Hypothalamic Long Noncoding RNA AK044061 Is Involved In The Development Of Dietary Obesity In Mice

Objective:Long noncoding RNAs（lnc RNAs）have exerted crucial effects on a variety of pivotal biological processes,but the effects of hypothalamic lnc RNAs on energy balance and obesity remain obscure.Thus,this study aimed to explore the role of hypothalamic lnc RNA AK044061 in the regulation of energy balance and the development of dietary obesity in mice.Methods:We established a diet-induced obesity（DIO）mouse model by feeding male C57 BL/6 mice with a 12-week high-fat diet（HFD）.Strand-specific RNA sequencing（RNA-Seq）was used for identifying differentially expressed（DE）lnc RNAs in the hypothalami of HFD-fed versus chow-fed mice.The characterization of hypothalamic lnc RNA AK044061 was assessed using the NCBI,Coding Potential Calculator 2 and in situ hybridization.Quantitative real-time PCR（q RT-PCR）was used to assess the effect of different nutritional statuses on the hypothalamic expression of AK044061.The role of lnc RNA AK044061 in energy balance was assessed via both gain-of-function and loss-of-function by injecting lentiviruses into the arcuate nucleus（ARC）in mice.We measured the indicators for energy metabolism in virus-injected mice.We explored the underlying mechanisms of hypothalamic AK044061 regulating energy metabolism at the molecular and animal levels.The effect of AK044061 on NF-κB activity was measured using dual luciferase reporter assay.RNA immunoprecipitation（RIP）and RNA pull-down assays were conducted to evaluate the association between AK044061 and Rel A.The q RT-PCR and immunofluorescent staining（IF）assays were performed to examine the effect of lnc RNA AK044061 overexpression on hypothalamic cytokine expressions and neuronal activity.We evaluated the effect of acute i.c.v.infusion of NF-κB inhibitor on the body weight and food intake of mice in which AK044061 was overexpressed in the ARC neurons.The RNA-Seq data was used for the differentially expressed analysis and bioinformatics analysis of m RNA in the hypothalami of DIO mice,and for the construction of a protein-protein interaction network.Results:RNA-seq data indicated that 94 lnc RNAs were differentially expressed,with 36 lnc RNAs being upregulated,and the remaining 58 lnc RNAs being downregulated.The CPC2 analysis showed that lnc RNA AK044061 lacked a peptide-coding capacity.The q RT-PCR results displayed that HFD and overnight fasting significantly increased hypothalamic AK044061 level,and that an acute i.c.v.injection of glucose markedly reduced the RNA level of AK044061 in mouse hypothalamus.Besides,2-deoxyglucose treatment could significantly inhibit the increase in hypothalamic AK044061 expression induced by HFD.Combined fluorescence in situ hybridization（FISH）and immunofluorescent staining（IF）images exhibited that AK044061 was expressed in the neurons of the ARC nucleus.Together,the above findings demonstrate that hypothalamic AK044061 may be involved in energy balance regulation and obesity formation in mice.We established an AK044061overexpression mouse model by injecting lentivirus expressing AK044061 into the ARC nucleus.Overexpression of AK044061 in the ARC promoted body weight gain,fat mass,adipocytes size in the epididymal white adipose tissue（e WAT）,plasma total cholesterol（TC）and triglycerides（TG）levels,food intake,and reduced locomotor activity,interscapular temperature,oxygen consumption（VO₂）and energy expenditure（EE）,as well as impaired glucose tolerance,and induced pancreatic islet hypertrophy and hepatic steatosis in mice.Collectively,these results indicate that overexpression of lnc RNA AK044061 in the ARC nucleus leads to an obesity-like phenotype and related metabolic disorders in mice.Also,we established an ARC neuron-specific,AK044061overexpression mouse model by injecting the lentivirus expressing AK044061 into the ARC neurons.The mice injected with Syn-AK044061-Lenti showed an increase in appetite and a decrease in energy expenditure compared with that in the control animals.Thus,overexpression of AK044061 in the ARC,especially in the ARC neurons,leads to an obesity-like phenotype and its related metabolic disorders in mice.We established an Ag RP neuron-specific,AK044061 knockdown mouse model by injecting lentivirus expressing sh AK044061 into the hypothalamic ARC of adult Ag RP-Cre mice.Under an HFD,the mice injected with sh AK044061-p Sico displayed a decrease in appetite and an increase in energy expenditure compared with that in the controls.Collectively,Ag RP neuron-specific knockdown of lnc RNA AK044061 ameliorates DIO and its related metabolic disorders.Dual luciferase reporter assay data showed that AK044061significantly promoted NF-κB activity.RIP and RNA pull-down assays indicated that AK044061 was physically associated with Rel A.The q RT–PCR data demonstrated that overexpression in the ARC neurons markedly elevated the hypothalamic cytokine expressions（TNF-α,IL-1βand IL-6）.IF images suggested lnc RNA AK044061 could modulate neuronal activities in the hypothalamus.Pharmacological inhibition of NF-κB significantly attenuated the effects of lnc RNA AK044061 on body weight and food intake in mice.Together,these data reveal that NF-k B may be important for lnc RNA AK044061to regulate energy balance.RNA-seq data identified that 347 m RNAs were differentially expressed,with 259 m RNAs being increased,and the remaining 88 m RNAs being decreased.Functional analysis showed the enrichment of differentially expressed m RNAs in specific molecular functions and pathway,including energy homeostasis and inflammatory signaling,emphasizing the importance of these differentially expressed m RNAs in the regulation of obesity.Conclusions:These results demonstrate that the excessive expression of AK044061 in the ARC nucleus,or specifically in the neurons of arcuate nucleus（ARC）induced an obesity-like phenotype and its related metabolic comorbidities in mice.Ag RP neuron-specific knockdown of AK044061 dramatically protected against HFD-induced obesity and its related metabolic dysregulations.Our data indicate that lnc RNA AK044061 is physically associated with Rel A,and can enhance the transcriptional activity of NF-κB.Therefore,we disclose that lnc RNA AK044061expressed in the hypothalamus,especially in the Ag RP neurons,is involved in the development of dietary obesity in mice.