Lipid synthesis is promoted by hypoxic adipocyte-derived exosomes in 3T3-L1 cells.

Hypoxia occurs within adipose tissues as a result of adipocyte hypertrophy and is associated with adipocyte dysfunction in obesity. Here, we examined whether hypoxia affects the characteristics of adipocyte-derived exosomes. Exosomes are nanovesicles secreted from most cell types as an information carrier between donor and recipient cells, containing a variety of proteins as well as genetic materials. Cultured differentiated 3T3-L1 adipocytes were exposed to hypoxic conditions and the protein content of the exosomes produced from these cells was compared by quantitative proteomic analysis. A total of 231 proteins were identified in the adipocyte-derived exosomes. Some of these proteins showed altered expression levels under hypoxic conditions. These results were confirmed by immunoblot analysis. Especially, hypoxic adipocyte-released exosomes were enriched in enzymes related to de novo lipogenesis such as acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and fatty acid synthase (FASN). The total amount of proteins secreted from exosomes increased by 3-4-fold under hypoxic conditions. Moreover, hypoxia-derived exosomes promoted lipid accumulation in recipient 3T3-L1 adipocytes, compared with those produced under normoxic conditions. FASN levels were increased in undifferentiated 3T3-L1 cells treated with FASN-containing hypoxic adipocytes-derived exosomes. This is a study to characterize the proteomic profiles of adipocyte-derived exosomes. Exosomal proteins derived from hypoxic adipocytes may affect lipogenic activity in neighboring preadipocytes and adipocytes.

Inhibition of stearoyl-CoA desaturase-1 in differentiating 3T3-L1 preadipocytes upregulates elongase 6 and downregulates genes affecting triacylglycerol synthesis.

BACKGROUND: Stearoyl-CoA desaturase-1 (SCD1) is rate limiting for the conversion of saturated fatty acids palmitate (16:0) and stearate (18:0) to monounsaturated fatty acids palmitoleate (16:1n7) and oleate (18:1n9), respectively. Given that reduced SCD1 activity is associated with improved insulin sensitivity and decreased body weight, there is considerable interest to elucidate the role of this enzyme in adipocytes. During adipogenesis, SCD1 levels increase concomitantly with the accumulation of triacylglycerol (TG); however, the extent to which reduced SCD1 activity can influence TG synthesis and metabolic pathways in differentiating adipocytes remains relatively unexplored. OBJECTIVE: The aim of this work was to delineate how reduced SCD1 activity affects gene expression, protein content and cellular fatty acids in differentiating murine preadipocytes. METHODS: 3T3-L1 preadipocytes were treated with an SCD1 inhibitor (10 nM) throughout differentiation. After 7 days, global gene expression, protein content and fatty acid profiles were examined using microarrays, western blotting and gas chromatography, respectively. RESULTS: SCD1 inhibition increased the abundance of 16:0 and 18:0 (45% and 194%, respectively) and decreased 16:1n7 and 18:1n7 (61% and 35%, respectively) in differentiated preadipocytes. Interestingly, 18:1n9 levels increased by 61%. The augmented 18:0 suggested a possible increase in elongase activity. Elongase 6 (Elovl6) gene expression was increased 2.8-fold (P = 0.04); however, changes were not detected for ELOVL6 protein content. Microarray analysis revealed that genes affecting TG synthesis were downregulated with SCD1 inhibition, which coincided with a 33% decrease in cellular TG content. CONCLUSION: These results provide new mechanistic insight into the role of SCD1 as a regulator of fatty acid profiles and TG synthesis in adipocytes, and reinforce that modulating SCD1 activity may help reduce the risk of obesity-related complications.

N-acetylcysteine inhibits induction of nitric oxide synthase in 3T3-L1 adipocytes.

The present study was designed to determine whether N-acetylcysteine (NAC), a potent antioxidant, modulates nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) in adipocytes. Stimulation by the combination of 5 microg/ml of LPS and 100 ng/ml of TNF-alpha (LT) significantly enhanced NO production in 3T3-L1 adipocytes. Preincubation of the cells with NAC (5-20 mM) for 24 h suppressed the increased NO production in a dose-dependent manner. The production of NO was decreased by 49% at the concentration of 20 mM of NAC. The decrease in NO production by NAC was accompanied by a decrease in inducible nitric oxide synthase (iNOS) protein, detected by immunoblot analysis, and iNOS mRNA, determined by real-time reverse-transcriptase coupled polymerase chain reaction analysis. Nuclear factor-kappa B (NF-kappa B) was significantly activated by LT-treatment, while the pretreatment with 20 mM of NAC prevented the activity by 42%. Pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, also inhibited the LT-mediated NO production dose-dependently. One hundred microM of PDTC inhibited the NO production by 46%. We also investigated the effect of NAC and PDTC on the production of interleukein-6 (IL-6), which is regulated transcriptionally by NF-kappa B in 3T3-L1 adipocytes. IL-6 production was markedly increased by LT stimulus, and the enhanced secretion of IL-6 was suppressed in a dose-dependent manner by pretreatment with NAC or PDTC. These results suggest that NAC regulates iNOS expression and NO production in adipocytes through the modulating activation of NF-kappa B.

Pitavastatin enhanced lipoprotein lipase expression in 3T3-L1 preadipocytes.

It is known that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) enhance the expression- of the low-density lipoprotein (LDL) receptor and lower the level of LDL cholesterol in the blood. But, a triglyceride (TG)-lowering effect is also observed during their administration. To clarify the possibility that statins enhance LPL activity and its mechanism, the effects of statins on the expression of LPL in adipocytes were studied. When statins (pravastatin, simvastatin, atorvastatin and pitavastatin) were added to the culture medium of mouse 3T3-L1 preadipocytes at final concentrations of 1 microM for 3 days, LPL activity increased. Pitavastatin increased the activity the most. Western and Northern blotting showed that LPL protein and m-RNA were strongly expressed on the addition of pitavastatin. With the addition of mevalonate (10 microM, 3 days), LPL activity weakened significantly. Statins, especially pitavastatin, increased the expression of LPL in 3T3-L1 preadipocytes. The TG-lowering effect of pitavastatin might be mediated by enhancement of LPL production in adipocytes.

Bisphenol a accelerates terminal differentiation of 3T3-L1 cells into adipocytes through the phosphatidylinositol 3-kinase pathway.

In order to identify whether bisphenol A (BPA) acts as an adipogenic agent, following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for six days with BPA alone. Treatment with BPA increased the triacylglycerol (TG) content of the cultures, increased the percentage of Oil Red O-staining cells in the cultures, and increased the levels of lipoprotein lipase (LPL) and adipocyte-specific fatty acid binding protein (aP2) mRNAs. These findings indicate that BPA was able to accelerate terminal differentiation of 3T3-L1 cells into adipocytes. LY294002, a chemical inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), blocked completely the increasing effect of BPA on TG accumulation and expression of LPL and aP2 mRNAs. Western blot analysis revealed that BPA increased the level of phosphorylated Akt kinase. Based on these findings, we concluded that BPA acted through the PI 3-kinase and Akt kinase pathway, resulting in increased TG accumulation and expression of adipocyte genes. The structure-activity relationship for BPA-related chemicals was examined. Eight derivatives of BPA (three diphenylalkanes with different substituents at the central carbon atom, three diphenylalkanes with ester bonds on hydroxyl groups in the phenolic rings, one bisphenol consisting of a sulphur atom at the central position, one chemical with cyanic groups, instead of hydroxyl groups, in the phenolic rings) accelerated terminal adipocyte differentiation and their potencies to increase TG accumulation were 73-97% of that of BPA. Two diphenylalkanes with ether bonds on hydroxyl groups and two alkylphenols (4-nonylphenol and 4-tert-octylphenol) did not have the ability to accelerate terminal adipocyte differentiation.

Regulation of GLUT1-mediated glucose uptake by PKClambda-PKCbeta(II) interactions in 3T3-L1 adipocytes.

Members of the PKC (protein kinase C) superfamily play key regulatory roles in glucose transport. How the different PKC isotypes are involved in the regulation of glucose transport is still poorly defined. PMA is a potent activator of conventional and novel PKCs and PMA increases the rate of glucose uptake in many different cell systems. In the present study, we show that PMA treatment increases glucose uptake in 3T3-L1 adipocytes by two mechanisms: a mitogen-activated protein kinase kinase-dependent increase in GLUT1 (glucose transporter 1) expression levels and a PKClambda-dependent translocation of GLUT1 towards the plasma membrane. Intriguingly, PKClambda co-immunoprecipitated with PKCbeta(II) and did not with PKCbeta(I). Previously, we have described that down-regulation of PKCbeta(II) protein levels or inhibiting PKCbeta(II) by means of the myristoylated PKCbetaC2-4 peptide inhibitor induced GLUT1 translocation towards the plasma membrane in 3T3-L1 adipocytes. Combined with the present findings, these results suggest that the liberation of PKClambda from PKCbeta(II) is an important factor in the regulation of GLUT1 distribution in 3T3-L1 adipocytes.

A simple and sensitive method for glutamine:fructose-6-phosphate amidotransferase assay.

For measuring glutamine:fructose-6-phosphate amidotransferase (GFAT) activity in cultured cells, an enzyme method -GDH method- was set up with high-efficiency, high-sensitivity and simple operation by determining the formed glutamate. During the process of making samples, reduced glutathione (GSH, 5 mM) and glucose-6-phosphate Na2 (5 mM) were added to the buffer for scraping the cells. The range of protein content in the samples was 80-150 microg. In the GFAT activity assay, the end product reduced acetylpyridine adenine dinucleotide (APADH) was determined at 370 nm directly. The suitable concentrations of the reactants fructose-6-phosphate (F-6-P), glutamine, acetylpyridine adenine dinucleotide (APAD) and glutamate dehydrogenase (GDH) were 0.8, 6 and 0.3 mM and 6 U, respectively. However, the excess of APAD may interfere with the APADH measurement. The reaction time course was 90 min. The GFAT activity in 3T3-L1, L6, HepG2 and HIRc cells were 1.84-8.51 nmol glutamate/mg protein.min.

Insulin stimulates expression of the pyruvate kinase M gene in 3T3-L1 adipocytes.

M2-type pyruvate kinase (M2-PK) mRNA is produced from the PKM gene by an alternative RNA splicing in adipocytes. We found that insulin increased the level of M2-PK mRNA in 3T3-L1 adipocytes in both time- and dose-dependent manners. This induction did not require the presence of glucose or glucosamine in the medium. The insulin effect was blocked by pharmacological inhibitors of insulin signaling pathways such as wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), and PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase. A stable reporter expression assay showed that the promoter activity of an about 2.2-kb 5'-flanking region of the rat PKM gene was stimulated by insulin, but the extents of these stimulations were lower than those of the mRNA stimulation. Thus, we suggest that insulin increases the level of M2-PK mRNA in adipocytes by acting at transcriptional and post-transcriptional levels through signaling pathways involving both PI3K and MAPK kinase.