| Objectives:Insulin resistance(IR) is the pre-diabetic state in which target tissues, including the liver, muscle and adipose, show reduced insulin responsiveness and lipid metabolism disorders. The phenotype is characterized by progressive IR, with the development of the full spectrum of the metabolic syndrome, including atherosclerosis and coronary heart disease. Currently, non-pharmacological intervention is one of the important means to prevent the occurrence and development of IR through ameliorating lipid metabolism disorders. High-fat diet can be used to induce ideal IR model which is similar with human IR pathogenesis and plays a key role in IR research.Utilizing n-3PUFA in prevention of hyperlipidemia through diet had been paid high attention abroad. Experiments showed that n-3PUFA can prevent the occurrence of IR, but the curative effect and the lipids modulation mechanism of n-3PUFA are still unkown. Although α-linolenic acid(ALA, C18: 3), eicosapentaenoic acid(EPA,C20: 5) and docosahexaenoic acid(DHA, C22: 6) belong to the n-3PUFA, EPA and DHA benefits have been well established and there is a lack of substantive evidence for the biological role of ALA.In combination of blood chemistry analysis, colorimetric analysis, gas chromatography, ultracentrifugation techniques, frozen section pathology staining pathological analysis of experimental techniques, real-time q PCR and Western-Blot and other molecular biology techniques, we investigate the effect of ALA on lipid biosynthesis-related genes in animal and cell IR models, and provide scientific data for the use of ALA in the treatment of IR.Methods:In this study, high-fat diet rich in perilla oil(PO) was given to IR rats, serum biochemical indexes, m RNA and protein expression level of key genes such as key genes of triglyceride(TG) synthesis SREBP-1c and FASN, insulin-induced genes,cholesterol synthesis genes SREBP-2 and HMGCR were analysed. Based on animal experiments, insulin resistant Hep G2(IR-Hep G2) cell model was induced by palmitic acid(PA) and cellular reseach aimed to analyse the effect of ALA into these genes. In combination of animal and cell results, we investigate the machanism of ALA ameliorating lipids disorders of IR.48 SPF rats were randomly divided into two groups, i.e. the normal control(NC)diet group(n=12 rats) and high fat(HF) diet group(n=36 rats). Up to week 12, 24 of the IR rats were given ad libitum access to one of two diets containing equal diet energy, HF diet(n=12 rats) or perilla oil(PO) diet(n=12 rats). After the intervention at week 4, serum and liver ALA content were assayed by gas chromatography, TCH and TG level of serum and liver were detected with enzymic method and hepatic m RNA and protein expression of genes related to lipid synthesis were examinated by real-time q PCR and Western-Blot.Hep G2 cells were divided into two groups, normal control group(NC) and palmitic acid group(PA) in which cells were firstly cultured for 24 h in the medium contained 0.25mmol/L palmitic acid. The glucose uptake, cellular TG and TCH level were determined and once there was significant difference in glucose uptake,20%ALA took the place of PA for another 12 h culture. Celluar TCH and TG level were detected and m RNA and protein expression of genes related to lipid synthesis were examinated by real-time q PCR and Western blot.Results:1 Rats(1) The energy intake and fat-energy intake of HF rats were significantly higher than those of the NC group(P<0.0001, each). Following the 14 h fasting period, the serum TG of the IR model rats was found to be remarkably higher than the rats fed with the NC diet(P<0.05), and there was no significant difference in TCH. The HF rats exhibited insulin resistance, due to the fact that the glucose infusion rate(GIR) in the HF were significantly lower than those of the NC(P=0.0086).(2) The histological examination of the livers indicated a significant decrease of large lipid accumulation in IR animals after PO intervention. This corresponded to the significant decrease of serum and hepatic TG levels, in comparison to those measured for rats maintained on the HF diet(P<0.05, each). Although PO decreased the hepatic TCH by >30%, the PO had no significant effect on the serum TCH or hepatic TCH.As expected, due to high n-3 PUFA intake, the PO increased the hepatic and serum ALA content remarkably in the IR rat liver(P<0.0001, P=0.0006).(3) When compared with HF, PO increased the INSIG-2 m RNA 1.5-fold and INSIG-2 protein 2.2-fold in the IR rat liver, whereas neither the INSIG-1 m RNA nor protein changed significantly in response to PO. In the IR rat liver, PO decreased the abundance of the transcriptionally active(65 k Da) form of SREBP-1c by >75%,whereas PO had no significant effect on the 125-k Da precursor form of SREBP-1c,resulting in a >70% decrease in the 65/125-k Da protein ratio. Since FASN is positively controlled by SREBP-1c, we found that the FASN protein reduced by 30%after PO intervention. Neither SREBP-1c nor FASN m RNA exhibited significant change in response to PO. When compared with HF, PO reduced SREBP-2 m RNA by >35%. As for protein expression, PO decreased the 55-k Da cleaved form of SREBP-2, 126-k Da precursor form of SREBP-2 and 55/126-k Da protein ratio by >30%, >70% and >60%, respectively. Although HMGCR m RNA did not change in response to PO, the HMGCR protein decreased by >35%.2 Hep G2 cells(1) After incubation with 0.25mmol/L PA for 24 h, the glucose uptake of PA group was significantly lowered than NC group indicating the IR-Hep G2 cell model was established characterized by extremely high TCH level.(2) Comparing with the PA group, although gene SREBP-2 m RNA expression were blocked by 32%, all the 6 genes m RNA expression of ALA group showed no significant difference.(3) In correspondence with IR rats results, ALA selectively upregulated the protein expression of INSIG-2 and decreased the TG level in IR-Hep G2 cells, and there were no statistically significant differences in TCH level and INSIG-1 protein expression.ALA had no significant effect on the abundance of the transcriptionally active(65k Da) form of SREBP-1c while the FASN protein reduced by 21% after ALA intervention. When compared with PA, ALA reduced 55-k Da cleaved form of SREBP-2 and HMGCR protein by 43% and 79%, respectively.Conclusions:1 Rats(1) In supplementation with ALA by 0.556g/d may reduce the abundance of post-translational form of SREBP-1c and its target FASN by increasing the expression of INSIG-2, resulting in significant decrease of TG in both liver and plasma of IR rats.(2) PO caused inhibition of SREBP-2 and HMGCR in the IR rat liver by increasing INSIG-2, whereas it exhibited no effect on TCH in both liver and plasma.(3) PO selectively upregulated the expression of INSIG-2 in IR rats, rather than INSIG-1.2 Hep G2 cells(1) Incubation with 0.25mmol/L PA for 24 h can establish IR-Hep G2 cell model characterized by lipids disorder.(2) ALA selectively decreased the TG level in IR-Hep G2 cells, and there were no statistically significant differences in TCH level and insulin sensitivity.(3) ALA selectively upregulated the protein expression of INSIG-2 in IR-Hep G2 cells,rather than INSIG-1.(4) ALA reduced the abundance of post-translational form of SREBP-2 and its target HMGCR and FASN by increasing the expression of INSIG-2, resulting in significant decrease of TG and TCH synthesis. |
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