Aims/hypothesis The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein called Wolfram syndrome 1 protein, homozygous mutations of which cause selective beta cell loss in humans. The function(s) of this protein and the mechanism by which the mutations of this gene cause beta cell death are still not fully understood. We hypothesised that increased insulin demand as a result of obesity/insulin resistance causes ER stress in pancreatic beta cells, thereby promoting beta cell death. Methods We studied the effect of breeding Wfs1 −/− mice on a C57BL/6J background with mild obesity and insulin resistance, by introducing the agouti lethal yellow mutation (A y /a). We also treated the mice with pioglitazone.

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using antiphosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulinstimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulinstimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.Akt, also known as protein kinase B or Rac-PK, is a Ser/Thr kinase that was originally identified as a transforming oncogene in a retrovirus from a spontaneous thymoma in an AKR mouse (1). Activation of Akt1 by growth factors, such as insulin and IGF-1, 1 has been shown to be necessary for various cellular processes including cell growth, differentiation, metabolism, and apoptosis (2-6). To date, two potential in vivo substrates of Akt1 have been identified, namely glycogen synthase kinase-3 (GSK3) and BAD (4). GSK3 is inhibited by Akt1, which is thought to contribute to the insulin-induced dephosphorylation (activation) of glycogen synthase and protein synthesis initiation factor eIF2B and thereby to the stimulation of glycogen synthesis and protein synthesis (4). In addition, one of the cellular targets that Akt1 may phosphorylate to protect cells from apoptosis is BAD (7,8). This protein, in its dephosphorylated form, interacts with the Bcl family member BclXL and induces apoptosis of some cells; however, BAD, which is phosphorylated by Akt1, dissociates from BclXL, instead of interacting with 14-3-3, to prevent apoptosis (9). Furthermore, in transfection-based experiments, constitutively active Akt1 also mimics other actions of insulin, such as the enhancement of glucose uptake in 3T3-L1 adipocytes (10) and L6 myotubes (11) that results in the translocation of GLUT4 from an intracellular compartment to the plasma membrane. Although several lines of evidence ...

A woman in her fifties showed symptoms of fever, loss of appetite, vomiting, and general fatigue 2 days after she was bitten by a sick cat, which had later died, in Yamaguchi prefecture, western Japan, in June 2016. She subsequently died of multiorgan failure, and an autopsy was performed to determine the cause of death. However, the etiological pathogens were not quickly identified. The pathological features of the patient were retrospectively re-examined, and the pathology of the regional lymph node at the site of the cat bite was found to show necrotizing lymphadenitis with hemophagocytosis. The pathological features were noted to be similar to those of patients reported to have severe fever with thrombocytopenia syndrome (SFTS). Therefore, the lymph node section was retrospectively tested immunohistochemically, revealing the presence of the SFTS virus (SFTSV) antigen. The sick cat showed similar symptoms and laboratory findings similar to those shown in human SFTS cases. The patient had no history of tick bites, and did not have skin lesions suggestive of these. She had not undertaken any outdoor activities. It is highly possible that the patient was infected with SFTSV through the sick cat’s bite. If a patient gets sick in an SFTS-endemic region after being bitten by a cat, SFTS should be considered in the differential diagnosis.

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-alpha, novel PKC-delta, and atypical PKC isoforms of PKC-lambda and PKC-zeta, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-alpha and PKC-lambda/zeta, but not of PKC-delta, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-alpha and exogenous PKC-delta but not atypical PKC-lambda/zeta. Insulin also activated the overexpressed PKC-delta but not PKC-alpha. Expression of the wild-type PKC-alpha or PKC-delta resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-alpha expression, which inhibited the PMA activation of PKC-alpha, decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-delta but not of PKC-alpha. These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-lambda/zeta was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the insulin-signaling pathway for glucose metabolism, wild-type (wt), the kinase-dead (kd), or the plecstrin homology (PH) domain deletion (⌬PH) mutant of PDK1 was expressed using an adenovirus gene transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were found in both membrane and cytosol fractions, whereas ⌬PH-PDK1, which exhibited PDK1 activity similar to that of wt-PDK1, was detected exclusively in the cytosol fraction. Insulin dose dependently activated protein kinase B (PKB) but did not change atypical protein kinase C (aPKC) activity in control cells. aPKC activity was not affected by expression of wt-, kd-, or ⌬PH-PDK1 in either the presence or the absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced activation of PKB as well as insulin-induced phosphorylation of glycogen synthase kinase (GSK)3␣/␤, a direct downstream target of PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither ⌬PH-PDK1 nor kd-PDK1 expression affected PKB activity, GSK3 phosphorylation, or glycogen synthesis. Thus membrane localization of PDK1 via its PH domain is essential for insulin signaling through the PDK1-PKB-GSK3␣/␤ pathway. Glucose transport activity was unaffected by expression of wt-PDK1, kd-PDK1, or ⌬PH-PDK1 in either the presence or the absence of insulin. These findings suggest the presence of a signaling pathway for insulinstimulated glucose transport in which PDK1 to PKB or aPKC is not involved. glycogen synthesis; glucose transport; GLUT4; protein kinase B; protein kinase C INSULIN STIMULATES GLUCOSE UPTAKE into muscle and adipose tissues by redistribution of the insulin-responsive glucose transporter GLUT4 from intracellular stores to the plasma membrane. Insulin transmits its signals through a cell surface tyrosine kinase receptor, which stimulates multiple intracellular signaling events (49). Activated insulin receptors phosphorylate adapter proteins, members of the insulin receptor substrate family, which recruit and activate downstream effector molecules. Among the downstream proteins, activation of phosphatidylinositol 3-kinase (PI 3-kinase) is required for insulin's acute glucose metabolism-regulating actions, such as acceleration of glucose transport and glycogen synthesis.PI 3-kinase (pharmacological) inhibitors, e.g., wortmannin and LY-294002, reportedly completely inhibit the insulin-induced activation of glycogen synthase in a variety of cell models (27,35,43,45). Glycogen synthase kinase 3 (GSK3) plays an important role in the regulation of glycogen synthesis via inhibitory phosphorylation of glycogen synthase (22,50). Two isoforms of GSK3, GSK3␣ and -␤, are broadly expressed and play multiple regulatory roles in development and metabolism (40). GSK3 is constitutively active in cells and is transiently inhibited after insulin treatment (17). Insulin-induced inactivation of GSK3 by insulin appears to be mediated by phosphorylation of GSK3 at...