FL83B Cells Research Articles

Protective effects of the crude extracts from yam (Dioscorea alata) peel on tert-butylhydroperoxide-induced oxidative stress in mouse liver cells

Researchers have shown that yam extracts contain antioxidative activity; however, there are few reports regarding the antioxidant activities of yam peel. The effects of water and 50% ethanolic extracts from Darsan yam (Dioscorea alata) peel on the oxidative status of tert-butylhydroperoxide (t-BHP)-treated mouse Hepa 1–6 and FL83B liver cell lines were investigated. The cytosols were analysed for H2O2 and malondialdehyde (MDA) levels and antioxidative enzymes activities, including superoxide dismutase, glutathione peroxidase (GPx) and catalase activities. Both water and 50% ethanolic extracts from yam peel did not affect cellular MDA level in t-BHP-treated cells, but they altered the level of H2O2. Water extract from yam peel amplified the t-BHP-induced cytotoxicity in Hepa 1–6 whilst the ethanolic extract showed protection in FL83B cells. GPx activity might play an important role in the protective effect associated with t-BHP-induced oxidative stress.

Starch Binding Domain-containing Protein 1/Genethonin 1 Is a Novel Participant in Glycogen Metabolism

Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.

Effects of Caffeic Acid and Cinnamic Acid on Glucose Uptake in Insulin-Resistant Mouse Hepatocytes

Tumor necrosis factor-alpha was used to induce insulin resistance of mouse liver FL83B cells. Two phenolic acids, caffeic acid and cinnamic acid, were then added separately to investigate their effects on glucose uptake of the insulin-resistant cells. The results suggest that these two phenolic acids may promote insulin receptor tyrosyl phosphorylation, up-regulate the expression of insulin signal associated proteins, including insulin receptor, phosphatidylinositol-3 kinase, glycogen synthase, and glucose transporter-2, increase the uptake of glucose, and alleviate insulin resistance in cells as a consequence.

GRK2-Deficiency Reduces Insulin Activation of ERK1/2 and Mitogenesis in Mouse Liver FL83B Cells

We have reported that G protein coupled receptor kinase-2 (GRK2) negatively regulates insulin receptor signaling leading to glycogen synthesis in mouse liver derived FL83B cells. Since insulin is a potent cellular growth hormone, present study investigated the effect of GRK2 on insulin-stimulated MAP kinase pathway leading to mitogenesis. Specific GRK2 siRNA was used to knock-down (>90%) GRK2 in FL83B cells. Effect of insulin on ERK1/2 activation and [ 3 H]-methyl thymidine incorporation were determined in GRK2 siRNA-treated and control cells. Insulinstimulated ERK1/2 activation was attenuated in GRK2-deficient as compared to control cells. Basal and insulinstimulated [ 3 H]-methyl thymidine incorporation, a measure of mitogenesis, was lower in GRK2-deficient cells. The data suggest that GRK2 may have positive regulatory role in insulin-stimulated MAP kinase pathway and mitogenesis. The present study together with our earlier report on insulin-induced glycogen synthesis indicates a dual role of GRK2 in insulin receptor signaling/function.

GRK2 Negatively Regulates Glycogen Synthesis in Mouse Liver FL83B Cells

G-protein-coupled receptor (GPCR) kinases (GRKs) are serine/threonine kinases that desensitize agonist-occupied classical GPCRs. Although the insulin receptor (IR) is a tyrosine kinase receptor, the IR also couples to G-proteins and utilizes G-protein signaling components. The present study was designed to test the hypothesis that GRK2 negatively regulates IR signaling. FL83B cells, derived from mouse liver, were treated with insulin and membrane translocation of GRK2 was determined using immunofluoresecence and Western blotting. Insulin caused an increase in the translocation of GRK-2 from cytosol to the plasma membrane. To determine the role of GRK2 in IR signaling, GRK2 was selectively down-regulated ( approximately by 90%) in FL83B cells using a small interfering RNA technique. Basal as well as insulin-induced glycogen synthesis (measured by d-[U-(14)C]glucose incorporation) was increased in GRK2-deficient cells compared with control cells. Similarly, GRK2 deficiency increased the basal and insulin-stimulated phosphorylation of Ser(21) in glycogen synthase kinase-3alpha. Insulin-induced tyrosine phosphorylation of the IR was similar in control and GRK2-deficient cells. Basal and insulin-stimulated phosphorylation of Tyr(612) in insulin receptor subunit 1 was significantly increased while phosphorylation of Ser(307) was decreased in GRK2-deficient FL83B cells compared with control cells. Chronic insulin treatment (24 h) in control cells caused an increase in GRK2 (56%) and a decrease in IR (50%) expression associated with the absence of an increase in glycogen synthesis, suggesting impairment of IR function. However, chronic insulin treatment (24 h) did not decrease IR expression or impair IR effects on glycogen synthesis in GRK2-deficient cells. We conclude that (i) GRK2 negatively regulates basal and insulin-stimulated glycogen synthesis via a post-IR signaling mechanism, and (ii) GRK2 may contribute to reduced IR expression and function during chronic insulin exposure.