HMGR overexpression and interference affects the expression of steroidogenic genes and cholesterol content in bovine intramuscular adipocytes

HMGR overexpression and interference affects the expression of steroidogenic genes and cholesterol content in bovine intramuscular adipocytes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Help Center Sign In Submit a Preprint Cite Share Download PDF Research article HMGR overexpression and interference affects the expression of steroidogenic genes and cholesterol content in bovine intramuscular adipocytes Xiaomu Liu, Wei You, Xianglun Zhang, Qing Jin, Xiuwen Tan, Chen Wei, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.10141/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background:Previously, we found that mevalonic acid stimulates HMGR expression in bovine intramuscular adipocytes, and influences adipocyte differentiation. However, it remains unclear whether there is any direct link between HMGR, steroidogenic genes, and cholesterol content. RNA-Seq was conducted to determine the differences between the gene expression profiles of bovine adipocytes containing different HMGR expression constructs. Results:In total, 10 234 differentially expressed genes (DEGs) were found. Of these, 35 and 6 DEGs between the control and the overexpression groups were functionally related to lipid and energy metabolisms, respectively. Additionally, 43 and 8 DEGs between the control and the HMGR inhibition groups were related to lipid and energy metabolism. Additionally, several DEGs related to lipid and energy metabolism were identified between the HMGR overexpression group and the HMGR interference group. Several DEGs correlated positively or negatively with overexpression or inhibition of HMGR. We also found that, following activation or inhibition of the HMGR gene, AMPK and SIRT1 had opposite expression patterns in bovine intramuscular adipocytes. Interestingly, the HMGR gene was downregulated when HMGR was overexpressed, and upregulated when HMGR was inhibited. Conclusion:Our findings establish a theoretical understanding of signaling pathways involved in cholesterol synthesis by elucidating the relationships between key genes. Epigenetics & Genomics HMGR Overexpression Interference Cholesterol Bovine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Intramuscular fat (IMF) is an important factor influencing meat quality in beef cattle [ 1-4 ]. The right amount of IMF can enhance meat quality traits such as flavor, juiciness, and tenderness [ 5 , 6 ]. One of the important traits in beef that researchers have focused on are the mechanisms and regulation of fat deposition in fat tissue following increased or decreased fat intake, which may be related to concentrations of circulating cholesterol [ 7 , 8 ]. With increasing concerns regarding the relationship between fat intake and health, consumers have become more conscious of what constitutes a healthy diet, and are increasingly demanding products with reduced cholesterol content [ 9 ]. Due to the high cholesterol content and the appealing taste of IMF, beef cattle are considered to be an attractive candidate for cholesterol reduction and dietary improvement. Three-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), is a rate-limiting enzyme of cholesterol synthesis. This protein can catalyze the conversion of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) to mevalonate, and is a key factor in the regulation of cholesterol content in vivo [ 10 , 11 ]. Sirtuin type 1 (SIRTl), AMP-activated protein kinase (AMPK), and lipoprotein lipase (LPL) play important roles in the regulation of the energy metabolism. Previous studies have shown that long-term moderate caloric restriction and resveratrol supplementation can improve lipid disorders and decrease fat accumulation, by regulating the SIRT1-autophagy pathway [ 12 ]. However, it remains unclear whether there is any direct link between HMGR, steroidogenic genes, and cholesterol content. Cholesterol content is a complex genetic trait. The genetics underlying the biochemical process and molecular background that determine cholesterol content are not yet fully understood, particularly with regard to local Chinese cattle breeds. Using RNA-Seq analysis to select differently expressed genes (DEGs) correlated with cholesterol levels, the present study aimed to explore the effects of HMGR overexpression and interference on the expression patterns of cholesterol-related genes, as well as on differential gene expression in bovine intramuscular adipose cells. Materials and methods Cell culture and induction of preadipocyte differentiation Cells from intramuscular fat tissue of Lilu cattle were isolated according to the method described by Liu et al. [ 13 ]. Cattles were raised in the Shandong Provincial Testing Center of Beef Cattle Performance (Shandong Province, Jinan, China). The cells were cultured and maintained in DMEM/F12 (HyClone, UT, USA) containing 10% fetal bovine serum (FBS; Gibco, NY, USA), an antibiotic-antimycotic agent (100 U/ml penicillin and 100 μg/ml streptomycin; Gibco). Cells were seeded at 2 × 10 4 cells/cm 2 and incubated at 37°C in a humidified 5% CO 2 environment. To induce adipogenesis, two-day post-confluent preadipocytes (designated as day zero) were incubated for 72 h in differentiation-inducing medium with DMEM/F12, 4 μg/ml insulin (Sigma, St. Louis, MO, USA), 0.1 mg/ml dexamethasone (DEX; Sigma), and 0.1 mg/ml 3-isobutyl-1-methylxanthine (IBMX; Sigma). The cells were then transferred into DMEM/F12 containing 1 mM octanoate (Sigma), 10 mM acetate (Sigma), 10 μg/ml transferrin (Sigma), 3 μg/ml cholesterol (Sigma), 17 mM biotin (Sigma), 100 mM calcium pantothenate (Sigma), and 0.5% bovine serum albumin (BSA). After induction, the experimental cell groups were defined as follows: 1) adipogenic group (control group), 2) adipogenic + overexpression negative control (NC) group (viral transduction control group), 3) adipogenic + HMGR overexpression group, 4) adipogenic + interference NC group (transfected with an unrelated sequence), and 5) adipogenic + HMGR interference group The animals used in this study were reared and sacrificed in compliance with national regulations for the humane care and use of animals in research (China Administration Rule of Laboratory Animals, Operating Procedure of Cattle Slaughtering GB/T 19477-2004). Oil Red O staining To examine lipid accumulation, cells were seeded in six-well culture plates at 5 × 10 4 /cm 2 . At eight days of incubation, the medium was removed, and the cells were washed three times with phosphate-buffered saline (PBS; Beijing Dingguo Changsheng Biotechnology Co. Ltd. Beijing, China) and fixed with 10% formaldehyde for 30 min at room temperature. After washing with PBS, the cells were stained for at least 1 h with 1% filtered Oil Red O (6:4 ratio of Oil Red O stock solution to H 2 O, whereby the Oil Red O stock solution comprised 0.5% Oil Red O in isopropyl alcohol). Oil Red O was obtained from Beijing Dingguo Changsheng Biotechnology Co. Ltd. Morphological features of cells were examined by microscopy (Olympus Corporation, Japan). Construction of the HMGR expression vector To determine the effects of HMGR on cholesterol synthesis, bovine intramuscular adipocytes were transfected with recombinant adenoviral vectors (Beijing Dingguo Changsheng Biotechnology Co. Ltd.). The recombinant vector ADV4-HMGR was then transfected into adipose cells via the method described by Hofgen and Willmitzer [ 14 ]. siRNA transfection The sequence of the small interfering RNA (siRNA) for HMGR inhibition was as follows (5'–3'): sense, GUUCUAACUCACAGGAUGATT; and antisense, UCAUCCUGUGAGUUAGAACTT. The siRNA oligonucleotides were designed by Beijing Dingguo Changsheng Biotechnology Co. Ltd. The siRNA inhibition group and siRNA negative control group were transfected for 48 h using the lipofectamine transfection reagent (11668-500; Invitrogen), according to the manufacturer's protocol. Detection of DEGs The generation and processing of raw RNA-seq data was conducted by the Beijing Genomics Institute (Beijing, China) using the Illumina HiSeq platform. We detected DEGs using the PossionDis algorithm, which is based on the Poisson distribution, and was performed as described by Audic et al. [ 15 ]. The parameters to determine significant changes in gene were as follows: fold change ≥ 2 and a false discovery rate (FDR) ≤ 0.001. Pathway analysis of DEGs DEGs were annotated according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We performed pathway enrichment analysis using phyper, a function in R based on hypergeometric distribution (https://cran.r-project.org/) . We then calculated the FDR for each p-value, and considered an FDR < 0.001 to represent significant enrichment. Quantification of total cholesterol in adipose cells Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured in adipose cells after seeding in a six-well culture plate, using detection kits specific to each cholesterol type (T-CHO kit, HDL-C kit, and LDL-C kit; Nanjing Jiancheng Biology Institute, Nanjing, China). Total RNA isolation and quantitative real-time PCR Total RNA was isolated form adipose cells using the TRIzol ® reagent (15596-018; Invitrogen, CA, USA), and then converted into cDNA using the PrimeScript ® RT Reagent Kit (DRR027A; TaKaRa, Shanghai, China). Expression of selected genes was then analyzed using the Fast SYBR® Green Master Mix Bulk Pack (4385614; Invitrogen, USA). The following primers were used for quantitative real-time PCR (qRT-PCR): HMGR sense, 5'-TGCTGGTGCTGAGTATGTGG-3' and antisense, 5'-CATTCTACAAGAGCATCCAGG-3'; LPL sense, 5'-GACACTTGCCACCTCATTC-3' and antisense, 5'-CATCCGCCATCCAGTTCATA-3'; AMPK sense, 5'-CCGTATTATTTGCGTGTTCG-3' and antisense, 5'-TGTGGCGTAGCAGTCCCT-3'; SIRT1 sense, 5'-TGCAATAGACTTTCCAGACC-3' and antisense, 5'-GTGTATCTATGTTCTGAGT-3'; and GAPDH sense, 5'-TGCTGGTGCTGAGTATGTGG-3' and antisense, 5'-GATGATGACCCTCTTGGCG-3' (Beijing Dingguo Changsheng Biotechnology Co. Ltd.). Three replicates were used for each experiment. Relative mRNA expression levels were measured using the comparative Ct method (∆∆Ct), with GAPDH as the internal control. Western blotting Cell extracts were isolated in RIPA buffer (1 ml per 10 7 cells/100 mm-dish/150 cm 2 -flask; 0.5 ml per 5 × 10 6 cells/60 mm-dish/75 cm 2 -flask; WB-0071; Beijing Dingguo Changsheng Biotechnology Co. Ltd.). The Pierce BCA Assay (BCA01; Beijing Dingguo Changsheng Biotechnology Co. Ltd.) was used to determine protein concentration. Samples (50–100 μg) were separated using 10% sodium dodecyl sulfate polyvinylidene fluoride (SDS-PVDF) gels, and then transferred to PVDF membranes (XLL092-2; Pall, USA). The membranes were incubated with anti-β-actin (SC-47778; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-HMGR (Ab174830; Abcam, Cambridge, United Kingdom), anti-AMPK (Ab3759; Abcam), anti-LPL (Ab21356; Abcam), and anti-SIRT1 (Bs-0921R; Bioss Biotechnology, Inc., Beijing, China) antibodies in blocking buffer for 2 h at room temperature. After the membranes were incubated with secondary antibodies (Santa Cruz Biotechnology, Inc.), the enhanced chemiluminescence system was used to detect immunoreactive bands. Statistical analysis Differences in gene expression between the experimental groups were calculated using an analysis of variance (ANOVA), followed by a Bonferroni test. Results were considered statistically significant when P < 0.05. Results Adipose cell morphology and lipid accumulation We first evaluated morphological changes in induced adipogenic bovine intramuscular adipocytes, and stained cells with Oil Red O to visualize lipid droplet accumulation after induction. As shown in Figure 1, induced adipocytes were large, round, and filled with fat droplets, compared to immature adipocytes. DEG detection We sequenced RNA from the five experimental groups using the Illumina HiSeq platform, and generated an average of 6.58 Gb for each sample. As showed in Figure 2, after mapping sequenced reads to the reference genome and reconstructing the transcripts, we obtained 13 264 novel transcripts from all samples. Of these, 9 444 were previously unknown splicing events for known genes, 862 were novel coding transcripts without any known features, and the remaining 2 958 were long noncoding RNAs. We then analyzed differential gene expression between samples. The summary of DEGs is shown in Figure 2. There were a total of 10 234 DEGs, and this number was higher when the HMGR gene was overexpressed or suppressed. There were 1 554 DEGs found in the HMGR overexpression group vs. the HMGR interference group, where genes were mostly upregulated. We also found that there were more upregulated genes within DEGs between all experimental groups. Pathway analysis of DEGs We performed KEGG pathway classification and functional enrichment analyses for DEGs (Figure 3). These DEGs were found to be mostly involved in signal transduction, cancers, infectious disease, global and overview maps, and the immune system. As shown in Figure 3a and 3b, 35 DEGs after HMGR overexpression, and 43 DEGs after HMGR inhibition, were functionally involved in lipid metabolism. Furthermore, 6 DEGs after HMGR overexpression, and 8 DEGs after HMGR inhibition, were functionally involved in energy metabolism. AMPK signaling Changes in gene expression relevant to the AMPK signaling pathway following over-expression of the HMGR gene are shown in Figure 4a and 4b. Interestingly, the HMGR gene was downregulated when HMGR was overexpressed, whereas the SIRT1 gene was upregulated. In contrast, the HMGR gene itself was upregulated following HMGR interference. TC, LDL-C, and HDL-C content in adipose cells To understand the impact of HMGR overexpression on the amount of TC, LDL-C, and HDL-C, we examined the levels of these types of cholesterol in the five experimental groups. In contrast with other groups, the HMGR overexpression group had the highest amounts of TC, LDL-C, and HDL-C, particularly for TC and HDL-C (Figure 5). qRT-PCR of HMGR, AMPK, SIRT1, and LPL To investigate the relationship between HMGR expression and cholesterol synthesis in bovine adipose cells, expression levels of HMGR, AMPK, SIRT1, and LPL, which are four steroidogenesis-related genes, were assessed in adipose tissue using qRT-PCR (Figure 6). For HMGR, the overexpression group showed higher mRNA expression levels than those in other groups, with the HMGR interference group showing the lowest levels out of all groups. AMPK mRNA expression was lowest when HMGR was overexpressed; even though AMPK protein expression levels were increased (as shown in Figure 7). SIRT1 showed similar results as AMPK, whereas LPL expression levels were higher when HMGR was overexpressed, and lower when HMGR was inhibited. Western blotting of HMGR, AMPK, SIRT1, and LPL To investigate changes in protein expression, we performed western blotting in the five experimental groups (Figure 7). The results showed that HMGR protein expression in the HMGR overexpression group was higher than that in the other four groups. AMPK protein expression levels showed a little differences between the groups. SIRT-1 and LPL protein expression levels were higher in the HMGR interference group, and lower in the HMGR overexpression group. Discussion Adipose tissue is the major depot for energy and cholesterol storage, with most of the intracellular cholesterol distributed in the form of lipid droplets [ 16 , 17 ]. Cholesterol homeostasis may have a role in the regulation of adipocyte size and function [ 18 , 19 ]. In addition, cellular cholesterol homeostasis is involved in various diseases of nongenetic origin, such as atherosclerosis, leading to lipid accumulation in target organs [ 18 , 20 ]. In the present study, DEG and KEGG pathway analyses were performed to explore the regulatory network underlying bovine adipose cell following HMGR overexpression and interference. As expected, several well-known pathways [ 21 ] related to lipid metabolism were identified, particularly the AMPK signaling pathway. In AMPK signaling, the HMGR, AMPK, and SITR1 genes play a functional role in lipid metabolism or cholesterol biosynthesis. In terms of cholesterol levels, we found that those of TC, LDL-C, and HDL-C were highest when the HMGR gene was overexpressed; however, their levels were not majorly decreased when HMGR was inhibited. When we investigated protein expression of HMGR, AMPK, SIRT-1, and LPL, we found that HMGR protein levels were higher in the HMGR overexpression group compared to those in the control group. However, SIRT-1 protein levels were lower in the HMGR overexpression group compared to those in the HMGR interference group. Therefore, the results obtained for protein expression were consistent with those obtained for gene expression. Given that HMGR is a rate-limiting enzyme in cholesterol biosynthesis [ 10 ], we generated an adipocyte gene expression dataset using RNA-Seq, and examined the relationship between steroidogenic genes and cholesterol levels by overexpressing or inhibiting the HMGR gene. Transcriptional and pathway analysis results showed that overexpression of HMGR correlated with downregulation of AMPK and SIRT1 gene expression. AMPK is a crucial energy sensor that maintains energy homeostasis. It is also a major regulator of the lipid metabolism through phosphorylation and inactivation of numerous metabolic enzymes, including HMGR [ 22 , 23 ]. In adipose tissue, the chronic activation of AMPK led to downregulation of HMGR expression in a new transgenic mouse model [ 24 ]. The 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide was shown to induce activation of AMPK, and directly inhibit the expression of HMGR, one of its target genes [ 22 ]. In the present study, HMGR mRNA expression was reduced when it was overexpressed in bovine adipose cells. In contrast, HMGR expression was increased when the HMGR gene was inhibited. These results are important, given the key role of the HMGR gene in the process of cholesterol synthesis. SIRT1 is a longevity-associated deacetylase enzyme that modulates metabolic homeostasis in response to cellular energy. AMPK and SIRT1 are related proteins and share common target pathways [ 25 ]. The present study showed that the expression of SIRT1 decreased when the HMGR gene was overexpressed. However, SIRT1 was upregulated when HMGR expression was modified. These results imply that the HMGR and SIRT1 genes have contrasting roles in the cholesterol synthesis pathway. The LPL gene encodes a rate-limiting enzyme that has a key role in the hydrolysis of triglycerides. LPL deficiency and dysfunction is associated with many disorders of the lipoprotein metabolism [26, 27 ]. In the present study, our results showed that the patterns of LPL gene expression were consistent with those of HMGR expression. Conclusions This study showed that both overexpression and interference of HMGR in bovine intramuscular adipocytes could regulate the expression levels of relevant genes. Several DEGs correlated positively or negatively with HMGR overexpression or inhibition. Furthermore, AMPK and SIRT1 showed opposite expression patterns in bovine intramuscular adipocytes following overexpression or inhibition of HMGR. These findings shed light on the signaling pathways involved in the cholesterol synthesis process, and elucidate how cholesterol levels may be controlled for future beef commercial production via a multi-gene pyramiding system. Declarations Abbreviations Not applicable. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and material Not applicable. Competing interests The authors declare that they have no competing interest. Funding This work was supported by Young Talents Training Program of Shandong Academy of Agricultural Science, Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences (CXGC2018E10), the National Natural Science Foundation of China (grants no. 31601966 and no. 31100890), and CARS-37. Authors' contributions Guifen Liu and Fachun Wan designed the experiments. Xiaomu Liu and Wei You drafted the manuscript. Xianglun Zhang, Qing Jin, Xiuwen Tan, Chen Wei, Hongbo Zhao, Chen Zhang and Yifan Liu performed the experiments. Acknowledgements Not Applicable. References Cui H, Liu XRR, Zhao GP, Zheng MQ, Chen JL, Wen J (2012) Identification of differentially expressed genes and pathways for intramuscular fat deposition in pectoralis major tissues of fast-and slow-growing chickens. BMC genomics 13:213 Fernandez X, Monin G, Talmant A, Mourot J, Lebret B (1999) Influence of intramuscular fat content on the quality of pig meat-1. Composition of the lipid fraction and sensory characteristics of m. longissimus lumborum. Meat science 53(1):59-65 Fernandez X, Monin G, Talmant A, Mourot J, Lebret B (1999) Influence of intramuscular fat content on the quality of pig meat - 2. Consumer acceptability of m. longissimus lumborum. Meat science 53(1):67-72 Rincker PJ, Killefer J, Ellis M, Brewer MS, McKeith FK (2008) Intramuscular fat content has little influence on the eating quality of fresh pork loin chops. Journal of animal science 86(3):730-737 Soret B, Mendizabal JA, Arana A, Alfonso L (2016) Expression of genes involved in adipogenesis and lipid metabolism in subcutaneous adipose tissue and longissimus muscle in low-marbled Pirenaica beef cattle. Animal : an international journal of animal bioscience 10(12):2018-2026 Hocquette JF, Gondret F, Baeza E, Medale F, Jurie C, Pethick DW (2010) Intramuscular fat content in meat-producing animals: development, genetic and nutritional control, and identification of putative markers. Animal : an international journal of animal bioscience 4(2):303-319 da Costa AS, Pires VM, Fontes CM, Mestre Prates JA (2013) Expression of genes controlling fat deposition in two genetically diverse beef cattle breeds fed high or low silage diets. BMC veterinary research 9:118 Haberka M, Okopien B, Gasior Z (2016) Obesity, ultrasound indexes of fat depots and lipid goal attainment in patients with high and very high cardiovascular risk: A novel approach towards better risk reduction. Nutrition, metabolism, and cardiovascular diseases : NMCD 26(2):123-133 Ospina EJ, Sierra CA, Ochoa O, Perez-Alvarez JA, Fernandez-Lopez J (2012) Substitution of saturated fat in processed meat products: a review. Critical reviews in food science and nutrition 52(2):113-122 Istvan ES, Deisenhofer J (2000) The structure of the catalytic portion of human HMG-CoA reductase. Biochimica et biophysica acta 1529(1-3):9-18 Jiang J, Kai G, Cao X, Chen F, He D, Liu Q (2006) Molecular cloning of a HMG-CoA reductase gene from Eucommia ulmoides Oliver. Bioscience reports 26(2):171-181 Ding S, Jiang J, Zhang G, Bu Y, Zhang G, Zhao X (2017) Resveratrol and caloric restriction prevent hepatic steatosis by regulating SIRT1-autophagy pathway and alleviating endoplasmic reticulum stress in high-fat diet-fed rats. PloS one 12(8):e0183541 Liu X, You W, Cheng H, Zhang Q, Song E, Wan F, Han H, Liu G (2016) Effect of mevalonic acid on cholesterol synthesis in bovine intramuscular and subcutaneous adipocytes. Journal of applied genetics 57(1):113-118 Hofgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic acids research 16(20):9877 Audic S, Claverie JM (1997) The significance of digital gene expression profiles. Genome research 7(10):986-995 Farkas J, Angel A, Avigan MI (1973) Studies on the compartmentation of lipid in adipose cells. II. Cholesterol accumulation and distribution in adipose tissue components. Journal of lipid research 14(3):344-356 Zhu Y, Chen CY, Li J, Cheng JX, Jang M, Kim KH (2018) In vitro exploration of ACAT contributions to lipid droplet formation during adipogenesis. Journal of lipid research 59(5):820-829 Le Lay S, Krief S, Farnier C, Lefrere I, Le Liepvre X, Bazin R, Ferre P, Dugail I (2001) Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene expression in adipocytes. The Journal of biological chemistry 276(20):16904-16910 Shao F, Wang X, Yu J, Jiang H, Zhu B, Gu Z (2014) Expression of miR-33 from an SREBF2 intron targets the FTO gene in the chicken. PloS one 9(3):e91236 Khera AV, Demler OV, Adelman SJ, Collins HL, Glynn RJ, Ridker PM, Rader DJ, Mora S (2017) Cholesterol Efflux Capacity, High-Density Lipoprotein Particle Number, and Incident Cardiovascular Events: An Analysis From the JUPITER Trial (Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin). Circulation 135(25):2494-2504 Kokta TA, Dodson MV, Gertler A, Hill RA (2004) Intercellular signaling between adipose tissue and muscle tissue. Domestic animal endocrinology 27(4):303-331 Liu S, Jing F, Yu C, Gao L, Qin Y, Zhao J (2015) AICAR-Induced Activation of AMPK Inhibits TSH/SREBP-2/HMGCR Pathway in Liver. PloS one 10(5):e0124951 Hardie DG, Ross FA, Hawley SA (2012) AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nature reviews Molecular cell biology 13(4):251-262 Knowles C, Liu ZM, Yang J (2011) Compensatory increase in lipogenic gene expression in adipose tissue of transgenic mice expressing constitutively active AMP-activated protein kinase-alpha1 in liver. Biochemical and biophysical research communications 412(2):249-252 Arab Sadeghabadi Z, Nourbakhsh M, Pasalar P, Emamgholipour S, Golestani A, Larijani B, Razzaghy-Azar M (2018) Reduced gene expression of sirtuins and active AMPK levels in children and adolescents with obesity and insulin resistance. Obesity research & clinical practice 12(2):167-173 Xie C, Wang ZC, Liu XF, Yang MS (2010) The common biological basis for common complex diseases: evidence from lipoprotein lipase gene. European journal of human genetics : EJHG 18(1):3-7 Socquard E, Durlach A, Clavel C, Nazeyrollas P, Durlach V (2006) Association of HindIII and PvuII genetic polymorphisms of lipoprotein lipase with lipid metabolism and macrovascular events in type 2 diabetic patients. Diabetes & metabolism 32(3):262-269 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1219","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":93986,"identity":"787e0334-86fd-4afc-9df1-0600b92e7c06","order_by":1,"name":"Xiaomu Liu","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaomu","middleName":"","lastName":"Liu","suffix":""},{"id":93987,"identity":"1ba69327-edda-4dfa-b628-ba2434323949","order_by":2,"name":"Wei You","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"You","suffix":""},{"id":93988,"identity":"edf4807e-e463-4729-9fb5-8d7bd9d25d39","order_by":3,"name":"Xianglun Zhang","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xianglun","middleName":"","lastName":"Zhang","suffix":""},{"id":93989,"identity":"4e6b83ef-1f4a-4592-afa5-62058e4e55f4","order_by":4,"name":"Qing Jin","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Jin","suffix":""},{"id":93990,"identity":"a030fddc-e0ca-4479-be37-572435b4f09a","order_by":5,"name":"Xiuwen Tan","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiuwen","middleName":"","lastName":"Tan","suffix":""},{"id":93991,"identity":"cfa7dd23-6d62-467c-a04e-a304c03143cc","order_by":6,"name":"Chen Wei","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Wei","suffix":""},{"id":93992,"identity":"0e98d735-2619-4d70-bd2b-3da2102945c1","order_by":7,"name":"Hongbo Zhao","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongbo","middleName":"","lastName":"Zhao","suffix":""},{"id":93993,"identity":"8a167573-2c65-450c-9bdf-1434373072eb","order_by":8,"name":"Chen Zhang","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Zhang","suffix":""},{"id":93994,"identity":"e9bdd2d7-0e45-4333-8275-4ba4066a6d21","order_by":9,"name":"Yifan Liu","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Liu","suffix":""},{"id":93995,"identity":"d20ac7da-9143-4089-8f57-9945bda3f816","order_by":10,"name":"Fachun Wan","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fachun","middleName":"","lastName":"Wan","suffix":""},{"id":93996,"identity":"397d051a-57fa-4c06-95bc-0b7597b693a2","order_by":11,"name":"guifen liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYBACPmYQaSDHwMDMfPDBBwMbOYJa2CBajBkY2NmSDWcUpBkT1gKhgAr5ecyEeT4cTiSshZ3H+DVPgYGcOTODGbONAXMCA/vhoxvwO4zHzJrHwMDYspkh7XGOAVseA09a2g1CWox5DP4kbjjMcNw4x4CnmEGCx4wYLQb1Gw4ztklbGEgkNhChxfgxUEuCwWFmNmkGAwNitLCVMc4xMDDccJiN2bDHIMGYjZBf+PkPb/7w5o+BvMH58x8f/PjzX46f/fAxvFpAFkmgcgkoBwHmD0QoGgWjYBSMgpEMAOefPBkq/BIWAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3300-8028","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"guifen","middleName":"","lastName":"liu","suffix":""}],"badges":[],"createdAt":"2019-06-05 13:31:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.2.10141/v1","doiUrl":"https://doi.org/10.21203/rs.2.10141/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":608262,"identity":"e39118e1-e03b-4655-8e6f-e22e93da5ae1","added_by":"auto","created_at":"2020-03-06 17:45:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":481936,"visible":true,"origin":"","legend":"Oil Red O staining of bovine adipose cells. Original magnification: × 400.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1219/v1/Figure 1.jpg"},{"id":608263,"identity":"e36779f0-8475-4e4f-be48-8ea9b0c76f4c","added_by":"auto","created_at":"2020-03-06 17:45:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126487,"visible":true,"origin":"","legend":"Summary of DEGs. The X axis shows the compared samples. The Y axis shows DEG numbers. The red color represents upregulated DEGs, and the blue color represents downregulated DEGs. 1: adipogenic group (control group), 2: adipogenic + overexpression NC, 3: adipogenic + HMGR overexpression group, 4: adipogenic + interference NC, and 5: adipogenic + HMGR interference group. DEGs, differentially expressed genes.","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1219/v1/Figure 2.jpg"},{"id":608264,"identity":"f786540d-9288-4917-bfd8-e0bd8882d6c5","added_by":"auto","created_at":"2020-03-06 17:45:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":723780,"visible":true,"origin":"","legend":"Pathway annotation of DEGs. The X axis shows the number of DEGs. The Y axis shows the pathway names. Left column: adipogenic group (control group) vs. adipogenic + HMGR overexpression group. Right column: adipogenic group (control group) vs. adipogenic + HMGR interference group. DEGs, differentially expressed genes.","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1219/v1/Figure 3.jpg"},{"id":608265,"identity":"c3f23f87-6d8f-4f17-b5c8-df3c91773e72","added_by":"auto","created_at":"2020-03-06 17:45:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2474344,"visible":true,"origin":"","legend":"The AMPK signaling pathway. Upregulated genes are marked with red borders and downregulated genes with green borders. Non-differentially expressed genes are marked with black borders.","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1219/v1/Figure 4.jpg"},{"id":608266,"identity":"59000fd4-5863-43dc-b6a8-4037bf9341f6","added_by":"auto","created_at":"2020-03-06 17:45:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":118228,"visible":true,"origin":"","legend":"The levels of TC, LDL-C, and HDL-C in five different experimental groups. 1: adipogenic group (control group), 2: adipogenic + overexpression NC, 3: adipogenic + HMGR overexpression group, 4: adipogenic + interference NC, and 5: adipogenic + HMGR interference group. NC, negative control.","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1219/v1/Figure 5.jpg"},{"id":608267,"identity":"3820dbf5-ad49-4470-aa57-719b60d69343","added_by":"auto","created_at":"2020-03-06 17:45:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":129530,"visible":true,"origin":"","legend":"Gene expression of HMGR, AMPK, SIRT1, and LPL in intramuscular adipose cells.","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1219/v1/Figure 6.jpg"},{"id":608268,"identity":"a75cac60-deab-4844-9022-e1bf83bda323","added_by":"auto","created_at":"2020-03-06 17:45:52","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":129552,"visible":true,"origin":"","legend":"Western blotting of HMGR, AMPK, SIRT1, and LPL in the five experimental groups. Column 1: adipogenic group (control group), column 2: adipogenic group + overexpression NC, column 3: adipogenic + HMGR overexpression group, column 4: adipogenic + interference NC, and column 5: adipogenic + HMGR interference group. NC, negative control.","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1219/v1/Figure 7.jpg"},{"id":13467353,"identity":"a615ae1a-a66a-481a-b097-43d8ec88aeae","added_by":"auto","created_at":"2021-09-16 20:55:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":945471,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1219/v1/685f1768-484e-48a5-9549-eefd451be727.pdf"}],"financialInterests":"","formattedTitle":"HMGR overexpression and interference affects the expression of steroidogenic genes and cholesterol content in bovine intramuscular adipocytes","fulltext":[{"header":"Background","content":"\u003cp\u003eIntramuscular fat (IMF) is an important factor influencing meat quality in beef cattle\n [\u003ca href=\"#_ENREF_1\"\u003e\n 1-4\u003c/a\u003e]. The right amount of IMF can enhance meat quality traits such as flavor, juiciness,\n and tenderness [\u003ca href=\"#_ENREF_5\"\u003e\n 5\u003c/a\u003e, \u003ca href=\"#_ENREF_6\"\u003e\n 6\u003c/a\u003e]. One of the important traits in beef that researchers have focused on are the mechanisms\n and regulation of fat deposition in fat tissue following increased or decreased fat\n intake, which may be related to concentrations of circulating cholesterol [\u003ca href=\"#_ENREF_7\"\u003e\n 7\u003c/a\u003e, \u003ca href=\"#_ENREF_8\"\u003e\n 8\u003c/a\u003e]. With increasing concerns regarding the relationship between fat intake and health,\n consumers have become more conscious of what constitutes a healthy diet, and are increasingly\n demanding products with reduced cholesterol content [\u003ca href=\"#_ENREF_9\"\u003e\n 9\u003c/a\u003e]. Due to the high cholesterol content and the appealing taste of IMF, beef cattle\n are considered to be an attractive candidate for cholesterol reduction and dietary\n improvement.\u003c/p\u003e\n \n\u003cp\u003eThree-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), is a rate-limiting enzyme\n of cholesterol synthesis. This protein can catalyze the conversion of 3-hydroxy-3-methyl-glutaryl-coenzyme\n A (HMG-CoA) to mevalonate, and is a key factor in the regulation of cholesterol content\n in vivo [\u003ca href=\"#_ENREF_10\"\u003e\n 10\u003c/a\u003e, \u003ca href=\"#_ENREF_11\"\u003e\n 11\u003c/a\u003e]. Sirtuin type 1 (SIRTl), AMP-activated protein kinase (AMPK), and lipoprotein lipase\n (LPL) play important roles in the regulation of the energy metabolism. Previous studies\n have shown that long-term moderate caloric restriction and resveratrol supplementation\n can improve lipid disorders and decrease fat accumulation, by regulating the SIRT1-autophagy\n pathway [\u003ca href=\"#_ENREF_12\"\u003e\n 12\u003c/a\u003e]. However, it remains unclear whether there is any direct link between HMGR, steroidogenic\n genes, and cholesterol content.\u003c/p\u003e\n \n\u003cp\u003eCholesterol content is a complex genetic trait. The genetics underlying the biochemical\n process and molecular background that determine cholesterol content are not yet fully\n understood, particularly with regard to local Chinese cattle breeds. Using RNA-Seq\n analysis to select differently expressed genes (DEGs) correlated with cholesterol\n levels, the present study aimed to explore the effects of HMGR overexpression and\n interference on the expression patterns of cholesterol-related genes, as well as on\n differential gene expression in bovine intramuscular adipose cells. \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch2\u003eCell culture and induction of preadipocyte differentiation \u003c/h2\u003e\n \n\u003cp\u003eCells from intramuscular fat tissue of Lilu cattle were isolated according to the\n method described by Liu et al. [\u003ca href=\"#_ENREF_13\"\u003e\n 13\u003c/a\u003e]. Cattles were raised in the Shandong Provincial Testing Center of Beef Cattle Performance\n (Shandong Province, Jinan, China). The cells were cultured and maintained in DMEM/F12\n (HyClone, UT, USA) containing 10% fetal bovine serum (FBS; Gibco, NY, USA), an antibiotic-antimycotic\n agent (100 U/ml penicillin and 100 μg/ml streptomycin; Gibco). Cells were seeded at\n 2 × 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e and incubated at 37°C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e environment. To induce adipogenesis, two-day post-confluent preadipocytes (designated\n as day zero) were incubated for 72 h in differentiation-inducing medium with DMEM/F12,\n 4 μg/ml insulin (Sigma, St. Louis, MO, USA), 0.1 mg/ml dexamethasone (DEX; Sigma),\n and 0.1 mg/ml 3-isobutyl-1-methylxanthine (IBMX; Sigma). The cells were then transferred\n into DMEM/F12 containing 1 mM octanoate (Sigma), 10 mM acetate (Sigma), 10 μg/ml transferrin\n (Sigma), 3 μg/ml cholesterol (Sigma), 17 mM biotin (Sigma), 100 mM calcium pantothenate\n (Sigma), and 0.5% bovine serum albumin (BSA). After induction, the experimental cell\n groups were defined as follows: 1) adipogenic group (control group), 2) adipogenic\n + overexpression negative control (NC) group (viral transduction control group), 3)\n adipogenic + HMGR overexpression group, 4) adipogenic + interference NC group (transfected\n with an unrelated sequence), and 5) adipogenic + HMGR interference group \u003c/p\u003e\n \n\u003cp\u003eThe animals used in this study were reared and sacrificed in compliance with national\n regulations for the humane care and use of animals in research (China Administration\n Rule of Laboratory Animals, Operating Procedure of Cattle Slaughtering GB/T 19477-2004).\u003c/p\u003e\n \n\u003ch2\u003eOil Red O staining\u003c/h2\u003e\n \n\u003cp\u003eTo examine lipid accumulation, cells were seeded in six-well culture plates at 5 ×\n 10\u003csup\u003e4\u003c/sup\u003e/cm\u003csup\u003e2\u003c/sup\u003e. At eight days of incubation, the medium was removed, and the cells were washed three\n times with phosphate-buffered saline (PBS; Beijing Dingguo Changsheng Biotechnology\n Co. Ltd. Beijing, China) and fixed with 10% formaldehyde for 30 min at room temperature.\n After washing with PBS, the cells were stained for at least 1 h with 1% filtered Oil\n Red O (6:4 ratio of Oil Red O stock solution to H\u003csub\u003e2\u003c/sub\u003eO, whereby the Oil Red O stock solution comprised 0.5% Oil Red O in isopropyl alcohol).\n Oil Red O was obtained from Beijing Dingguo Changsheng Biotechnology Co. Ltd. Morphological\n features of cells were examined by microscopy (Olympus Corporation, Japan).\u003c/p\u003e\n \n\u003ch2\u003eConstruction of the HMGR expression vector\u003c/h2\u003e\n \n\u003cp\u003eTo determine the effects of HMGR on cholesterol synthesis, bovine intramuscular adipocytes\n were transfected with recombinant adenoviral vectors (Beijing Dingguo Changsheng Biotechnology\n Co. Ltd.). The recombinant vector ADV4-HMGR was then transfected into adipose cells\n via the method described by Hofgen and Willmitzer [\u003ca href=\"#_ENREF_14\"\u003e\n 14\u003c/a\u003e]. \u003c/p\u003e\n \n\u003ch2\u003esiRNA transfection \u003c/h2\u003e\n \n\u003cp\u003eThe sequence of the small interfering RNA (siRNA) for HMGR inhibition was as follows (5'–3'): sense, GUUCUAACUCACAGGAUGATT; and antisense,\n UCAUCCUGUGAGUUAGAACTT. The siRNA oligonucleotides were designed by Beijing Dingguo\n Changsheng Biotechnology Co. Ltd. The siRNA inhibition group and siRNA negative control\n group were transfected for 48 h using the lipofectamine transfection reagent (11668-500;\n Invitrogen), according to the manufacturer's protocol. \u003c/p\u003e\n \n\u003ch2\u003eDetection of DEGs \u003c/h2\u003e\n \n\u003cp\u003eThe generation and processing of raw RNA-seq data was conducted by the Beijing Genomics\n Institute (Beijing, China) using the Illumina HiSeq platform. We detected DEGs using\n the PossionDis algorithm, which is based on the Poisson distribution, and was performed\n as described by Audic et al. [\u003ca href=\"#_ENREF_15\"\u003e\n 15\u003c/a\u003e]. The parameters to determine significant changes in gene were as follows: fold change\n ≥ 2 and a false discovery rate (FDR) ≤ 0.001.\u003c/p\u003e\n \n\u003ch2\u003ePathway analysis of DEGs\u003c/h2\u003e\n \n\u003cp\u003eDEGs were annotated according to the Kyoto Encyclopedia of Genes and Genomes (KEGG)\n database. We performed pathway enrichment analysis using phyper, a function in R based\n on hypergeometric distribution \u003ca href=\"http://(https://cran.r-project.org/)\"\u003e(https://cran.r-project.org/)\u003c/a\u003e. We then calculated the FDR for each p-value, and considered an FDR \u0026lt; 0.001 to represent\n significant enrichment.\u003c/p\u003e\n \n\u003ch2\u003eQuantification of total cholesterol in adipose cells\u003c/h2\u003e\n \n\u003cp\u003eTotal cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density\n lipoprotein cholesterol (LDL-C) levels were measured in adipose cells after seeding\n in a six-well culture plate, using detection kits specific to each cholesterol type\n (T-CHO kit, HDL-C kit, and LDL-C kit; Nanjing Jiancheng Biology Institute, Nanjing,\n China).\u003c/p\u003e\n \n\u003ch2\u003eTotal RNA isolation and quantitative real-time PCR\u003c/h2\u003e\n \n\u003cp\u003eTotal RNA was isolated form adipose cells using the TRIzol\u003csup\u003e®\u003c/sup\u003e reagent (15596-018; Invitrogen, CA, USA), and then converted into cDNA using the\n PrimeScript\u003csup\u003e®\u003c/sup\u003e RT Reagent Kit (DRR027A; TaKaRa, Shanghai, China). Expression of selected genes was\n then analyzed using the Fast SYBR® Green Master Mix Bulk Pack (4385614; Invitrogen,\n USA). The following primers were used for quantitative real-time PCR (qRT-PCR): HMGR\n sense, 5'-TGCTGGTGCTGAGTATGTGG-3' and antisense, 5'-CATTCTACAAGAGCATCCAGG-3'; LPL\n sense, 5'-GACACTTGCCACCTCATTC-3' and antisense, 5'-CATCCGCCATCCAGTTCATA-3'; AMPK sense,\n 5'-CCGTATTATTTGCGTGTTCG-3' and antisense, 5'-TGTGGCGTAGCAGTCCCT-3'; SIRT1 sense, 5'-TGCAATAGACTTTCCAGACC-3'\n and antisense, 5'-GTGTATCTATGTTCTGAGT-3'; and GAPDH sense, 5'-TGCTGGTGCTGAGTATGTGG-3'\n and antisense, 5'-GATGATGACCCTCTTGGCG-3' (Beijing Dingguo Changsheng Biotechnology\n Co. Ltd.). Three replicates were used for each experiment. Relative mRNA expression\n levels were measured using the comparative Ct method (∆∆Ct), with GAPDH as the internal\n control.\u003c/p\u003e\n \n\u003ch2\u003eWestern blotting \u003c/h2\u003e\n \n\u003cp\u003eCell extracts were isolated in RIPA buffer (1 ml per 10\u003csup\u003e7\u003c/sup\u003e cells/100 mm-dish/150 cm\u003csup\u003e2\u003c/sup\u003e-flask; 0.5 ml per 5 × 10\u003csup\u003e6\u003c/sup\u003e cells/60 mm-dish/75 cm\u003csup\u003e2\u003c/sup\u003e-flask; WB-0071; Beijing Dingguo Changsheng Biotechnology Co. Ltd.). The Pierce BCA\n Assay (BCA01; Beijing Dingguo Changsheng Biotechnology Co. Ltd.) was used to determine\n protein concentration. Samples (50–100 μg) were separated using 10% sodium dodecyl\n sulfate polyvinylidene fluoride (SDS-PVDF) gels, and then transferred to PVDF membranes\n (XLL092-2; Pall, USA). The membranes were incubated with anti-β-actin (SC-47778; Santa\n Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-HMGR (Ab174830; Abcam, Cambridge,\n United Kingdom), anti-AMPK (Ab3759; Abcam), anti-LPL (Ab21356; Abcam), and anti-SIRT1 (Bs-0921R; Bioss\n Biotechnology, Inc., Beijing, China) antibodies in blocking buffer for 2 h at room\n temperature. After the membranes were incubated with secondary antibodies (Santa Cruz\n Biotechnology, Inc.), the enhanced chemiluminescence system was used to detect immunoreactive\n bands.\u003c/p\u003e\n \n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \n\u003cp\u003eDifferences in gene expression between the experimental groups were calculated using\n an analysis of variance (ANOVA), followed by a Bonferroni test. Results were considered\n statistically significant when P \u0026lt; 0.05. \u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eAdipose cell morphology and lipid accumulation\u003c/h2\u003e\n \n\u003cp\u003eWe first evaluated morphological changes in induced adipogenic bovine intramuscular\n adipocytes, and stained cells with Oil Red O to visualize lipid droplet accumulation\n after induction. As shown in Figure 1, induced adipocytes were large, round, and filled\n with fat droplets, compared to immature adipocytes.\u003c/p\u003e\n \n\u003ch2\u003eDEG detection \u003c/h2\u003e\n \n\u003cp\u003eWe sequenced RNA from the five experimental groups using the Illumina HiSeq platform,\n and generated an average of 6.58 Gb for each sample. As showed in Figure 2, after\n mapping sequenced reads to the reference genome and reconstructing the transcripts,\n we obtained 13 264 novel transcripts from all samples. Of these, 9 444 were previously\n unknown splicing events for known genes, 862 were novel coding transcripts without\n any known features, and the remaining 2 958 were long noncoding RNAs.\u003c/p\u003e\n \n\u003cp\u003eWe then analyzed differential gene expression between samples. The summary of DEGs\n is shown in Figure 2. There were a total of 10 234 DEGs, and this number was higher\n when the HMGR gene was overexpressed or suppressed. There were 1 554 DEGs found in\n the HMGR overexpression group vs. the HMGR interference group, where genes were mostly\n upregulated. We also found that there were more upregulated genes within DEGs between\n all experimental groups.\u003c/p\u003e\n \n\u003ch2\u003ePathway analysis of DEGs\u003c/h2\u003e\n \n\u003cp\u003eWe performed KEGG pathway classification and functional enrichment analyses for DEGs\n (Figure 3). These DEGs were found to be mostly involved in signal transduction, cancers,\n infectious disease, global and overview maps, and the immune system. As shown in Figure\n 3a and 3b, 35 DEGs after HMGR overexpression, and 43 DEGs after HMGR inhibition, were\n functionally involved in lipid metabolism. Furthermore, 6 DEGs after HMGR overexpression,\n and 8 DEGs after HMGR inhibition, were functionally involved in energy metabolism.\u003c/p\u003e\n \n\u003ch2\u003eAMPK signaling \u003c/h2\u003e\n \n\u003cp\u003eChanges in gene expression relevant to the AMPK signaling pathway following over-expression\n of the HMGR gene are shown in Figure 4a and 4b. Interestingly, the HMGR gene was downregulated\n when HMGR was overexpressed, whereas the SIRT1 gene was upregulated. In contrast,\n the HMGR gene itself was upregulated following HMGR interference.\u003c/p\u003e\n \n\u003ch2\u003eTC, LDL-C, and HDL-C content in adipose cells\u003c/h2\u003e\n \n\u003cp\u003eTo understand the impact of HMGR overexpression on the amount of TC, LDL-C, and HDL-C,\n we examined the levels of these types of cholesterol in the five experimental groups.\n In contrast with other groups, the HMGR overexpression group had the highest amounts\n of TC, LDL-C, and HDL-C, particularly for TC and HDL-C (Figure 5). \u003c/p\u003e\n \n\u003ch2\u003eqRT-PCR of HMGR, AMPK, SIRT1, and LPL\u003c/h2\u003e\n \n\u003cp\u003eTo investigate the relationship between HMGR expression and cholesterol synthesis\n in bovine adipose cells, expression levels of HMGR, AMPK, SIRT1, and LPL, which are four steroidogenesis-related genes, were assessed in adipose tissue using\n qRT-PCR (Figure 6). \u003c/p\u003e\n \n\u003cp\u003eFor HMGR, the overexpression group showed higher mRNA expression levels than those\n in other groups, with the HMGR interference group showing the lowest levels out of\n all groups. AMPK mRNA expression was lowest when HMGR was overexpressed; even though\n AMPK protein expression levels were increased (as shown in Figure 7). SIRT1 showed\n similar results as AMPK, whereas LPL expression levels were higher when HMGR was overexpressed,\n and lower when HMGR was inhibited.\u003c/p\u003e\n \n\u003ch2\u003eWestern blotting of HMGR, AMPK, SIRT1, and LPL \u003c/h2\u003e\n \n\u003cp\u003eTo investigate changes in protein expression, we performed western blotting in the\n five experimental groups (Figure 7). The results showed that HMGR protein expression\n in the HMGR overexpression group was higher than that in the other four groups. AMPK\n protein expression levels showed a little differences between the groups. SIRT-1 and\n LPL protein expression levels were higher in the HMGR interference group, and lower\n in the HMGR overexpression group.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAdipose tissue is the major depot for energy and cholesterol storage, with most of\n the intracellular cholesterol distributed in the form of lipid droplets [\u003ca href=\"#_ENREF_16\"\u003e\n 16\u003c/a\u003e, \u003ca href=\"#_ENREF_17\"\u003e\n 17\u003c/a\u003e]. Cholesterol homeostasis may have a role in the regulation of adipocyte size and\n function [\u003ca href=\"#_ENREF_18\"\u003e\n 18\u003c/a\u003e, \u003ca href=\"#_ENREF_19\"\u003e\n 19\u003c/a\u003e]. In addition, cellular cholesterol homeostasis is involved in various diseases of\n nongenetic origin, such as atherosclerosis, leading to lipid accumulation in target\n organs [\u003ca href=\"#_ENREF_18\"\u003e\n 18\u003c/a\u003e, \u003ca href=\"#_ENREF_20\"\u003e\n 20\u003c/a\u003e].\u003c/p\u003e\n \n\u003cp\u003eIn the present study, DEG and KEGG pathway analyses were performed to explore the\n regulatory network underlying bovine adipose cell following HMGR overexpression and\n interference. As expected, several well-known pathways [\u003ca href=\"#_ENREF_21\"\u003e\n 21\u003c/a\u003e] related to lipid metabolism were identified, particularly the AMPK signaling pathway.\n In AMPK signaling, the HMGR, AMPK, and SITR1 genes play a functional role in lipid\n metabolism or cholesterol biosynthesis.\u003c/p\u003e\n \n\u003cp\u003eIn terms of cholesterol levels, we found that those of TC, LDL-C, and HDL-C were highest\n when the HMGR gene was overexpressed; however, their levels were not majorly decreased\n when HMGR was inhibited. When we investigated protein expression of HMGR, AMPK, SIRT-1,\n and LPL, we found that HMGR protein levels were higher in the HMGR overexpression\n group compared to those in the control group. However, SIRT-1 protein levels were\n lower in the HMGR overexpression group compared to those in the HMGR interference\n group. Therefore, the results obtained for protein expression were consistent with\n those obtained for gene expression. \u003c/p\u003e\n \n\u003cp\u003eGiven that HMGR is a rate-limiting enzyme in cholesterol biosynthesis [\u003ca href=\"#_ENREF_10\"\u003e\n 10\u003c/a\u003e], we generated an adipocyte gene expression dataset using RNA-Seq, and examined the\n relationship between steroidogenic genes and cholesterol levels by overexpressing\n or inhibiting the HMGR gene. Transcriptional and pathway analysis results showed that\n overexpression of HMGR correlated with downregulation of AMPK and SIRT1 gene expression.\n \u003c/p\u003e\n \n\u003cp\u003eAMPK is a crucial energy sensor that maintains energy homeostasis. It is also a major\n regulator of the lipid metabolism through phosphorylation and inactivation of numerous\n metabolic enzymes, including HMGR [\u003ca href=\"#_ENREF_22\"\u003e\n 22\u003c/a\u003e, \u003ca href=\"#_ENREF_23\"\u003e\n 23\u003c/a\u003e]. In adipose tissue, the chronic activation of AMPK led to downregulation of HMGR\n expression in a new transgenic mouse model [\u003ca href=\"#_ENREF_24\"\u003e\n 24\u003c/a\u003e]. The 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide was shown to induce activation\n of AMPK, and directly inhibit the expression of HMGR, one of its target genes [\u003ca href=\"#_ENREF_22\"\u003e\n 22\u003c/a\u003e]. In the present study, HMGR mRNA expression was reduced when it was overexpressed\n in bovine adipose cells. In contrast, HMGR expression was increased when the HMGR\n gene was inhibited. These results are important, given the key role of the HMGR gene\n in the process of cholesterol synthesis. \u003c/p\u003e\n \n\u003cp\u003eSIRT1 is a longevity-associated deacetylase enzyme that modulates metabolic homeostasis\n in response to cellular energy. AMPK and SIRT1 are related proteins and share common\n target pathways [\u003ca href=\"#_ENREF_25\"\u003e\n 25\u003c/a\u003e]. The present study showed that the expression of SIRT1 decreased when the HMGR gene\n was overexpressed. However, SIRT1 was upregulated when HMGR expression was modified.\n These results imply that the HMGR and SIRT1 genes have contrasting roles in the cholesterol\n synthesis pathway. \u003c/p\u003e\n \n\u003cp\u003eThe LPL gene encodes a rate-limiting enzyme that has a key role in the hydrolysis\n of triglycerides. LPL deficiency and dysfunction is associated with many disorders\n of the lipoprotein metabolism [26, \u003ca href=\"#_ENREF_27\"\u003e\n 27\u003c/a\u003e]. In the present study, our results showed that the patterns of LPL gene expression\n were consistent with those of HMGR expression. \u003c/p\u003e\n \n\u003ch1\u003eConclusions\u003c/h1\u003e\n \n\u003cp\u003eThis study showed that both overexpression and interference of HMGR in bovine intramuscular\n adipocytes could regulate the expression levels of relevant genes. Several DEGs correlated positively or negatively with HMGR overexpression or inhibition.\n Furthermore, AMPK and SIRT1 showed opposite expression patterns in bovine intramuscular\n adipocytes following overexpression or inhibition of HMGR. These findings shed light\n on the signaling pathways involved in the cholesterol synthesis process, and elucidate\n how cholesterol levels may be controlled for future beef commercial production via\n a multi-gene pyramiding system.\u003c/p\u003e\n \n"},{"header":"Declarations","content":" \n\u003ch1\u003eAbbreviations\u003c/h1\u003e\n \n\u003cp\u003eNot applicable.\u003c/p\u003e\n \n\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n \n\u003cp\u003eNot applicable.\u003c/p\u003e\n \n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n \n\u003cp\u003eNot applicable.\u003c/p\u003e\n \n\u003ch2\u003eAvailability of data and material\u003c/h2\u003e\n \n\u003cp\u003eNot applicable.\u003c/p\u003e\n \n\u003ch2\u003eCompeting interests \u003c/h2\u003e\n \n\u003cp\u003eThe authors declare that they have no competing interest.\u003c/p\u003e\n \n\u003ch2\u003eFunding\n \n \u003c/h2\u003e\n \n\u003cp\u003eThis work was supported by Young Talents Training Program of Shandong Academy of Agricultural\n Science, Agricultural Science and Technology Innovation Project of Shandong Academy\n of Agricultural Sciences (CXGC2018E10), the National Natural Science Foundation of China (grants no. 31601966 and no. 31100890), and CARS-37.\u003c/p\u003e\n \n\u003ch2\u003eAuthors' contributions\u003c/h2\u003e\n \n\u003cp\u003eGuifen Liu and Fachun Wan designed the experiments. Xiaomu Liu and Wei You drafted\n the manuscript. Xianglun Zhang, Qing Jin, Xiuwen Tan, Chen Wei, Hongbo Zhao, Chen\n Zhang and Yifan Liu performed the experiments.\u003c/p\u003e\n \n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n \n\u003cp\u003eNot Applicable.\u003c/p\u003e\n \n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCui H, Liu XRR, Zhao GP, Zheng MQ, Chen JL, Wen J (2012) Identification of\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003edifferentially expressed genes and pathways for intramuscular fat deposition in pectoralis major\u003c/p\u003e\n\u003cp\u003etissues of fast-and slow-growing chickens. BMC genomics 13:213\u003c/p\u003e\n\u003col start=\"2\"\u003e\n\u003cli\u003eFernandez X, Monin G, Talmant A, Mourot J, Lebret B (1999) Influence of\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eintramuscular fat content on the quality of pig meat-1. Composition of the lipid fraction and\u003c/p\u003e\n\u003cp\u003esensory characteristics of m. longissimus lumborum. Meat science 53(1):59-65\u003c/p\u003e\n\u003col start=\"3\"\u003e\n\u003cli\u003eFernandez X, Monin G, Talmant A, Mourot J, Lebret B (1999) Influence of\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eintramuscular fat content on the quality of pig meat - 2. Consumer acceptability of m. longissimus\u003c/p\u003e\n\u003cp\u003elumborum. Meat science 53(1):67-72\u003c/p\u003e\n\u003col start=\"4\"\u003e\n\u003cli\u003eRincker PJ, Killefer J, Ellis M, Brewer MS, McKeith FK (2008) Intramuscular fat\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003econtent has little influence on the eating quality of fresh pork loin chops. Journal of animal science 86(3):730-737\u003c/p\u003e\n\u003col start=\"5\"\u003e\n\u003cli\u003eSoret B, Mendizabal JA, Arana A, Alfonso L (2016) Expression of genes involved in\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eadipogenesis and lipid metabolism in subcutaneous adipose tissue and longissimus muscle in\u003c/p\u003e\n\u003cp\u003elow-marbled Pirenaica beef cattle. Animal : an international journal of animal bioscience\u003c/p\u003e\n\u003cp\u003e10(12):2018-2026\u003c/p\u003e\n\u003col start=\"6\"\u003e\n\u003cli\u003eHocquette JF, Gondret F, Baeza E, Medale F, Jurie C, Pethick DW (2010)\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eIntramuscular fat content in meat-producing animals: development, genetic and nutritional control,\u003c/p\u003e\n\u003cp\u003eand identification of putative markers. Animal : an international journal of animal bioscience\u003c/p\u003e\n\u003cp\u003e4(2):303-319\u003c/p\u003e\n\u003col start=\"7\"\u003e\n\u003cli\u003eda Costa AS, Pires VM, Fontes CM, Mestre Prates JA (2013) Expression of genes\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003econtrolling fat deposition in two genetically diverse beef cattle breeds fed high or low silage diets.\u003c/p\u003e\n\u003cp\u003eBMC veterinary research 9:118\u003c/p\u003e\n\u003col start=\"8\"\u003e\n\u003cli\u003eHaberka M, Okopien B, Gasior Z (2016) Obesity, ultrasound indexes of fat depots and\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003elipid goal attainment in patients with high and very high cardiovascular risk: A novel approach\u003c/p\u003e\n\u003cp\u003etowards better risk reduction. Nutrition, metabolism, and cardiovascular diseases : NMCD\u003c/p\u003e\n\u003cp\u003e26(2):123-133\u003c/p\u003e\n\u003col start=\"9\"\u003e\n\u003cli\u003eOspina EJ, Sierra CA, Ochoa O, Perez-Alvarez JA, Fernandez-Lopez J (2012)\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eSubstitution of saturated fat in processed meat products: a review. Critical reviews in food science\u003c/p\u003e\n\u003cp\u003eand nutrition 52(2):113-122\u003c/p\u003e\n\u003col start=\"10\"\u003e\n\u003cli\u003eIstvan ES, Deisenhofer J (2000) The structure of the catalytic portion of human\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eHMG-CoA reductase. Biochimica et biophysica acta 1529(1-3):9-18\u003c/p\u003e\n\u003col start=\"11\"\u003e\n\u003cli\u003eJiang J, Kai G, Cao X, Chen F, He D, Liu Q (2006) Molecular cloning of a HMG-CoA\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003ereductase gene from Eucommia ulmoides Oliver. Bioscience reports 26(2):171-181\u003c/p\u003e\n\u003col start=\"12\"\u003e\n\u003cli\u003eDing S, Jiang J, Zhang G, Bu Y, Zhang G, Zhao X (2017) Resveratrol and caloric\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003erestriction prevent hepatic steatosis by regulating SIRT1-autophagy pathway and alleviating\u003c/p\u003e\n\u003cp\u003eendoplasmic reticulum stress in high-fat diet-fed rats. PloS one 12(8):e0183541\u003c/p\u003e\n\u003col start=\"13\"\u003e\n\u003cli\u003eLiu X, You W, Cheng H, Zhang Q, Song E, Wan F, Han H, Liu G (2016) Effect of\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003emevalonic acid on cholesterol synthesis in bovine intramuscular and subcutaneous adipocytes.\u003c/p\u003e\n\u003cp\u003eJournal of applied genetics 57(1):113-118\u003c/p\u003e\n\u003col start=\"14\"\u003e\n\u003cli\u003eHofgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003etransformation. Nucleic acids research 16(20):9877\u003c/p\u003e\n\u003col start=\"15\"\u003e\n\u003cli\u003eAudic S, Claverie JM (1997) The significance of digital gene expression profiles.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eGenome research 7(10):986-995\u003c/p\u003e\n\u003col start=\"16\"\u003e\n\u003cli\u003eFarkas J, Angel A, Avigan MI (1973) Studies on the compartmentation of lipid in\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eadipose cells. II. Cholesterol accumulation and distribution in adipose tissue components. Journal\u003c/p\u003e\n\u003cp\u003eof lipid research 14(3):344-356\u003c/p\u003e\n\u003col start=\"17\"\u003e\n\u003cli\u003eZhu Y, Chen CY, Li J, Cheng JX, Jang M, Kim KH (2018) In vitro exploration of ACAT\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003econtributions to lipid droplet formation during adipogenesis. Journal of lipid\u003c/p\u003e\n\u003cp\u003eresearch 59(5):820-829\u003c/p\u003e\n\u003col start=\"18\"\u003e\n\u003cli\u003eLe Lay S, Krief S, Farnier C, Lefrere I, Le Liepvre X, Bazin R, Ferre P, Dugail I\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e(2001) Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene\u003c/p\u003e\n\u003cp\u003eexpression in adipocytes. The Journal of biological chemistry 276(20):16904-16910\u003c/p\u003e\n\u003col start=\"19\"\u003e\n\u003cli\u003eShao F, Wang X, Yu J, Jiang H, Zhu B, Gu Z (2014) Expression of miR-33 from an\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eSREBF2 intron targets the FTO gene in the chicken. PloS one 9(3):e91236\u003c/p\u003e\n\u003col start=\"20\"\u003e\n\u003cli\u003eKhera AV, Demler OV, Adelman SJ, Collins HL, Glynn RJ, Ridker PM, Rader DJ,\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eMora S (2017) Cholesterol Efflux Capacity, High-Density Lipoprotein Particle Number, and\u003c/p\u003e\n\u003cp\u003eIncident Cardiovascular Events: An Analysis From the JUPITER Trial (Justification for the Use of\u003c/p\u003e\n\u003cp\u003eStatins in Prevention: An Intervention Trial Evaluating Rosuvastatin). Circulation\u003c/p\u003e\n\u003cp\u003e135(25):2494-2504\u003c/p\u003e\n\u003col start=\"21\"\u003e\n\u003cli\u003eKokta TA, Dodson MV, Gertler A, Hill RA (2004) Intercellular signaling between\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eadipose tissue and muscle tissue. Domestic animal endocrinology 27(4):303-331\u003c/p\u003e\n\u003col start=\"22\"\u003e\n\u003cli\u003eLiu S, Jing F, Yu C, Gao L, Qin Y, Zhao J (2015) AICAR-Induced Activation of\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eAMPK Inhibits TSH/SREBP-2/HMGCR Pathway in Liver. PloS one 10(5):e0124951\u003c/p\u003e\n\u003col start=\"23\"\u003e\n\u003cli\u003eHardie DG, Ross FA, Hawley SA (2012) AMPK: a nutrient and energy sensor that\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003emaintains energy homeostasis. Nature reviews Molecular cell biology 13(4):251-262\u003c/p\u003e\n\u003col start=\"24\"\u003e\n\u003cli\u003eKnowles C, Liu ZM, Yang J (2011) Compensatory increase in lipogenic gene expression in\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eadipose tissue of transgenic mice expressing constitutively active AMP-activated protein\u003c/p\u003e\n\u003cp\u003ekinase-alpha1 in liver. Biochemical and biophysical research communications 412(2):249-252\u003c/p\u003e\n\u003col start=\"25\"\u003e\n\u003cli\u003eArab Sadeghabadi Z, Nourbakhsh M, Pasalar P, Emamgholipour S, Golestani A,\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eLarijani B, Razzaghy-Azar M (2018) Reduced gene expression of sirtuins and active AMPK\u003c/p\u003e\n\u003cp\u003elevels in children and adolescents with obesity and insulin resistance. Obesity research \u0026amp; clinical\u003c/p\u003e\n\u003cp\u003epractice 12(2):167-173\u003c/p\u003e\n\u003col start=\"26\"\u003e\n\u003cli\u003eXie C, Wang ZC, Liu XF, Yang MS (2010) The common biological basis for\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003ecommon complex diseases: evidence from lipoprotein lipase gene. European journal of human\u003c/p\u003e\n\u003cp\u003egenetics : EJHG 18(1):3-7\u003c/p\u003e\n\u003col start=\"27\"\u003e\n\u003cli\u003eSocquard E, Durlach A, Clavel C, Nazeyrollas P, Durlach V (2006) Association of\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eHindIII and PvuII genetic polymorphisms of lipoprotein lipase with lipid metabolism and\u003c/p\u003e\n\u003cp\u003emacrovascular events in type 2 diabetic patients. Diabetes \u0026amp; metabolism 32(3):262-269\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HMGR, Overexpression, Interference, Cholesterol, Bovine","lastPublishedDoi":"10.21203/rs.2.10141/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.10141/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Background:Previously, we found that mevalonic acid stimulates HMGR expression in bovine intramuscular adipocytes, and influences adipocyte differentiation. However, it remains unclear whether there is any direct link between HMGR, steroidogenic genes, and cholesterol content. RNA-Seq was conducted to determine the differences between the gene expression profiles of bovine adipocytes containing different HMGR expression constructs.\nResults:In total, 10 234 differentially expressed genes (DEGs) were found. Of these, 35 and 6 DEGs between the control and the overexpression groups were functionally related to lipid and energy metabolisms, respectively. Additionally, 43 and 8 DEGs between the control and the HMGR inhibition groups were related to lipid and energy metabolism. Additionally, several DEGs related to lipid and energy metabolism were identified between the HMGR overexpression group and the HMGR interference group. Several DEGs correlated positively or negatively with overexpression or inhibition of HMGR. We also found that, following activation or inhibition of the HMGR gene, AMPK and SIRT1 had opposite expression patterns in bovine intramuscular adipocytes. Interestingly, the HMGR gene was downregulated when HMGR was overexpressed, and upregulated when HMGR was inhibited.\nConclusion:Our findings establish a theoretical understanding of signaling pathways involved in cholesterol synthesis by elucidating the relationships between key genes.","manuscriptTitle":"HMGR overexpression and interference affects the expression of steroidogenic genes and cholesterol content in bovine intramuscular adipocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2019-06-12 15:21:29","doi":"10.21203/rs.2.10141/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"23268b5f-cfa4-43ca-83c2-6c38e7371d45","owner":[],"postedDate":"June 12th, 2019","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":13188,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"","versionOfRecord":[],"versionCreatedAt":"2019-06-12 15:21:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1219","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-1219","version":["v1"]},"buildId":"re_ckhLnmML6MCF96OHNJ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}