GLUT4 in murine bone growth: from uptake and translocation to proliferation and differentiation

Skeletal growth, taking place in the cartilaginous growth plates of long bones, consumes high levels of glucose for both metabolic and anabolic purposes. We previously showed that Glut4 is present in growing bone and is decreased in diabetes. In the present study, we examined the hypothesis that in bone, GLUT4 gene expression and function are regulated via the IGF-I receptor (IGF-IR) and that Glut4 plays an important role in bone growth. Insulin and IGF-I actions on skeletal growth and glucose uptake were determined using mandibular condyle (MC) organ cultures and MC-derived primary cell cultures (MCDC). Chondrogenesis was determined by following proliferation and differentiation activities using immunohistochemical (IHC) analysis of proliferating cell nuclear antigen and type II collagen expression, respectively. Overall condylar growth was assessed morphometrically. GLUT4 mRNA and protein levels were determined using in situ hybridization and IHC, respectively. Glut4 translocation to the cell membrane was assessed using confocal microscopy analysis of GFP-Glut4 fusion-transfected cells and immunogold and electron microscopy on MC sections; glucose uptake was assayed by 2-deoxyglucose (2-DOG) uptake. Both IGF-I and insulin-stimulated glucose uptake in MCDC, with IGF-I being tenfold more potent than insulin. Blockage of IGF-IR abrogated both IGF-I- and insulin-induced chondrogenesis and glucose metabolism. IGF-I, but not insulin, induced Glut4 translocation to the plasma membrane. Additionally, insulin induced both GLUT4 and IGF-IR gene expression and improved condylar growth in insulin receptor knockout mice-derived MC. Moreover, silencing of GLUT4 gene in MCDC culture abolished both IGF-I-induced glucose uptake and chondrocytic proliferation and differentiation. In growing bone, the IGF-IR pathway stimulates Glut4 translocation and enhances glucose uptake. Moreover, intact Glut4 cellular levels and translocation machinery are essential for early skeletal growth.     

Insulin and IGF-I actions on IGF-I receptor in seminiferous tubules from immature rats

Insulin and insulin-like growth factor 1 (IGF-I) are capable of activating similar intracellular pathways. Insulin acts mainly through its own receptor, but can also activate the IGF-I receptor (IGF-IR). The aim of this study was to investigate the involvement of the IGF-IR in the effects of insulin and IGF-I on the membrane potential of immature Sertoli cells in whole seminiferous tubules, as well as on calcium, amino acid, and glucose uptake in testicular tissue of immature rats. The membrane potential of the Sertoli cells was recorded using a standard single microelectrode technique. In calcium uptake experiments, the testes were pre-incubated with ⁴⁵Ca^2 +, with or without JB1 (1 μg/mL), and then incubated with insulin (100 nM) or IGF-I (15 nM). In amino acid and glucose uptake experiments, the gonads were pre-incubated with or without JB1 (1 μg/mL) and then incubated with radiolabeled amino acid or glucose analogues in the presence of insulin (100 nM) or IGF-I (15 nM). The blockade of IGF-IR with JB1 prevented the depolarising effects of both insulin and IGF-I on membrane potential, as well as the effect of insulin on calcium uptake. JB1 also inhibited the effects of insulin and IGF-I on glucose uptake. The effect of IGF-I on amino acid transport was inhibited in the presence of JB1, whereas the effect of insulin was not. We concluded that while IGF-I seems to act mainly through its cognate receptor to induce membrane depolarisation and calcium, amino acid and glucose uptake, insulin appears to be able to elicit its effects through IGF-IR, in seminiferous tubules from immature rats.     

RACK1, an insulin-like growth factor I (IGF-I) receptor-interacting protein, modulates IGF-I-dependent integrin signaling and promotes cell spreading and contact with extracellular matrix

The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to regulate a variety of cellular processes including cell proliferation, cell survival, cell differentiation, and cell transformation. IRS-1 and Shc, substrates of the IGF-IR, are known to mediate IGF-IR signaling pathways such as those of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K), which are believed to play important roles in some of the IGF-IR-dependent biological functions. We used the cytoplasmic domain of IGF-IR in a yeast two-hybrid interaction trap to identify IGF-IR-interacting molecules that may potentially mediate IGF-IR-regulated functions. We identified RACK1, a WD repeat family member and a Gbeta homologue, and demonstrated that RACK1 interacts with the IGF-IR but not with the closely related insulin receptor (IR). In several types of mammalian cells, RACK1 interacted with IGF-IR, protein kinase C, and beta1 integrin in response to IGF-I and phorbol 12-myristate 13-acetate stimulation. Whereas most of RACK1 resides in the cytoskeletal compartment of the cytoplasm, transformation of fibroblasts and epithelial cells by v-Src, oncogenic IR or oncogenic IGF-IR, but not by Ros or Ras, resulted in a significantly increased association of RACK1 with the membrane. We examined the role of RACK1 in IGF-IR-mediated functions by stably overexpressing RACK1 in NIH 3T3 cells that expressed an elevated level of IGF-IR. RACK1 overexpression resulted in reduced IGF-I-induced cell growth in both anchorage-dependent and anchorage-independent conditions. Overexpression of RACK1 also led to enhanced cell spreading, increased stress fibers, and increased focal adhesions, which were accompanied by increased tyrosine phosphorylation of focal adhesion kinase and paxillin. While IGF-I-induced activation of IRS-1, Shc, PI3K, and MAPK pathways was unaffected, IGF-I-inducible beta1 integrin-associated kinase activity and association of Crk with p130(CAS) were significantly inhibited by RACK1 overexpression. In RACK1-overexpressing cells, delayed cell cycle progression in G(1) or G(1)/S was correlated with retinoblastoma protein hypophophorylation, increased levels of p21(Cip1/WAF1) and p27(Kip1), and reduced IGF-I-inducible Cdk2 activity. Reduction of RACK1 protein expression by antisense oligonucleotides prevented cell spreading and suppressed IGF-I-dependent monolayer growth. Our data suggest that RACK1 is a novel IGF-IR signaling molecule that functions as a positive mediator of cell spreading and contact with extracellular matrix, possibly through a novel IGF-IR signaling pathway involving integrin and focal adhesion signaling molecules.     

IGF-I and insulin regulate glucose transport in mouse blastocysts via IGF-I receptor

The roles of glucose deprivation, insulin, and insulin-like growth factor I (IGF-I) in the regulation of glucose transport in the mouse blastocyst were examined. Glucose transport, measured by uptake of 3-O-methyl glucose (3-OMG), was increased by 19% (P < 0.01) in response to glucose deprivation. Both IGF-I and insulin stimulated uptake, but IGF-I was 1,000-fold more potent than insulin, increasing uptake by 51% at 1.7 pM (P < 0.001). These effects began to appear after 20 min of incubation with growth factors, and required the simultaneous presence of glucose. The relative potencies of insulin and IGF-I suggest that the actions of IGF-I and insulin were both mediated via the IGF-I receptor. The inactivity of a specific agonistic insulin receptor antibody (B10) confirms this and suggests that this action may be independent of signalling through IRS-1. Cycloheximide decreased growth factor-stimulated transport by about 40%, indicating that both protein synthesis and transporter recruitment from cytoplasmic stores are responsible for maximal stimulation. These characteristics are consistent with GLUT1-facilitated glucose uptake and suggest that GLUT1 is the regulatable transporter in mouse blastocysts. Stimulation of GLUT1 may be a ubiquitous feature of the autocrine/paracrine activity of IGF-I in cell growth and proliferation. © 1996 Wiley-Liss, Inc.     

RACK1-mediated integration of adhesion and insulin-like growth factor I (IGF-I) signaling and cell migration are defective in cells expressing an IGF-I receptor mutated at tyrosines 1250 and 1251

The scaffolding protein receptor for activated C kinase (RACK1) has been proposed to mediate the integration of insulin-like growth factor I receptor (IGF-IR) and adhesion signaling. Here we investigated the mechanism of this integration of signaling, by using an IGF-IR mutant (Y1250F/Y1251F) that is deficient in anti-apoptotic and transforming function. RACK1 was found to associate with the IGF-IR only in adherent cells and did not associate with the IGF-IR in nonadherent cells, lymphocytic cells, or cells expressing the Y1250F/Y1251F mutant. In R- cells transiently expressing the Y1250F/Y1251F mutant RACK1 became constitutively associated with beta1 integrin and did not associate with Shc, Src, or Shp2. This was accompanied by the loss of formation of a complex containing the IGF-IR, RACK1, and beta1 integrin; loss of migratory capacity; enhanced Src and FAK activity; enhanced Akt phosphorylation; and decreased p38 mitogen-activated protein kinase activity. Shc was not phosphorylated in response to IGF-I in cells expressing the Y1250F/Y1251F mutant and remained associated with protein phosphatase 2A. Similar alterations in signaling were observed in cells that were stimulated with IGF-I in nonadherent cultures. Our data suggest that disruption of RACK1 scaffolding function in cells expressing the Y1250F/Y1251F mutant results in the loss of adhesion signals that are necessary to regulate Akt activity and to promote turnover of focal adhesions and cell migration.     

Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin

Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc N-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this N-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications.     

Adipose-specific deletion of stearoyl-CoA desaturase 1 up-regulates the glucose transporter GLUT1 in adipose tissue

Stearoyl-CoA desaturase 1 (SCD1) deficiency protects mice from diet-induced obesity and insulin resistance. To understand the tissue-specific role of SCD1 in energy homeostasis, we have generated mice with an adipose-specific knockout of Scd1 (AKO), and report here that SCD1 deficiency increases GLUT1 expression in adipose tissue of AKO mice, but not global SCD1 knockout (GKO) mice. In 3T3-L1 adipocytes treated with an SCD inhibitor, basal glucose uptake and the cellular expression of GLUT1 were significantly increased while GLUT4 expression remained unchanged. Consistently, adipose-specific SCD1 knockout (AKO) mice had significantly elevated GLUT1 expression, but not GLUT4, in white adipose tissue compared to Lox counterparts. Concurrently, adiponectin expression was significantly diminished, whereas TNF-α expression was elevated. In contrast, in adipose tissue of GKO mice, GLUT4 and adiponectin expression were significantly elevated with lowered TNF-α expression and little change in GLUT1 expression, suggesting a differential responsiveness of adipose tissue to global- or adipose-specific SCD1 deletion. Taken together, these results indicate that adipose-specific deletion of SCD1 induces GLUT1 up-regulation in adipose tissue, associated with decreased adiponectin and increased TNF-α production, and suggest that GLUT1 may play a critical role in controlling glucose homeostasis of adipose tissue in adipose-specific SCD1-deficient conditions.     

Acute and long-term effects of insulin-like growth factor I on glucose transporters in muscle cells. Translocation and biosynthesis.

Insulin-like growth factor I (IGF-I) rapidly (less than 10 min) stimulated glucose uptake into myotubes of the L6 muscle cell line, at concentrations that act specifically on IGF-I receptors. Uptake remained stimulated at a steady level for 1-2 h, after which a second stimulation occurred. The first phase was insensitive to inhibition of protein synthesis. Subcellular fractionation demonstrated that it was accompanied by translocation of glucose transporters (both GLUT1 and GLUT4) to the plasma membrane from intracellular membranes. Translocation sufficed to explain the first phase increase in glucose transport, and there was no change in the total cellular content of GLUT1 or GLUT4 glucose transporters. The second phase of stimulation was inhibitable by cycloheximide, and involved a net increase in either GLUT1 or GLUT4 transporter content, which was reflected in an increase in transporter number in plasma membranes. These results define a cellular mechanism of metabolic action of IGF-I in muscle cells; furthermore, they suggest that IGF-I has acute metabolic effects that mimic those of insulin, bypassing action on the insulin receptor.     

Insulin and insulin-like growth factor I receptors utilize different G protein signaling components

We examined the role of heterotrimeric G protein signaling components in insulin and insulin-like growth factor I (IGF-I) action. In HIRcB cells and in 3T3L1 adipocytes, treatment with the Galpha(i) inhibitor (pertussis toxin) or microinjection of the Gbetagamma inhibitor (glutathione S-transferase-betaARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, Galpha(i) and Gbeta were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with Galpha(i) increased concomitantly with a decrease in Gbeta association. No association of Galpha(i) was found with either the insulin or EGF receptor. Microinjection of anti-beta-arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. beta-Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that Galpha(i), betagamma subunits, and beta-arrestin-1 all play a critical role in IGF-I mitogenic signaling. In contrast, neither metabolic, such as GLUT4 translocation, nor mitogenic signaling by insulin is dependent on these protein components. These results suggest that insulin receptors and IGF-IRs can function as G protein-coupled receptors and engage different G protein partners for downstream signaling.     

Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and beta1 integrin to promote cell migration

The WD repeat scaffolding protein RACK1 can mediate integration of the insulin-like growth factor I receptor (IGF-IR) and integrin signaling in transformed cells. To address the mechanism of RACK1 function, we searched for regulatory proteins that associate with RACK1 in an IGF-I-dependent manner. The serine threonine phosphatase protein phosphatase 2A (PP2A) was found associated with RACK1 in serum-starved cells, and it dissociated immediately upon stimulation with IGF-I. This dissociation of PP2A from RACK1 and an IGF-I-mediated decrease in cellular PP2A activity did not occur in cells expressing either the serine 1248 or tyrosine 1250/1251 mutants of the IGF-IR that do not interact with RACK1. Recombinant RACK1 could bind to PP2A in vitro and restore phosphatase activity to PP2A from IGF-I-stimulated cells. Ligation of integrins with fibronectin or Matrigel was sufficient to facilitate IGF-I-mediated dissociation of PP2A from RACK1 and also to recruit beta1 integrin as PP2A dissociated. By using TAT-fused N-terminal and C-terminal deletion mutants of RACK1, we determined that both PP2A and beta1 integrin interact in the C terminus of RACK1 within WD repeats 4 to 7. This suggests that integrin ligation displaces PP2A from RACK1. MCF-7 cells overexpressing RACK1 exhibited enhanced motility, which could be reversed by the PP2A inhibitor okadaic acid. Small interfering RNA-mediated suppression of RACK1 also decreased the migratory capacity of DU145 cells. Taken together, our findings indicate that RACK1 enhances IGF-I-mediated cell migration through its ability to exclusively associate with either beta1 integrin or PP2A in a complex at the IGF-IR.     

Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells

Micro- and macroangiopathy are major causes of morbidity and mortality in patients with diabetes. Our aim was to characterize IGF-I receptor (IGF-IR) and insulin receptor (IR) in human micro- and macrovascular endothelial cells. Cultured human dermal microvascular endothelial cells (HMVEC) and human aortic endothelial cells (HAEC) were used. Gene expression was measured by quantitative real-time RT-PCR and receptor protein by ligand-binding assay. Phosphorylation of IGF-IR beta-subunit was analyzed by immunoprecipitation and Western blot. Glucose metabolism and DNA synthesis was assessed using [(3)H]glucose and [(3)H]thymidine incorporation, respectively. We detected gene expression of IGF-IR and IR in HAEC and HMVEC. IGF-IR gene expression was severalfold higher than that of IR. The specific binding of (125)I-IGF-I was higher than that of (125)I-insulin in HAEC and HMVEC. Insulin and the new, long-acting insulin analog glargine interacted with the IGF-IR with thousand- and hundred-fold less potency than IGF-I itself. Phosphorylation of the IGF-IR beta-subunit was shown in HAEC for IGF-I (10(-8) M) and insulin (10(-6) M) and in HMVEC for IGF-I and glargine (10(-8) M, 10(-6) M). IGF-I 10(-7) M stimulated incorporation of [(3)H]thymidine into DNA, and 10(-9)-10(-7) M also the incorporation of [(3)H]glucose in HMVEC, whereas glargine and insulin had no significant effects at 10(-9)-10(-7) M. Human micro- and macrovascular endothelial cells express more IGF-IR than IR. IGF-I and high concentrations of glargine and insulin activates the IGF-IR. Glargine has a higher affinity than insulin for the IGF-IR but probably has no effect on DNA synthesis at concentrations reached in vivo.     

Effects of RACK1 on cell migration and IGF-I signalling in cardiomyoctes are not dependent on an association with the IGF-IR

RACK1 can act as a scaffolding protein to integrate IGF-IR and integrin signalling in transformed cells but its actions in regulating IGF-IR signalling in non-transformed cells are less well understood. Here, we investigated the function of RACK1 in the non-transformed cardiomyocyte cell line H9c2. Overexpression of RACK1 in H9c2 cells was sufficient to increase cell size, increase adhesion to collagen 1, enhance protection from hydrogen peroxide-induced cell death, and increase cell migration. However, cell proliferation was decreased in these cells. Small interfering RNA (siRNA)-mediated suppression of RACK1 in H9c2 cells resulted in decreased cell adhesion and migration, but had no effect on cell proliferation or size. Increased basal and IGF-I-mediated Erk phosphorylation was observed in RACK1-overexpressing H9c2 cells. Interestingly, contrary to observations in transformed cells, RACK1 was not observed to interact with the IGF-IR in H9c2 cells. Also in contrast to observations in transformed cells, IGF-I promoted recruitment of Src to RACK1 as well as recruitment of PKC, and PKC to RACK1. Overall, the data indicate that in H9c2 cells RACK1 can influence cell size, cell survival, adhesion, migration, but its responses to IGF-I are independent of an association with the IGF-IR. Thus, the composition of the RACK1 scaffolding complex and its effects on IGF-I signalling may be different in transformed and non-transformed cells.     

PID1 regulates insulin-dependent glucose uptake by controlling intracellular sorting of GLUT4-storage vesicles

The phosphotyrosine interacting domain-containing protein 1 (PID1) serves as a cytosolic adaptor protein of the LDL receptor-related protein 1 (LRP1). By regulating its intracellular trafficking, PID1 controls the hepatic, LRP1-dependent clearance of pro-atherogenic lipoproteins. In adipose and muscle tissues, LRP1 is present in endosomal storage vesicles containing the insulin-responsive glucose transporter 4 (GLUT4). This prompted us to investigate whether PID1 modulates GLUT4 translocation and function via its interaction with the LRP1 cytosolic domain. We initially evaluated this in primary brown adipocytes as we observed an inverse correlation between brown adipose tissue glucose uptake and expression of LRP1 and PID1. Insulin stimulation in wild type brown adipocytes induced LRP1 and GLUT4 translocation from endosomal storage vesicles to the cell surface. Loss of PID1 expression in brown adipocytes prompted LRP1 and GLUT4 sorting to the plasma membrane independent of insulin signaling. When placed on a diabetogenic high fat diet, systemic and adipocyte-specific PID1-deficient mice presented with improved hyperglycemia and glucose tolerance as well as reduced basal plasma insulin levels compared to wild type control mice. Moreover, the improvements in glucose parameters associated with increased glucose uptake in adipose and muscle tissues from PID1-deficient mice. The data provide evidence that PID1 serves as an insulin-regulated retention adaptor protein controlling translocation of LRP1 in conjunction with GLUT4 to the plasma membrane of adipocytes. Notably, loss of PID1 corrects for insulin resistance-associated hyperglycemia emphasizing its pivotal role and therapeutic potential in the regulation of glucose homeostasis.     

GLUT1-mediated glucose transport and its regulation by IGF-I in cultured bovine chromaffin cells

Regulation of glucose transport was studied in primary cultures of bovine chromaffin cells (BCC) using the glucose analogue 2-deoxyglucose (DOG) as a model substrate. The glucose transporter in freshly isolated and cultured BCC was identified as GLUT1 by Western immunoblots. The level of GLUT1 increased by time in culture and was followed by an enhancement in uptake of DOG. The DOG uptake was stimulated by insulin-like growth factor I (IGF-I) with an EC₅₀ of 1 nM and a maximal response (∼2-fold) was obtained at 10–100 nM IGF-I. Insulin was at least 100-fold less potent than IGF-I. Exposure to 10⁻⁸ M IGF-I also caused a redistribution of GLUT1 from an intracellular compartment to a plasma membrane-enriched fraction. Our results demonstrate a GLUT1-mediated glucose uptake in adrenomedullary cells. An enhanced glucose transport in response to IGF-I appears to be coupled to activation of IGF receptor type 1 and GLUT1 translocation. © 1996 Wiley-Liss, Inc.     

MKR mice are resistant to the metabolic actions of both insulin and adiponectin: discordance between insulin resistance and adiponectin responsiveness

Most rodent models of insulin resistance are accompanied by decreased circulating adiponectin levels. Adiponectin treatment improves the metabolic phenotype by increasing fatty acid oxidation in skeletal muscle and suppressing hepatic glucose production. Muscle IGF-I receptor (IGF-IR)-lysine-arginine (MKR) mice expressing dominant-negative mutant IGF-IRs in skeletal muscle are diabetic with insulin resistance in muscle, liver, and adipose tissue. Adiponectin levels are elevated in MKR mice, suggesting an unusual discordance between insulin resistance and adiponectin responsiveness. Therefore, we investigated the metabolic actions of adiponectin in MKR mice. MKR and ob/ob mice were treated both acutely (28 microg/g) and chronically (for 2 wk) with full-length adiponectin. Acute hypoglycemic effects of adiponectin were evident only in ob/ob mice but not in MKR mice. Chronic adiponectin treatment significantly improved both insulin sensitivity and glucose tolerance in ob/ob but not in MKR mice. Adiponectin receptor mRNA levels and adiponectin-stimulated phosphorylation of AMPK in skeletal muscle and liver were similar among MKR, wild-type, and ob/ob mice. Thus MKR mice are adiponectin resistant despite normal expression of adiponectin receptors and normal AMPK phosphorylation in muscle and liver. MKR mice may be a useful model for dissecting relationships between insulin resistance and adiponectin action in regulation of glucose homeostasis.     

Decorin differentially modulates the activity of insulin receptor isoform A ligands

The proteoglycan decorin, a key component of the tumor stroma, regulates the action of several tyrosine-kinase receptors, including the EGFR, Met and the IGF-IR. Notably, the action of decorin in regulating the IGF-I system differs between normal and transformed cells. In normal cells, decorin binds with high affinity to both the natural ligand IGF-I and the IGF-I receptor (IGF-IR) and positively regulates IGF-IR activation and downstream signaling. In contrast, in transformed cells, decorin negatively regulates ligand-induced IGF-IR activation, downstream signaling and IGF-IR-dependent biological responses. Whether decorin may bind another member of the IGF-I system, the insulin receptor A isoform (IR-A) and its cognate ligands, insulin, IGF-II and proinsulin, have not been established. Here we show that decorin bound with high affinity insulin and IGF-II and, to a lesser extent, proinsulin and IR-A. We utilized as a cell model system mouse embryonic fibroblasts homozygous for a targeted disruption of the Igf1r gene (designated R⁻ cells) which were stably transfected with a human construct harboring the IR-A isoform of the receptor. Using these R⁻/IR-A cells, we demonstrate that decorin did not affect ligand-induced phosphorylation of the IR-A but enhanced IR-A downregulation after prolonged IGF-II stimulation without affecting insulin and proinsulin-dependent effects on IR-A stability. In addition, decorin significantly inhibited IGF-II-mediated activation of the Akt pathways, without affecting insulin and proinsulin-dependent signaling. Notably, decorin significantly inhibited IGF-II-mediated cell proliferation of R⁻/IR-A cells but affected neither insulin- nor proinsulin-dependent mitogenesis. Collectively, these results suggest that decorin differentially regulates the action of IR-A ligands. Decorin preferentially inhibits IGF-II-mediated biological responses but does not affect insulin- or proinsulin-dependent signaling. Thus, decorin loss may contribute to tumor initiation and progression in malignant neoplasms which depend on an IGF-II/IR-A autocrine loop.     

The signaling potential of the receptors for insulin and insulin-like growth factor I (IGF-I) in 3T3-L1 adipocytes: comparison of glucose transport activity, induction of oncogene c-fos, glucose transporter mRNA, and DNA-synthesis.

The receptors for insulin and insulin-like growth factor I (IGF-I) have in common a high sequence homology and diverse overlapping functions, (e.g., the stimulation of acute metabolic events and the induction of cell growth.). In the present study, we have compared the potential of insulin and IGF-I receptors in stimulating glucose transport activity, glucose transporter gene expression, DNA-synthesis, and expression of proto-oncogene c-fos in 3T3-L1 adipocytes which express high levels of both receptors. Binding of both hormones to their own receptors was highly specific as compared with binding to the respective other receptor (insulin receptor: KD = 3.6 nM, KI of IGF-I greater than 500 nM; IGF-I receptor, KD = 1.1 nM, KI of insulin = 191 nM). Induction of proto-oncogene c-fos mRNA by insulin and IGF-I paralleled their respective receptor occupancy and was thus induced by both hormones via their own receptor (EC50 of insulin, 3.7; IGF-I, 3.9 nM). Similarly, both insulin and IGF-I increased DNA synthesis (EC50 of insulin, 5.8 nM; IGF-I, 4.0 nM), glucose transport activity (EC50 of insulin, 1.7 nM; IGF-I, 1.4 nM), and glucose transporter (GLUT4) mRNA levels in concentrations corresponding with their respective receptor occupancy. These data indicate that in 3T3-L1 cells the alpha-subunits of insulin and IGF-I receptors have an equal potential to stimulate a metabolic and a mitogenic response.     

Effects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.     

Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and beta1 integrin and for tumor cell proliferation and migration

Insulin-like growth factor (IGF)-I regulates a mutually exclusive interaction of PP2A and beta1 integrin with the WD repeat scaffolding protein RACK1. This interaction is required for the integration of IGF-I receptor (IGF-IR) and adhesion signaling. Here we investigated the nature of the binding site for PP2A and beta1 integrin in RACK1. A WD7 deletion mutant of RACK1 did not associate with PP2A but retained some interaction with beta1 integrin, whereas a WD6/WD7 mutant lost the ability to bind to both PP2A and beta1 integrin. Using immobilized peptide arrays representing the entire RACK1 protein, we identified a common cluster of amino acids (FAGY) at positions 299-302 within WD7 of RACK1 which were essential for binding of both PP2A and beta1 integrin to RACK1. PP2A showed a higher level of association with a peptide in which Tyr-302 was phosphorylated compared with an unphosphorylated peptide, whereas beta1 integrin binding was not affected by phosphorylation. RACK1 mutants in which either the FAGY cluster or Tyr-302 were mutated to AAAF, or Phe, respectively, did not interact with either PP2A or beta1 integrin. These mutants were unable to rescue the decrease in PP2A activity caused by suppression of RACK1 in MCF-7 cells with small interfering RNA. MCF-7 cells and R+ (IGF-IR-overexpressing fibroblasts) expressing these mutants exhibited decreased proliferation and migration, whereas R- cells (IGF-IR null fibroblasts) were unaffected. Taken together, the data demonstrate that Tyr-302 in RACK1 is required for interaction with PP2A and beta1 integrin, for regulation of PP2A activity, and for IGF-I-mediated cell migration and proliferation.