| BackgroundAt present, the incidence of obesity is rising year by year, which has become a worldwide public health problem. Obesity is not only increasing fat cells but also the increase of volume. Obesity induced metabolic syndrome including insulin resistance, nonalcoholic fatty liver disease(NAFLD) and type 2 diabetes mellitus(DM2) and inflammatory-induced autoimmune disease. In the view of current obesity and diabetes, the molecular mechanisms of insulin resistance are causal to the development of these metabolic disorders. The traditional concepts think that muscle, fat, and liver are classic targets of insulin action. But there are evidences that hormone signaling pathways in the hypothalamus-fat tissue not only intervene in occurrence and development of obesity but also become potential targets of insulin action.The hypothalamus contains a variety of ghrelin and fat factor receptor and acts the important role of energy balance. NPY is one of the most abundant neurotransmitter in the central, the neurons are mainly distributed in the arcuate nucleus and dorsal medial nucleus, it is important to promote appetite signals. Recently, accumulated evidence also suggested that NPY acting as a orexigenic signal may contribute to obesity, hyperinsulinemia and hyperglycemia. A initial report showed that knockdown of NPY expression in the ARC via AAV-mediated RNAi attenuated the feeding response to food deprivation. Cumulative reports had demonstrated the genetic deletion of NPY in rats resulted in a lean and hypophagic phenotype. A study demonstrated that DMH NPY knockdown increased energy expenditure and physical activity, enhanced the thermogenic response to a cold environment, and promoted development of brown adipocytes in white adipose tissue. It can be seen that NPY is also responsible for the metabolic disturbance. Similarly to insulin action in peripheral target tissues through the PI3K/AKT and MAPK/ERK signaling pathway, how does NPY maintain energy homeostasis? NPY mediates its pleiotropic effects through a family of G protein-coupled receptors including Y1, Y2, Y4, Y5, and Y6.Y1 and Y5 receptor paly a role in the regulation food intake, however, Y5 receptor had been shown to be strongly involved in food intake. A report showed that NPY has been shown to stimulate feeding via the Y5 receptor, which is found in a series of insulin-mediated peripheral target tissues. It is well unclear, however, whether molecular mechanism of NPY contributes to insulin resistance in adipose tissue and glucose homeostasis in peripheral target tissues. ObjectivesWe make an attempt to investigate wheather the knockdown of NPY expression in the DMH improved obesity-induced insulin resistance and β-cell proliferation in rats through PI3K/Akt/GSK-3βby adeno-associated virus(AAV)-mediated RNAi. In addition, we also observe whether the knockdown of NPY affect the glucose consumption and glucose intake in 3T3-L1 adipocyte cells and further explore whether the Y5 receptor participate in the mechanism of glusoce metabolism disorders with S- 2367 and L – 152,804 in3T3-L1 adipocyte cells. Methods1.The knockdown of NPY expression in the dorsomedial hypothalamus improve obesity-induced insulin resistance.Adeno-associated virus(AAV)-mediated RNAi was utilized to down-regulation Npy gene expression with rats on regular chow or high diet. By investigating the difference of rat body weight and food intake, we assessed the effect of knockdown ofNPY expression on alienation ability ofinsulin sensitivity,β-cellproliferation and involved signal transduction--PI3K、Akt and GSK-3β.2. Knockdown of NPY expression in 3T3-L1 adipocytess affect the glucose consumption and intake.In addition, in vitro experiment, we not only tested that knockdown of NPY expression affect the glucose consumption and 2-[3H]DGuptake in 3T3-L1 adipocytes incubated with Y5 receptor agonist(S- 2367) and the inhibitor(L-152804), but also the protein levels of PI3 K, Akt and GSK-3β were assessed. Results1. Compared with AAVshCTL, DMH NPY knockdown decreased food intake and body weight, and the differency were statistically significant(P < 0.05). High sugar clamps experiment suggests that compared with AAVshCTL, GIR in AAVshNPY group increased obviously and the differency were statistically significant(P < 0.05). WB showed that the phosphorylation of PI3 K, and Akt and GSK – 3βin the rats with AAVshNPY was significantly higher than the controls, the differency were statistically significant(P < 0.05). EdU counting showed that theproliferation of β-cell in AAVshCTL and AAVshNPY did not obvious changes, the differency was meaningless(P > 0.05).2. In vitro, compared with AAVshCTL and insulin, basal and insulin induced-glucose consumption in AAVshNPY(109) increased significantly, glucose uptake was significantly reduced, and the differency was statistically significant(P < 0.05). The glucose consumption and glucose uptake in AAVshNPY(107 and 108) did not change, and the differency was meaningless. Compared with AAVshNPY, glucose consumption increased significantly in AAVshNPY +L-152804, glucose uptake decreased significantly in AAVshNPY +L-152804, the phosphorylation of PI3 K, Akt and GSK3β increased significantly in AAVshNPY +L-152804, and the differency was statistically significant(P<0.05). While glucose consumption decreased significantly in AAVshNPY+S-2367, glucose uptake increased significantly in AAVshNPY+S-2367, the phosphorylation of PI3 K, Akt and GSK3β decreased significantly in AAVshNPY+S-2367, and the differency was statistically significant(P<0.05). However, the total protein of PI3 K, Akt and GSK3β did not change in each group,and the differency was meaningless(P > 0.05). Conclusions1.Adeno-associated virus(AAV)-mediated RNAi can tranfect stability durably the hypothalamus DMH.2.Knockdown of NPY expression in the dorsomedial hypothalamus improve obesity-induced insulin resistance by promoting the phosphorylation of PI3 K, Akt and GSK-3β. However, knockdown of NPY did not promote the proliferation of β-cell.3.Knockdown of NPY expression increases glucose consumption and decrease 2-[3H]DG uptake in 3T3-L1 adipocytes via PI3K/Akt/GSK-3βsignaling pathways and the NPY Y5 receptor. |
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