Search for type 2 diabetes susceptibility genes on chromosomes 1q, 3q and 12q.

To systematically evaluate genetic susceptibility to type 2 diabetes (T2D) in "candidate" regions on chromosomes 1q, 3q and 12q, we examined disease association by using a total of 2,083 SNPs in two-step screening; a screening panel comprised 473 cases and 285 controls and an extended (or combined) panel involved 658 cases and 474 controls. For the total interval screened (40.9 Mb), suggestive evidence of association was provided for several annotated gene loci. For example, in the MCF2L2 gene on 3q, a significant association (a nominal P value of 0.00009) was observed when logistic regression analysis was performed for three associated SNPs (rs684846, rs35069869 and rs35368790) that belonged to different LD groups. Also, in the SLC15A4 gene on 12q, rs3765108 showed a marginally significant association with an overall estimated odds ratio of 0.79 (P=0.001). No significant association was found for known candidate gene loci on 3q, such as ADIPOQ and IGF2BP2. Using the available samples, we have observed disease associations of SNPs derived from two novel gene loci in the Japanese population through high-density searches of diabetes susceptibility in three chromosomal regions. Independent replication will clarify the etiological relevance of these genomic loci to T2D.

Search of type 2 diabetes susceptibility gene on chromosome 20q.

Significant evidence of linkage to type 2 diabetes (T2D) has been shown in a relatively broad region on chromosome 20q, where the hepatocyte nuclear factor-4alpha (HNF4A) has been noted as a positional candidate. To systematically evaluate genetic susceptibility to T2D in the relevant region, we examined the disease association by using 1145 SNPs in two-step screening in the Japanese population. The marker screening enabled us to identify significant disease association in the lipopolysaccharide binding protein (LBP) but not in the HNF4A locus. In a 17.7-Mb interval screened, the strongest association was identified for a SNP, rs2232592, located in the intron of LBP, with an estimated odds ratio of 1.73 (95% CI 1.30-2.31) (P=0.0002) in the whole study panel involving 675 case and 474 control subjects. Our data suggest that the LBP gene may confer genetic susceptibility to T2D and this warrants further replication study.

Enhanced production of CC-chemokines (RANTES, MCP-1, MIP-1alpha, MIP-1beta, and eotaxin) in patients with atopic dermatitis.

CC-chemokines are potent molecules that direct the migration of leukocytes to inflammatory foci. To determine their role in inflammation associated with atopic dermatitis (AD), we determined serum levels and spontaneous production of CC-chemokines by peripheral blood mononuclear cells (PBMC) in AD patients using an ELISA. Serum levels of RANTES, MCP-1, MIP-1beta, and eotaxin were increased in AD patients (n = 52) compared with normal controls (n = 22). Serum levels of RANTES, MCP-1, and MIP-1beta were increased in AD patients with severe disease (n = 19) compared with normal controls (n = 22). Spontaneous production of RANTES, MCP-1, MIP-1alpha and MIP-1beta by PBMC was augmented in AD patients (n = 39) and in patients with severe AD (n = 14) compared with normal controls (n = 20). Serum RANTES levels correlated with total serum IgE levels, eosinophil numbers, and serum lactate dehydrogenase levels. Our results suggest that augmented production of CC-chemokines correlates with inflammation associated with AD.

Insulin-independent and wortmannin-resistant targeting of IRS-3 to the plasma membrane via its pleckstrin homology domain mediates a different interaction with the insulin receptor from that of IRS-1.

AIMS/HYPOTHESIS: In primary adipocytes, although IRS-1 and IRS-3 are expressed in comparable amounts, these proteins manifest distinct distribution and significance in insulin signalling. We investigated the molecular basis of the difference between these two proteins. METHODS: In Cos-1 cells transiently expressing rat IRS-1, IRS-3, or chimeric proteins of these two proteins we examined the tyrosine phosphorylation via the wild-type or mutant insulin receptors and evaluated their targeting to the plasma membrane by immunostaining the membrane ghost. RESULTS: In contrast to IRS-1, IRS-3 was tyrosine-phosphorylated by the insulin receptor altering Tyr960 to Phe (Y960F), which disrupts the binding site of the PTB domain of IRSs, to an extent comparable to the wild-type receptor. The tyrosine phosphorylation of IRS-3 with the PH domain replacement via the Y960F insulin receptor markedly decreased, whereas that of IRS-3 with the PTB domain alteration was mildly impaired. Insulin-stimulated translocation of IRS-1 to the plasma membrane, as well as that of IRS-3 with the PH domain replacement, was wortmannin-sensitive, although that of IRS-3 was insulin-independent and wortmannin-resistant. CONCLUSIONS/INTERPRETATION: The affinity of the PH domain for the phospholipids in the plasma membrane seems to influence the receptor-substrate interaction required for IRS tyrosine phosphorylation, indicating that the PH domain and the PTB domain of IRSs cooperatively function in insulin-stimulated tyrosine phosphorylation of these proteins.

Subcellular localization of insulin receptor substrate family proteins associated with phosphatidylinositol 3-kinase activity and alterations in lipolysis in primary mouse adipocytes from IRS-1 null mice.

To clarify the roles of insulin receptor substrate (IRS) family proteins in phosphatidylinositol (PI) 3-kinase activation and insulin actions in adipocytes, we investigated the intracellular localization of IRS family proteins and PI 3-kinase activation in response to insulin by fractionation of mouse adipocytes from wild-type and IRS-1 null mice. In adipocytes from wild-type mice, tyrosine-phosphorylated IRS-1 and IRS-2, which were found to associate with PI 3-kinase in response to insulin, were detected in the plasma membrane (PM) and low-density microsome (LDM) fractions. By contrast, tyrosine-phosphorylated IRS-3 (pp60), which was found to associate with PI 3-kinase, was predominantly localized in the PM fraction. In adipocytes from IRS-1-null mice, insulin-stimulated PI 3-kinase activity in anti-phosphotyrosine (alphaPY) immunoprecipitates in the LDM fraction was almost exclusively mediated via IRS-2 and was reduced to 25%; however, insulin-stimulated PI 3-kinase activity in the PM fraction was primarily mediated via IRS-3 and was reduced to 60%. To determine the potential functional impact of the distinct subcellular localization of IRSs and associating PI 3-kinase activity on adipocyte-specific metabolic actions, we examined lipolysis in IRS-1 null mice. The level of isoproterenol-induced lipolysis was increased 5.1-fold in adipocytes from IRS-1 null mice as compared with wild-type mice. Moreover, hormone-sensitive lipase (HSL) protein was increased 4.3-fold in adipocytes from IRS-1-null mice compared with wild-type mice, and HSL mRNA expression was also increased. The antilipolytic effect of insulin in IRS-1 null adipocytes, however, was comparable to that in wild-type mice. Thus, discordance between these two insulin actions as well as the transcriptional and translational effect (HSL mRNA and protein regulation) and the PM effect (antilipolysis) of insulin may be explained by distinct roles of both PI 3-kinase activity associated with IRS-1/IRS-2 and PI 3-kinase activity associated with IRS-3 in insulin actions related to their subcellular localization.

Essential role of insulin receptor substrate 1 (IRS-1) and IRS-2 in adipocyte differentiation.

To investigate the role of insulin receptor substrate 1 (IRS-1) and IRS-2, the two ubiquitously expressed IRS proteins, in adipocyte differentiation, we established embryonic fibroblast cells with four different genotypes, i.e., wild-type, IRS-1 deficient (IRS-1(-/-)), IRS-2 deficient (IRS-2(-/-)), and IRS-1 IRS-2 double deficient (IRS-1(-/-) IRS-2(-/-)), from mouse embryos of the corresponding genotypes. The abilities of IRS-1(-/-) cells and IRS-2(-/-) cells to differentiate into adipocytes are approximately 60 and 15%, respectively, lower than that of wild-type cells, at day 8 after induction and, surprisingly, IRS-1(-/-) IRS-2(-/-) cells have no ability to differentiate into adipocytes. The expression of CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) is severely decreased in IRS-1(-/-) IRS-2(-/-) cells at both the mRNA and the protein level, and the mRNAs of lipoprotein lipase and adipocyte fatty acid binding protein are severely decreased in IRS-1(-/-) IRS-2(-/-) cells. Phosphatidylinositol 3-kinase (PI 3-kinase) activity that increases during adipocyte differentiation is almost completely abolished in IRS-1(-/-) IRS-2(-/-) cells. Treatment of wild-type cells with a PI 3-kinase inhibitor, LY294002, markedly decreases the expression of C/EBPalpha and PPARgamma, a result which is associated with a complete block of adipocyte differentiation. Moreover, histologic analysis of IRS-1(-/-) IRS-2(-/-) double-knockout mice 8 h after birth reveals severe reduction in white adipose tissue mass. Our results suggest that IRS-1 and IRS-2 play a crucial role in the upregulation of the C/EBPalpha and PPARgamma expression and adipocyte differentiation.

CCR4 memory CD4+ T lymphocytes are increased in peripheral blood and lesional skin from patients with atopic dermatitis.

BACKGROUND: Recent studies have reported that TH1 and TH2 cells express CXCR3 and CCR4, respectively. OBJECTIVE: Our goal was to assess the association of CCR4 and CXCR3 expression with TH2 and TH1 cells and association of CCR4 and CXCR3 expression with inflammation in patients with atopic dermatitis (AD). METHODS: Intracellular cytokine production and chemokine receptor expression in blood T cells were examined by flow cytometry. Immunohistochemical expression of chemokine receptors was also investigated in chronically lesional skin. RESULTS: CCR4+ and CXCR3+ CD4+ T cells predominantly produced IL-4 and IFN-gamma, respectively. Although the frequency of CXCR3+ cells among CD4+ CD45RO+ T cells was similar for patients with AD (n = 29) and healthy control subjects (n = 19), patients with severe AD (n = 14) had a reduced frequency of CXCR3+ cells. In contrast, the frequency of CCR4+ cells and the CCR4/CXCR3 ratio were higher in patients with AD (n = 22) than healthy control subjects (n = 16) and correlated with disease severity of AD. The frequency of CCR4+ cells correlated positively with eosinophil numbers and serum IgE levels, whereas the frequency of CXCR3+ cells correlated inversely with eosinophil numbers. The frequency of CCR4+ or CXCR3+ cells was similar in patients with psoriasis (n = 6) and healthy control subjects. Immunohistochemical analysis showed that the frequency of CCR4+ cells among CD4+ T cells in chronically lesional skin of patients with AD (n = 9) was higher than that of patients with psoriasis (n = 4). CONCLUSION: Our data suggest the association of CCR4 expression with TH2 cells, the predominance of CCR4+ cells in blood from patients with AD, and an important role of CCR4 in the migration of TH2 cells from blood into AD lesional skin.

Leukocytoclasis: ultrastructural in situ nick end labeling study in anaphylactoid purpura.

In order to characterize leukocytoclasis of polymorphonuclear neutrophils (PMNs), the method of in situ nick end labeling for DNA breakdown was applied on tissue samples from 36 patients with anaphylactoid purpura at ultrastructural, as well as light microscopic, level. Light microscopic immuno-peroxidase technique showed positively labeled PMNs infiltrating in the dermis of 24 cases in which leukocytoclastic vasculitis was fully developed, suggesting that breakdown of DNA strands is triggered in the PMNs. Electron microscopic immuno-gold technique employed in six patients with the fully developed stage of inflammation identified the DNA breaks in the nuclei of PMNs. Ultrastructure of these cells, however, showed that only a minor population ( approximately 1/60) of PMNs showed the condensed and marginated nuclei, being compatible with typical apoptotic change. However, the majority of immuno-gold-labeled cells showed relatively intact nuclei without margination of condensed heterochromatin and with disintegrated cytoplasmic organelles and plasma membrane, suggesting that apoptotic cell removal mechanism may be incomplete. The immuno-gold-positive nuclear debris scattering in the tissue is most likely the remnants of unsatisfactory disposal by apoptosis of potentially injurious PMNs, resulting in the vascular and perivascular damage in leukocytoclastic vasculitis in anaphylactoid purpura.

Overexpression of the glycogen targeting (G(M)) subunit of protein phosphatase-1.

The G(M) glycogen-targeting subunit of protein phosphatase-1 (PP1) is believed to be involved in dephosphorylation of the enzymes of glycogen metabolism. To assess the roles of G(M) on glycogen metabolism, we created site-directed G(M) mutants and overexpressed them in Chinese hamster ovary (CHO) cells expressing human insulin receptor. Overexpressed G(M) recruited glycogen synthase as well as PP1 to the glycogen pellet, and upregulated basal glycogen synthase activity. Overexpressed G(M)-67A (Ser-67 replaced with alanine) exhibited decreased sensitivity to suppression of glycogen synthase activity by forskolin, while overexpression of G(M)-48A (Ser-48 replaced with alanine) preserved glycogen synthase activation in response to insulin. These observations indicate that in CHO cells overexpressing G(M); (1) G(M) translocates glycogen synthase to the glycogen pellet and affected basal glycogen synthase, (2) Ser-67 might be involved in the suppression of glycogen synthase activity by glycogenolytic agents, and (3) Ser-48 might not commit to activation of glycogen synthase by insulin.

Delayed wound healing in the absence of intercellular adhesion molecule-1 or L-selectin expression.

Inflammatory cells play a crucial role in wound healing, but the role of adhesion molecules including L-selectin and intercellular adhesion molecule-1 (ICAM-1) is not known in this process. We examined skin wound repair of excisional wounds in mice lacking L-selectin, ICAM-1, or both. The loss of ICAM-1 inhibited wound healing, keratinocyte migration from the edges of the wound toward the center, and granulation tissue formation. By contrast, L-selectin deficiency alone did not affect any of these parameters. However, the loss of both L-selectin and ICAM-1 resulted in inhibition of keratinocyte migration and granulation tissue formation beyond those caused by loss of ICAM-1 alone. Treatment of platelet-derived growth factor to the wounds normalized delayed wound healing in ICAM-1(-/-) mice, but not in L-selectin/ICAM-1(-/-) mice. Therefore, although ICAM-1 contributes to wound repair to a greater extent than L-selectin, a role for L-selectin was revealed in the absence of ICAM-1. The impaired wound repair was associated with reduced infiltration of neutrophils and macrophages in ICAM-1(-/-) and L-selectin/ICAM-1(-/-) mice. These results demonstrate a distinct role of ICAM-1 and L-selectin in wound healing and that the delayed wound healing in the absence of these molecules is likely because of decreased leukocyte accumulation into the wound site.

Restored insulin-sensitivity in IRS-1-deficient mice treated by adenovirus-mediated gene therapy.

Insulin resistance is commonly observed both in overt diabetes and in individuals prone to, but not yet manifesting, diabetes. Hence the maintenance or restoration of insulin sensitivity may prevent the onset of this disease. We previously showed that homozygous disruption of insulin receptor substrate-1 (IRS-1) in mice resulted in insulin resistance but not diabetes. Here, we have explored the mechanism of systemic insulin resistance in these mice and used adenovirus-mediated gene therapy to restore their insulin sensitivity. Mice expressing the IRS-1transgene showed almost normal insulin sensitivity. Expression of an IRS-1 mutant (IRS-1Deltap85) lacking the binding site for the p85 subunit of phosphatidylinositol 3-kinase (PI3K) also restored insulin sensitivity, although PI3K is known to play a crucial role in insulin's metabolic responses. Protein kinase B (PKB) activity in liver was decreased in null mice compared with the wild-type and the null mice expressing IRS-1 or IRS-1Deltap85. In primary hepatocytes isolated from null mice, expression of IRS-1 enhanced both PI3K and PKB activities, but expression of IRS-1Deltap85 enhanced only PKB. These data suggest that PKB in liver plays a pivotal role in systemic glucose homeostasis and that PKB activation might be sufficient for reducing insulin resistance even without full activation of PI3K.

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.

The mechanism of insulin-induced signal transduction mediated by the insulin receptor substrate family.

Distinct from other growth factor receptors, insulin and insulin-like growth factor-I (IGF-I) receptors phosphorylate endogenous substrates on tyrosine residues which in turn associate with the SH2 domain-containing proteins transducing signals to downstream pathways. Among the cellular substrates of insulin and IGF-I receptors, insulin receptor substrate (IRS)-1 has been shown to play an important role in mediating the actions of these hormones. Recently, several proteins with similar structures and different tissue distributions were cloned as IRS-2, -3 and -4. To study the roles of these IRSs in mediating insulin actions, we analyzed liver, muscle and adipocytes, the major targets of insulin actions, from IRS-1 null mice which we previously generated, and showed that: 1) insulin-stimulated activation of PI 3-kinase, mitogen-activated protein kinase and glucose transport were impaired in muscles from IRS-1 null mice which was in contrast to the grossly normal signaling and actions in livers from these mice; 2) the difference in the degree of insulin resistance in these two major insulin targets appeared to depend on the amount of tyrosine phosphorylation of IRS-2 compensating for IRS-1 deficiency; 3) insulin-induced activation of PI 3-kinase, glucose transport and GLUT4 translocation were impaired but not abolished in adipocytes from these mice in which IRS-3 was the major tyrosine-phosphorylated protein activating PI 3-kinase and at least partially mediating some residual insulin actions in the absence of IRS-1. These data suggest that the members of the IRS family redundantly regulate insulin actions in each target organ in a distinct fashion.

Pigmented dermatofibrosarcoma protuberans (Bednár tumor) occurring in a Japanese infant.

A case of pigmented dermatofibrosarcoma protuberans (Bednár tumor) occurring in a Japanese infant is reported. A 6-month-old girl developed a 16 x 10 mm erythematous tumor with a slight elevation on the lower back at 1 month of age. Histological examination revealed proliferation of spindle-shaped, fibroblast-like cells in the myxoid interstitium. Subsequently the tumor grew gradually to a red-purplish tumor measuring 45 x 36 mm. The second biopsy specimen presented hypercellular proliferation composed of spindle-shaped cells showing slight nuclear atypia and a characteristic storiform pattern, with scattered slender dendritic cells containing a large amount of brown pigment. Immunohistochemical studies of the second biopsy specimen showed that the spindle-shaped tumor cells were positive for vimentin and CD34 and negative for factor XIIIa. The number of CD34-reactive spindle-shaped tumor cells increased in the second biopsy specimen compared with the first biopsy.

Growth hormone and prolactin stimulate tyrosine phosphorylation of insulin receptor substrate-1, -2, and -3, their association with p85 phosphatidylinositol 3-kinase (PI3-kinase), and concomitantly PI3-kinase activation via JAK2 kinase.

Growth hormone (GH) and prolactin (PRL) binding to their receptors, which belong to the cytokine receptor superfamily, activate Janus kinase (JAK) 2 tyrosine kinase, thereby leading to their biological actions. We recently showed that GH mainly stimulated tyrosine phosphorylation of epidermal growth factor receptor and its association with Grb2, and concomitantly stimulated mitogen-activated protein kinase activity in liver, a major target tissue. Using specific antibodies, we now show that GH was also able to induce tyrosine phosphorylation of insulin receptor substrate (IRS)-1/IRS-2 in liver. In addition, the major tyrosine-phosphorylated protein in anti-p85 phosphatidylinositol 3-kinase (PI3-kinase) immunoprecipitate from liver of wild-type mice was IRS-1, and IRS-2 in IRS-1 deficient mice, but not epidermal growth factor receptor. These data suggest that tyrosine phosphorylation of IRS-1 may be a major mechanism for GH-induced PI3-kinase activation in physiological target organ of GH, liver. We also show that PRL was able to induce tyrosine phosphorylation of both IRS-1 and IRS-2 in COS cells transiently transfected with PRLR and in CHO-PRLR cells. Moreover, we show that tyrosine phosphorylation of IRS-3 was induced by both GH and PRL in COS cells transiently transfected with IRS-3 and their cognate receptors. By using the JAK2-deficient cell lines or by expressing a dominant negative JAK2 mutant, we show that JAK2 is required for the GH- and PRL-dependent tyrosine phosphorylation of IRS-1, -2, and -3. Finally, a specific PI3-kinase inhibitor, wortmannin, completely blocked the anti-lipolytic effect of GH in 3T3 L1 adipocytes. Taken together, the role of IRS-1, -2, and -3 in GH and PRL signalings appears to be phosphorylated by JAK2, thereby providing docking sites for p85 PI3-kinase and activating PI3-kinase and its downstream biological effects.

Potential role of protein kinase B in insulin-induced glucose transport, glycogen synthesis, and protein synthesis.

Various biological responses stimulated by insulin have been thought to be regulated by phosphatidylinositol 3-kinase, including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between phosphatidylinositol 3-kinase and these biological responses has been poorly understood. Recently, it has been shown that protein kinase B (PKB/c-Akt/Rac) lies immediately downstream from phosphatidylinositol 3-kinase. Here, we show that expression of a constitutively active form of PKB induced glucose uptake, glycogen synthesis, and protein synthesis in L6 myotubes downstream of phosphatidylinositol 3-kinase and independent of Ras and mitogen-activated protein kinase activation. Introduction of constitutively active PKB induced glucose uptake and protein synthesis but not glycogen synthesis in 3T3L-1 adipocytes, which lack expression of glycogen synthase kinase 3 different from L6 myotubes. Furthermore, we show that deactivation of glycogen synthase kinase 3 and activation of rapamycin-sensitive serine/threonine kinase by PKB in L6 myotubes might be involved in the enhancement of glycogen synthesis and protein synthesis, respectively. These results suggest that PKB acts as a key enzyme linking phosphatidylinositol 3-kinase activation to multiple biological functions of insulin through regulation of downstream kinases in skeletal muscle, a major target tissue of insulin.

Role of insulin receptor substrate-1 and pp60 in the regulation of insulin-induced glucose transport and GLUT4 translocation in primary adipocytes.

In muscle and fat, glucose transport occurs through the translocation of GLUT4 from an intracellular pool to the cell surface. Phosphatidylinositol (PI) 3-kinase has been shown to be required in this process. Insulin is thought to activate this enzyme by stimulating its association with tyrosine-phosphorylated proteins such as insulin receptor substrate (IRS)-1, IRS-2, Grb2-associated binder-1, and pp60. To study the role of these endogenous substrates in glucose transport, we analyzed adipocytes from IRS-1 null mice that we previously generated (Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y. , Ikawa, Y., Kasuga, M., Yazaki Y., and Aizawa S. (1994) Nature 372, 182-186). In adipocytes from these mice, we showed that: 1) insulin-induced PI 3-kinase activity in the antiphosphotyrosine immunoprecipitates was 54% of wild-type; 2) pp60 was the major tyrosine-phosphorylated protein that associated with PI 3-kinase, whereas tyrosine phosphorylaion of IRS-2 as well as its association with this enzyme was almost undetectable; and 3) glucose transport and GLUT4 translocation at maximal insulin stimulation were decreased to 52 and 68% of those from wild-type. These data suggest that both IRS-1 and pp60 play a major role in insulin-induced glucose transport in adipocytes, and that pp60 is predominantly involved in regulating this process in the absence of IRS-1.