC zeta lack GLUT4 translocation and glucose uptake. Our results support the essential role played by PKC zeta in the insulin resistance and impaired glucose uptake observed in IRS-2-deficient brown adipocytes.     

Tumor-suppressive activity of 1,25-dihydroxyvitamin D3 against kidney cancer cells via up-regulation of FOXO3

1,25-Dihydroxyvitamin D3 has been known to have the tumor-suppressive activity in various kinds of tumors. However, the exact effect and working mechanism of 1,25-dihydroxyvitamin D3 on the tumor-suppressive activity in human kidney cancer cells remains poorly understood. 1,25-Dihydroxyvitamin D3 has cytotoxicity to ACHN cells and inhibited ACHN cell proliferation compared to the vehicle control. 1,25-Dihydroxyvitamin D3 increased the expression of the cleaved PARP1, active Caspase3, Bax, and Bim but decreased the expression of Bcl2 in ACHN cells. Moreover, 1,25-dihydroxyvitamin D3 down-regulated the phosphorylated Akt and Erk which might lead to apoptosis through activation of FOXO3 in ACHN cells. Transfection of siRNA against FOXO3 attenuated the pro-apoptotic BimEL expression in ACHN cells treated with 1,25-dihydroxyvitamin D3. These results suggest that FOXO3 is involved in the apoptosis induced by 1,25-dihydroxyvitamin D3.     

A phosphatidylinositol 3-kinase-independent insulin signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling.

Recruitment of intracellular glucose transporter 4 (GLUT4) to the plasma membrane of fat and muscle cells in response to insulin requires phosphatidylinositol (PI) 3-kinase as well as a proposed PI 3-kinase-independent pathway leading to activation of the small GTPase TC10. Here we show that in cultured adipocytes insulin causes acute cortical localization of the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) and actin-related protein-3 (Arp3) as well as cortical F-actin polymerization by a mechanism that is insensitive to the PI 3-kinase inhibitor wortmannin. Expression of the dominant inhibitory N-WASP-DeltaWA protein lacking the Arp and actin binding regions attenuates the cortical F-actin rearrangements by insulin in these cells. Remarkably, the N-WASP-DeltaWA protein also inhibits insulin action on GLUT4 translocation, indicating dependence of GLUT4 recycling on N-WASP-directed cortical F-actin assembly. TC10 exhibits sequence similarity to Cdc42 and has been reported to bind N-WASP. We show the inhibitory TC10 (T31N) mutant, which abrogates insulin-stimulated GLUT4 translocation and glucose transport, also inhibits both cortical localization of N-WASP and F-actin formation in response to insulin. These findings reveal that N-WASP likely functions downstream of TC10 in a PI 3-kinase-independent insulin signaling pathway to mobilize cortical F-actin, which in turn promotes GLUT4 responsiveness to insulin.     

Effects of 1,25-dihydroxyvitamin D3 on proliferation and differentiation of porcine preadipocyte in vitro

1,25-Dihydroxyvitamin D3, the physiologically active form of vitamin D3, exerts its functions through a receptor-mediated mechanism and plays an important role in the cell differentiation. This study investigated the effects of 1,25-dihydroxyvitamin D3 on the proliferation and differentiation of porcine preadipocyte. Stromal-vascular cells containing preadipocytes were prepared from dorsal subcutaneous adipose tissue of approximately 3-day-old Chinese male crossbred pigs. After confluence, the differentiation was induced by transferrin, dexamethasone and insulin for 2 days, and then subsequently cultured for 6 days. The cells were treated with 1,25-dihydroxyvitamin D3 during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. The terminal differentiation markers, such as glycerol-3-phosphate dehydrogenase activity and lipid accumulation were measured during the process of cultures. The treatment with 1,25-dihydroxyvitamin D3 severely affected the induction of all differentiation markers throughout the differentiation period. 1,25-Dihydroxyvitamin D3 suppressed the expression of peroxisome proliferator-activated receptor gamma mRNA and interfered with the induction of retinoid X receptor alpha mRNA. The mRNAs of the adipogenesis-related genes, lipoprotein lipase, stearoyl-CoA desaturase, phosphoenolpyruvate carboxykinase, glycerol-3-phosphate dehydrogenase and glucose transporter 4 were reduced when 1,25-dihydroxyvitamin D3 was added into differentiation medium. Also, 1,25-dihydroxyvitamin D3 inhibited preadipocyte differentiation in dose-dependent manner. These results suggested that 1,25-dihydroxyvitamin D3 inhibited porcine preadipocyte differentiation through suppressing PPAR gamma and RXR alpha mRNA expressions and then down regulating the expression of adipogenesis-related genes.     

Epinephrine inhibits insulin-stimulated muscle glucose transport.

We recently demonstrated that epinephrine could inhibit the activation by insulin of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-kinase) in skeletal muscle (Hunt DG, Zhenping D, and Ivy JL. J Appl Physiol 92: 1285-1292, 2002). Activation of PI3-kinase is recognized as an essential step in the activation of muscle glucose transport by insulin. We therefore investigated the effect of epinephrine on insulin-stimulated glucose transport in both fast-twitch (epitrochlearis) and slow-twitch (soleus) muscle of the rat by using an isolated muscle preparation. Glucose transport was significantly increased in the epitrochlearis and soleus when incubated in 50 and 100 microU/ml insulin, respectively. Activation of glucose transport by 50 microU/ml insulin was inhibited by 24 nM epinephrine in both muscle types. This inhibition of glucose transport by epinephrine was accompanied by suppression of IRS-1-associated PI3-kinase activation. However, when muscles were incubated in 100 microU/ml insulin, 24 nM epinephrine was unable to inhibit IRS-1-associated PI3-kinase activation or glucose transport. Even when epinephrine concentration was increased to 500 nM, no attenuating effect was observed on glucose transport. Results of this study indicate that epinephrine is capable of inhibiting glucose transport activated by a moderate, but not a high, physiological insulin concentration. The inhibition of glucose transport by epinephrine appears to involve the inhibition of IRS-1-associated PI3-kinase activation.     

Effect of 1,25-dihydroxyvitamin D3 on pancreatic B and D cell function

To investigate the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on pancreatic B and D cell function in normal rats, 1 microgram of 1,25(OH)2D3 was administered intravenously 20 hours before the experiment. The plasma 1,25(OH)2D3 and calcium concentrations were significantly elevated, and plasma insulin levels also increased in 1,25(OH)2D3-administered rats compared with controls. Glucose-induced insulin and somatostatin release from the isolated pancreas perfused with lower calcium, however, was the same between the 1,25(OH)2D3-administered group and the controls. On the other hand, when the isolated pancreas was perfused with higher calcium, the glucose-induced insulin release was significantly increased in the 1,25(OH)2D3-administered group, while no significant difference in somatostatin release was observed in any group. These results suggest that the sensitivity of pancreatic B cells to glucose perfused with more calcium may increase when 1,25(OH)2D3 has been previously administered. In addition, 1,25(OH)2D3 does not seem to affect the somatostatin release from the pancreatic D cells.     

C(24)- and C(23)-oxidation, converging pathways of intestinal 1,25-dihydroxyvitamin D3 metabolism: identification of 24-keto-1,23,25-trihydroxyvitamin D3

24-Keto-1,23,25-trihydroxyvitamin D3 has been identified as a major 1,25-dihydroxyvitamin D3 metabolite, produced by intestinal mucosa cells isolated from rats dosed chronically with 1,25-dihydroxyvitamin D3. The identification was based on ultraviolet absorbance spectroscopy, mass spectroscopy, and chemical derivatization. The pathway of biosynthesis proceeded through 1,24,25-trihydroxyvitamin D3 and 24-keto-1,25-dihydroxyvitamin D3, which are physiological metabolites of 1,25-dihydroxyvitamin D3. Previous work [Napoli, J. L., Pramanik, B. C., Royal, P. M., Reinhardt, T. A., & Horst, R. L. (1983) J. Biol. Chem. 258, 9100-9107] had shown that the amount of 24-keto-1,23,25-trihydroxyvitamin D3 in intestine in vivo, relative to its C(24)-oxidized precursors, is enhanced by chronically dosing rats with 1,25-dihydroxyvitamin D3. These results establish the C(24)-oxidation pathway as a predominant route of intestinal 1,25-dihydroxyvitamin D3 metabolism under physiological conditions and indicate that treatment of the rat with exogenous 1,25-dihydroxyvitamin D3 causes expression of C(23)-hydroxylase activity, which uses C(24)-oxidized 1,25-dihydroxyvitamin D3 metabolites as substrates.     

Ginsenoside Re reduces insulin resistance through inhibition of c-Jun NH2-terminal kinase and nuclear factor-kappaB

Ginsenoside Re (Re), a compound derived from Panax ginseng, shows an antidiabetic effect. However, the molecular basis of its action remains unknown. We investigated insulin signaling and the antiinflammatory effect by Re in 3T3-L1 adipocytes and in high-fat diet (HFD) rats to dissect its anti-hyperglycemic mechanism. Glucose uptake was measured in 3T3-L1 cells and glucose infusion rate determined by clamp in HFD rats. The insulin signaling cascade, including insulin receptor (IR) beta-subunit, IR substrate-1, phosphatidylinositol 3-kinase, Akt and Akt substrate of 160 kDa, and glucose transporter-4 translocation are examined. Furthermore, c-Jun NH(2)-terminal kinase (JNK), MAPK, and nuclear factor (NF)-kappaB signaling cascades were also assessed. The results show Re increases glucose uptake in 3T3-L1 cells and glucose infusion rate in HFD rats. The activation of insulin signaling by Re is initiated at IR substrate-1 and further passes on through phosphatidylinositol 3-kinase and downstream signaling cascades. Moreover, Re demonstrates an impressive suppression of JNK and NF-kappaB activation and inhibitor of NF-kappaBalpha degradation. In conclusion, Re reduces insulin resistance in 3T3-L1 adipocytes and HFD rats through inhibition of JNK and NF-kappaB activation.     

Agents affecting adenylate cyclase activity modulate the stimulatory action of 1,25-dihydroxyvitamin D3 on the production of osteocalcin by human bone cells

The stimulation of osteocalcin synthesis by human osteoblast-like cells in response to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is antagonised by several bone regulatory agents. We have shown that agents which activate adenylate cyclase inhibit this action of 1,25(OH)2D3 on human osteoblast-like cells. Activation of adenylate cyclase, either via the stimulatory GTP-binding protein using cholera toxin, or directly at the catalytic via the stimulatory GTP-binding protein using cholera toxin, or directly at the catalytic subunit using forskolin, results in a suppression of osteocalcin synthesis. Whilst the activation of adenylate cyclase induces this inhibitory response, neither exogenous dibutyryl cyclic AMP nor the phosphodiesterase inhibitor, IBMX, exerted any apparent effect on the production of osteocalcin. The tumour promoting phorbol ester, 4 beta-phorbol 12,13-dibutyrate, also inhibited 1,25(OH)2D3-stimulated osteocalcin production. This was not apparent in response to the non-tumour promoting phorbol ester 4 beta-phorbol suggesting the involvement of protein kinase C.     

1α,25-dihydroxyvitamin D(3) mechanism of action: modulation of L-type calcium channels leading to calcium uptake and intermediate filament phosphorylation in cerebral cortex of young rats

The involvement of calcium-mediated signaling pathways in the mechanism of action of 1α,25-dihydroxyvitamin D(3) (1,25D) is currently demonstrated. In this study we found that 1,25D induces nongenomic effects mediated by membrane vitamin D receptor (VDRm) by modulating intermediate filament (IF) phosphorylation and calcium uptake through L-type voltage-dependent calcium channels (L-VDCC) in cerebral cortex of 10 day-old rats. Results showed that the mechanism of action of 1,25D involves intra- and extracellular calcium levels, as well as the modulation of chloride and potassium channels. The effects of L-VDCCs on membrane voltage occur over a broad potential range and could involve depolarizing or hyperpolarizing coupling modes, supporting a cross-talk among Ca(2+) uptake and potassium and chloride channels. Also, the Na(+)/K(+)-ATPase inactivation by ouabain mimicked the 1,25D action on (45)Ca(2+) uptake. The Na(+)/K(+)-ATPase inhibition observed herein might lead to intracellular Na(+) accumulation with subsequent L-VDCC opening and consequently increased (45)Ca(2+) (calcium, isotope of mass 45) uptake. Moreover, the 1,25D effect is dependent on the activation of the following protein kinases: cAMP-dependent protein kinase (PKA), Ca(2+)/calmodulin-dependent protein kinase (PKCaMII), phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase p38 (p38(MAPK)). The modulation of calcium entry into neural cells by the 1,25D we are highlighting, might take a role in the regulation of a plethora of intracellular processes. Considering that vitamin D deficiency can lead to brain illness, 1,25D may be a possible candidate to be used, at least as an adjuvant, in the pharmacological therapy of neuropathological conditions.     

Modulation by the steroid/thyroid hormone superfamily of TGF-beta-stimulated VEGF release from vascular smooth muscle cells

We previously reported that transforming growth factor-beta (TGF-beta) stimulates the release of vascular endothelial growth factor (VEGF) from aortic smooth muscle A10 cells via activation of p38 mitogen-activated protein (MAP) kinase. In the present study, we investigated whether nuclear hormone receptor superfamily members affect TGF-beta-stimulated VEGF release from A10 cells. Retinoic acid or 1,25-dihydroxyvitamin D3 enhanced TGF-beta-induced VEGF release in a concentration-dependent manner, whereas dexamethasone or corticosterone suppressed TGF-beta-induced VEGF release. 1,25-Dihydroxyvitamin D3 and TGF-beta stimulated phosphorylation of p38 MAP kinase in an additive manner. SB203580, an inhibitor of p38 MAP kinase, decreased the VEGF release induced by TGF-beta or 1,25-dihydroxyvitamin D3. However, retinoic acid, dexamethasone, or corticosterone did not affect phosphorylation of p38 MAP kinase. These results indicate that retinoic acid, 1,25-dihydroxyvitamin D3, and glucocorticoids affect TGF-beta-stimulated VEGF release from aortic smooth muscle cells. The stimulatory effect of 1,25-dihydroxyvitamin D3 occurs, in part, via modification of TGF-beta-induced activation of p38 MAP kinase.     

Insulin and exercise differentially regulate PI3-kinase and glycogen synthase in human skeletal muscle.

The purpose of this study was to determine the separate and combined effects of exercise and insulin on the activation of phosphatidylinositol 3-kinase (PI3-kinase) and glycogen synthase in human skeletal muscle in vivo. Seven healthy men performed three trials in random order. The trials included 1) ingestion of 2 g/kg body wt carbohydrate in a 10% solution (CHO); 2) 75 min of semirecumbent cycling exercise at 75% of peak O(2) consumption; followed by 5 x 1-min maximal sprints (Ex); and 3) Ex, immediately followed by ingestion of the carbohydrate solution (ExCHO). Plasma glucose and insulin were increased (P < 0.05) at 15 and 30 (Post-15 and Post-30) min after the trial during CHO and ExCHO, although insulin was lower for ExCHO. Hyperinsulinemia during recovery in CHO and ExCHO led to an increase (P < 0.001) in PI3-kinase activity at Post-30 compared with basal, although the increase was lower (P < 0. 004) for ExCHO. Furthermore, PI3-kinase activity was suppressed (P < 0.02) immediately after exercise (Post-0) during Ex and ExCHO. Area under the insulin response curve for all trials was positively associated with PI3-kinase activity (r = 0.66, P < 0.001). Glycogen synthase activity did not increase during CHO but was increased (P < 0.05) at Post-0 and Post-30 during Ex and ExCHO. Ingestion of the drink increased (P < 0.05) carbohydrate oxidation during CHO and ExCHO, although the increase after ExCHO was lower (P < 0.05) than CHO. Carbohydrate oxidation was directly correlated with PI3-kinase activity for all trials (r = 0.63, P < 0.001). In conclusion, under resting conditions, ingestion of a carbohydrate solution led to activation of the PI3-kinase pathway and oxidation of the carbohydrate. However, when carbohydrate was ingested after intense exercise, the PI3-kinase response was attenuated and glycogen synthase activity was augmented, thus facilitating nonoxidative metabolism or storage of the carbohydrate. Activation of glycogen synthase was independent of PI3-kinase.     

Protein kinase C modulates insulin action in human skeletal muscle

There is good evidence from cell lines and rodents that elevated protein kinase C (PKC) overexpression/activity causes insulin resistance. Therefore, the present study determined the effects of PKC activation/inhibition on insulin-mediated glucose transport in incubated human skeletal muscle and primary adipocytes to discern a potential role for PKC in insulin action. Rectus abdominus muscle strips or adipocytes from obese, insulin-resistant, and insulin-sensitive patients were incubated in vitro under basal and insulin (100 nM)-stimulated conditions in the presence of GF 109203X (GF), a PKC inhibitor, or 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a PKC activator. PKC inhibition had no effect on basal glucose transport. GF increased (P < 0.05) insulin-stimulated 2-deoxyglucose (2-DOG) transport approximately twofold above basal. GF plus insulin also increased (P < 0.05) insulin receptor tyrosine phosphorylation 48% and phosphatidylinositol 3-kinase (PI 3-kinase) activity approximately 50% (P < 0.05) vs. insulin treatment alone. Similar results for GF on glucose uptake were observed in human primary adipocytes. Further support for the hypothesis that elevated PKC activity is related to insulin resistance comes from the finding that PKC activation by dPPA was associated with a 40% decrease (P < 0.05) in insulin-stimulated 2-DOG transport. Incubation of insulin-sensitive muscles with GF also resulted in enhanced insulin action ( approximately 3-fold above basal). These data demonstrate that certain PKC inhibitors augment insulin-mediated glucose uptake and suggest that PKC may modulate insulin action in human skeletal muscle.     

The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis.

The Drosophila insulin receptor (DIR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins which binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric DIR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediated tyrosine phosphorylation of Shc and activated mitogen-activated protein kinase. IRS-1 was required by the human insulin receptor to activate PI 3-kinase and p70s6k, whereas hDIR associated with PI 3-kinase and activated p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the DIR possesses additional signaling capabilities compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells.