The Effect Of LncRNAH19 On Glucose And Lipid Metabolism In Obese Mice And Its Underlying Molecular Mechanisms

Obesity is a severe global public health problem closely relevant to the occurrence and development of chronic metabolic diseases.An in-depth study of the pathogenesis of obesity helps establish effective prevention and treatment measures for these chronic diseases.Obesity results from a long-term imbalance between energy intake and energy expenditure,leading to excessive accumulation of lipids in various body tissues.Both type 2 diabetes and obesity are associated with insulin resistance.Hence,it is essential to get a further understanding of the mechanism of insulin resistance.An increasing number of studies have shown that long non-coding RNA(lncRNA)plays a vital role in glycolipid metabolism.Studies have reported that lnc RNA H19(H19),the earliest discovered imprint gene,is highly expressed in skeletal muscle and can promote skeletal muscle glucose uptake.Other studies have shown that H19 is involved in regulating liver lipid metabolism,but there are still contradictions in these studies,and no clear conclusion has been reached.Also,the effect of H19 on lipid metabolism in skeletal muscle is rarely reported,and a large number of studies are needed to explore its specific role and potential mechanism.In this study,we evaluated the effects of H19 on systemic glucose and lipid metabolism of obese mice and its potential mehanism via in vivo and in vitro experiment.In vivo,the study intended to use db/db mice as obesity and insulin resistance models.We overexpressed H19 in db/db mice via tail vein injection of H19 overexpression adenovirus.Then we observed the effect of H19 on glucose and lipid metabolism of obese mice.In vitro,our study overexpressed and knocked down H19 in primary skeletal muscle cells,and then observed the effects of H19 on lipid metabolism and mitochondrial function of primary skeletal muscle cells.Furthermore,we explored the underlying molecular mechanism of H19 regulating the lipid metabolism and mitochondrial functions.Part I.The effects of H19 overexpression on glucose and lipid metabolism and ectopic lipid accumulation and its potential mechanism in obese miceObjectivesTo investigate the effects of long non-coding RNA H19(H19)on glucose and lipid metabolism,hepatic lipid accumulation and skeletal muscle lipid metabolism and its potential mechanism in obese mice.Methods8-week-old db/db mice were randomly divided into ad-GFP group(n=6)and ad-H19 group(n=6).In ad-GFP group,the mice were injected with negative virus through the tail vein,and mice in ad-H19 group were injected with overexpressed H19 adenovirus.After two weeks of injection,oral glucose tolerance test(OGTT)was performed.Moreover,we detected body weight,fasting blood glucose,insulin level,serum glycolated albumin,and serum triglyceride to evaluate the functions of H19 in regulating systemic glucose and lipid metabolism of obese mice.Furthermore,we performed oil red O staining and hematoxylin-eosin(H&E)staining in skeletal muscle.Western blotting and q RT-PCR were used to detect the changes of genes related to lipid metabolism in the skeletal muscle.Results1.Successful construction of db/db mice with H19 overexpression: the expression of H19 in the liver and skeletal muscle of mice in the ad-H19 group was 7 times and 2.6 times higher than that in the ad-GFP group,respectively(p<0.05),suggesting the successful construction of db/db mouse model with H19 overexpression.2.Overexpression of H19 improved glucose metabolism of db/db mice: compared with the ad-GFP group,ad-H19 group had lower serum glycolated albumin levels(4.2mmol/L vs 5.0mmol/L),and OGTT showed that the AUC of mice in the ad-H19 group(2642 ± 615mmol/L·min)was significantly lower than that of mice in the ad-GFP group(3303 ± 340mmol/L·min)(p<0.05),suggesting that overexpression of H19 improved glucose metabolism of db/db mice.3.Overexpression of H19 improved lipid ectopic accumulation in skeletal muscle of db/db mice: compared with the ad-GFP group,lipid accumulation in skeletal muscle was improved,skeletal muscle and serum triglyceride were decreased in the ad-H19 group(p<0.05),suggesting that overexpression of H19 improved lipid ectopic accumulation in skeletal muscle of db/db mice.4.Overexpression of H19 improved lipid ectopic accumulation in liver of db/db mice: compared with the ad-GFP group,the hepatic fat infiltration was ameliorated,and the contents of cholesterol and triglyceride in the liver were decreased in the ad-H19 group.No significant changes were observed in serum total cholesterol,body weight and serum ALT and AST levels,suggesting that overexpression of H19 improved lipid ectopic accumulation in liver of db/db mice.5.Overexpression of H19 up-regulated fatty acid metabolism-related genes in skeletal muscle of db/db mice: compared with the ad-GFP group,the m RNA levels of fatty acid metabolism-related genes(Pgcα,Cpt1 b and Cd36)in skeletal muscle of mice in the ad-H19 group were up-regulated by 2.2,1.6 and 2.5 fold,and the protein levels were increased by 1.9,1.8 and 1.9 folds,suggesting that overexpression of H19 upregulated fatty acid metabolism-related genes in skeletal muscle of db/db mice.ConclusionsOverexpression of H19 improves hyperglycemia and reduced lipid deposition in liver and skeletal muscle in db/db mice.This effect may occur through up-regulation of fatty acid metabolism in skeletal muscle.Part II.The regulation of H19 on lipid metabolism and mitochondrial function of skeletal muscleObjectivesIn the first part,we found that overexpression of H19 improved hyperglycemia,decreased serum triglycerides levels,reduced the deposition of lipids in liver and skeletal muscle,and increased the expression levels of genes related to fatty acid metabolism(Pgc1α,Cpt1 b,and Cd36)in skeletal muscle of obese mice.Therefore,we hypothesized that H19 alleviated lipid deposition mainly by enhancing fatty acid metabolism in skeletal muscle.To further verify our conjecture,this part of the study overexpressed and knocked down H19 in primary skeletal muscle cells to observe the effects of H19 on fatty acid metabolism and mitochondrial function.MethodsBioinformatics analysis was used to detect the differentially expressed genes(DEGs)between wide type mice and whole-body H19 knockout mice.GO and KEGG analyses were used to explore the related biological functions of these genes.The primary muscle satellite cells were isolated and cultured from C57BL/6J neonatal mice and induced into mature primary skeletal muscle cells by 2% horse serum medium.H19 overexpression and knockdown were performed on primary skeletal muscle cells using H19 overexpression adenovirus and si RNA,respectively.Free fatty acid(FFA)was used to construct in vitro model of lipid deposition.Then we conducted Nile red staining and detection of intracellular triglyceride(TG)and diacylglycerol(DAG)levels to evaluate H19 lipid metabolism on primary skeletal muscle cells.The respiration rate(OCR),transmission electron microscopy,mitochondrial staining,and mitochondrial DNA copy number were detected to assess mitochondrial functions.Western blotting and q RT-PCR were carried out to detect the expression of mitochondrial electron transport chain complex and genes related to fatty acid metabolism.Results1.H19 could regulate mitochondrial functions in skeletal muscle: 554 DEGs were found between wide type mice and whole-body H19 knockout mice,these genes were mainly related to mitochondrial functions.2.Successful construction of primary skeletal muscle cells with H19 overexpression and knockdown: Primary muscle satellite cells were successfully isolated and induced into mature primary skeletal muscle cells.Compared with primary skeletal muscle cells treated with ad-GFP,the expression of H19 in cells treated with overexpressed H19 adenovirus increased by 2.7 times(p<0.01).The si RNA targeting H19 knock downed the expression of H19 by 60%(p<0.001).3.H19 overexpression in primary skeletal muscle cells ameliorated the lipid accumulation while H19 knockdown aggravated the effect: in primary skeletal muscle cells with H19 overexpression,FFA-induced TG and DAG levels were significantly reduced(p<0.05);while in primary skeletal muscle cells with H19 knockdown,FFAinduced TG and DAG levels were increased(p<0.05).4.H19 overexpression in primary skeletal muscle cells increased mitochondrial respiration while H19 knockdown inhibited mitochondrial respiration: in primary skeletal muscle cells with H19 overexpression,basal respiratory rate,maximum respiratory rate,and ATP production were increased(p<0.05),and FFA-induced mitochondrial swelling,and cristae fractured were significantly improved;H19 depletion reduced basal respiratory rate,maximum respiratory rate,and ATP production(p<0.05).Mitochondrial copy numbers were detected and the results showed that H19 overexpression or knockdown did not change the number of mitochondria.5.H19 overexpression in primary skeletal muscle cells increased the expression of fatty acid metabolism-related genes while H19 knockdown exhibited the opposite effect: overexpression of H19 in primary skeletal muscle cells increased protein levels of the mitochondrial electron transport chain complex(ATP5A and MTCO1)(p<0.01)and m RNA levels of fatty acid metabolism-related genes(Pgc1α,Cd36,Cpt1 b,Pdk4 and Sirt1)(p<0.05);knockdown of H19 reduced m RNA levels of genes related to fatty acid metabolism(Pgc1α,Cd36,Cpt1 b,Pdk4 and Sirt1)(p<0.05).ConclusionsOverexpression of H19 can improve lipid accumulation in primary skeletal muscle cells induced by FFA,enhance mitochondrial respiration and improve mitochondrial morphology.Knockdown of H19 increases intracellular lipid level and inhibits mitochondrial respiration.These effects are mainly caused by the regulation of mitochondrial electron transport chain complex and fatty acid metabolism-related genes,suggesting that H19 regulates lipid metabolism and mitochondrial function in primary skeletal muscle cells.Part III.The potential molecular mechanisms of H19 on the regulation of lipid metabolism and mitochondrial functions in skeletal muscleObjectivesIn the second part,we revealed the significant regulatory effects of H19 on lipid metabolism and mitochondrial function in primary skeletal muscle cells,but the specific mechanism remained unclear.Lnc RNA can often affect the expression of downstream target genes through the interaction with RBPs,thus causes functional changes.Therefore,we hypothesized that H19 could interact with a certain RBP and influence downstream genes related to fatty acid metabolism.The purpose of this part of the study is to find the RBP that directly binds to H19 and verify the biological function of this RBP and the mutual regulation between H19 and the RBP.MethodsRNA-protein pull down test combined with mass spectrometry was used to find the RBP directly binding to H19.Western blotting and RNA immunoprecipitation were used to further verify the binding between the two.The si RNA targeting RBP was used to knock down its expression in primary skeletal muscle cells.The levels of TG and DAG in the muscle cells were subsequently detected.The influence of RBP on mitochondrial respiration of primary skeletal muscle cells was detected by hippocampal experiment.The expression of genes related to fatty acid metabolism was detected by Western blotting.The overexpressed RBP plasmid was used to overexpress the RBP in primary skeletal muscle cells with knockdown of H19,and then genes related to mitochondrial respiration and fatty acid metabolism were detected to clarify the regulation between H19 and RBP.Results1.H19 could bind directly with hn RNPA1: RNA-protein pull down assay and mass spectrometry indicated the binding of H19 and hn RNPA1.This interaction was further verified by Western blotting and RNA immunoprecipitation.2.Hn RNPA1 antibodies could bind with fatty acid metabolism-related genes: hn RNPA1 antibodies bind to m RNA of Cpt1 b,Pgc1α and Cd36.3.Hn RNPA1 knockdown increased lipid contents,inhibited mitochondrial respiration and reduced fatty acid metabolism-related genes in primary skeletal muscle cells: hn RNPA1 was successfully knocked down in primary skeletal muscle cells using si RNA(p<0.05).Knockdown of hn RNPA1 increased the contents of TG and DAG in primary skeletal muscle cells(p<0.05),inhibited mitochondrial maximum respiration rate and ATP production(p<0.05),decreased the protein levels of fatty acid metabolismrelated genes(CPT1b,SIRT1 and PGC1α)and the phosphorylation level of AMPK(p<0.05).4.Overexpression of hn RNPA1 could partially rescue the effect induced by H19 depletion: overexpression of hn RNPA1 can reverse the mitochondrial respiratory inhibition of H19 on primary skeletal muscle cells and increase the protein levels of CPT1 b and PGC1α.ConclusionsH19 can directly bind to the hn RNPA1 protein.Knocking down hn RNPA1 increases lipid accumulation and inhibits mitochondrial function in primary skeletal muscle cells.Overexpression of hn RNPA1 partially rescues decreased mitochondrial respiration and the decreased protein levels of CPT1 b and PGC1α caused by H19 knockdown,suggesting that H19 and hn RNPA1 interact and regulate lipid metabolism and mitochondrial function in primary skeletal muscle cells.The possible mechanism is that H19 recruits hn RNPA1 to regulate the downstream translation levels of CPT1 b and PGC1α.