游离脂肪酸对3T3-L1脂肪细胞糖代谢的影响

目的:通过培养3T3-L1前脂肪细胞,并诱导其分化至成熟,研究游离脂肪酸对脂肪细胞糖代谢的影响。方法:培养诱导3T3-L1脂肪细胞,用油红O染色鉴定并比较其形态结构的变化。LPS、EPA、SA、PA干预成熟脂肪细胞,收集不同时间的培养基,葡萄糖氧化酶法算出各组脂肪细胞的葡萄糖消耗量。用Western blot检测不同时间各组干预后细胞AMPK、GLUT4蛋白含量。结果:油红O染色鉴定成熟脂肪细胞胞浆中的脂滴染成红色,并出现戒环样结构;诱导分化第8天,90%以上细胞均分化成熟。含LPS、EPA、SA、PA的培养基作用于成熟脂肪细胞,随着时间的延长,显著抑制脂肪细胞对葡萄糖的吸收(P<0.05),同时,脂肪细胞AMPK、GLUT4蛋白含量在减少(P<0.05)。结论:游离脂肪酸可以诱导胰岛素抵抗的分子机制可能是通过胰岛素信号通路激活蛋白激酶(AMPK),进而影响GLUT4的蛋白表达,使脂肪细胞的葡萄糖吸收率减低,影响脂肪细胞的糖代谢。     

Role of PDK1 in insulin-signaling pathway for glucose metabolism in 3T3-L1 adipocytes

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the insulin-signaling pathway for glucose metabolism, wild-type (wt), the kinase-dead (kd), or the plecstrin homology (PH) domain deletion (DeltaPH) mutant of PDK1 was expressed using an adenovirus gene transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were found in both membrane and cytosol fractions, whereas DeltaPH-PDK1, which exhibited PDK1 activity similar to that of wt-PDK1, was detected exclusively in the cytosol fraction. Insulin dose dependently activated protein kinase B (PKB) but did not change atypical protein kinase C (aPKC) activity in control cells. aPKC activity was not affected by expression of wt-, kd-, or DeltaPH-PDK1 in either the presence or the absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced activation of PKB as well as insulin-induced phosphorylation of glycogen synthase kinase (GSK)3alpha/beta, a direct downstream target of PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither DeltaPH-PDK1 nor kd-PDK1 expression affected PKB activity, GSK3 phosphorylation, or glycogen synthesis. Thus membrane localization of PDK1 via its PH domain is essential for insulin signaling through the PDK1-PKB-GSK3alpha/beta pathway. Glucose transport activity was unaffected by expression of wt-PDK1, kd-PDK1, or DeltaPH-PDK1 in either the presence or the absence of insulin. These findings suggest the presence of a signaling pathway for insulin-stimulated glucose transport in which PDK1 to PKB or aPKC is not involved.     

Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N ¹-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis.     

Fargesin improves lipid and glucose metabolism in 3T3-L1 adipocytes and high-fat diet-induced obese mice

This study examined the effects of fargesin, a neolignan isolated from Magnolia plants, on obesity and insulin resistance and the possible mechanisms involved in these effects in 3T3-L1 adipocytes and high-fat diet (HFD)-induced obese mice. Fargesin promoted the glucose uptake in 3T3-L1 adipocytes. In HFD-induced obese mice, fargesin decreased the body weight gain, white adipose tissue (WAT), and plasma triglyceride, non-esterified fatty acid and glucose levels, and improved the glucose tolerance. Fargesin increased glucose transporter 4 (GLUT4) protein expression and phosphorylation of Akt, AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC) in both 3T3-L1 adipocytes and WAT of HFD-induced obese mice. Fargesin also decreased the mRNA expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase-1 (CPT-1), uncoupling protein-2 (UCP-2) and leptin in WAT. Taken together, the present findings suggest that fargesin improves dyslipidemia and hyperglycemia by activating Akt and AMPK in WAT. ? 2012 International Union of Biochemistry and Molecular Biology, Inc.     

Development of an intracellular pool of glucose transporters in 3T3-L1 cells.

The membrane-impermeant bis-mannose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) has been used to study the development of an intracellular pool of glucose transporters in 3T3-L1 cells. The subcellular distributions of the transporter isoforms GLUT1 and GLUT4 were determined by comparing the labeling obtained in cells in which the impermeant reagent only had access to the cell surface and the labeling obtained in digitonin-permeabilized cells. ATB-BMPA labeling showed that only GLUT1 was present in preconfluent fibroblasts and that most of the transporters were distributed to the cell surface. In preconfluent fibroblasts, the 2-deoxy-D-glucose transport activity was approximately 5 times higher than in confluent fibroblasts. ATB-BMPA labeling showed that the decrease in transport as cells reached confluence was associated with a decrease in the proportion of GLUT1 distributed to the cell surface. The sequestration of these transporters was associated with the development of an insulin-responsive transport activity which increased by approximately 2.5-fold compared with unstimulated confluent cells. ATB-BMPA labeling showed that insulin stimulation resulted in an approximately 2-fold increase in surface GLUT1 so that about one-half of the available transporters became recruited to the cell surface. Measurements of the changes in the distribution of both GLUT1 and GLUT4 throughout the differentiation of confluent fibroblasts into adipocytes showed that both transporters were sequestered in parallel. Basal levels of transport and photolabeling remained low throughout the differentiation period when the total pool of transporters (GLUT1 plus GLUT4) was increased by approximately 5-fold. These results suggest that the sequestration process was present before new transporters were synthesized. Thus, the sequestration mechanism develops in confluent growth-arrested fibroblasts although the capacity to sequester additional transporters may increase as differentiation proceeds.     

Gallic acid induces GLUT4 translocation and glucose uptake activity in 3T3-L1 cells

GLUT4, a 12 transmembrane protein, plays a major role in insulin mediated glucose transport in muscle and adipocytes. For glucose transport, the GLUT4 protein needs to be translocated to the plasma membrane from the intracellular pool and it is possible that certain compounds may be able to enhance this process. In the present work, we have shown that gallic acid can increase GLUT4 translocation and glucose uptake activity in an Akt-independent but wortmannin-sensitive manner. Further analysis suggested the role of atypical protein kinase Cζ/λ in gallic acid mediated GLUT4 translocation and glucose uptake.     

The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy- methyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA-AM. While the ionophores or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degrees C, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/calmodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.     

Nigericin inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes

We used nigericin, a K+/H+ exchanger, to test whether glucose transport in 3T3-L1 adipocytes was modulated by changes in intracellular pH. Our results showed that nigericin increased basal but decreased insulin-stimulated glucose uptake in a time- and dose-dependent manner. Whereas the basal translocation of GLUT1 was enhanced, insulin-stimulated GLUT4 translocation was inhibited by nigericin. On the other hand, the total amount of neither transporter protein was altered. The finding that insulin-stimulated phosphoinositide 3-kinase (PI 3-kinase) activity was not affected by nigericin implies that nigericin exerted its inhibition at a step downstream of PI 3-kinase activation. At maximal dose, nigericin rapidly lowered cytosolic pH to 6.7; however, this effect was transient and cytosolic pH was back to normal in 20 min. Removal of nigericin from the incubation medium after 20 min abolished its enhancing effect on basal but had little influence on its inhibition of insulin-stimulated glucose transport. Moreover, lowering cytosolic pH to 6.7 with an exogenously added HCl solution had no effect on glucose transport. Taken together, it appears that nigericin may inhibit insulin-stimulated glucose transport mainly by interfering with GLUT4 translocation, probably by a mechanism not related to changes in cytosolic pH.     

Adenovirus transduction of 3T3-L1 cells

3T3-L1 cells offer an excellent model system for studies of differentiation and biochemistry of fat cells. However, these cells are limited in their utility by the low efficiency with which DNA can be introduced by transfection. Gene delivery by viral vectors, particularly adenovirus, has proven a powerful means for introduction of genes into certain cell types. Furthermore, adenovirus transduction has been used to study mechanisms involved in the differentiation of 3T3-L1 cells into mature fat cells. We show in this study that 3T3-L1 cells are inefficiently transduced by adenovirus. The potential advantages offered by adenovirus transduction led us to examine methods designed to enhance transduction of 3T3-L1 cells by adenovirus. Of these methods, polylysine-mediated enhancement demonstrates considerable promise because it permits up to 100% of cells to be transduced and because it does not inhibit differentiation of 3T3-L1 cells. -- Orlicky D. J., and J. Schaack. Adenovirus transduction of 3T3-L1 cells. J. Lipid Res. 2001. 42: 460--466.     

Bilobalide attenuates hypoxia induced oxidative stress,inflammation, and mitochondrial dysfunctions in 3T3-L1 adipocytes via its antioxidant potential

Abstract

Excessive expansion of white adipose tissue leads to hypoxia which is considered as a key factor responsible for adipose tissue dysfunction in obesity. Hypoxia induces inflammation, insulin resistance, and other obesity related complications. So the hypoxia-signalling pathway is expected to provide a new target for the treatment of obesity-associated complications. Inhibition or downregulation of the HIF-1 pathway could be an effective target for the treatment of obesity related hypoxia. In the present study, we evaluated the effect of hypoxia on functions of 3T3-L1 adipocytes emphasising on oxidative stress, antioxidant status, inflammation and mitochondrial functions. We have also evaluated the protective role of bilobalide, a bioactive from Gingko biloba, on hypoxia induced alterations. The results revealed that hypoxia significantly altered all the vital parameters of adipocyte biology like HIF-1α expression (103.47% ↑), lactate and glycerol release (184.34% and 69.1% ↑, respectively), reactive oxygen species (ROS) production (432.53% ↑), lipid and protein oxidation (376.6% and 566.6% ↑, respectively), reduction in antioxidant enzymes (superoxide dismutase and catalase) status, secretion of inflammatory markers (TNF-α, IL-6, IL-1β and IFN-γ) and mitochondrial functions (mitochondrial mass, membrane potential, permeability transition pore integrity, superoxide generation). Bilobalide significantly protected adipocytes from adverse effects of hypoxia in a dose-dependent manner by attenuating oxidative stress, inflammation and protecting mitochondria. Acriflavine (HIF-1 inhibitor) was used as positive control. On the basis of this study, a detailed investigation is needed to delineate the mechanism of action of bilobalide to develop it as therapeutic target for obesity.     

Somatotropin antagonism of insulin-stimulated glucose utilization in 3T3-L1 adipocytes

It is well established that somatotropin (GH) antagonizes insulin action in vivo and that supraphysiologic concentrations of GH frequently result in insulin resistance and glucose intolerance. However, the demonstration of an anti-insulin activity by GH in vitro has been difficult. This study, therefore, set out to determine whether cultures of 3T3-L1 adipocytes could be used to examine the anti-insulin activity of GH. The ability of insulin to stimulate glucose utilization by 3T3-L1 adipocytes increases approximately five-fold during the first 4 days following treatment of the cells with a differentiation medium. It was found that glucose utilization in 3T3-L1 adipocytes is regulated in a reciprocal fashion by insulin and GH. Bovine or human GH directly inhibit up to 50% of insulin-stimulated [14C]-glucose incorporation into lipids in a concentration-dependent manner. The 3T3-L1 sensitivity to GH appears to be at the maximum (50% inhibition of an insulin response) immediately following removal of the cells from the differentiation medium and remains essentially constant during the subsequent 4 days. The GH inhibition of insulin action does not appear to be due GH enhancement of cellular degradation of insulin, competitive binding of GH to the insulin receptor, or GH-induced decrease in cell number. The 3T3-L1 adipocyte system appears to be a sensitive and reliable in vitro model with which to study the molecular mechanisms involved in both GH antagonism of insulin action and development of hormone responsiveness during cellular differentiation into adipocytes.     

Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes

This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1 adipogenesis, was reduced in the presence of MacCM (89%; n = 3; p < 0.001), and BrdU incorporation was impaired by 55% (n = 3; p < 0.01). Activation of ERK1/2 was not affected by MacCM, and neither was the expression of p27^kip1, a cyclin-dependent kinase inhibitor. However, phosphorylation of the retinoblastoma protein (Rb), required for cell cycle progression, was impaired by MacCM (94% inhibition; n = 3; p < 0.01). Differentiation-dependent expression, nuclear localization, and DNA binding ability of C/EBPβ were not inhibited by MacCM. Alterations in cell cycle-associated proteins may be important with respect to the anti-adipogenic action of MacCM.     

Overexpression of H ferritin and up-regulation of iron regulatory protein genes during differentiation of 3T3-L1 pre-adipocytes

The role of iron-dependent oxidative metabolism in protecting the oxidable substrates contained in mature adipocytes is still unclear. Because differentiation increases ferritin formation in several cell types, thereby leading to an accumulation of H-rich isoferritins, we investigated whether differentiation affects iron metabolism in 3T3-L1 pre-adipocytes. To this aim, we evaluated the expression of the genes coding for the H and L ferritin subunits and for cytoplasmic iron regulatory protein (IRP) during the differentiation of 3T3-L1 cells in adipocytes induced by the addition of isobutylmethylxanthine, insulin, and dexamethasone. Differentiation enhanced ferritin formation and caused overexpression of the H subunit, thus altering the H/L subunit ratio. Northern blot analysis showed increased levels of H subunit mRNA. A gel retardation assay of cytoplasmic extract from differentiated cells, using an iron-responsive element as a probe, revealed enhanced an RNA binding capacity of IRP1, which correlated with the increase of IRP1 mRNA. The observed correlation between differentiation and iron metabolism in adipocytes suggests that an accumulation of H-rich isoferritin may limit the toxicity of iron in adipose tissue, thus exerting an antioxidant function.     

MEK inhibitors impair insulin-stimulated glucose uptake in 3T3-L1 adipocytes

In 3T3-L1 adipocytes, insulin activates three major signaling cascades, the phosphoinositide 3-kinase (PI3K) pathway, the Cbl pathway, and the mitogen-activated protein kinase (MAPK) pathway. Although PI3K and Cbl mediate insulin-stimulated glucose uptake by promoting the translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane, the MAPK pathway does not have an established role in insulin-stimulated glucose uptake. We demonstrate in this report that PI3K inhibitors also inhibit the MAPK pathway. To investigate the role of the MAPK pathway separately from that of the PI3K pathway in insulin-stimulated glucose uptake, we used two specific inhibitors of MAPK kinase (MEK) activity, PD-98059 and U-0126, which reduced insulin-stimulated glucose uptake by approximately 33 and 50%, respectively. Neither MEK inhibitor affected the activation of Akt or PKCzeta/lambda, downstream signaling molecules in the PI3K pathway. Inhibition of MEK with U-0126 did not prevent GLUT4 from translocating to the plasma membrane, nor did it inhibit the subsequent docking and fusion of GLUT4-myc with the plasma membrane. MEK inhibitors affected glucose transport mediated by GLUT4 but not GLUT1. Importantly, the presence of MEK inhibitors only at the time of the transport assay markedly impaired both insulin-stimulated glucose uptake and MAPK signaling. Conversely, removal of MEK inhibitors before the transport assay restored glucose uptake and MAPK signaling. Collectively, our studies suggest a possible role for MEK in the activation of GLUT4.     

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.     

Effects of forced uncoupling protein 1 expression in 3T3-L1 cells on mitochondrial function and lipid metabolism

Obesity-related increase in body fat mass is a risk factor for many diseases, including type 2 diabetes. Controlling adiposity by targeted modulation of adipocyte enzymes could offer an attractive alternative to current dietary approaches. Brown adipose tissue, which is present in rodents but not in adult humans, expresses the mitochondrial uncoupling protein 1 (UCP1) that promotes cellular energy dissipation as heat. Here, we report on the direct metabolic effects of forced UCP1 expression in white adipocytes derived from a murine (3T3-L1) preadipocyte cell line. After stable integration, the ucp1 gene product was continuously expressed during differentiation and reduced the total lipid accumulation by approximately 30% without affecting other adipocyte markers, such as cytosolic glycerol-3-phosphate dehydrogenase activity and leptin production. The expression of UCP1 also decreased glycerol output and increased glucose uptake, lactate output, and the sensitivity of cellular ATP content to nutrient removal. However, oxygen consumption and beta-oxidation were minimally affected. Together, our results suggest that the reduction in intracellular lipid by constitutive expression of UCP1 reflects a downregulation of fat synthesis rather than an upregulation of fatty acid oxidation.