Cloning of Cotton Delta-12 Oleate Desaturase Gene FAD2-1 and Construction of Its ihpRNA and amiRNA Interference Vectors

Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.

mi R-192-5p regulates lipid synthesis in non-alcoholic fatty liver disease through SCD-1

AIM To evaluate the levels of mi R-192-5 p in non-alcoholic fatty liver disease(NAFLD) models and demonstrate the role of mi R-192-5 p in lipid accumulation. METHODS Thirty Sprague Dawley rats were randomly divided into three groups, which were given a standard diet, a high-fat diet(HFD), and an HFD with injection of liraglutide. At the end of 16 weeks, hepatic mi R-192-5 p and stearoyl-Co A desaturase 1(SCD-1) levels were measured. Mi R-192-5 p mimic and inhibitor and SCD-1 si RNA were transfected into Huh7 cells exposed to palmitic acid(PA). Lipid accumulation was evaluated by oil red O staining and triglyceride assays. Direct interaction was validated by dual-luciferase reporter gene assays.RESULTS The HFD rats showed a 0.46-fold decrease and a 3.5-fold increase in hepatic mi R-192-5 p and SCD-1 protein levels compared with controls, respectively, which could be reversed after disease remission by liraglutide injection(P < 0.01). The Huh7 cells exposed to PA also showed down-regulation and up-regulation of mi R-192-5 p and SCD-1 protein levels, respectively(P < 0.01). Transfection with mi R-192-5 p mimic and inhibitor in Huh7 cells induced dramatic repression and promotion of SCD-1 protein levels, respectively(P < 0.01). Luciferase activity was suppressed and enhanced by mi R-192-5 p mimic and inhibitor, respectively, in wild-type SCD-1(P < 0.01) but not in mutant SCD-1. Mi R-192-5 p overexpression reduced lipid accumulation significantly in PA-treated Huh7 cells, and SCD-1 si RNA transfection abrogated the lipid deposition aggravated by mi R-192-5 p inhibitor(P < 0.01).CONCLUSION This study demonstrates that mi R-192-5 p has a negative regulatory role in lipid synthesis, which is mediated through its direct regulation of SCD-1.

Arachidyl amido cholanoic acid improves liver glucose and lipid homeostasis in nonalcoholic steatohepatitis via AMPK and mTOR regulation

BACKGROUND Arachidyl amido cholanoic acid(Aramchol)is a potent downregulator of hepatic stearoyl-CoA desaturase 1(SCD1)protein expression that reduces liver triglycerides and fibrosis in animal models of steatohepatitis.In a phase IIb clinical trial in patients with nonalcoholic steatohepatitis(NASH),52 wk of treatment with Aramchol reduced blood levels of glycated hemoglobin A1c,an indicator of glycemic control.AIM To assess lipid and glucose metabolism in mouse hepatocytes and in a NASH mouse model[induced with a 0.1%methionine and choline deficient diet(0.1MCD)]after treatment with Aramchol.METHODS Isolated primary mouse hepatocytes were incubated with 20μmol/L Aramchol or vehicle for 48 h.Subsequently,analyses were performed including Western blot,proteomics by mass spectrometry,and fluxomic analysis with 13C-uniformly labeled glucose.For the in vivo part of the study,male C57BL/6J mice were randomly fed a control or 0.1MCD for 4 wk and received 1 or 5 mg/kg/d Aramchol or vehicle by intragastric gavage for the last 2 wk.Liver metabolomics were assessed using ultra-high-performance liquid chromatography-time of flight-MS for the determination of glucose metabolism-related metabolites.RESULTS Combination of proteomics and Western blot analyses showed increased AMPK activity while the activity of nutrient sensor mTORC1 was decreased by Aramchol in hepatocytes.This translated into changes in the content of their downstream targets including proteins involved in fatty acid(FA)synthesis and oxidation[PACCα/β(S79),SCD1,CPT1A/B,HADHA,and HADHB],oxidative phosphorylation(NDUFA9,NDUFB11,NDUFS1,NDUFV1,ETFDH,and UQCRC2),tricarboxylic acid(TCA)cycle(MDH2,SUCLA2,and SUCLG2),and ribosome(P-p70S6K[T389]and P-S6[S235/S236]).Flux experiments with 13Cuniformely labeled glucose showed that TCA cycle cataplerosis was reduced by Aramchol in hepatocytes,as indicated by the increase in the number of rounds that malate remained in the TCA cycle.Finally,liver metabolomic analysis showed that glucose homeostasis was improved by Aramchol in 0.1MCD fed mice in a dose-dependent manner,showing normalization of glucose,G6P,F6P,UDP-glucose,and Rbl5P/Xyl5P.CONCLUSION Aramchol exerts its effect on glucose and lipid metabolism in NASH through activation of AMPK and inhibition of mTORC1,which in turn activate FAβ-oxidation and oxidative phosphorylation.