An enzymatic photometric assay for 2-deoxyglucose uptake in insulin-responsive tissues and 3T3-L1 adipocytes

An enzymatic assay adapted to photometric analysis with 96-well microplates was evaluated for the measurement of 2-deoxyglucose (2DG) uptake in insulin-responsive tissues and differentiated 3T3-L1 adipocytes. For in vivo measurements, a small amount of nonradiolabeled 2DG was injected into mice without affecting glucose metabolism. For photometric quantification of the small amount of 2-deoxyglucose 6-phosphate (2DG6P) that accumulates in cells, we introduced glucose-6-phosphate dehydrogenase, glutathione reductase, and 5,5′-dithiobis(2-nitrobenzoic acid) to the recycling amplification reaction of NADPH. We optimized the enzyme reaction for complete oxidation of endogenous glucose 6-phosphate (G6P) and glucose in mouse tissues in vivo and serum as well as in 3T3-L1 adipocytes in vitro. All reactions are performed in one 96-well microplate by consecutive addition of reagents, and the assay is able to quantify 2DG and 2DG6P in the range of 5–80 pmol. The results obtained with the assay for 2DG uptake in vitro and in vivo in the absence or presence of insulin stimulation was similar to those obtained with the standard radioisotopic method. Thus, the enzymatic assay should prove to be useful for measurement of 2DG uptake in insulin-responsive tissues in vivo as well as in cultured cells.     

Simvastatin enhances induction of inducible nitric oxide synthase in 3T3-L1 adipocytes

The present study was designed to determine whether hydroxymethylglutaryl-CoA reductase inhibitors (statins) modulate the NO production via iNOS in adipocytes stimulated by lipopolysaccharide (L) and tumour necrosis factor-alpha (T). Well-differentiated 3T3-L1 adipocytes significantly produced NO by LT-treatment. Pre-incubation with simvastatin, a lipophilic statin, pravastatin, a hydrophilic one, or Y27632, an inhibitor of Rho kinase, further enhanced the production of NO. The effect of simvastatin was offset by mevalonate and geranylgeranyl pyrophosphate (GGPP) but not by squalene. The mRNA level for iNOS parallelled the NO production. The NF-kappaB was activated by the LT-treatment and was further enhanced by simvastatin, pravastatin or Y27632 addition. Mevalonate and GGPP completely offset the effect of simvastatin. Statins and Y27632 also further increased the interleukin-6 secretion in the LT-treated 3T3-L1 adipocytes. These results suggest that statins, especially lipophilic type, enhance induction of iNOS by inhibiting the small GTP-binding protein signal in adipocytes.     

Regulation of haptoglobin gene expression in 3T3-L1 adipocytes by cytokines, catecholamines, and PPARgamma

Factors which regulate expression of the haptoglobin (acute phase reactant) gene in adipocytes have been examined using 3T3-L1 cells. Haptoglobin expression was observed by Northern blotting in each of the major white adipose tissue depots of mice (epididymal, subcutaneous, mesenteric, and perirenal) and in interscapular brown fat. Expression occurred in mature adipocytes, but not in the stromal-vascular fraction. In 3T3-L1 cells, haptoglobin mRNA was detected from day 4 after the induction of differentiation into adipocytes. Lipopolysaccharide and the cytokines, TNFalpha and interleukin-6, resulted in substantial increases in haptoglobin mRNA in 3T3-L1 adipocytes; the increase (7-fold) was highest with TNFalpha. Increases in haptoglobin mRNA level were also induced by dexamethasone, noradrenaline, isoprenaline, and a beta3-adrenoceptor agonist. In contrast, haptoglobin mRNA was reduced by nicotinic acid and the PPARgamma agonist, rosiglitazone. RT-PCR showed that the haptoglobin gene was expressed in human adipose tissue (subcutaneous, omental). It is concluded that haptoglobin gene expression in adipocytes is stimulated by inflammatory cytokines, glucocorticoids, and the sympathetic system, while activation of the PPARgamma nuclear receptor is strongly inhibitory.     

Simvastatin enhances induction of inducible nitric oxide synthase in 3T3-L1 adipocytes

The present study was designed to determine whether hydroxymethylglutaryl-CoA reductase inhibitors (statins) modulate the NO production via iNOS in adipocytes stimulated by lipopolysaccharide (L) and tumour necrosis factor-α (T). Well-differentiated 3T3-L1 adipocytes significantly produced NO by LT-treatment. Pre-incubation with simvastatin, a lipophilic statin, pravastatin, a hydrophilic one, or Y27632, an inhibitor of Rho kinase, further enhanced the production of NO. The effect of simvastatin was offset by mevalonate and geranylgeranyl pyrophosphate (GGPP) but not by squalene. The mRNA level for iNOS parallelled the NO production. The NF-κB was activated by the LT-treatment and was further enhanced by simvastatin, pravastatin or Y27632 addition. Mevalonate and GGPP completely offset the effect of simvastatin. Statins and Y27632 also further increased the interleukin-6 secretion in the LT-treated 3T3-L1 adipocytes. These results suggest that statins, especially lipophilic type, enhance induction of iNOS by inhibiting the small GTP-binding protein signal in adipocytes.     

Pioglitazone upregulates adiponectin receptor 2 in 3T3-L1 adipocytes

AimsPioglitazone, a full peroxisome proliferator-activated receptor (PPAR)-γ agonist, improves insulin sensitivity by increasing circulating adiponectin levels. However, the molecular mechanisms by which pioglitazone induces insulin sensitization are not fully understood. In this study, we investigated whether pioglitazone improves insulin resistance via upregulation of either 2 distinct receptors for adiponectin (AdipoR1 or AdipoR2) expression in 3T3-L1 adipocytes.Main methodsGlucose uptake was evaluated by 2-[³H] deoxy-glucose uptake assay in 3T3-L1 adipocytes with pioglitazone treatment. AdipoR1 and AdipoR2 mRNA expressions were analyzed by qRT–PCR.Key findingsWe first confirmed that pioglitazone significantly increased insulin-induced 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes. Next, we investigated the mRNA expression and regulation of AdipoR1 and AdipoR2 after treatment with pioglitazone. Interestingly, pioglitazone significantly induced AdipoR2 expression but it did not affect AdipoR1 expression. In addition, adenovirus-mediated PPARγ expression significantly enhanced the effects of pioglitazone on insulin-stimulated 2-DOG uptake and AdipoR2 expression in 3T3-L1 adipocytes. These data suggest that pioglitazone enhances adiponectin's autocrine and paracrine actions in 3T3-L1 adipocytes via upregulation of PPARγ-mediated AdipoR2 expression. Furthermore, we found that pioglitazone significantly increased AMP-activated protein kinase (AMPK) phosphorylation in insulin-stimulated 3T3-L1 adipocytes, but it did not lead to the phosphorylation of IRS-1, Akt, or protein kinase Cλ/ζ.SignificanceOur results suggest that pioglitazone increases insulin sensitivity, at least partly, by PPARγ-AdipoR2-mediated AMPK phosphorylation in 3T3-L1 adipocytes. In conclusion, the upregulation of AdipoR2 expression may be one of the mechanisms by which pioglitazone improves insulin resistance in 3T3-L1 adipocytes.     

Dual action of isoprenols from herbal medicines on both PPARgamma and PPARalpha in 3T3-L1 adipocytes and HepG2 hepatocytes

Several herbal medicines improve hyperlipidemia, diabetes and cardiovascular diseases. However, the molecular mechanism underlying this improvement has not yet been clarified. In this study, we found that several isoprenols, common components of herbal plants, activate human peroxisome proliferator-activated receptors (PPARs) as determined using the novel GAL4 ligand-binding domain chimera assay system with coactivator coexpression. Farnesol and geranylgeraniol that are typical isoprenols in herbs and fruits activated not only PPARgamma but also PPARalpha as determined using the chimera assay system. These compounds also activated full-length human PPARgamma and PPARalpha in CV1 cells. Moreover, these isoprenols upregulated the expression of some lipid metabolic target genes of PPARgamma and PPARalpha in 3T3-L1 adipocytes and HepG2 hepatocytes, respectively. These results suggest that herbal medicines containing isoprenols with dual action on both PPARgamma and PPARalpha can be of interest for the amelioration of lipid metabolic disorders associated with diabetes.     

Methotrexate enhances 3T3-L1 adipocytes hypertrophy

Methotrexate (MTX) is broadly used in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA). The prevalence of metabolic syndrome (MeS) in patients with this condition is relatively high. Given the importance of adipose tissue in the development of obesity metabolic complications, this study aimed to investigate the effect of methotrexate on preadipocyte proliferation, adipogenesis, and glucose uptake by adipocytes. 3T3-L1 preadipocytes proliferation was evaluated by sulforhodamine B staining and ³H-thymidine incorporation, after 24 or 48 h of treatment with MTX (0.1 and 10 μM). Preadipocytes were induced to differentiate with an appropriate adipogenic cocktail in the presence or absence of MTX. Adipogenesis was determined by measuring lipid accumulation after staining with oil red O. ³H-Deoxyglucose (³H-DG) uptake was determined by liquid scintillation counting. MTX treatment reduced culture protein content in a concentration-dependent manner and ³H-thymidine incorporation (P?<?0.05). MTX (0.1 μM) treatment increased lipid accumulation and basal ³H-DG uptake by adipocytes (P?<?0.05). In 0.1 μM MTX-treated adipocytes, insulin stimulation did not result in an increase of ³H-DG uptake, contrarily to what was observed in control cells. These results demonstrate that methotrexate interferes with adipocyte proliferation and promotes the hypertrophic growth of adipocytes. These molecular effects may have implications on metabolic profile of RA patients treated with MTX.     

Endothelin-1 induces lipolysis in 3T3-L1 adipocytes

Endothelin-1 (ET-1) affects glucose uptake in adipocytes and may play an important role in adipose physiology. One of the principal functions of adipose tissue is the provision of energy substrate through lipolysis. In the present study, we investigated the effects of ET-1 on lipolysis in 3T3-L1 adipocytes. When glycerol release in the culture medium was measured as an index of lipolysis, the results showed that ET-1 caused a significant increase that was time and dose dependent. With a concentration of 10 nM ET-1, stimulation of glycerol release plateaued after 4 h of exposure. This effect was inhibited by the ETA receptor antagonist BQ-610 (10 microM) but not by the ETB receptor antagonist BQ-788 (10 microM). To further explore the underlying mechanisms of ET-1 action, we examined the involvement of the cAMP-dependent protein kinase A-mediated, phospholipase A2 (PLA2)-mediated, protein kinase C (PKC)-mediated, phosphatidylinositol 3 (PI 3)-kinase-mediated, and the mitogen-activated protein kinase (MAPK)-mediated pathways. Inhibition of adenylyl cyclase activation by SQ-22536 (100 microM) did not block ET-1-induced lipolysis. Pretreatment of adipocytes with the PLA2 inhibitor dexamethasone (100 nM), the PKC inhibitor H-7 (6 microM), or the PI 3-kinase inhibitor wortmannin (100 nM) also had no effect. ET-1-induced lipolysis was blocked by inhibition of extracellular signal-regulated kinase (ERK) activation using PD-98059 (75 microM), whereas a p38 MAPK inhibitor (SB-203580; 20 microM) had no effect. Results of Western blot further demonstrated that ET-1 induced ERK phosphorylation. These data show that ET-1 induces lipolysis in 3T3-L1 adipocytes via a pathway that is different from the conventional cAMP-dependent pathway used by isoproterenol and that involves ERK activation.     

Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes

The insulin-responsive aminopeptidase (IRAP/VP165/gp160) was identified originally in GLUT4-containing vesicles and shown to translocate in response to insulin, much like the glucose transporter 4 (GLUT4). This study characterizes the trafficking and kinetics of IRAP in exocytosis, endocytosis, and recycling to the membrane in 3T3-L1 adipocytes. After exposure of 3T3-L1 adipocytes to insulin, IRAP translocated to the plasma membrane as assessed by either cell fractionation, surface biotinylation, or the plasma membrane sheet assay. The rate of exocytosis closely paralleled that of GLUT4. In the continuous presence of insulin, IRAP was endocytosed with a half-time of about 3-5 min. IRAP endocytosis is inhibited by cytosol acidification, a property of clathrin-mediated endocytosis, but not by the expression of a constitutively active Akt/PKB. Arrival in an LDM fraction derived via subcellular fractionation exhibited a slower time course than disappearance from the cell surface, suggesting additional endocytic intermediates. As assayed by membrane "sheets," GLUT4 and IRAP showed similar internalization rates that are wortmannin-insensitive and occur with a half-time of roughly 5 min. IRAP remaining on the cell surface 10 min following insulin removal was both biotin- and avidin-accessible, implying the absence of thin-necked invaginations. Finally, endocytosed IRAP quickly recycled back to the plasma membrane in a wortmannin-sensitive process. These results demonstrate rapid endocytosis and recycling of IRAP in the presence of insulin and trafficking that matches GLUT4 in rate.     

2'-benzyloxychalcone derivatives stimulate glucose uptake in 3T3-L1 adipocytes

By a cell-based glucose uptake screening assay, a chalcone derivative, 3-nitro-2'-benzyloxychalcone (compound 1) was identified. Compound 1 stimulated glucose uptake and potentiated insulin-stimulated glucose uptake in a concentration-dependent manner in 3T3-L1 adipocytes. When cells were treated with various concentrations of insulin in the presence of compound 1, marked enhancement of insulin-stimulated glucose uptake was observed at each concentration, suggesting that the compound might function as an insulin sensitizer. Preliminary study on the structure-activity relationships revealed that two aromatic benzene rings tolerated several substituents, but substitution by acidic or highly polar groups abolished the activity. Among several chalcone derivatives, 4-chloro-2'-benzyloxychalcone (compound 8) showed the highest level of activity. Compound 8-stimulated glucose uptake was almost completely inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). These results suggest that the action of chalcone derivatives is mediated via a pathway involving PI3K.     

Glyburide action in cultured 3T3-L1 adipocytes

During adipocyte differentiation of 3T3-L1 cells, glyburide increased the specific activity (mU/mg protein) of glycerol-3-P dehydrogenase (by at least 14-fold) and glutamine synthetase (by 5-fold). The glyburide-mediated increases in enzyme activities were greater in the presence than in the absence of insulin. Our data indicate that glyburide either potentiates or mimics the actions of insulin to increase the activity of glycerol-3-P dehydrogenase during adipocyte differentiation of cultured 3T3-L1 cells.     

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes

To study molecular mechanisms for glucosamine-induced insulin resistance, we induced complete and reversible insulin resistance in 3T3-L1 adipocytes with glucosamine in a dose- and time-dependent manner (maximal effects at 50 mM glucosamine after 6 h). In these cells, glucosamine impaired insulin-stimulated GLUT-4 translocation. Glucosamine (6 h) did not affect insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 and -2 and weakly, if at all, impaired insulin stimulation of phosphatidylinositol 3-kinase. Glucosamine, however, severely impaired insulin stimulation of Akt. Inhibition of insulin-stimulated glucose transport was correlated with that of Akt activity. In these cells, glucosamine also inhibited insulin stimulation of p70 S6 kinase. Glucosamine did not alter basal glucose transport and insulin stimulation of GLUT-1 translocation and mitogen-activated protein kinase. In summary, glucosamine induced complete and reversible insulin resistance in 3T3-L1 adipocytes. This insulin resistance was accompanied by impaired insulin stimulation of GLUT-4 translocation and Akt activity, without significant impairment of upstream molecules in insulin-signaling pathway.     

Physiological significance of the taurine transporter and taurine biosynthetic enzymes in 3T3-L1 adipocytes

The intracellular level of taurine is maintained both by the taurine transporter (TAUT) and by endogenous synthesis from Met and Cys. We investigated in the present study the regulation of TAUT and of cysteine dioxygenase (CDO), one of the major taurine biosynthetic enzymes, in 3T3-L1 adipocytes. The TAUT activity, expression of TAUT and CDO mRNA were up-regulated by hypertonicity. In contrast, the TAUT activity, expression of TAUT and CDO mRNA were down-regulated by taurine-rich conditions. Furthermore, it was indicated that the up-regulation of TAUT activity resulted from the increased number of expressed TAUT, and not by the change in affinity of TAUT. On the other hand, the taurine-induced down-regulation of TAUT activity resulted not only from a decrease in the number of expressed TAUT but also from a decrease in their affinity. These results suggest that murine TAUT and CDO were cooperatively regulated in response to hypertonicity and taurine-rich conditions.     

Insulin increases tristetraprolin and decreases VEGF gene expression in mouse 3T3-L1 adipocytes

Objectives: Tristetraprolin (TTP) family proteins (TTP/ZFP36; ZFP36L1, ZFP36L2, ZFP36L3) destabilize adenylate uridylate‐rich element‐containing mRNAs encoding cytokines, such as tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Little is known about the expression and insulin regulation of TTP and related genes in adipocytes. We analyzed the relative abundance of TTP family mRNAs in 3T3‐L1 adipocytes compared to RAW264.7 macrophages and investigated insulin effects on the expression of 43 genes in 3T3‐L1 adipocytes. Methods and Procedures: Insulin was added to mouse 3T3‐L1 adipocytes. Relative abundance of mRNA levels was determined by quantitative real‐time PCR. TTP and ZFP36L1 proteins were detected by immunoblotting. Results: Zfp36l1 and Zfp36l2 genes were expressed at eight‐ to tenfold higher than Ttp in adipocytes. Zfp36l3 mRNA was detected at ～1% of Ttp mRNA levels in adipocytes and its low level expression was confirmed in RAW cells. Insulin at 10 and 100 nmol/l increased Ttp mRNA levels by five‐ to sevenfold, but decreased those of Zfp36l3 by 40% in adipocytes after a 30‐min treatment. Immunoblotting showed that insulin induced TTP but did not affect ZFP36L1 protein levels in adipocytes. Insulin decreased mRNA levels of Vegf and a number of other genes in adipocytes. Discussion: Insulin induced Ttp mRNA and protein expression and decreased Vegf mRNA levels in adipocytes. Zfp36l3 mRNA was detected, for the first time, in cells other than mouse placenta and extraembryonic tissues. This study established a basis for the investigation of TTP and VEGF genes in the regulation of obesity and suggested that Vegf mRNA may be a target of TTP in fat cells.     

LPS and proinflammatory cytokines decrease lipin-1 in mouse adipose tissue and 3T3-L1 adipocytes

Infection and inflammation affect adipose triglyceride metabolism, resulting in increased plasma free fatty acid (FFA) and VLDL levels during the acute-phase response. Lipin-1, a multifunctional protein, plays a critical role in adipose differentiation, mitochondrial oxidation, and triglyceride synthesis. Here, we examined whether LPS [a Toll-like receptor (TLR)-4 activator], zymosan (a TLR-2 activator), and proinflammatory cytokines regulate lipin-1 in adipose tissue. LPS administration caused a marked decrease in the levels of lipin-1 mRNA and protein in adipose tissue. The decrease in lipin-1 mRNA levels occurred rapidly and lasted for at least 24 h. In contrast, lipin-2 and -3 mRNA levels did not change, suggesting specific repression of lipin-1. Zymosan similarly decreased lipin-1 mRNA without affecting lipin-2 or lipin-3 mRNA levels. To determine the pathways by which LPS repressed lipin-1, we examined the effect of proinflammatory cytokines on cultured adipocytes. In 3T3-L1 adipocytes, TNF-alpha, IL-1beta, and IFN-gamma, but not LPS or IL-6, caused a decrease in lipin-1 mRNA levels. Furthermore, TNF-alpha and IL-1beta administration also decreased mRNA levels of lipin-1 in adipose tissue in mice. Importantly, the LPS-induced decrease in lipin-1 mRNA levels was significantly but not totally blunted in TNF-alpha/IL-1 receptor-null mice compared with controls, suggesting key roles for TNF-alpha/IL-1beta and other cytokines in mediating LPS-induced repression of lipin-1. Together, our results demonstrate that expression of lipin-1, one of the essential triglyceride synthetic enzymes, was suppressed by LPS, zymosan, and proinflammatory cytokines in mouse adipose tissue and in cultured 3T3-L1 adipocytes, which could contribute to a decrease in the utilization of FFA to synthesize triglycerides in adipose tissue, thus promoting the release of FFA into the circulation.