Naringenin inhibits phosphoinositide 3-kinase activity and glucose uptake in 3T3-L1 adipocytes

Previous studies have shown that flavonoids inhibit glucose uptake in cultured cells. In this report, we show that the grapefruit flavanone naringenin inhibited insulin-stimulated glucose uptake in 3T3-L1 adipocytes in a dose-dependent manner. Naringenin acts by inhibiting the activity of phosphoinositide 3-kinase (PI3K), a key regulator of insulin-induced GLUT4 translocation. Although naringenin did not alter the phosphotyrosine status of the insulin receptor, insulin receptor substrate proteins, or PI3K, it did inhibit the phosphorylation of the downstream signaling molecule Akt. In an in vitro kinase assay, naringenin inhibited PI3K activity. A physiologically attainable dose of 6 microM naringenin reduced insulin-stimulated glucose uptake by approximately 20%. This inhibitory effect remained 24h after the removal of naringenin from the culture medium. Collectively, our findings suggest that the regular consumption of naringenin in grapefruit may exacerbate insulin resistance in susceptible individuals via impaired glucose uptake in adipose tissue.     

Localization and insulin-regulated relocation of phosphoinositide 5-kinase PIKfyve in 3T3-L1 adipocytes

The mammalian phosphoinositide kinase PIKfyve catalyzes the synthesis of phosphatidylinositol 5-P and phosphatidylinositol 3,5-P(2), thought essential in cellular functions, including membrane trafficking. To discern the intracellular loci of PIKfyve products' formation, we have examined the localization of PIKfyve protein versus enzymatic activity and a possible acutely regulated redistribution in 3T3-L1 adipocytes. Subcellular fractions of resting cells that were positive for immunoreactive PIKfyve, such as cytosol ( approximately 76%), internal structures (low density microsomal fraction (LDM), composed of recycling endosomes, GLUT4 storage compartment, Golgi, and cytoskeletal elements) ( approximately 20%), and plasma membrane (( approximately )4%), expressed enzymatically active PIKfyve. While the presence of a FYVE finger in PIKfyve predicts early endosome targeting, density gradient sedimentation, immunoadsorption, and fluorescence microscopy analyses segregated the LDM-associated PIKfyve from the membranes of the recycling endosomes and GLUT4. PIKfyve fluorescence staining largely coincided with trans-Golgi network/multivesicular body markers, indicating PIKfyve's role in the late endocytic/biosynthetic pathways. A subfraction of particulate PIKfyve resisted nonionic detergent treatment, implying association with cytoskeletal structures, previously found positive for key members of the insulin signaling cascade. Upon acute stimulation of 3T3-L1 adipocytes with insulin or pervanadate, a portion of the cytosolic PIKfyve was recruited onto LDM, which was coupled with a commensurate increase of PIKfyve lipid kinase activity and an electrophoretic mobility shift. We suggest the recruited PIKfyve specifies the site and timing of phosphoinositide signals that are relevant to the acute insulin action.     

PTEN, but not SHIP2, suppresses insulin signaling through the phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 adipocytes

Glucose homeostasis is controlled by insulin in part through the stimulation of glucose transport in muscle and fat cells. This insulin signaling pathway requires phosphatidylinositol (PI) 3-kinase-mediated 3'-polyphosphoinositide generation and activation of Akt/protein kinase B. Previous experiments using dominant negative constructs and gene ablation in mice suggested that two phosphoinositide phosphatases, SH2 domain-containing inositol 5'-phosphatase 2 (SHIP2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulate this insulin signaling pathway. Here we directly tested this hypothesis by selectively inhibiting the expression of SHIP2 or PTEN in intact cultured 3T3-L1 adipocytes through the use of short interfering RNA (siRNA). Attenuation of PTEN expression by RNAi markedly enhanced insulin-stimulated Akt and glycogen synthase kinase 3alpha (GSK-3alpha) phosphorylation, as well as deoxyglucose transport in 3T3-L1 adipocytes. In contrast, depletion of SHIP2 protein by about 90% surprisingly failed to modulate these insulin-regulated events under identical assay conditions. In control studies, no diminution of insulin signaling to the mitogen-activated protein kinases Erk1 and Erk2 was observed when either PTEN or SHIP2 were depleted. Taken together, these results demonstrate that endogenous PTEN functions as a suppressor of insulin signaling to glucose transport through the PI 3-kinase pathway in cultured 3T3-L1 adipocytes.     

Involvement of phosphoinositide 3-kinase and PTEN protein in mechanism of activation of TRPC6 protein in vascular smooth muscle cells

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry after the stimulation of a G(q)-protein-coupled or tyrosine-kinase receptor. TRPC6 translocates to the plasma membrane upon stimulation and remains there as long as the stimulus is present. However, the mechanism that regulates the trafficking and activation of TRPC6 are unclear. In this study we showed phosphoinositide 3-kinase and its antagonistic phosphatase, PTEN, are involved in the activation of TRPC6. The inhibition of PI3K by PIK-93, LY294002, or wortmannin decreased carbachol-induced translocation of TRPC6 to the plasma membrane and carbachol-induced net Ca(2+) entry into T6.11 cells. Conversely, a reduction of PTEN expression did not affect carbachol-induced externalization of TRPC6 but increased Ca(2+) entry through TRPC6 in T6.11 cells. We also showed that the PI3K/PTEN pathway regulates vasopressin-induced translocation of TRPC6 to the plasma membrane and vasopressin-induced Ca(2+) entry into A7r5 cells, which endogenously express TRPC6. In summary, we provided evidence that the PI3K/PTEN pathway plays an important role in the translocation of TRPC6 to the plasma membrane and may thus have a significant impact on Ca(2+) signaling in cells that endogenously express TRPC6.     

Phosphoinositide lipid phosphatases: natural regulators of phosphoinositide 3-kinase signaling in T lymphocytes

The phosphoinositide 3-kinase signaling pathway has been implicated in a range of T lymphocyte cellular functions, particularly growth, proliferation, cytokine secretion, and survival. Dysregulation of phosphoinositide 3-kinase-dependent signaling and function in leukocytes, including B and T lymphocytes, has been implicated in many inflammatory and autoimmune diseases. As befits a pivotal signaling cascade, several mechanisms exist to ensure that the pathway is tightly regulated. This minireview focuses on two lipid phosphatases, viz. the 3'-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP (Src homology 2 domain-containing inositol-5-phosphatase). We discuss their role in regulating T lymphocyte signaling as well their potential as future therapeutic targets.     

Effect of differentiation on the adenylate cyclase system of 3T3-C2 and 3T3-L1 cells. Determination of choleragen substrates in differentiating 3T3-L1 and nondifferentiating 3T3-C2 cells

3T3-L1 preadipocytes, when treated with 3-isobutyl-1-methylxanthine, dexamethasone, and insulin, differentiate into cells with the morphological and biochemical properties of adipocytes; the closely related 3T3-C2 cells, under identical conditions, exhibit a low frequency of adipocyte conversion. During differentiation, 3T3-L1 preadipocytes acquire an increased responsiveness to certain agonists (e.g. isoproterenol and adrenocorticotropic hormone) that influence lipolysis and lipogenesis through activation of adenylate cyclase, whereas 3T3-C2 cells do not. It has been suggested that changes in hormone responsiveness of 3T3-L1 cells during differentiation result from increased amounts of the guanyl nucleotide-binding protein of adenylate cyclase, as demonstrated by choleragen-catalyzed [32P]ADP ribosylation of 42 and 49-50-kilodalton particulate peptides. Particulate fractions from nondifferentiating 3T3-C2 cells, like those from 3T3-L1 cells, contained choleragen substrates of 42 and 46-47 (doublet) kilodaltons. Incubation of intact 3T3-L1 or 3T3-C2 cells with choleragen prior to preparation of particulate fractions prevented the subsequent in vitro choleragen-dependent [32P]ADP ribosylation of only these peptides. Increased incorporation of radioactivity into both the 42 and 46-47-kilodalton peptides was observed during differentiation of 3T3-L1 cells. However, a similar increase was also observed in nondifferentiating 3T3-C2 cells subjected to the differentiation protocol. Therefore, increased hormone responsiveness of 3T3-L1 adipocytes cannot be explained solely on the basis of increased labeling, and perhaps increased amounts, of the guanyl nucleotide-binding protein.     

Regulation of ribosomal S6 kinase 2 by effectors of the phosphoinositide 3-kinase pathway

Ribosomal S6 kinase (S6K1), through phosphorylation of the 40 S ribosomal protein S6 and regulation of 5'-terminal oligopyrimidine tract mRNAs, is an important regulator of cellular translational capacity. S6K1 has also been implicated in regulation of cell size. We have recently identified S6K2, a homolog of S6K1, which phosphorylates S6 in vitro and is regulated by the phosphatidylinositide 3-kinase (PI3-K) and mammalian target of rapamycin pathways in vivo. Here, we characterize S6K2 regulation by PI3-K signaling intermediates and compare its regulation to that of S6K1. We report that S6K2 is activated similarly to S6K1 by the PI3-K effectors phosphoinositide-dependent kinase 1, Cdc42, Rac, and protein kinase Czeta but that S6K2 is more sensitive to basal activation by myristoylated protein kinase Czeta than is S6K1. The C-terminal sequence of S6K2 is divergent from that of S6K1. We find that the S6K2 C terminus plays a greater role in S6K2 regulation than does the S6K1 C terminus by functioning as a potent inhibitor of activation by various agonists. Removal of the S6K2 C terminus results in an enzyme that is hypersensitive to agonist-dependent activation. These data suggest that S6K1 and S6K2 are similarly activated by PI3-K effectors but that sequences unique to S6K2 contribute to stronger inhibition of its kinase activity. Understanding the regulation of the two S6K homologs may provide insight into the physiological roles of these kinases.     

Characterization of heterokaryons between skeletal myoblasts and preadipocytes: myogenic potential of 3T3-L1 preadipocytes

It has been shown previously that heterokaryons between myoblasts and non-myogenic cells disturb myogenic differentiation (Hirayama et al. (2001); Cell Struct. Funct. 26, 37-47), suggesting that some myogenesis inhibitory factors exist in non-myogenic cells. Skeletal myoblasts and adipose cells are derived from a common mesodermal stem cell, indicating that both cells have a closer relationship in the developmental lineage than the other somatic cells. To investigate the functional relationship between myoblasts and adipose cells, heterokaryons between quail myoblasts and 3T3-L1 cells, a mouse preadipocyte cell line, were prepared and examined for characteristics of myogenic differentiation. Myogenic differentiation was inhibited in the heterokaryons between quail myoblasts and well-differentiated (adipocytes) 3T3-L1 cells. On the contrary, normal myogenic differentiation proceeded in the heterokaryons between quail myoblasts and undifferentiated (preadipocytes) 3T3-L1 cells. Further investigation showed that the mouse myogenin gene from 3T3-L1 cells was transactivated in the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells. The results demonstrated that undifferentiated 3T3-L1 cells have no myogenesis inhibitory factors but acquire these during terminal differentiation into adipocytes.     

Phosphatidylinositol 3-kinase is required for insulin-stimulated tyrosine phosphorylation of Shc in 3T3-L1 adipocytes

The interactions between the phosphatidylinositol 3-kinase (PI 3-kinase) and Ras/MAPK kinase pathways have been the subject of considerable interest. In the current studies, we find that epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) lead to rapid phosphorylation of Shc (maximum at 1-2 min), whereas insulin-mediated Shc phosphorylation is relatively delayed (maximum at 5-10 min), suggesting that an intermediary step may be necessary for insulin stimulation of Shc phosphorylation. The Src homology-2 (SH2) domain of Shc is necessary for PDGF- and EGF-mediated Shc phosphorylation, whereas the phosphotyrosine binding (PTB) domain is critical for the actions of insulin. Because the Shc PTB domain can interact with phospholipids, we postulated that PI 3-kinase might be a necessary intermediary step facilitating insulin-stimulated phosphorylation of Shc. In support of this, we found that the PI 3-kinase inhibitors, wortmannin and LY294002, blocked insulin-stimulated but not EGF- or PDGF-stimulated Shc phosphorylation. Furthermore, overexpression of a dominant negative PI 3-kinase construct (p85N-SH2) blocked insulin, but not EGF- or PDGF-induced Shc phosphorylation. All three growth factors cause localization of Shc to the plasma membrane, but only the effect of insulin was inhibited by wortmannin, supporting the view that PI 3-kinase-generated phospholipids mediate insulin-stimulated Shc phosphorylation. Consistent with this, expression of a constitutively active PI 3-kinase (p110(C)(AAX)) increased membrane localization of Shc, and this was completely blocked by wortmannin. A mutant Shc with a disrupted PTB domain (Shc S154) did not localize to the membrane in p110(C)(AAX)-expressing cells or after insulin stimulation and was not phosphorylated by insulin. In summary, 1) PI 3-kinase is a necessary early step in insulin-stimulated Shc phosphorylation, whereas the effects of EGF and PDGF on Shc phosphorylation are independent of PI 3-kinase. 2) PI 3-kinase-stimulated generation of membrane phospholipids can localize Shc to the plasma membrane through the Shc PTB domain facilitating phosphorylation by the insulin receptor.     

Heterogeneity in the physiological states and pharmacological responses of differentiating 3T3-L1 preadipocytes

Increases in key components of adipogenesis and lipolysis pathways correlate at the population-averaged level during adipogenesis. However, differentiating preadipocytes are highly heterogeneous in cellular and lipid droplet (LD) morphologies, and the degree to which individual cells follow population-averaged trends is unclear. In this study, we analyze the molecular heterogeneity of differentiating 3T3-L1 preadipocytes using immunofluorescence microscopy. Unexpectedly, we only observe a small percentage of cells with high simultaneous expression of markers for adipogenesis (peroxisome proliferator-activated receptor γ [PPARγ], CCAAT/enhancer-binding protein α, and adiponectin) and lipid accumulation (hormone-sensitive lipase, perilipin A, and LDs). Instead, we identify subpopulations of cells with negatively correlated expressions of these readouts. Acute perturbation of adipocyte differentiation with PPARγ agonists, forskolin, and fatty acids induced subpopulation-specific effects, including redistribution of the percentage of cells in observed subpopulations and differential expression levels of PPARγ. Collectively, our results suggested that heterogeneity observed during 3T3-L1 adipogenesis reflects a dynamic mixture of subpopulations with distinct physiological states.     

The catalytic subunit of phosphoinositide 3-kinase: requirements for oncogenicity

The retroviral oncogene p3k (v-p3k) of avian sarcoma virus 16 (ASV16) codes for the catalytic subunit of phosphoinositide (PI) 3-kinase, p110alpha. The v-P3k protein is oncogenic in vivo and in vitro; its cellular counterpart, c-P3k, lacks oncogenicity. Fusion of viral Gag sequences to the amino terminus of c-P3k activates the transforming potential. Activation can also be achieved by the addition of a myristylation signal to the amino terminus or of a farnesylation signal to the carboxyl terminus of c-P3k. A mutated myristylation signal was equally effective; it also caused a strong increase in the kinase activity of P3k. Mutations that inactivate lipid kinase activity abolish oncogenicity. The transforming activity of P3k is correlated with the ability to induce activating phosphorylation in Akt. Point mutations and amino-terminal deletions recorded in v-P3k were shown to be irrelevant to the activation of oncogenic potential. Interactions of P3k with the regulatory subunit of PI 3-kinase, p85, or with Ras are not required for transformation. These results support the conclusion that the oncogenicity of P3k depends on constitutive lipid kinase activity. Akt is an important and probably essential downstream component of the oncogenic signal from P3k.     

Effects of sterculic acid on stearoyl-CoA desaturase in differentiating 3T3-L1 adipocytes

The effects of sterculic acid on cell size, adiposity, and fatty acid composition of differentiating 3T3-L1 adipocytes are correlated with stearoyl-CoA desaturase (SCD) expression (mRNA and protein levels) and enzyme activity. Fluorescence-activated cell scanning (FACS) analysis showed that adipocytes differentiated with methylisobutylxanthine, dexamethasone, and insulin (MDI) plus 100 microM sterculic acid comprised a population of predominantly large cells with reduced adiposity compared to MDI-treated cells. Although both groups had similar amounts of total fat, their fatty acid profiles were strikingly different: MDI-treated cells had high levels of the unsaturated palmitoleic (Delta(9)-16:1) and oleic (Delta(9)-18:1) acids, whereas the cells cultured with MDI plus sterculic acid accumulated palmitic (16:0) and stearic (18:0) acids together with a marked reduction in Delta(9)-16:1. Although the cells treated with MDI plus sterculic acid had similar levels of scd1 and scd2 mRNAs and antibody-detectable SCD protein as the MDI-treated cells, the SCD enzyme activity was inhibited more than 90%. The accumulation of 16:0 and 18:0, together with normal levels of fatty acid synthase (FAS) and aP2 mRNAs, shows that de novo synthesis and elongation of fatty acids, as well as cell differentiation, were not affected by sterculic acid. Because of the increase in cell size in the sterculic acid-treated cells, the insulin-stimulated 2-deoxyglucose (2-DOG) uptake was determined. Compared to MDI-treated cells, the 2-DOG uptake in the cells treated with sterculic acid was not affected. These results indicate that sterculic acid directly inhibits SCD activity, possibly by a turnover-dependent reaction, without affecting the processes required for adipocyte differentiation, scd gene expression or SCD protein translation.     

Cdc42 and phosphoinositide 3-kinase drive Rac-mediated actin polymerization downstream of c-Met in distinct and common pathways

Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise contributions of distinct Rho-GTPases, the phosphatidylinositol 3-kinases, and actin assembly regulators to c-Met-mediated actin reorganization are still elusive. Here we report that HGF-induced membrane ruffling and Listeria invasion mediated by the bacterial c-Met ligand internalin B (InlB) were significantly impaired but not abrogated upon genetic removal of either Cdc42 or pharmacological inhibition of phosphoinositide 3-kinase (PI3-kinase). While loss of Cdc42 or PI3-kinase function correlated with reduced HGF- and InlB-triggered Rac activation, complete abolishment of actin reorganization and Rac activation required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N-WASP. Instead, actin polymerization was driven by Arp2/3 complex activation through the WAVE complex downstream of Rac. Together, our data establish an intricate signaling network comprising as key molecules Cdc42 and PI3-kinase, which converge on Rac-mediated actin reorganization essential for Listeria invasion and membrane ruffling downstream of c-Met.     

The role of phosphoinositide 3-kinase C2alpha in insulin signaling

The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2alpha, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2alpha involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2alpha and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2alpha contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.     

Functional interactions of phosphatidylinositol 3-kinase with GTPase-activating protein in 3T3-L1 adipocytes.

The role of phosphatidylinositol (PI) 3-kinase in specific aspects of insulin signaling was explored in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity by LY294002 or wortmannin significantly enhanced basal and insulin-stimulated GTPase-activating protein (GAP) activity in 3T3-L1 adipocytes. Furthermore, removal of the inhibitory influence of PI 3-kinase on GAP resulted in dose-dependent decreases in the ability of insulin to stimulate p21ras. This effect was specific to adipocytes, as inhibition of PI 3-kinase did not influence GAP in either 3T3-L1 fibroblasts, Rat-1 fibroblasts, or CHO cells. Immunodepletion of either of the two subunits of the PI 3-kinase (p85 or p110) yielded similar activation of GAP, suggesting that catalytic activity of p110 plays an important role in controlling GAP activity in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity in 3T3-L1 adipocytes resulted in abrogation of insulin-stimulated glucose uptake and thymidine incorporation. In contrast, effects of insulin on glycogen synthase and mitogen-activated protein kinase activity were inhibited only at higher concentrations of LY294002. It appears that in adipocytes, P1 3-kinase prevents activation of GAP. Inhibition of PI 3-kinase activity or immunodepletion of either one of its subunits results in activation of GAP and decreases in GTP loading of p21ras.     

Trigonelline attenuates the adipocyte differentiation and lipid accumulation in 3T3-L1 cells

Trigonelline is a natural alkaloid mainly found in Trigonella Foenum Graecum (fenugreek) Fabaceae and other edible plants with a variety of medicinal applications. Therefore, we investigated the molecular mechanism of trigonelline (TG) on the inhibition of adipocyte differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline suppressed lipid droplet accumulation in a concentration (75 and 100 μM) dependent manner. Treatment of adipocyte with of TG down regulates the peroxisome proliferator-activated receptor (PPARγ) and CCAAT element binding protein (C/EBP-α) mRNA expression, which leads to further down regulation of other gene such as adiponectin, adipogenin, leptin, resistin and adipocyte fatty acid binding protein (aP2) as compared with respective control cells on 5th and 10th day of differentiation. Further, addition of triognelline along with troglitazone to the adipocyte attenuated the troglitazone effects on PPARγ mediated differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline might compete against troglitazone for its binding to the PPARγ. In addition, adipocyte treated with trigonelline and isoproterenol separately. Isoproterenol, a lipolytic agent which inhibits the fatty acid synthase and GLUT-4 transporter expression via cAMP mediated pathway, we found that similar magnitude response of fatty acid synthase and GLUT-4 transporter expression in trigonelline treated adipocyte. These results suggest that the trigonelline inhibits the adipogenesis by its influences on the expression PPARγ, which leads to subsequent down regulation of PPAR-γ mediated pathway during adipogenesis. Our findings provide key approach to the mechanism underlying the anti-adipogenic activity of trigonelline.