Kazuhiro Kishi

GLUT4 TRANSLOCATION BY INSULIN IN INTACT MUSCLE CELLS : DETECTION BY A FAST AND QUANTITATIVE ASSAY

We report a rapid and sensitive colorimetric approach to quantitate the amount of glucose transporters exposed at the surface of intact cells, using L6 muscle cells expressing GLUT4 containing an exofacial myc epitope. Unstimulated cells exposed to the surface 5 fmol GLUT4myc per mg protein. This value increased to 10 fmol/mg protein in response to insulin as 2‐deoxyglucose (10 μM) uptake doubled. The results are substantiated by immunofluorescent detection of GLUT4myc in unpermeabilized cells and by subcellular fractionation. We further show that wortmannin and the cytoskeleton disruptors cytochalasin D and latrunculin B completely blocked these insulin effects. The rapid quantitative assay described here could be of high value to study insulin signals and to screen for potential anti‐diabetic drugs.

Soluble insulin receptor ectodomain is elevated in the plasma of patients with diabetes

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.

Reconstitution of Insulin Signaling Pathways in Rat 3Y1 Cells Lacking Insulin Receptor and Insulin Receptor Substrate-1 EVIDENCE THAT ACTIVATION OF Akt IS INSUFFICIENT FOR INSULIN-STIMULATED GLYCOGEN SYNTHESIS OR GLUCOSE UPTAKE IN RAT 3Y1 CELLS

Rat 3Y1 cells have endogenous insulin-like growth factor-1 receptors and insulin receptor substrate (IRS)-2, but lack both insulin receptor (IR) and IRS-1. To investigate the role of IR and IRS-1 in effects of insulin, we transfected IR and IRS-1 expression plasmids into cells and reconstituted the insulin signaling pathways. 3Y1 cells stably expressing the c-myc epitope-tagged glucose transporter type 4 (3Y1-GLUT4myc) exhibit no effects of insulin, at physiological concentrations. The 3Y1-GLUT4myc-IR cells expressing GLUT4myc and IR responded to phosphatidylinositol 3,4,5-trisphosphate (PI-3,4,5-P3) accumulation, Akt activation, the stimulation of DNA synthesis, and membrane ruffling but not to glycogen synthesis, glucose uptake, or GLUT4myc translocation. The further expression of IRS-1 in 3Y1-GLUT4myc-IR cells led to stimulation of glycogen synthesis but not to glucose uptake or GLUT4myc translocation in response to insulin, although NaF or phorbol 12-myristate 13-acetate did trigger GLUT4myc translocation in the cells. These results suggest that, in rat 3Y1 cells, (i) IRS-1 is essential for insulin-stimulated glycogen synthesis but not for DNA synthesis, PI-3,4,5-P3 accumulation, Akt phosphorylation, or membrane ruffling, and (ii) the accumulation of PI-3,4,5-P3 and activation of Akt are insufficient for glycogen synthesis, glucose uptake or for GLUT4 translocation.

Effects of losartan in combination with or without exercise on insulin resistance in Otsuka Long–Evans Tokushima Fatty rats

Hypertension often complicates type 2 diabetes mellitus, and angiotensin converting enzyme inhibitor treatment has been shown to improve insulin resistance in such cases. However, the effect of angiotensin II type-1 (AT(1)) receptor antagonists on insulin resistance is still controversial. To gain further information on this effect, we examined the effect of losartan on insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus. Losartan administration alone lowered systolic blood pressure, but did not improve oral glucose tolerance test or insulin resistance in OLETF rats. However, the administration of losartan with exercise significantly improved both systolic blood pressure and insulin resistance relative to control OLETF rats. On the other hand, losartan treatment, regardless of exercise, increased glucose uptake in excised soleus muscle and fat cells. To explore the beneficial effect of losartan on skeletal muscle glucose uptake, we examined intracellular signaling of soleus muscle. Although Akt activity and glucose transporter type 4 (GLUT4) expressions were not affected by losartan with or without exercise, extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein (MAP) kinase activities were increased by both interventions. These results indicate that angiotensin AT(1) receptor antagonist improved local insulin resistance, but not systemic insulin resistance. These findings may explain the controversy over the effect of angiotensin AT(1) receptor antagonists on insulin resistance in clinical use. The enhancing effect of angiotensin AT(1) receptor antagonist on skeletal muscle glucose uptake may be attributable to MAP kinase activation or other mechanisms rather than phosphatidylinositol 3-kinase activation.

ENDOSCOPIC ABLATION THERAPY FOR GASTROINTESTINAL SUPERFICIAL NEOPLASIA

Aim:  In Japan, endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) have been widely accepted and standardized for the treatment of gastrointestinal superficial neoplasia.

Cortactin, an actin binding protein, regulates GLUT4 translocation via actin filament remodeling

Insulin regulates glucose uptake into fat and skeletal muscle cells by modulating the translocation of GLUT4 between the cell surface and interior. We investigated a role for cortactin, a cortical actin binding protein, in the actin filament organization and translocation of GLUT4 in Chinese hamster ovary (CHO-GLUT4myc) and L6-GLUT4myc myotube cells. Overexpression of wild-type cortactin enhanced insulin-stimulated GLUT4myc translocation but did not alter actin fiber formation. Conversely, cortactin mutants lacking the Src homology 3 (SH3) domain inhibited insulin-stimulated formation of actin stress fibers and GLUT4 translocation similar to the actin depolymerizing agent cytochalasin D. Wortmannin, genistein, and a PP1 analog completely blocked insulin-induced Akt phosphorylation, formation of actin stress fibers, and GLUT4 translocation indicating the involvement of both PI3-K/Akt and the Src family of kinases. The effect of these inhibitors was even more pronounced in the presence of overexpressed cortactin suggesting that the same pathways are involved. Knockdown of cortactin by siRNA did not inhibit insulin-induced Akt phosphorylation but completely inhibited actin stress fiber formation and glucose uptake. These results suggest that the actin binding protein cortactin is required for actin stress fiber formation in muscle cells and that this process is absolutely required for translocation of GLUT4-containing vesicles to the plasma membrane.

Activation of G‑protein coupled fMLP or PAF receptor directly triggers glucose transporter type 1 (GLUT1 translocation in Chinese hamster ovary (CHO cells stably expressing fMLP or PAF receptor

The chemoattractants, fMLP and PAF, stimulate glucose uptake in phagocytes to obtain an energy source for host defense. Glucose uptake in phagocytes is mainly regulated via glucose transporter type 1 (GLUT1). To examine molecular mechanisms of facilitated glucose uptake in response to fMLP or PAF, we established CHO cells stably expressing fMLP or PAF receptor with c-myc epitope tagged GLUT1 which could immunologically detect GLUT1 on the cell surface. In the CHO cells, both fMLP and PAF directly triggered GLUT1 translocation from the intracellular pool to the cell surface, and stimulated glucose uptake. Therefore, in phagocytes, we propose that fMLP and PAF also trigger GLUT1 translocation to stimulate glucose uptake as an energy source for host defense.

Paraneoplastic Hypocalcemia Developed in Gastric Cancer Accompanied by Osteoblastic Metastasis

Paraneoplastic syndromes are generally defined as clinical disorders associated with malignant diseases, and hypocalcemia associated with cancer is a rare condition. A woman in her 60s was referred to our hospital for the further examination of massive ascites due to carcinoma of unknown primary origin. She complained of numbness around her lips, and marked hypocalcemia of 5.0 mg/dL was noted. After two courses of chemotherapy, computed tomography showed a decrease in the ascites, and her serum calcium level increased. Although hypocalcemia is a very rare condition in patients with gastric cancer, serum calcium values should be evaluated when neurological symptoms are observed.

Poly-(ADP-Ribose) Polymerase-1 Promotes Prothrombin Gene Transcription and Produces Des-Gamma-Carboxy Prothrombin in Hepatocellular Carcinoma

Background and Aim: Although des-gamma-carboxy prothrombin (DCP) is a well-known tumor marker for hepatocellular carcinoma (HCC), the mechanism of DCP production is unclear. This study aimed to investigate the mechanism how DCP is produced in HCC cells. Methods: Levels of mRNA and DCP were analyzed by real-time polymerase chain reaction and electro-chemiluminescence immunoassay respectively. Secreted alkaline phosphatase (SEAP) expression vectors including deletion mutants of the prothrombin gene promoter were constructed for reporter gene assay. The transcription factors bound to DNA fragments were analyzed by mass spectrometry. An electrophoretic mobility shift assay (EMSA) was performed using a biotin end-labeled DNA. Results: The prothrombin mRNA levels in all 5 DCP producing cell lines were appreciably high. However, those in 2 DCP non-producing cell lines were below detectable levels. A SEAP vector with -2985 to +27 showed a very high transcription activity in DCP-producing Huh-1 cells. However, transcription abruptly decreased when the vector with -2955 to +27 was transfected, and then remained at the similar levels with larger deletion mutants, indicating the existence of a cis-element at -2985 to -2955 (31-bp). Mass spectrometry analysis identified the protein that bound to the 31-bp DNA as poly-(ADP-ribose) polymerase-1 (PARP-1). Knockdown of the PARP-1 gene by small interfering RNA in Huh-1 cells induced marked inhibition of prothrombin gene transcription. The EMSA clearly showed that PARP-1 specifically binds to the 31-bp DNA fragment in the prothrombin gene promoter. Conclusions: Our data suggest that PARP-1 activates prothrombin gene transcription and that the excessive prothrombin gene transcription induces DCP production in DCP-producing HCC cells.

Unusual endoscopic findings of gastric neuroendocrine tumor.

Gastric neuroendocrine tumor (NET) is sometimes found as a submucosal tumor on upper gastrointestinal endoscopy. Gastric NET with malignant profile and neuroendocrine carcinoma (NEC) show various forms which are difficult to distinguish from gastric cancer and other disease. We report a case of a cauliflower-shaped NET of the stomach. A 61-year-old man was referred to our hospital with a complaint of abdominal fullness. Upper gastrointestinal endoscopic examination revealed an unusual, whitish cauliflower-shaped tumor that belongs to Borrmann type I on the lesser curvature of the gastric antrum. Histological examination of the biopsy specimen revealed NET G2, because the tumor cells were CD56- and synaptophysin-positive by immunohistochemical analysis. A distal gastrectomy with D2 lymphadenectomy was performed. A recurrence in the liver was revealed by follow up computed tomography after 11 months from operation. Combined chemotherapy with irinotecan (CPT-11) plus cisplatin (CDDP) was treated. The patient achieved a partial response, but he died after 31 months from gastrectomy. There is no independent, large-scaled prospective study and no standard treatment for gastric NETs with distant metastases. Our case is reported with a literature review of the treatment of metastatic gastric NET G2.