The Regulatory Mechanism Of HDAC11 On Animal Fat Deposition

As global demand for meat continues to rise,meat quality has become an important factor influencing consumer choice.The level of subcutaneous fat,an important component of the body’s energy metabolism and endocrine system,is an important indicator of pork quality.Fat deposition in pigs is a key factor in healthy pig farming and economic development,and pigs are also an ideal model for exploring obesity-related diseases in humans.Therefore,the search for novel targets and potential mechanisms of fat deposition may open a new regulatory switch,which is of great value to systematically reveal the regulatory mechanisms of fat deposition in pigs,to select high-quality meat breeds and to prevent and treat human obesity-related diseases.In this study,histone deacetylase 11(HDAC11)was identified as a potential target for regulating fat deposition in animals by sequencing non-histone acetylation modifications in combination with online RNA-seq database analysis.The correlation between HDAC11 and porcine adipose traits was further analyzed by molecular and cell biological assays in combination with STRING,Ensenble,and ISwine databases using mouse adipose-derived stem cel s(ADSCs),pre-adipocytes 3T3-L1 and porcine primary adipocytes as test materials.The effect of HDAC11 on adipose deposition was explored from in vivo to cellular.The study revealed the role and mechanism of HDAC11 in driving ADSCs to differentiate into mesoderm and become brown adipose-like cel s to regulate fat deposition,and evaluated HDAC11 as a genetic breeding target for regulating adipogenesis in pigs.The main findings are as listed below:1.Screening of HDAC11 as a candidate gene for fat deposition in animals.Proteomic sequencing analysis of non-histone acetylation modifications in obese(ob/ob)and indirect fasting-induced anti-obese(fasting)mice revealed that a large number of non-histone proteins in inguinal white adipose tissue(i WAT)are dynamically modified by acetylation during the process of fat deposition,and that acetylation modifications of these non-histone proteins are involved in regulating various biological processes related to fat deposition.Further analysis of the acetylated protein interactions network using STRING revealed that the expression of some HDACs was differentially altered during adipogenesis,and the addition of the HDACs inhibitor SAHA inhibited adipogenesis,suggesting that HDACs are involved in regulating adipogenesis in mice.The combination of porcine RNA-seq online data further screened HDAC11 as a potential target for regulating fat deposition in animals.2.HDAC11 knockdown inhibits high-fat diet(HFD)-induced fat deposition in mice.HDAC11 was significantly overexpressed in the i WAT of ob/ob and HFD-induced obese mice(P<0.01).After lentiviral interference with HDAC11,HFD-induced weight gain was significantly suppressed and the mean fat area of i WAT was significantly reduced(P<0.05);serum cholesterol and triglyceride levels were significantly decreased(P<0.001),glucose tolerance was improved and insulin sensitivity was increased(P<0.05);hepatic steatosis was slowed;serum pro-inflammatory cytokine levels were reduced and fat and liver tissue inflammation was reduced(P<0.05).At the same time the browning marker genes UCP1,PRDM16 and PGC1α appeared significantly upregulated in i WAT to varying degrees(P<0.05),showing significant browning.In addition,knockdown of HDAC11 at 3T3-L1 promoted its differentiation towards brown adipose-like cel s.3.HDAC11 inhibits the differentiation of ADSCs into brown adipocyte-like cells,while promoting proliferation and maintenance of stemness.During the differentiation of ADSCs towards adipocytes,HDAC11 expression appeared significantly upregulated(P<0.01).The mechanism of HDAC11 regulation of adipogenesis was explored using lentivira l interference or overexpression of HDAC11 in ADSCs.Inhibition of HDAC11 promoted differentiation towards brown adipocytes,whereas the opposite trend was observed when HDAC11 was overexpressed.The proliferation and pluripotency of ADSCs with disrupted HDAC11 expression were examined and found that the expression levels of the pluripotenc y marker genes Oct4,Sox2,and the proliferation markers Cyclin D1 and PCNA were significantly down-regulated in ADSCs with HDAC11 knockdown(P <0.01),while overexpression of HDAC11 showed the opposite trend.EBs formation and spontaneous differentiation assays showed that HDAC11 overexpression had a maintenance effect on cell stemness,and that knockdown of HDAC11 inhibited the differentiation of ADSCs towards the ectoderm and promoted their differentiation towards the mesoderm.Inhibition of Sox2 alone diminished the ability of ADSCs to form EBs and upregulated the mesodermal marker PAX3 in spheroids;further overexpression of HDAC11 would reverse this situation.In addition,HDAC11 overexpression significantly increased β-catenin protein content in cells(P<0.001),while simultaneous downregulation of Sox2 caused a regression of intracellular β-catenin protein content(P<0.01),somewhat inhibiting the activation of Wnt/β-catenin signaling by HDAC11 overexpression.These results suggest that HDAC11 is involved in the proliferation and pluripotent gene regulatory network of ADSCs and that Sox2/Wnt/β-catenin signaling is involved in regulating the role of HDAC11 on the proliferation and stemness maintenance of ADSCs.4.The expression pattern of HDAC11 in porcine correlates with adiposity traits.Analysis of the interspecies conservation of HDAC11 using the Ensenble database and COBALT showed that HDAC11 was highly conserved between mammalia n species.Correlation analysis of HDAC11 with fat traits in pigs using ISwine showed that HDAC11 was strongly associated with fat development and meat quality traits in pigs such as daily weight gain,backfat thickness,fat water content and muscle content.The expression pattern of HDAC11 was analyzed in the Joint Porcine Genome Database and it was found that the content of HDAC11 in i WAT increased with the increase of obesity index.The expression profile of HDAC11 and key regulatory genes associated with adipogenesis was further validated by using transcriptomic data from subcutaneous preadipocytes of native pig breeds and lipogenic differentiation induction experiments.The results showed that HDAC11 increased and then decreased during adipogenic differentiation,peaking on the second day of differentiation,with an overall upward trend.HDAC11 expression trends converged with the markers of adipocyte differentiation,PPARγ and CEBPα;the expression pattern was identical to that of the browning marker gene PGC1α;and the expression pattern of the cell cycle-related marker Cyclin D1 was completely opposite.This suggests that HDAC11 may regulate pig adipogenesis by affecting lipogenic differentiation and adipocyte proliferation,and could be a potential target in pig quality selection.In conclusion,the present study identifies HDAC11 as a novel target for regulating fat deposition in animals,preliminarily investigates the mechanism by which HDAC11 affects lipogenic differentiation,and evaluates HDAC11 as a genetic target for regulating adipogenesis in pigs,which provides a basis and a reliable entry point for optimizing pig breeding and treating obesity-related metabolic diseases in humans.