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. Diabetes 52: [2657][2658][2659][2660][2661][2662][2663][2664][2665] 2003 I nsulin signaling begins with the binding of insulin to its receptor present on the cell surface, and activation of the insulin receptor tyrosine kinase results in tyrosine phosphorylation of a number of intracellular substrates. These molecules, including the insulin receptor substrate (IRS) family (1), src homology and collagen (2), Gab1 (3), and Grb10 (4), act as adaptor molecules that link between the insulin receptor and downstream signaling effectors. Adaptor protein containing a pleckstrin homology and SH2 domain (APS) is also one of the substrates that tyrosine phosphorylated by insulin receptor kinase (5,6).APS was first described to interact with an oncogenic mutant of the tyrosine kinase receptor c-Kit (7), and APS was isolated by the two-hybrid system using the cytoplasmic domain of the human insulin receptor as bait (5,6). APS (66.5 kDa) forms an adaptor protein family together with Lnk (8,9) and SH2-B (SH2-B␣, SH2-B␤, SH2-B␥, and SH2-B␦) (10 -13), whose members share a homologous NH 2 -terminal region with proline-rich stretches, pleckstrin homology and SH2 domains, and a conserved COOHterminal tyrosine phosphorylation site. It has been demonstrated that some members of this adaptor protein family act as modulators of signaling through various tyrosine kinase receptors. Lnk plays a role in regulating production of B-cell precursors and hematopoietic progenitor cells (8,14). SH2-B is an important signaling molecule in the insulin-like growth factor I (IGF-1) mediated reproductive pathway (13).APS is highly expressed in insulin-r...

Certain nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against HIV-1 and HBV. However, both viruses often acquire NRTI resistance, making it crucial to develop more potent agents that offer profound viral suppression. We report here that 4′-C-cyano-2-amino-2′-deoxyadenosine (CAdA) is a novel highly potent inhibitor of both HBV (IC50=0.4 nM) and HIV-1 (IC50=0.4 nM). In contrast, the approved anti-HBV NRTI entecavir (ETV) potently inhibits HBV (IC50=0.7 nM) but is much less active against HIV-1 (IC50=1,000 nM). Similarly, the highly potent HIV-1 inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) (IC50=0.3 nM) is less active against HBV (IC50=160 nM). Southern analysis using Huh-7 cells transfected with HBV-containing plasmids demonstrated that CAdA was potent against both wild-type (IC50=7.2 nM) and ETV-resistant HBV (IC50=69.6 nM for HBVETV-RL180M/S202G/M204V), whereas ETV failed to reduce HBVETV-RL180M/S202G/M204V DNA even at 1 μM. Once daily peroral administration of CAdA reduced HBVETV-RL180M/S202G/M204V viremia (p=0.0005) in human-liver-chimeric/HBVETV-RL180M/S202G/M204V-infected mice, while ETV completely failed to reduce HBVETV-RL180M/S202G/M204V viremia. None of the mice had significant drug-related body-weight or serum human-albumin concentration changes. Molecular modeling suggests that a shallower HBV-RT hydrophobic pocket at the polymerase active site can better accommodate the slightly shorter 4′-cyano of CAdA-triphosphate (TP), but not the longer 4′-ethynyl of EFdA-TP. In contrast, the deeper HIV-1-RT pocket can efficiently accommodate the 4′-substitutions of both NRTIs. The ETV-TP’s cyclopentyl ring can bind more efficiently at the shallow HBV-RT binding pocket. Conclusion: These data provide insights on the structural and functional associations of HBV- and HIV-1-RTs and show that CAdA may offer new therapeutic options for HBV patients.

Diabetic nephropathy (DN) is the major cause of end-stage renal failure and is associated with increased morbidity and mortality as compared to other causes of renal disease. Albuminuria is often the first clinical indicator of the presence of DN. However, albuminuria or proteinuria is a common symptom in patients with various renal disorders. Therefore, specific biomarkers for the diagnosis of DN are required. A primary hallmark of DN is the progressive damage and death of glomerular podocytes, resulting in the leaking of proteins into the urine. Urinary exosomes released by podocytes are microvesicles containing information of the originated cells. Podocyte-derived signal transduction factors (PDSTFs) are good candidates to assess podocyte injuries. The profile of PDSTFs in urinary exosomes from patients with DN is different from that from patients with minimal change nehrotic syndrome. In addition, PDSTFs molecules in exosomes were derived from primary murine podocytes under high glucose conditions. Among PDSTFs in urinary exosomes, Wilms tumor 1 (WT1) levels reflected damage of diabetic glomeruli in the patients. Urinary exosomal WT1 can predict the decline in eGFR for the following several years. In conclusion, urinary exosomal WT1 is a useful biomarker to improve risk stratification in patients with DN.

Summary The aim of this study was to investigate the effect of dietary lemon polyphenols on high-fat diet-induced obesity in mice, and on the regulation of the expression of the genes involved in lipid metabolism to elucidate the mechanisms. Mice were divided into three groups and fed either a low fat diet (LF) or a high fat diet (HF) or a high fat diet supplemented with 0.5% w/w lemon polyphenols (LP) extracted from lemon peel for 12 weeks. Body weight gain, fat pad accumulation, the development of hyperlipidemia, hyperglycemia, and insulin resistance were significantly suppressed by lemon polyphenols. Supplementation with lemon polyphenols also significantly up-regulated the mRNA level of the peroxisome proliferator activated receptor-α (PPARα) compared to the LF and HF groups in the liver. Furthermore, the mRNA level of acyl-CoA oxidase (ACO) was up-regulated in the LP group compared to the LF group, but not HF group in the liver, and was also significantly increased in the epididymal white adipose tissue. Thus, feeding with lemon polyphenols suppressed body weight gain and body fat accumulation by increasing peroxisomal β-oxidation through upregulation of the mRNA level of ACO in the liver and white adipose tissue, which was likely mediated via up-regulation of the mRNA levels of PPARα.