Erratum. Increased Insulin Sensitivity and Hypoinsulinemia in APS Knockout Mice. Diabetes 2003;52:2657—2665 DOI: 10.2337/db15-er08 Asako Minami, Masanori Iseki, Kazuhiro Kishi, Miao Wang, Makoto Ogura, Noboru Furukawa, Sanae Hayashi, Mizuki Yamada, Toshiyuki Obata, Yukari Takeshita, Yutaka Nakaya, Yoshimi Bando, Keisuke Izumi, Shonna A. Moodie, Fumiko Kajiura, Mitsuru Matsumoto, Kiyoshi Takatsu, Satoshi Takaki, and Yousuke Ebina As reported by the authors of the above-listed article, Fig. 3B contains two corrections:

A tyrosine kinase adaptor protein containing pleckstrin homology and SH2 domains (APS) is rapidly and strongly tyrosine phosphorylated by insulin receptor kinase upon insulin stimulation. The function of APS in insulin signaling has heretofore remained unknown. APS-deficient (APS(-/-)) mice were used to investigate its function in vivo. The blood glucose-lowering effect of insulin, as assessed by the intraperitoneal insulin tolerance test, was increased in APS(-/-) mice. Plasma insulin levels during fasting and in the intraperitoneal glucose tolerance test were lower in APS(-/-) mice. APS(-/-) mice showed an increase in the whole-body glucose infusion rate as assessed by the hyperinsulinemic-euglycemic clamp test. These findings indicated that APS(-/-) mice exhibited increased sensitivity to insulin. However, overexpression of wild-type or dominant-negative APS in 3T3L1 adipocytes did not affect insulin receptor numbers, phosphorylations of insulin receptor, insulin receptor substrate-1, or Akt and mitogen-activated protein kinase. The glucose uptake and GLUT4 translocation were not affected by insulin stimulation in these cells. Nevertheless, the insulin-stimulated glucose transport in isolated adipocytes of APS(-/-) mice was increased over that of APS(+/+) mice. APS(-/-) mice also showed increased serum levels of leptin and adiponectin, which might explain the increased insulin sensitivity of adipocytes.

Soluble insulin receptor (sIR), the ectodomain of IR, has been detected in human plasma, and its concentration parallels that of blood glucose in patients with diabetes. IR has a pivotal role in glucose homeostasis and diabetes development; therefore, cleavage of IR promoted by hyperglycemia is involved in insulin resistance and glucose toxicity. To elucidate the physiology of sIR, we developed an in vitro model mimicking the changes in sIR levels in plasma from patients with diabetes. Among four human cell lines that expressed IR, spontaneous cleavage of IR occurred only in HepG2 cells. The molecular characteristics of sIR derived from HepG2 cells were similar to those of sIR detected in human plasma. The concentration of sIR in the medium did not differ between basal and high-glucose conditions in the initial 24-h period, but increasing the duration of pre-stimulation (>48 h) led to a significant increase in sIR levels in cells exposed to high glucose. Additionally, glucose-dependent increment of sIR was reversible in this model. These results are consistent with the observation of plasma sIR in patients with diabetes. Using this model, O-linked N-acetylglucosamine modification was determined to be involved in high-glucose-induced IR cleavage. A calcium-dependent protease was shown to cleave IR extracellularly. These findings show that this in vitro model could be useful for determining the molecular mechanism underlying IR cleavage.

plays a key role in the acute regulation of blood

Objective: For the early identification of patients at risk of developing diabetes mellitus, and to prevent the onset of diabetes by performing dietary counseling and exercise guidance, we have developed an ultra-sensitive immune complex transfer enzyme immunoassay (ICT-EIA) to measure soluble human insulin receptor ectodomain (sIR alpha) in urine which is collected non-invasively.Design and methods: We developed ICT-EIA for sIR alpha and measured urinary sIR alpha from 106 healthy volunteers, 35 obese volunteers and 42 patients with diabetes.Results: The detection limit of ICT-EIA (0.04 pg/mL), using a urine sample of as little as 100 mu L, was a few hundred-fold higher than that of conventional ELISA. Using ICT-EIA, the urinary sIR alpha level in patients with diabetes (9.7 +/- 20.1 pg/mg creatinine) was significantly higher than those in healthy volunteers (1.4 +/- 0.9; P < 0.001).Conclusion: ICT-EIA for sIR alpha may be useful as a good marker for evaluating diabetes risk. (C) 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Akt substrate of 160kDa (AS160) is a Rab GTPase activating protein (GAP) and was recently identified as a component of the insulin signaling pathway of glucose transporter type 4 (GLUT4) translocation. We and others, previously reported that the activation of Galphaq protein-coupled receptors (GalphaqPCRs) also stimulated GLUT4 translocation and glucose uptake in several cell lines. Here, we report that the activation of GalphaqPCRs also promoted phosphorylation of AS160 by the 5'-AMP activated protein kinase (AMPK). The suppression of AS160 phosphorylation by the siRNA mediated AMPKalpha1 subunit knockdown promoted GLUT4 vesicle retention in intracellular compartments. This suppression did not affect the ratio of non-induced cell surface GLUT4 to Galphaq-induced it. Rat 3Y1 cells lacking AS160 did not show insulin-induced GLUT4 translocation. The cells stably expressing GLUT4 revealed GLUT4 vesicles that were mainly localized in the perinuclear region and less frequently on the cell surface. After expression of exogenous AS160, GLUT4 on the cell surface decreased and GLUT4 vesicles were redistributed throughout the cytoplasm. Although PMA-induced or sodium fluoride-induced GLUT4 translocation was significantly increased in these cells, insulin did not affect GLUT4 translocation. These results suggest that AS160 is a common regulator of insulin- and GalphaqPCR activation-mediated GLUT4 distribution in the cells.

Insulin resistance is a common disorder caused by a wide variety of physiological insults, some of which include poor diet, inflammation, anti-inflammatory steroids, hyperinsulinemia, and dyslipidemia. The common link between these diverse insults and insulin resistance is widely considered to involve impaired insulin signaling, particularly at the level of the insulin receptor substrate (IRS). To test this model, we utilized a heterologous system involving the platelet-derived growth factor (PDGF) pathway that recapitulates many aspects of insulin action independently of IRS. We comprehensively analyzed six models of insulin resistance in three experimental systems and consistently observed defects in both insulin and PDGF action despite a range of insult-specific defects within the IRS-Akt nexus. These findings indicate that while insulin resistance is associated with multiple deficiencies, the most deleterious defects and the origin of insulin resistance occur independently of IRS.

APS, a tyrosine kinase adaptor protein with pleckstrin homology and Src homology 2 domains, is rapidly and strongly tyrosine-phosphorylated by insulin receptor kinase upon insulin stimulation. We have previously shown that APS knockout mice have increased insulin sensitivity, and that this enhancement is possibly due to increased insulin-response on adipose tissues. However, the function of APS in insulin signaling has so far been controversial. Here, we report that APS enhanced ligand-dependent multi-ubiquitination of the insulin receptor (IR) in CHO cells overexpressing the IR. APS-mediated ubiquitination of the IR induced enhancement of the IR internalization, but did not affect the IR degradation. This finding shows one of the pleiotropic functions of APS in insulin signaling.

OBJECTIVE—Insulin binds to the α-subunit of the insulin receptor (IRα) and subsequently exerts its effects in the cells. The soluble ectodomains of several receptors have been found to circulate in the plasma. Therefore, we hypothesized that soluble human insulin receptor (hIR) ectodomain (α-subunit and a part of β-subunit) may exist in the plasma of diabetic patients. RESEARCH DESIGN AND METHODS—We identified soluble hIR ectodomain in human plasma by a two-step purification followed by immunoblotting and gel-filtration chromatography. Furthermore, we established an hIRα-specific enzyme-linked immunosorbent assay and measured the plasma IRα levels in patients with diabetes. We also investigated this phenomenon in streptozotocin-induced diabetic hIR transgenic mice. RESULTS—Soluble hIRα, but not intact hIRβ or whole hIR, exists in human plasma. The plasma IRα levels were significantly higher in type 1 (2.00 ± 0.60 ng/ml; n = 53) and type 2 (2.26 ± 0.80; n = 473) diabetic patients than in control subjects (1.59 ± 0.40 ng/ml; n = 123 (P < 0.001 vs. control). Plasma IRα level was positively correlated with blood glucose level, and 10–20% of the insulin in plasma bound to hIRα. In the in vivo experiments using diabetic hIR transgenic mice, hyperglycemia was confirmed to increase the plasma hIRα level and the half-life estimated to be ∼6 h. CONCLUSIONS—We propose that the increased soluble IR ectodomain level appears to be a more rapid glycemic marker than A1C or glycoalbumin.

OBJECTIVE—c-Cbl plays an important role in whole-body fuel homeostasis by regulating insulin action. In the present study, we examined the role of Cbl-b, another member of the Cbl family, in insulin action. RESEARCH DESIGN AND METHODS—C57BL/6 (Cbl-b+/+) or Cbl-b-deficient (Cbl-b−/−) mice were subjected to insulin and glucose tolerance tests and a hyperinsulinemic-euglycemic clamp test. Infiltration of macrophages into white adipose tissue (WAT) was assessed by immunohistochemistry and flow cytometry. We examined macrophage activation using co-cultures of 3T3-L1 adipocytes and peritoneal macrophages. RESULTS—Elderly Cbl-b−/− mice developed glucose intolerance and peripheral insulin resistance; serum insulin concentrations after a glucose challenge were always higher in elderly Cbl-b−/− mice than age-matched Cbl-b+/+ mice. Deficiency of the Cbl-b gene significantly decreased the uptake of 2-deoxyglucose into WAT and glucose infusion rate, whereas fatty liver was apparent in elderly Cbl-b−/− mice. Cbl-b deficiency was associated with infiltration of macrophages into the WAT and expression of cytokines, such as tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein (MCP)-1. Co-culture of Cbl-b−/− macrophages with 3T3-L1 adipocytes induced leptin expression and dephosphorylation of insulin receptor substrate 1, leading to impaired glucose uptake in adipocytes. Furthermore, Vav1, a key factor in macrophage activation, was highly phosphorylated in peritoneal Cbl-b−/− macrophages compared with Cbl-b+/+ macrophages. Treatment with a neutralizing anti–MCP-1 antibody improved peripheral insulin resistance and macrophage infiltration into WAT in elderly Cbl-b−/− mice. CONCLUSIONS—Cbl-b is a negative regulator of macrophage infiltration and activation, and macrophage activation by Cbl-b deficiency contributes to the peripheral insulin resistance and glucose intolerance via cytokines secreted from macrophages.

There is crosstalk in intracellular signaling between Angiotensin II (Ang II) and insulin. We hypothesized that the underlying mechanism might be related to changes in cytoskeleton. In the presence of 100 nM of Ang II, insulin-induced glucose uptake was decreased and insulin-induced actin filament organization was inhibited. PKC inhibitors, including GF109203x and p38MAPK inhibitor (SB203580) neither improved insulin-induced actin reorganization nor glucose uptake. In contrast, the Ang II-induced inhibition of glucose uptake and actin filament disorganization was reversed by 10 micromol ERK 1/2 MAPK inhibitor (PD98059). Pretreatment of Ang II increased ERK1/2 phosphorylation and inhibited insulin-induced Akt phosphorylation. The effect of Ang II on ERK1/2 phosphorylation was blocked by Ang II type 1 receptor antagonists, RNH6270 and PD98059 but not by SB203580 or Guanosine-5'-O-(2-ThioDiphosphate), a G-protein inhibitor. We next tested the effect of broad-spectrum matrix metalloproteinase (MMP) inhibitor (GM6001) on Ang II-inhibition of insulin signaling pathway. GM6001 did not improve Ang II-induced actin filament disorganization and did not inhibit ERK1/2 phosphorylation. From these data in L6 myotube, we conclude that Ang II negatively regulates the insulin signal not through MMP signaling pathway but specifically through MMP-independent ERK1/2 activation pathway, providing an alternative molecular mechanism for angiotensin-induced insulin resistance.

On a trouve que des sous-unites α du recepteur d'insuline libres sont presentes dans le sang. L'invention concerne un procede de mesure des sous-unites α du recepteur d'insuline, consistant a amener les sous-unites α du recepteur d'insuline d'un echantillon de sang, en contact avec un anticorps capable de reconnaitre les sous-unites α du recepteur d'insuline, puis a detecter la liaison entre eux. La mesure des sous-unites α du recepteur d'insuline libres dans le sang est utilisee pour l'estimation du facteur de risque du diabete. En outre, il a ete mis en evidence, par le procede de mesure, que la concentration des sous-unites α du recepteur d'insuline libres dans le sang de patients souffrant de diabete ou d'un cancer est remarquablement elevee. Les sous-unites α du recepteur d'insuline libres dans le sang sont utilisees comme marqueurs pour le diabete ou le cancer.

ABSTRACT Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase which has multiple functions, including inhibition of the mitogen-activated protein (MAP) kinase pathway. Simian virus 40 small t antigen specifically inhibits PP2A function by binding to the PP2A regulatory subunit, interfering with the ability of PP2A to associate with its cellular substrates. We have reported that the expression of small t antigen inhibits PP2A association with Shc, leading to augmentation of insulin and epidermal growth factor-induced Shc phosphorylation with enhanced activation of the Ras/MAP kinase pathway. However, the potential involvement of PP2A in insulin's metabolic signaling pathway is presently unknown. To assess this, we overexpressed small t antigen in 3T3-L1 adipocytes by adenovirus-mediated gene transfer and found that the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3β, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C λ (PKC λ) activity was also augmented in small-t-antigen-expressing 3T3-L1 adipocytes. Consistent with this result, both basal and insulin-stimulated glucose uptake were enhanced in these cells. In support of this result, when inhibitory anti-PP2A antibody was microinjected into 3T3-L1 adipocytes, we found a twofold increase in GLUT4 translocation in the absence of insulin. The small-t-antigen-induced increase in Akt and PKC λ activities was not inhibited by wortmannin, while the ability of small t antigen to enhance glucose transport was inhibited by dominant negative Akt (DN-Akt) expression and Akt small interfering RNA (siRNA) but not by DN-PKC λ expression or PKC λ siRNA. We conclude that PP2A is a negative regulator of insulin's metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC λ and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.