The Mechanism Of Apelin On Insulin Resistance In 3t3-l1 Adipocytes

Background:Insulin resistance (IR) is a pathological state that insulin targeting tissues and cells, including fat, muscle and liver cells fail to response adequately to insulin, which contributes to many physiological disorders. IR is the core pathophysiological mechanism for the metabolic syndrome (MS). It can not only lead to type 2 diabetes mellitus (T2DM), but also result in hypertesion, dyslipodemia, atherosclerosis (AS) and other disorders.Apelin, which was first isolated from bovine stomach extracts[1], is an endogenous ligand for the orphaned G protein-coupled receptor APJ. Apelin and its receptor APJ exerts various biological effects in various systems, including cardiovascular system, hypothalamus, gastrointestinal system, skin, muscle, mammary gland, and placenta and so on. It was reported that in the newly-diagnosed T2DM patients, serum apelin was increased while in the developed T2DM patients, serum apelin was decreased. In addition, in another study, after intraperitoneal injection of apelin in mice, the serum glucose was decreased accompanied with the decrease of serum insulin level. Apelin and glucose metabolism had intimate relationship, however, the underlining mechanism of how apelin affects the glucose level in vitro and in vivo was not fully elucidated. A recently report found that in the isolated insulin resistant skeleton muscle cells, apelin increased the glucose uptake. However, as an adipocytokine, the exact mechanism of apelin on insulin resisitant cells, especially insulin resisitant adipocytes, has not been reported.Research aims:1. Could apelin directly increase glucose uptake in insulin resistant 3T3-L1 adipocytes?2. Could apelin enhance insulin sensitivity and inderectly increase glucose uptake in insulin resistant 3T3-L1 adipocytes?3. Could apelin mediate inflammatory responses in insulin resistant state?4. How apelin increase glucose uptake? What is the underlining mechanism?Research methods:1. Inducing 3T3-L1 pre-adiopocytes into mature adipocytes, and evaluating mature adipocytes by oil red o staining.2. Building insulin resistant adipocyte model by TNF-α, and assessing this model by 3H-2DG glucose uptake assay.3. Observing the effects of apelin on glucose uptake, determining whether apelin could increase glucose uptake in insulin resistant 3T3-L1 adipocytes directly or through enhancing the insulin sensitivity of adipocytes and increase glucose uptake indirectly.4. Observing the mRNA expression of related inflammatory facters by RT-PCR, analyzing related pathway proteins expression by western blotting, elucidating the possible mechanism of how apelin exerts its effects in insulin resistant 3T3-L1 adipocytes. 5. Observing GLUT4 by immuocytochemistry and determining the protein of GLUT4 plasma membrane fraction by western blotting. Elucidating the role of GLUT4 after apelin treatment in insulin resistant 3T3-L1 adipocytes.Research results:1. After confluence, 3T3-L1 adipocytes were incubated in the cocktail medium: 10% FCS DMEM supplemented with 0.5 mM isobutylmethylxanthine, 1μM dexamethasone, and 10μg/ml insulin for 2 days. Subsequently, cells were treated with 10μg/ml insulin in 10% FCS DMEM for an additional 24 hours, and then maintained with 10% FCS fresh media every other day for 8 days. With this protocol, more than 85% of the pre-adipocytes differentiated into adipocytes. Oil red O staining confirmed that more than 80% of the pre-adipocytes differentiated into adipocytes.2. After treatment of 10ng/ml TNF-αfor 24h, insulin-stimulated glucose uptake were suppressed by 47% (P<0.05). The TNF-α-treated cells were considered to be insulin-resistant cells and were used for the following experiments.3. The effect of apelin on glucose transport in insulin-resistant 3T3-L1 adipocytes was evaluated using the 3H-2-DG uptake assay. After incubation with 10ng/ml TNF-αfor 24 h, cells were washed three times in PBS and incubated with apelin. Treatment with different concentrations of apelin for 60 min resulted in increased glucose uptake in insulin-resistant 3T3-L1 adipocytes. Apelin exerted an initial significant effect on glucose uptake at 100 nM (P<0.05) and a maximal effect at 1000 nM (P<0.01). Higher concentrations of apelin (104 nM and 105 nM) showed a decreased effect on glucose uptake compared to low concentrations of apelin. To perform a time course analysis, 1000 nM apelin was added to the insulin-resistant adipocytes at different time points. Results showed that glucose uptake was maximally augmented at 60 min after incubation with apelin (P<0.01); apelin still retained its effect at 120 min (P<0.05). After 120 min, the improvement in glucose uptake declined and was no longer significant compared to basal levels.4. Furthermore, we examined whether apelin could increase insulin sensitivity in insulin-resistant cells. Administration of 100 nM insulin for 30 min after stimulation with 100 or 1000 nM apelin significantly increased insulin-stimulated 3H-2-DG uptake, and 1000 nM apelin showed greater effects (P<0.05 versus apelin alone, P<0.01 versus apelin alone and P<0.05 for 1000 nM apelin versus 100 nM apelin). These results indicated that apelin alone could enhance glucose uptake in insulin-resistant cells. Apelin may also serve as an insulin-sensitizing agent because it can markedly improve insulin-stimulated glucose uptake in insulin-resistant 3T3-L1 adipocytes.5. To examine whether the PI3K/Akt pathway was responsible for the apelin-mediated improvements in glucose uptake, the cells were pre-treated with 100 nM wortmannin, a PI3K specific inhibitor, for 30 min prior to apelin treatment. Wortmannin pre-incubation significantly decreased apelin-induced glucose uptake as well as insulin-stimulatd glucose uptake in insulin-resistant 3T3-L1 adipocytes (P<0.05 versus 100 nM wortmannin, P<0.01 versus 100 nM wortmannin). We also determined the activation of phosphorylated Akt (Ser 473) in the presence or absence of wortmannin. As expected, insulin stimulation resulted in augmentation of pAkt (Ser 473). Interestingly, apelin also significantly enhanced Akt phosphorylation (Ser 473). In regards to enhancement of Akt phosphorylation, apelin was almost as potent as insulin. Pre-incubation with wortmannin blocked the enhancement of phosphorylated Akt (Ser 473).6. We observed plasma membrane (PM) localization of GLUT4 using an immunofluorescence confocal microscopy technique to determine whether the observed improvement in glucose uptake was due to increased GLUT4 translocation. Apelin and insulin increased the translocation of GLUT4 from the cytoplasm to the plasma membrane in insulin-resistant 3T3-L1 adipocytes. In regards to GLUT4 translocation, apelin was almost as potent as that of insulin. Furthermore, apelin pre-incubation significantly increased insulin-stimulated GLUT4 translocation. Consistent with immunofluorescence images, western blotting analysis of GLUT4 in the plasma membrane fraction also confirmed that apelin and insulin could increase GLUT4 expression on the plasma membrane.7. We investigated the effects of apelin on the mRNA expression of the pro-inflammatory factor IL-6 in insulin-resistant 3T3-L1 adipocytes. Cells were treated with or without 1000 nM apelin for 60 min, and total RNA was extracted using TRIzol reagent. Real-time PCR showed that mRNA expression of IL-6 was significantly down-regulated by apelin (P<0.01). In addition, apelin significantly up-regulated mRNA expression of the anti-inflammatory adipokine adiponectin (P<0.05). These results suggest that apelin could improve the chronic low-grade inflammatory state of insulin resistance.Conclusion:1. Apelin increases glucose uptake in a time- and dose-dependent manner, and improves insulin sensitivity in insulin resistant 3T3-L1 adipocytes.2. Apelin increases glucose uptake in insulin resistant 3T3-L1 adipocytes is through the PI3K/Akt pathway, with augment of phosphorylated Akt (Ser473) and GLUT4 translocantion to the plasma membrane.3. Apelin enhances the mRNA expression level of anti-inflammatory factor adiponectin and reduces the gene expression of the pro-inflammatory cytokine IL-6, thus mediates the inflammatory response in insulin-resistant 3T3-L1 adipocytes.