Role And Mechanism Of Liver OGA In Regulating HSL O-GlcNAcylation During NAFLD

Background:With the global epidemic of obesity,non-alcoholic fatty liver disease（NAFLD）has become a global public health issue.Due to its complex pathogenesis and disease heterogeneity,there are no specific drugs approved clinically for the treatment of NAFLD.Among the various mechanisms of NAFLD,imbalance between hepatic lipid synthesis and catabolism is the central mechanism that causes hepatic lipid accumulation and promotes the progression of NAFLD.Therefore,elucidating the key molecular mechanisms of hepatic lipid synthesis and catabolism is a fundamental basis for finding therapeutic targets for NAFLD.Recent studies have shown that protein O-GlcNAcylation plays an important role in the regulation of hepatic lipid metabolism.A single O-linked N-acetylglucosamine（O-GlcNAc）moieties are added to Ser and Thr residues on proteins via O-GlcNAc transferase（OGT）,while O-GlcNAc hydrolase（OGA）hydrolyzes it,affecting protein content and function.Studies have shown that several key transcription factors and related proteins in hepatic lipid metabolism are subject to OGT-mediated O-GlcNAcylation.Lipid synthesis-related transcription factors such as Ch REBP and LXR promote hepatic lipid synthesis after being O-GlcNAcylated.O-GlcNAc HCF-1 protein also increases Ch REBP transcriptional activity and causes activation of hepatic lipogenic pathways.Although numerous studies have confirmed that abnormally activated OGT promotes hepatic lipid anabolism,the mechanism of O-GlcNAcylation on hepatic lipid catabolism is unknown.Moreover,as the only hydrolase of O-GlcNAcylation,the function of OGA in hepatic lipid metabolism has not been reported.Our previous work has observed a significant increase in hepatic O-GlcNAcylation and a significant decrease in OGA protein levels in patients with NAFLD,especially in patients with non-alcoholic steatohepatitis（NASH）with massive inflammatory cell infiltration.Based on these preliminary results,we believe that hepatocyte OGA protein may play an important role in the progression of NAFLD,and its potential mechanisms remain to be explored.Hormone-sensitive lipase（HSL）is a key rate-limiting enzyme for lipid hydrolysis,and its activity is mainly regulated by phosphorylation modifications.Activated HSL can hydrolyze a variety of lipids such as triacylglycerols（TAGs）,diacylglycerols（DAGs）,and monoacylglycerols（MAGs）,but not phospholipids.HSL is mainly highly expressed in adipose tissue and lowly in hepatocytes.In mouse adipocytes,pro-lipolytic hormones（e.g.corticosteroids,epinephrine,norepinephrine and glucagon）induces PKA to phosphorylate HSL at the S563 site via a c AMP signaling cascade,which significantly enhances HSL lipolytic activity,whereas anti-lipolytic hormones（e.g.insulin）have the opposite effect.In a clinical study,homozygous carriers with a frameshift mutation in the gene encoding HSL exhibits fatty liver,insulin resistance,and hyperlipemia.In addition,a study also found that hepatic HSL overexpression in mice attenuated high-fat diet-induced fatty liver.These studies suggest that HSL plays an important role in lipid catabolism in the adipocytes and liver.The available literature suggests that post-translational modifications of HSL is regulated only by phosphorylation modifications,and the existence of other post-translational modifications is unclear.Whether O-GlcNAcylation regulates HSL and the role of HSL in a mouse liver OGA knockout model of NAFLD and the molecular regulatory mechanisms remain unclear.Aims:The aim of this study was to investigate the role of liver-specific OGA knockout（OGA-LKO）in a high-fat diet-induced mouse NAFLD model and its molecular mechanism,and to provide a new theoretical basis for clinical intervention in the NAFLD progression.1.To observe the effect of OGA-LKO on glucose and lipid metabolism,obesity,and energy metabolism.2.To find potential substrate proteins of OGA,and explore the effect of O-GlcNAcylation on the function of the substrate proteins.3.To identify the of O-GlcNAcylation sites of new substrate protein and to reveal the exact mechanism of O-GlcNAcylation on substrate molecule.Methods:1.Establishment of a model of high-fat diet-induced obesity and NAFLDUsing the Cre-Loxp knockout system,we were the first to construct hepatocyte OGA-specific knockout(Alb-Cre OGA^flox/flox)mice of C57BL/6J background.On this basis,male OGA-LKO and WT mice from the same-brood were obtained as subsequent experimental mice by crossing Alb-Cre OGA^flox/flox（OGA-LKO）mice with OGA flox/flox（Wild type,WT）mice.The experimental mice（aged 7w）were given a high-fat diet（HFD,60%fat）for 16 weeks to construct a model of obesity and NAFLD.The corresponding control diet group was given normal control diet（NCD）.The effects of hepatocyte OGA knockout on adipose tissue,energy metabolism,fatty liver and glucose metabolism were systematically observed during this period.2.Assessment of obesity-related phenotypesBody weight of mice was measured at fixed time periods each week,and body composition analyzer（Echo MRITM-100H,USA）of live small animals was used in combination to precisely measure whole body fat（Fat）and lean body tissue（Lean）content while the mice were awake.When the experiment reached the observed endpoint,the effect of hepatocyte OGA knockout on diet-induced obesity was observed by weighing scapular BAT,three types of white fat content（i WAT,e WAT,and p WAT）and subsequent HE staining.3.Assay of energy metabolismThe effects of hepatocyte OGA knockout on whole-body oxidative catabolism in mice were indirectly assessed by calculating oxygen consumption（VO₂）,carbon dioxide production（VCO₂）,and respiratory entropy（RER）in mice at different time periods（Light,Dark）and under different treatments（Fed,Fasted）using the experimental animal metabolic cage assay technique（CLAMS,USA）.The effect of hepatocyte OGA knockout on brown adipose thermogenesis in mice was assessed by measuring heat production（Heat）in mice and by detecting protein expression of FGF21 and UCP1 in scapular brown adipose tissue.4.Assessment of fatty liver and glucose metabolismLiver weight,liver-body mass index,blood lipids（TG,TC,FFA）were measured in mice,combined with liver tissue HE and oil red O staining to determine the effect of hepatocyte OGA knockout on fatty liver.By detecting insulin resistance（IPITT,IPGTT,insulin,HOMA-IR）,blood endocrine hormones（GIP,GLP-1,Glucagon,Ghrelin,Leptin,PAI-1,Resistin）and other indices,as well as detecting p-AKT（Ser473）in three white adipose tissues and total-AKT content to determine the effect of hepatocyte OGA knockout on glucose metabolism in mice.5.Multi-omics assay analysisThe changes of 13 major lipid classes,including TAGs,DAGs and CE,were quantified in mouse liver using lipidomics（Biotree,Shanghai,China）to determine what types of lipids were affected by OGA-LKO.We used transcriptomics technology（Energy,Shanghai,China）to detect the expression of differential genes in the liver of HFD groups,and combined with bioinformatics and lipidomics to systematically analyze the effects of OGA-LKO on lipid synthesis and catabolism,which laid a solid basis for subsequent screening of potential molecules.6.Screening and functional identification of substrate proteins of O-GlcNAcylationO-GlcNAc levels were elevated in Hep G2 and SK-hep-1 cells using the OGA inhibitor TMG（100 n M,6h）and sh OGA lentivirus,respectively.And potentially bound O-GlcNAc proteins were enriched by IP experiments using pan-O-GlcNAc antibodies followed by LC-MS liquid-phase mass spectrometry detection.Protein KEGG analysis and molecular biology experiments identified and found that O-GlcNAcylation affect HSL protein content and its activity.Analysis and prediction of potential O-GlcNAc modification sites of HSL protein（Ser565、Ser643、Ser792、Ser961）using Yin OYang 1.2and OGT site network database.The O-GlcNAc modification sites of HSL and their potential functions were determined by point mutation and IP experiments to determine their effects on lipid catabolism.7.Effects of PPARαagonist fenofibrate on obesity and energy metabolism in mice on high-fat dietAfter 5 weeks of HFD diet,mice in both HFD+WT（N=14）and HFD+OGA-LKO（N=18）groups were divided into 4 groups,named HFD+WT+Vehicle,HFD+OGA-LKO+Vehicle,HFD+WT+FN,and HFD+OGA-LKO+FN,respectively.And then these mice were treated with PPARαagonist Fenofibrate（FN,100 mg/kg/day,by gavage）or Vehicle（2%starch solution as vehicle）.The effects of FN on body weight,body mass,and energy metabolism of mice were observed during this period.Results:1.Hepatocyte OGA knockout promotes high-fat diet-induced obesity phenotypeUnder NCD diet conditions,there were no significant differences in body weight and body fat（Fat）content between mice in the WT and OGA-LKO groups.However,after the high-fat diet,the body weight and body fat content,as well as the content of the three types of white adipose tissues（i WAT,e WAT,and p WAT）were significantly higher in the OGA-LKO+HFD group compared to the WT+HFD group mice.Moreover,HE staining of the adipose tissues showed that the mice in the OGA-LKO+HFD group had larger adipocyte diameters.Notably,although there was no significant difference in the content of scapular brown fat,HE staining showed a significant whitening of brown fat.These results demonstrate that mouse hepatocyte OGA knockout promotes high-fat diet-induced obesity and brown fat whitening.2.Hepatocyte OGA knockout reduces oxidative metabolism and thermogenesis in high-fat diet miceBefore high-fat diet treatment,OGA knockout of hepatocytes had no significant effect on oxygen consumption（VO₂）,carbon dioxide exhalation（VCO₂）and respiratory entropy（RER）in mice under both Fed and Fasted conditions.However,after 4 weeks of high-fat diet,all of these indices except RER were significantly reduced.In particular,after 12 weeks of high-fat diet,the mice in the OGA-LKO+HFD group showed lower oxygen consumption and carbon dioxide exhalation compared with the WT+HFD group at different time periods of Fed and Fasted status.This suggests that hepatocyte OGA knockout reduces oxidative metabolism in high-fat diet mice.Although there was no difference in respiratory entropy（RER）between the above two groups,the values converged to 0.8,suggesting that the substrates of oxidative catabolism in both groups of mice were predominantly fat and carbohydrates.In conclusion,hepatocyte OGA knockout reduced fat-and carbohydrate-based oxidative catabolism in high-fat diet mice.In addition,the OGA-LKO+HFD group mice produced less heat,while there were no significant differences in food intake and activity.Immunoblotting experiments further confirmed that the levels of FGF21 and its downstream target protein UCP1 were significantly decreased in the scapular brown adipose tissue of HFD+OGA-LKO group mice.These results suggest that hepatocyte OGA knockout reduces brown adipose thermogenesis in high-fat diet mice.3.Hepatocyte OGA knockout promotes the progression of fatty liver in mice under NCD and HFD conditionsAfter 23 weeks of NCD,there were no significant differences in liver weight,liver-body weight ratio,and lipids（TG,TC,FFA）between WT and OGA-LKO groups of mice.However,oil red O staining of liver tissues suggested that hepatocytes in the NCD+OGA-LKO group mice aggregated a large number of lipid droplets,which suggested that hepatocyte OGA knockout could cause mice to spontaneously form fatty liver after a certain period of NCD.Moreover,after 16 weeks of HFD,hepatocyte OGA knockout exacerbated the abnormalities of the above indicators and promoted the progression of fatty liver.4.Hepatocyte OGA knockout exacerbates glucose metabolism disorders in high-fat diet miceAt 16 weeks of HFD,mice in the HFD+OGA-LKO group exhibited reduced glucose tolerance（IPGTT）and insulin sensitivity（IPITT）,as well as increased insulin resistance（HOMA-IR）compared to the HFD+WT group.The protein expression of p-AKT（Ser473）was significantly decreased in white adipose tissues（i WAT,e WAT,and p WAT）in the HFD+OGA-LKO group,suggesting that OGA-LKO attenuated the insulin signaling pathway in white adipose tissue and promoted insulin resistance in peripheral adipose tissue.In addition,the levels of serum Leptin,PAI-1 and Resistin,which synergize insulin resistance,were significantly increased in the HFD+OGA-LKO group of mice,while the levels of GLP-1,which counteract insulin resistance,were significantly decreased.5.OGA-LKO promotes hepatic TAGs aggregation in miceLiver lipidomic results showed that OGA-LKO under NCD 23w resulted in elevated levels of 38 TAGs.In contrast,OGA-LKO under HFD contributed to an increased level of266 TAGs,27 DAGs and 6 cholesterol esters.These results suggested that OGA-LKO affected lipid synthesis and catabolism,causing massive aggregation of hepatic TAG,DAG and other lipids.Liver transcriptome results from WT+HFD and OGA-LKO+HFD groups showed that OGA-LKO caused down-regulation of 1142 genes and up-regulation of 616 genes in response to HFD.KEGG pathway analysis showed that the genes involved in fatty acid elongation and biosynthesis of unsaturated fatty acids pathways were significantly downregulated.The down-regulated genes were Elovl7,Acot3,Scd1,Acot2,Elovl5,Acot4,Hacd4.These results indicated that OGA-LKO may inhibit TAGs and DAGs synthesis pathways,and that reduced hepatic lipolysis may be the main cause of lipid aggregation.6.OGA-LKO inhibits HSL lipolytic activityUsing pan O-GlcNAc antibodies to enrich potential O-GlcNAcylated proteins in hepatocellular carcinoma cell lines,combined with LC-MS spectroscopy,HSL was identified as a potential O-GlcNAc protein.IP experiments confirmed the interaction of HSL with OGA and OGT.Under NCD and HFD conditions,OGA-LKO resulted in a significant increase in HSL protein content and a significant decrease in Ser563 HSL which represents lipolytic activity,suggesting that OGA-LKO inhibits HSL lipolytic activity.Free fatty acids produced by hepatic lipolysis are common ligands for the activation of PPARɑ.And under NCD and HFD conditions,OGA-LKO resulted in a significant decrease in hepatic PPARɑ-FGF21 signaling and serum FGF21.In mouse liver primary cells treated with high-fat medium,either OGA knockdown or activity inhibition（TMG）promoted lipid aggregation in hepatocytes,and led to a significant increase in HSL protein content and a significant decrease in Ser563 HSL protein content,as well as inhibited Ser563 HSL transport from the cytoplasm to the surface of lipid droplets.In addition,in SK-Hep-1 cells,lentiviral overexpression of OGT or knockdown of OGA,as well as TMG to elevated O-GlcNAc levels caused a significant increase in HSL protein content,while Ser563 HSL content was significantly reduced.7.O-GlcNAcylation of HSL at S565 site inhibits S563 phosphorylation and ubiquitination of total HSLIn mouse primary hepatocytes,IP experiments demonstrated the presence of O-GlcNAc motifs in endogenous HSL proteins.In addition,a series of IP experiments in human hepatocellular carcinoma cell line SK-Hep-1 confirmed the interaction of HSL with OGA and OGT proteins,and the presence of O-GlcNAc motifs in endogenous HSL proteins.These experiments provide ample evidence that HSL is a potential O-glycosylation-modified substrate protein.Modification site prediction by 2 independent O-GlcNAcylation databases,modification site mutation,and IP experiments were performed to confirm that the S565 site may be a potential modification site for HSL.In SK-Hep-1 cells,HSL O-GlcNAcylation at the S565 site inhibited its phosphorylation at the Ser563 site,which resulted in diminished HSL lipolytic activity.Furthermore,HSL O-GlcNAcylation at the S565 site promotes HSL protein stability by inhibiting the proteasome-mediated ubiquitination degradation pathway,leading to increased HSL protein content.8.PPARαagonist Fenofibrate（FN）attenuates obesity and energy metabolism disorder in HFD+OGA-LKO miceThe diminished HSL lipolytic activity resulted in a decrease of free fatty acid（FFA）formed by hepatic lipolysis,which is an important ligand for activation of hepatocyte PPARɑ.Under NCD and HFD conditions,OGA-LKO significantly reduced HSL activity and decreased hepatic PPARαprotein expression.The production of the liver-derived hormone FGF21 was mainly regulated by PPARα.OGA-LKO also significantly reduced the protein expression of FGF21 in the liver.Moreover,the protein expression of FGF21was significantly decreased in all three types of white adipose and scapular brown adipose in the HFD+OGA-LKO group mice.These results suggest that hepatocyte OGA knockout attenuates the PPARα-FGF21 signaling pathway possibly by inhibiting HSL lipolytic activity,thereby exacerbating fatty liver,obesity,and brown fat thermogenesis in high-fat diet mice.After HFD treated for 5 weeks,the WT and OGA-LKO mice were gavaged daily with PPARαagonists FN or Vehicle for a period of time.FN significantly reduced body weight and body fat,and enhanced oxidative catabolism and thermogenesis in the HFD+OGA-LKO+Vehicle group compared to the HFD+OGA-LKO+Vehicle group.These results suggest that obesity and energy metabolism disorders in high-fat diet mice caused by hepatocyte OGA knockdown are likely dependent on PPARα-mediated signaling pathways,and that FGF21 protein may be a key factor linking fatty liver and obesity.Conclusion:This study is the first to elucidate the important role of hepatocyte OGA in hepatic lipid catabolism,glucose metabolism,energy metabolism,and obesity,further improving the theoretical system of O-GlcNAcylation in regulating hepatic lipid metabolism.Moreover,our study demonstrates for the first time that HSL is regulated by protein O-GlcNAc modifications in addition to hormone and phosphorylation modifications.HSL O-GlcNAcylation at the S565 site affects its lipolytic activity and protein stability.Finally,our findings extend the understanding of post-translational modifications of HSL,and provide a corresponding theoretical basis for HSL as a potential therapeutic target for fatty liver.