Flavonoids, polyphenolic compounds that exist widely in plants, inhibit cell proliferation and increase cell differentiation in many cancerous and noncancerous cell lines. Because terminal differentiation of preadipocytes to adipocytes depends on proliferation of both pre- and postconfluent preadipocytes, we predicted that flavonoids would inhibit adipogenesis in the 3T3-L1 preadipocyte cell line. The flavonoids genistein and naringenin inhibited proliferation of preconfluent preadipocytes in a time- and dose-dependent manner. When added to 2-day postconfluent preadipocytes at the induction of differentiation, genistein inhibited mitotic clonal expansion, triglyceride accumulation, and peroxisome proliferator-activated receptor-gamma expression, but naringenin had no effect. The antiadipogenic effect of genistein was not due to inhibition of insulin receptor subtrate-1 tyrosine phosphorylation. When added 3 days after induction of differentiation, neither flavonoid inhibited differentiation. In fully differentiated adipocytes, genistein increased basal and epinephrine-induced lipolysis, but naringenin had no significant effects. These data demonstrate that genistein and naringenin, despite structural similarity, have differential effects on adipogenesis and adipocyte lipid metabolism.

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BackgroundIncreased prevalences of diabetes mellitus have been reported among individuals chronically exposed to inorganic arsenic (iAs). However, the mechanisms underlying the diabetogenic effects of iAs have not been characterized. We have previously shown that trivalent metabolites of iAs, arsenite (iAsIII) and methylarsonous acid (MAsIII) inhibit insulin-stimulated glucose uptake (ISGU) in 3T3-L1 adipocytes by suppressing the insulin-dependent phosphorylation of protein kinase B (PKB/Akt).ObjectivesOur goal was to identify the molecular mechanisms responsible for the suppression of PKB/Akt phosphorylation by iAsIII and MAsIII.MethodsThe effects of iAsIII and MAsIII on components of the insulin-activated signal transduction pathway that regulate PKB/Akt phosphorylation were examined in 3T3-L1 adipocytes.ResultsSubtoxic concentrations of iAsIII or MAsIII had little or no effect on the activity of phosphatidylinositol 3-kinase (PI-3K), which synthesizes phosphatidylinositol-3,4,5-triphosphate (PIP3), or on phosphorylation of PTEN (phosphatase and tensin homolog deleted on chromosome ten), a PIP3 phosphatase. Neither iAsIII nor MAsIII interfered with the phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK-1) located downstream from PI-3K. However, PDK-1 activity was inhibited by both iAsIII and MAsIII. Consistent with these findings, PDK-1-catalyzed phosphorylation of PKB/Akt(Thr308) and PKB/Akt activity were suppressed in exposed cells. In addition, PKB/Akt(Ser473) phosphorylation, which is catalyzed by a putative PDK-2, was also suppressed. Notably, expression of constitutively active PKB/Akt restored the normal ISGU pattern in adipocytes treated with either iAsIII or MAsIII.ConclusionsThese results suggest that inhibition of the PDK-1/PKB/Akt-mediated transduction step is the key mechanism for the inhibition of ISGU in adipocytes exposed to iAsIII or MAsIII, and possibly for impaired glucose tolerance associated with human exposures to iAs.

The enzymatic methylation of inorganic As (iAs) is catalyzed by As(+3 oxidation state)-methyltransferase (AS3MT). AS3MT is expressed in rat liver and in human hepatocytes. However, AS3MT is not expressed in UROtsa, human urothelial cells that do not methylate iAs. Thus, UROtsa cells are an ideal null background in which the role of iAs methylation in modulation of toxic and cancer-promoting effects of this metalloid can be examined. A retroviral gene delivery system was used in this study to create a clonal UROtsa cell line (UROtsa/F35) that expresses rat AS3MT. Here, we characterize the metabolism and cytotoxicity of arsenite (iAs III ) and methylated trivalent arsenicals in parental cells and clonal cells expressing AS3MT. In contrast to parental cells, UROtsa/ F35 cells effectively methylated iAs III , yielding methylarsenic (MAs) and dimethylarsenic (DMAs) containing either As III or As V . When exposed to MAs III , UROtsa/F35 cells produced DMAs III and DMAs V . MAs III and DMAs III were more cytotoxic than iAs III in UROtsa and UROtsa/F35 cells. The greater cytotoxicity of MAs III or DMAs III than of iAs III was associated with greater cellular uptake and retention of each methylated trivalent arsenical. Notably, UROtsa/F35 cells were more sensitive than parental cells to the cytotoxic effects of iAs III but were more resistant to cytotoxicity of MAs III . The increased sensitivity of UROtsa/F35 cells to iAs III was associated with inhibition of DMAs production and intracellular accumulation of MAs. The resistance of UROtsa/F35 cells to moderate concentrations of MAs III was linked to its rapid conversion to DMAs and efflux of DMAs. However, concentrations of MAs III that inhibited DMAs production by UROtsa/F35 cells were equally toxic for parental and clonal cell lines. Thus, the production and accumulation of MAs III is a key factor contributing to the toxicity of acute iAs exposures in methylating cells.

The tyrosine kinase inhibitor genistein inhibits 3T3-L1 adipogenesis when present during the first 72 h of differentiation. In this report, we investigated the underlying mechanisms involved in the anti-adipogenic effects of genistein. We found that genistein blocked the DNA binding and transcriptional activity of CCAAT/enhancer-binding protein beta (C/EBPbeta) during differentiation by promoting the expression of C/EBP homologous protein, a dominant-negative member of the C/EBP family. Loss of C/EBPbeta activity was manifested as a loss of differentiation-induced C/EBPalpha and peroxisome-proliferator-activated receptor gamma protein expression and a dramatic reduction in lipid accumulation. Further, we documented for the first time that C/EBPbeta was tyrosine-phosphorylated in vivo during differentiation and in vitro by activated epidermal growth factor receptor. Genistein inhibited both of these events. Collectively, these results indicate that genistein blocks adipogenesis and C/EBPbeta activity by increasing the level of C/EBP homologous protein and possibly by inhibiting the tyrosine phosphorylation of C/EBPbeta.