Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

A case of insulin allergy successfully managed using multihexamer-forming insulin degludec combined with liraglutide.

BACKGROUND: Insulin allergy, one of insulin's adverse effects, is rare, especially in patients with Type 2 diabetes, but management is difficult and no effective strategy has yet been established. We experienced an insulin allergy case successfully managed with a novel combination of insulins. CASE REPORT: A 38-year-old woman started insulin therapy when diabetes was diagnosed at age 19 years. Despite poorly controlled diabetes because of poor adherence, she hoped to conceive a child and continuous subcutaneous insulin infusion was introduced using insulin aspart at age 32 years. One month thereafter, she developed skin reactions at the subcutaneous insulin infusion catheter insertion site. The patient was then tested for all rapid-acting insulin formulations, all of which triggered local reactions. She decided to continue the continuous subcutaneous infusion of human regular insulin, accompanied by oral cetirizine hydrochloride and betamethasone valerate ointment. The patient was admitted to our hospital at age 38 years with high HbA1c levels. She was tested for all long-acting insulin analogues. All results, except for insulin degludec, were positive. She discontinued continuous subcutaneous insulin infusion and switched to insulin degludec combined with liraglutide. The allergic reactions had completely disappeared and her blood glucose was well controlled by the time of discharge. CONCLUSION: Our patient was allergic to all insulin formulations except insulin degludec. Her allergic reactions completely disappeared after switching to insulin degludec. The crystallized structure of this insulin might mask its skin allergen antigenicity. Furthermore, her postprandial hyperglycaemia was successfully controlled with liraglutide. We propose multihexamer-forming ultra-long-acting insulin plus glucagon-like peptide-1 analogues as a therapeutic option for patients with insulin allergy.

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.

Monocyte chemoattractant protein-1 (MCP-1) deficiency enhances alternatively activated M2 macrophages and ameliorates insulin resistance and fatty liver in lipoatrophic diabetic A-ZIP transgenic mice.

AIMS/HYPOTHESIS: Monocyte chemoattractant protein-1 (MCP-1)/chemokine (C-C motif) ligand (CCL) 2 (CCL2) secreted from white adipose tissue (WAT) in obesity has been reported to contribute to tissue macrophage accumulation and insulin resistance by inducing a chronic inflammatory state. MCP-1 has been shown to be elevated in the fatty liver of lipoatrophic A-ZIP-transgenic (A-ZIP-Tg) mice. Treatment of these mice with the CC chemokine receptor (CCR) 2 antagonist has been shown to ameliorate the hyperglycaemia, hyperinsulinaemia and hepatomegaly, in conjunction with reducing liver inflammation. However, since CCR2 antagonists can block not only MCP-1 but also MCP-2 (CCL8) and MCP-3 (CCL7), it remains unclear whether MCP-1 secreted from the liver could contribute to hyperglycaemia, hyperinsulinaemia and hepatomegaly in conjunction with liver inflammation, as well as to the M1 and M2 states of macrophage polarisation. METHODS: To address these issues, we analysed the effects of targeted disruption of MCP-1 in A-ZIP-Tg mice. RESULTS: MCP-1 deficiency alone or per se resulted in a significant amelioration of insulin resistance in A-ZIP-Tg mice, which was associated with a suppression of extracellular signal-regulated protein kinase (ERK)-1/2 and p38 mitogen-activated protein kinase (p38MAPK) phosphorylation in liver. Although MCP-1 deficiency did not reduce the expression of macrophage markers, it increased the expression of the genes encoding M2 macrophage markers such as Arg1 and Chi3l3, as well as significantly reducing the triacylglycerol content of livers from A-ZIP-Tg mice. CONCLUSIONS/ INTERPRETATION: Our data clearly indicated that MCP-1 deficiency improved insulin resistance and hepatic steatosis in A-ZIP-Tg mice and was associated with switching macrophage polarisation and suppressing ERK-1/2 and p38MAPK phosphorylation.

Depletion of homeodomain-interacting protein kinase 3 impairs insulin secretion and glucose tolerance in mice.

AIMS/HYPOTHESIS: Insufficient insulin secretion and reduced pancreatic beta cell mass are hallmarks of type 2 diabetes. Here, we focused on a family of serine-threonine kinases known as homeodomain-interacting protein kinases (HIPKs). HIPKs are implicated in the modulation of Wnt signalling, which plays a crucial role in transcriptional activity, and in pancreas development and maintenance. The aim of the present study was to characterise the role of HIPKs in glucose metabolism. METHODS: We used RNA interference to characterise the role of HIPKs in regulating insulin secretion and transcription activity. We conducted RT-PCR and western blot analyses to analyse the expression and abundance of HIPK genes and proteins in the islets of high-fat diet-fed mice. Glucose-induced insulin secretion and beta cell proliferation were measured in islets from Hipk3 ( -/- ) mice, which have impaired glucose tolerance owing to an insulin secretion deficiency. The abundance of pancreatic duodenal homeobox (PDX)-1 and glycogen synthase kinase (GSK)-3ß phosphorylation in Hipk3 ( -/- ) islets was determined by immunohistology and western blot analyses. RESULTS: We found that HIPKs regulate insulin secretion and transcription activity. Hipk3 expression was most significantly increased in the islets of high-fat diet-fed mice. Furthermore, glucose-induced insulin secretion and beta cell proliferation were decreased in the islets of Hipk3 ( -/- ) mice. Levels of PDX1 and GSK-3ß phosphorylation were significantly decreased in Hipk3 ( -/- ) islets. CONCLUSIONS/INTERPRETATION: Depletion of HIPK3 impairs insulin secretion and glucose tolerance. Decreased levels of HIPK3 may play a substantial role in the pathogenesis of type 2 diabetes.

Influence of lingual plate fracture pattern on remodelling site during the healing process of sagittal split ramus osteotomy.

The purpose of this study was to investigate whether differences in the pattern of the lingual plate split in sagittal split ramus osteotomy (SSRO) affect the remodelling of the split site. Sixty-one patients with mandibular prognathism (122 sides) underwent SSRO. Computed tomography (CT) was performed at 1 week and 1 year after SSRO. Bone splits were classified according to the lingual split scale (LSS) and the lateral bone cut end (LBCE) by evaluating CT images at 1 week. The remodelling at the split sites was evaluated by superimposing the CT images obtained at 1 week and 1 year. Regarding the LSS pattern, significant differences were observed in the distance between anteroposterior ramus points (P = 0.033) and the ramus area in the axial image (P = 0.011). The LBCE pattern also showed a significant difference in the distance between anteroposterior ramus points (P = 0.043). In conclusion, the differences in the lingual plate split and ramus cut end of the SSRO influence the postoperative remodelling in the anteroposterior region of the split site.

Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase.

Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. However, the signaling pathways that mediate the metabolic effects of Ad remain poorly identified. Here we show that phosphorylation and activation of the 5'-AMP-activated protein kinase (AMPK) are stimulated with globular and full-length Ad in skeletal muscle and only with full-length Ad in the liver. In parallel with its activation of AMPK, Ad stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid oxidation, glucose uptake and lactate production in myocytes, phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. Blocking AMPK activation by dominant-negative mutant inhibits each of these effects, indicating that stimulation of glucose utilization and fatty-acid oxidation by Ad occurs through activation of AMPK. Our data may provide a novel paradigm that an adipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directly regulating glucose metabolism and insulin sensitivity in vitro and in vivo.

Antibiotic prophylaxis for sagittal split ramus osteotomy using resorbable plate and screw fixation: a randomised trial to compare extended dual-agent and inpatient single-agent regimens.

Resorbable materials are used to fix bony fragments after sagittal split ramus osteotomy (SSRO), but to our knowledge, there is no clear regimen for antibiotic prophylaxis when such materials are used. The purpose of this study therefore was to compare inpatient single-agent and extended dual-agent antibiotic prophylaxis for the prevention of surgical site infections (SSI) in patients after SSRO. This prospective study included 100 patients who underwent SSRO for deformities of the jaw. Cephalometric analysis was performed preoperatively, and at one month and one year postoperatively. Patients were divided into two groups of 50 each. Group A was given cefazolin sodium 1g preoperatively and every eight hours for 48 hours postoperatively. Group B was given cefazolin sodium 1g preoperatively and every eight hours for 48 hours postoperatively, after which they were provided with oral amoxicillin for three days. Both groups were assessed for SSIs for one year postoperatively using the Clavien-Dindo classification. SSIs were documented in seven patients in Group A and six in Group B, with no significant difference between the groups. The two regimens had no significant association with SSI. In conclusion, our results suggest that the inpatient, single-agent, postoperative antibiotic regimen is sufficient to prevent SSI in patients who have SSRO with resorbable plate and screw fixation.

Simplified stress analysis on the temporomandibular joint in Class III patients with and without mandibular asymmetry using a rigid body spring model.

OBJECTIVE: Aim of this study was to investigate the differences in stress on the temporomandibular joint (TMJ) between Class III patients with and without mandibular asymmetry using a rigid body spring model (RBSM). DESIGN: Menton (Me), the centre point of occlusal force on the line that connected the bilateral buccal cusps of the second molars and the most lateral, superior and medial points of the condyle were plotted on frontal cephalograms, and stress on the condyles was calculated with the 2-dimensional RBSM program of fortran. SETTING AND SAMPLE POPULATION: Eighty Japanese patients with diagnosed mandibular prognathism were divided into two groups, a symmetry group and asymmetry group on the basis of the Mx-Md midline position. OUTCOME MEASURE: The degree (force partition) of the resultant force, the direction (angulation) and displacement (X, Y) of each condyle were calculated. The horizontal displacement vector (u), the vertical displacement vector (v) and rotation angle (theta) of the mandibular body at Menton were also calculated. RESULTS: There were significant differences between the deviated and non-deviated sides of both groups regarding resultant force (symmetry group: p = 0.0372, asymmetry group: p = 0.0054), X (symmetry group: p < 0.0001, asymmetry group: p = 0.0001) and Y (symmetry group: p = 0.0354, asymmetry group: p = 0.0043). For angulation, there was a significant difference between the deviated and non-deviated sides in the asymmetry group (p = 0.0095). CONCLUSION: The results of this study suggest that difference in stress angulation on the condyles could be associated with asymmetry in mandibular prognathism.

Revised classification of lupus nephritis is valuable in predicting renal outcome with an indication of the proportion of glomeruli affected by chronic lesions.

OBJECTIVES: To determine if the International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification of lupus nephritis (LN) is helpful in predicting renal outcome. METHODS: A total of 92 patients with LN who underwent renal biopsy in our hospital were re-classified according to the ISN/RPS 2003 criteria. RESULTS: The mean patient age was 36.8 yrs and the median observation period was 65 months. The relative frequency for each class was as follows: Class I (minimal mesangial LN) 0%, Class II (mesangial proliferative LN) 13%, Class III (focal LN) 17%, Class IV (diffuse LN) 60% and Class V (membranous LN) 10%. Within Class IV, diffuse segmental (Class IV-S) was 25% and diffuse global (Class IV-G) 75%. During the observation period, renal function was more likely to deteriorate in Class IV-G cases than in Class IV-S cases. Importantly, when Class IV-G was subdivided into cases involving active lesion alone [IV-G (A)] or chronic lesion [IV-G (A/C)], the majority of cases in IV-G (A) was nephrotic, but responded well to therapy. In contrast, renal function declined only in IV-G (A/C) cases. Patients with Class IV-G (A/C) had persistent proteinuria in spite of intensified therapies. Moreover, the higher proportion of chronic lesions was related with the deterioration of renal function. CONCLUSIONS: This study showed that in Class IV-G cases, renal outcome differed in the presence of chronicity. Chronicity could be a critical factor in predicting outcome. Thus, the revised classification of LN is clinically valuable in identifying different renal outcomes among patients with diffuse LN.

Pleomorphic xanthoastrocytoma: a comparative pathological study between conventional and anaplastic types.

AIMS: To facilitate the understanding and correct diagnosis of the anaplastic variant of pleomorphic xanthoastrocytoma (PXA). METHODS AND RESULTS: Twelve cases of PXA were divided into six conventional and six anaplastic types. Three anaplastic PXAs developed in recurrent tumours and three occurred as the primary tumour. Anaplastic PXAs were microscopically characterized by monotonous proliferation of atypical cells, increased mitotic activity, necrosis and microvascular proliferation. Characteristic features of conventional PXA are also variously included in all anaplastic PXAs. No remarkable differences were detected in the immunohistochemical profiles including the neuronal phenotype between the conventional and anaplastic types. Ki67 labelling indices of the anaplastic type were significantly higher than those of the conventional type, whereas p53 showed no difference. Immunohistochemical and fluorescence in situ hybridization analyses on epidermal growth factor receptor did not demonstrate overexpression or gene amplification. CONCLUSIONS: The anaplastic PXA, which occurs de novo or through recurrence, should be distinguished from glioblastoma by identifying the salient microscopic features of conventional PXA even in the anaplastic areas; and by demonstrating the expression of neuronal markers, in that the former is expected to have longer survival.

Crucial role of insulin receptor substrate-2 in compensatory beta-cell hyperplasia in response to high fat diet-induced insulin resistance.

In type 2 diabetes, there is a defect in the regulation of functional beta-cell mass to overcome high-fat (HF) diet-induced insulin resistance. Many signals and pathways have been implicated in beta-cell function, proliferation and apoptosis. The co-ordinated regulation of functional beta-cell mass by insulin signalling and glucose metabolism under HF diet-induced insulin-resistant conditions is discussed in this article. Insulin receptor substrate (IRS)-2 is one of the two major substrates for the insulin signalling. Interestingly, IRS-2 is involved in the regulation of beta-cell proliferation, as has been demonstrated using knockout mice models. On the other hand, in an animal model for human type 2 diabetes with impaired insulin secretion because of insufficiency of glucose metabolism, decreased beta-cell proliferation was observed in mice with beta-cell-specific glucokinase haploinsufficiency (Gck(+/) (-)) fed a HF diet without upregulation of IRS-2 in beta-cells, which was reversed by overexpression of IRS-2 in beta-cells. As to the mechanism underlying the upregulation of IRS-2 in beta-cells, glucose metabolism plays an important role independently of insulin, and phosphorylation of cAMP response element-binding protein triggered by calcium-dependent signalling is the critical pathway. Downstream from insulin signalling via IRS-2 in beta-cells, a reduction in FoxO1 nuclear exclusion contributes to the insufficient proliferative response of beta-cells to insulin resistance. These findings suggest that IRS-2 is critical for beta-cell hyperplasia in response to HF diet-induced insulin resistance.

Mechanical stress activates protein kinase cascade of phosphorylation in neonatal rat cardiac myocytes.

We have previously shown that stretching cardiac myocytes evokes activation of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and 90-kD ribosomal S6 kinase (p90rsk). To clarify the signal transduction pathways from external mechanical stress to nuclear gene expression in stretch-induced cardiac hypertrophy, we have elucidated protein kinase cascade of phosphorylation by examining the time course of activation of MAP kinase kinase kinases (MAPKKKs), MAP kinase kinase (MAPKK), MAPKs, and p90rsk in neonatal rat cardiac myocytes. Mechanical stretch transiently increased the activity of MAPKKKs. An increase in MAPKKKs activity was first detected at 1 min and maximal activation was observed at 2 min after stretch. The activity of MAPKK was increased by stretch from 1-2 min, with a peak at 5 min after stretch. In addition, MAPKs and p90rsk were maximally activated at 8 min and at 10 approximately 30 min after stretch, respectively. Raf-1 kinase (Raf-1) and (MAPK/extracellular signal-regulated kinase) kinase kinase (MEKK), both of which have MAPKKK activity, were also activated by stretching cardiac myocytes for 2 min. The angiotensin II receptor antagonist partially suppressed activation of Raf-1 and MAPKs by stretch. The stretch-induced hypertrophic responses such as activation of Raf-1 and MAPKs and an increase in amino acid uptake was partially dependent on PKC, while a PKC inhibitor completely abolished MAPK activation by angiotensin II. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1 and MEKK, MAPKK, MAPKs and p90rsk, and that angiotensin II, which may be secreted from stretched myocytes, may be partly involved in stretch-induced hypertrophic responses by activating PKC.

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.

Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats.

Troglitazone (CS-045) is one of the thiazolidinediones that activate the peroxisome proliferator-activated receptor gamma (PPARgamma), which is expressed primarily in adipose tissues. To elucidate the mechanism by which troglitazone relieves insulin resistance in vivo, we studied its effects on the white adipose tissues of an obese animal model (obese Zucker rat). Administration of troglitazone for 15 d normalized mild hyperglycemia and marked hyperinsulinemia in these rats. Plasma triglyceride level was decreased by troglitazone in both obese and lean rats. Troglitazone did not change the total weight of white adipose tissues but increased the number of small adipocytes (< 2,500 micron2) approximately fourfold in both retroperitoneal and subcutaneous adipose tissues of obese rats. It also decreased the number of large adipocytes (> 5,000 micron2) by approximately 50%. In fact, the percentage of apoptotic nuclei was approximately 2.5-fold higher in the troglitazone-treated retroperitoneal white adipose tissue than control. Concomitantly, troglitazone normalized the expression levels of TNF-alpha which were elevated by 2- and 1.4-fold in the retroperitoneal and mesenteric white adipose tissues of the obese rats, respectively. Troglitazone also caused a dramatic decrease in the expression levels of leptin, which were increased by 4-10-fold in the white adipose tissues of obese rats. These results suggest that the primary action of troglitazone may be to increase the number of small adipocytes in white adipose tissues, presumably via PPARgamma. The increased number of small adipocytes and the decreased number of large adipocytes in white adipose tissues of troglitazone-treated obese rats appear to be an important mechanism by which increased expression levels of TNF-alpha and higher levels of plasma lipids are normalized, leading to alleviation of insulin resistance.

Positive and negative regulation of phosphoinositide 3-kinase-dependent signaling pathways by three different gene products of the p85alpha regulatory subunit.

Phosphoinositide (PI) 3-kinase is a key mediator of insulin-dependent metabolic actions, including stimulation of glucose transport and glycogen synthesis. The gene for the p85alpha regulatory subunit yields three splicing variants, p85alpha, AS53/p55alpha, and p50alpha. All three have (i) a C-terminal structure consisting of two Src homology 2 domains flanking the p110 catalytic subunit-binding domain and (ii) a unique N-terminal region of 304, 34, and 6 amino acids, respectively. To determine if these regulatory subunits differ in their effects on enzyme activity and signal transduction from insulin receptor substrate (IRS) proteins under physiological conditions, we expressed each regulatory subunit in fully differentiated L6 myotubes using adenovirus-mediated gene transfer with or without coexpression of the p110alpha catalytic subunit. PI 3-kinase activity associated with p50alpha was greater than that associated with p85alpha or AS53. Increasing the level of p85alpha or AS53, but not p50alpha, inhibited both phosphotyrosine-associated and p110-associated PI 3-kinase activities. Expression of a p85alpha mutant lacking the p110-binding site (Deltap85) also inhibited phosphotyrosine-associated PI 3-kinase activity but not p110-associated activity. Insulin stimulation of two kinases downstream from PI-3 kinase, Akt and p70 S6 kinase (p70(S6K)), was decreased in cells expressing p85alpha or AS53 but not in cells expressing p50alpha. Similar inhibition of PI 3-kinase, Akt, and p70(S6K) was observed, even when p110alpha was coexpressed with p85alpha or AS53. Expression of p110alpha alone dramatically increased glucose transport but decreased glycogen synthase activity. This effect was reduced when p110alpha was coexpressed with any of the three regulatory subunits. Thus, the three different isoforms of regulatory subunit can relay the signal from IRS proteins to the p110 catalytic subunit with different efficiencies. They also negatively modulate the PI 3-kinase catalytic activity but to different extents, dependent on the unique N-terminal structure of each isoform. These data also suggest the existence of a mechanism by which regulatory subunits modulate the PI 3-kinase-mediated signals, independent of the kinase activity, possibly through subcellular localization of the catalytic subunit or interaction with additional signaling molecules.

Insulin receptor substrate 3 (IRS-3) and IRS-4 impair IRS-1- and IRS-2-mediated signaling.

To investigate the roles of insulin receptor substrate 3 (IRS-3) and IRS-4 in the insulin-like growth factor 1 (IGF-1) signaling cascade, we introduced these proteins into 3T3 embryonic fibroblast cell lines prepared from wild-type (WT) and IRS-1 knockout (KO) mice by using a retroviral system. Following transduction of IRS-3 or IRS-4, the cells showed a significant decrease in IRS-2 mRNA and protein levels without any change in the IRS-1 protein level. In these cell lines, IGF-1 caused the rapid tyrosine phosphorylation of all four IRS proteins. However, IRS-3- or IRS-4-expressing cells also showed a marked decrease in IRS-1 and IRS-2 phosphorylation compared to the host cells. This decrease was accounted for in part by a decrease in the level of IRS-2 protein but occurred with no significant change in the IRS-1 protein level. IRS-3- or IRS-4-overexpressing cells showed an increase in basal phosphatidylinositol 3-kinase activity and basal Akt phosphorylation, while the IGF-1-stimulated levels correlated well with total tyrosine phosphorylation level of all IRS proteins in each cell line. IRS-3 expression in WT cells also caused an increase in IGF-1-induced mitogen-activated protein kinase phosphorylation and egr-1 expression ( approximately 1.8- and approximately 2.4-fold with respect to WT). In the IRS-1 KO cells, the impaired mitogenic response to IGF-1 was reconstituted with IRS-1 to supranormal levels and was returned to almost normal by IRS-2 or IRS-3 but was not improved by overexpression of IRS-4. These data suggest that IRS-3 and IRS-4 may act as negative regulators of the IGF-1 signaling pathway by suppressing the function of other IRS proteins at several steps.

Essential role of insulin receptor substrate 1 in differentiation of brown adipocytes.

The most widely distributed members of the family of insulin receptor substrate (IRS) proteins are IRS-1 and IRS-2. These proteins participate in insulin and insulin-like growth factor 1 signaling, as well as the actions of some cytokines, growth hormone, and prolactin. To more precisely define the specific role of IRS-1 in adipocyte biology, we established brown adipocyte cell lines from wild-type and IRS-1 knockout (KO) animals. Using differentiation protocols, both with and without insulin, preadipocyte cell lines derived from IRS-1 KO mice exhibited a marked decrease in differentiation and lipid accumulation (10 to 40%) compared to wild-type cells (90 to 100%). Furthermore, IRS-1 KO cells showed decreased expression of adipogenic marker proteins, such as peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer-binding protein alpha (C/EBPalpha), fatty acid synthase, uncoupling protein-1, and glucose transporter 4. The differentiation deficit in the KO cells could be reversed almost completely by retrovirus-mediated reexpression of IRS-1, PPARgamma, or C/EBPalpha but not the thiazolidinedione troglitazone. Phosphatidylinositol 3-kinase (PI 3-kinase) assays performed at various stages of the differentiation process revealed a strong and transient activation in IRS-1, IRS-2, and phosphotyrosine-associated PI 3-kinase in the wild-type cells, whereas the IRS-1 KO cells showed impaired phosphotyrosine-associated PI 3-kinase activation, all of which was associated with IRS-2. Akt phosphorylation was reduced in parallel with the total PI 3-kinase activity. Inhibition of PI 3-kinase with LY294002 blocked differentiation of wild-type cells. Thus, IRS-1 appears to be an important mediator of brown adipocyte maturation. Furthermore, this signaling molecule appears to exert its unique role in the differentiation process via activation of PI 3-kinase and its downstream target, Akt, and is upstream of the effects of PPARgamma and C/EBPalpha.

The extracellular signal-regulated kinase pathway phosphorylates AML1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability.

AML1 (also called PEBP2alphaB, CBFA2, or CBFalpha2) is one of the most frequently disrupted genes in chromosome abnormalities seen in human leukemias. It has been reported that AML1 plays several pivotal roles in myeloid hematopoietic differentiation and other biological phenomena, probably through the transcriptional regulation of various relevant genes. Here, we investigated the mechanism of regulation of AML1 functions through signal transduction pathways. The results showed that AML1 is phosphorylated in vivo on two serine residues within the proline-, serine-, and threonine-rich region, with dependence on the activation of extracellular signal-regulated kinase (ERK) and with interleukin-3 stimulation in a hematopoietic cell line. These in vivo phosphorylation sites of AML1 were phosphorylated directly in vitro by ERK. Although differences between wild-type AML1 and phosphorylation site mutants in DNA-binding affinity were not observed, we have shown that ERK-dependent phosphorylation potentiates the transactivation ability of AML1. Furthermore the phosphorylation site mutations reduced the transforming capacity of AML1 in fibroblast cells. These data indicate that AML1 functions are potentially regulated by ERK, which is activated by cytokine and growth factor stimuli. This study provides some important clues for clarifying unidentified facets of the regulatory mechanism of AML1 function.

Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins.

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.