Characterization of key genes of the renin–angiotensin system in mature feline adipocytes and during in vitro adipogenesis

Obesity is a growing health problem in humans as well as companion animals. In the development and progression of obesity‐associated diseases, the members of the renin–angiotensin system (RAS) are proposed to be involved. Particularly, the prevalence of type 2 diabetes mellitus in cats has increased enormously which is often been linked to obesity as well as to RAS. So far, reports about the expression of a local RAS in cat adipocytes are missing. Therefore, we investigated the mRNA expression of various RAS genes as well as the adipocyte marker genes adiponectin, leptin and PPAR‐γ in feline adipocytes using quantitative PCR. To characterize the gene expression during adipogenesis, feline pre‐adipocytes were differentiated into adipocytes in a primary cell culture and the expression of RAS key genes measured. All major RAS components were expressed in feline cells, but obvious differences in the expression between pre‐adipocytes and the various differentiation stages were found. Interestingly, the two enzymes ACE and ACE2 showed an opposite expression course. In addition to the in vitro experiments, mature adipocytes were isolated from subcutaneous and visceral adipose tissue. Significant differences between both fat depots were found for ACE as well as AT1 receptor with greater expression in subcutaneous than in visceral adipocytes. Visceral adipocytes had significantly higher adiponectin and PPAR‐γ mRNA level compared to the subcutaneous fat cells. Concerning the nutritional status, a significant lower expression of ACE2 was measured in subcutaneous adipocytes of overweight cats. In summary, the results show the existence of a potentially functional local RAS in feline adipose tissue which is differentially regulated during adipogenesis and dependent on the fat tissue depot and nutritional status. These findings are relevant for understanding the development of obesity‐associated diseases in cats such as diabetes mellitus.     

Adiponectin‐impaired adipocyte differentiation negatively regulates fat deposition in chicken

Adiponectin is a protein hormone secreted exclusively by adipocytes that plays an important role in the modulation of glucose and lipid metabolism. To investigate the effect of adiponectin on lipid metabolism in chicken, rosiglitazone (agonist of adiponectin) and dexamethasone (inhibitor of adiponectin) were used to treat 23‐day‐old broilers in vivo. To verify the functionality of adiponectin on fat deposition, chicken pre‐adipocytes were cultured in the medium containing 10 μg/ml adiponectin. Serum adiponectin and lipids and fat distribution were analysed. Oil Red O staining was used to determine lipid deposition in adipocytes. The expression levels of adiponectin, adiponectin receptors (AdipoR) and lipid metabolism–related genes in different tissues and pre‐adipocytes were measured using real‐time PCR, and the abundance of lipid metabolism–related proteins was measured by Western blot. Rosiglitazone increased serum adiponectin concentration and the expression levels of adiponectin and adiponectin receptor 1 (AdipoR1) in tissues and significantly decreased levels of serum lipids and fat deposition. Rosiglitazone significantly increased the expression levels of adipose triglyceride lipase (ATGL) and AdipoR1 and decreased the expression levels of fatty acid synthase (FAS). Dexamethasone had the converse effects compared with rosiglitazone. Oil red O staining results showed a marked decrease in fat deposition in cells treated with adiponectin. In adipocytes, adiponectin could decrease the expression levels of CCAAT/enhancer‐binding protein α (C/EBPα) and FAS and increased the expression levels of ATGL and AdipoR1. These results indicate that adiponectin has a remarkable effect on impairment of adipocyte differentiation, which contributes to the negative regulation of fat deposition in chicken.     

猪miR-199a-5p对脂质生成的影响及其靶基因的预测和分析

本研究旨在分析miR-199a-5p对猪肌内脂肪细胞脂质生成的影响及作用机制。采集淮南猪不同育肥时期(育肥前期、中期和后期)的背最长肌和皮下脂肪组织,通过实时荧光定量PCR分析其表达变化趋势;合成miR-199a-5p的mimics,转染猪原代肌内脂肪细胞,诱导分化后通过油红O染色观察过表达miR-199a-5p对脂质生成的影响;结合之前筛选到的脂肪型和瘦肉型猪差异表达mRNAs、lncRNAs和circRNAs,使用miRanda软件筛选miR-199a-5p的靶分子,并用GO和KEGG富集分析法对这些mRNAs、lncRNAs的共表达基因和circRNAs的来源基因进行功能分析。结果显示,随着育肥进程,miR-199a-5p在背最长肌中表达量持续上调,而在皮下脂肪组织中表达量先下调后上调;过表达miR-199a-5p可抑制肌内脂肪细胞的脂质生成。共筛选到9个mRNAs、5个lncRNAs和1258个circRNAs包含miR-199a-5p结合位点,GO分析主要富集于内质网和肌动蛋白结合,KEGG分析发现主要富集于糖、脂质和蛋白质代谢。提示miR-199a-5p可能通过与lncRNAs和circRNAs互作间接调控靶基因,参与调控肌肉发育、脂质生成和代谢,可作为影响肉质性状的候选miRNA。     

猪miR-199a-5p对脂质生成的影响及其靶基因的预测和分析

本研究旨在分析miR-199a-5p对猪肌内脂肪细胞脂质生成的影响及作用机制。采集淮南猪不同育肥时期（育肥前期、中期和后期）的背最长肌和皮下脂肪组织,通过实时荧光定量PCR分析其表达变化趋势;合成miR-199a-5p的mimics,转染猪原代肌内脂肪细胞,诱导分化后通过油红O染色观察过表达miR-199a-5p对脂质生成的影响;结合之前筛选到的脂肪型和瘦肉型猪差异表达mRNAs、lncRNAs和circRNAs,使用miRanda软件筛选miR-199a-5p的靶分子,并用GO和KEGG富集分析法对这些mRNAs、lncRNAs的共表达基因和circRNAs的来源基因进行功能分析。结果显示,随着育肥进程,miR-199a-5p在背最长肌中表达量持续上调,而在皮下脂肪组织中表达量先下调后上调;过表达miR-199a-5p可抑制肌内脂肪细胞的脂质生成。共筛选到9个mRNAs、5个lncRNAs和1 258个circRNAs包含miR-199a-5p结合位点,GO分析主要富集于内质网和肌动蛋白结合,KEGG分析发现主要富集于糖、脂质和蛋白质代谢。提示miR-199a-5p可能通过与lncRNAs和circRNAs互作间接调控靶基因,参与调控肌肉发育、脂质生成和代谢,可作为影响肉质性状的候选miRNA。     

Castration induced circRNA expressional changes in subcutaneous adipose tissue of male pigs

Circular RNAs (circRNAs) participated in regulation of lipid metabolism; however, its functional role on castration-induced lipid deposition has not been deeply researched. So in this research, we firstly compared circRNAs expressional differences in subcutaneous adipose tissue between intact and castrated male Huainan pigs. A total of 6116 differentially expressed circRNAs (DECs) were detected between these two groups (|log₂foldchange| ≥ 1 and p_adj ≤ 0.05); GO and KEGG analysis showed that their parent genes were mainly enriched in metabolism-related pathway. And TGF-beta, insulin, AMPK, and MAPK pathways might play vital role in castration-induced lipid deposition. The miRNAs enriched in the constructed circRNA–miRNA network were mainly participated in adipogenesis and lipid metabolism, such as miR-143a-3p, miR-378, and miR-195. And it was verified that testosterone upregulated miR-181a but downregulated circ_0005912 expression in a dose-dependent manner in porcine intramuscular adipocytes, and overexpression of miR-181a inhibited circ_0005912. Taken together, these DECs may participate in the regulation of lipid metabolism after castration by reaction with miRNAs, which indicated the novel role of circRNAs in castration-induced lipid deposition.     

Adipogenic differentiation state-specific gene expression as related to bovine carcass adiposity

Genetic regulation of the site of fat deposition is not well defined. The objective of this study was to investigate adipogenic differentiation state-specific gene expression in feedlot cattle (>75% Angus; <25% Simmental parentage) of varying adipose accretion patterns. Four groups of 4 steers were selected via ultrasound for the following adipose tissue characteristics: low subcutaneous-low intramuscular (LSQ-LIM), low subcutaneous-high intramuscular (LSQ-HIM), high subcutaneous-low intramuscular (HSQ-LIM), and high subcutaneous-high intramuscular (HSQ-HIM). Adipose tissue from the subcutaneous (SQ) and intramuscular (IM) depots was collected at slaughter. The relative expression of adipogenic genes was evaluated using quantitative PCR. Data were analyzed using the mixed model of SAS, and gene expression data were analyzed using covariate analysis with ribosomal protein L19 as the covariate. No interactions (P > 0.10) were observed between IM and SQ adipose tissue depots for any of the variables measured. Therefore, only the main effects of high and low accretion within a depot and the effects of depot are reported. Steers with LIM had smaller mean diameter IM adipocytes (P < 0.001) than HIM steers. Steers with HSQ had larger mean diameter SQ adipocytes (P < 0.001) than LSQ. However, there were no differences (P > 0.10) in any of the genes measured due to high or low adipose accretion. Preadipogenic delta-like kinase1 mRNA was greater in the IM than the SQ adipose tissue; conversely, differentiating and adipogenic genes, lipoprotein lipase, PPARγ, fatty acid synthetase, and fatty acid binding protein 4 were greater (P < 0.001) in the SQ than the IM depot. Intramuscular adipocytes were smaller than SQ adipocytes and had greater expression of the preadipogenic gene, indicating that more hyperplasia was occurring. Meanwhile, SQ adipose tissue contained much larger (P < 0.001) adipocytes that had a greater expression (P < 0.001) of differentiating and adipogenic genes than did the IM adipose tissue, indicating more cells were undergoing differentiation and hypertrophy. Adipogenic differentiation state-specific gene expression was not different in cattle with various phenotypes, but adipogenesis in the SQ and IM adipose tissues seems to occur independently.     

维生素A对肉牛脂肪沉积及其调控的研究进展

维生素A是肉牛维持正常生理功能、生化代谢及生长发育所必需的一类脂溶性维生素，机体自身不能合成，必须由饲粮供给。饲粮中维生素A不仅影响肉牛视觉和骨骼发育，也对肉牛脂肪沉积和肌肉大理石花纹形成等发挥着重要调控作用。在生产中，肉牛处在不同生理阶段对维生素A的需要量也不同，胎牛和犊牛阶段补充维生素A可增强肌内脂肪细胞发育和脂肪细胞增生，促进肌内脂肪沉积；育肥期限饲维生素A可提高肉牛的肌内脂肪沉积和大理石花纹等级。大理石花纹与牛肉的嫩度和风味密切相关，是衡量牛肉品质的重要指标。维生素A在肉牛体内通过其活性代谢产物视黄醇、视黄醛和视黄酸促进脂肪的形成，并在成脂定型、成脂分化和脂质蓄积的每个阶段都发挥重要作用。脂肪沉积过程受转录因子过氧化物酶体增殖物激活受体（peroxisome proliferator-activated receptors，PPARs）、CCAAT增强子结合蛋白（CCAAT-enhancer binding proteins，C/EBPs）和Janus激酶-信号转导及转录激活因子（JAK-STAT）信号转导通路等调控。表观遗传修饰的DNA甲基化和去甲基化也可通过调控脂肪形成过程中相关基因的表达参与脂肪细胞的分化和脂肪组织的生长发育，从而影响肉牛的脂肪沉积。作者主要介绍了脂肪组织不同阶段的形成过程和维生素A的生物学功能；重点阐述了肉牛在不同生理阶段补充和限饲维生素A，通过转录因子的表达、表观遗传修饰等途径来影响成脂相关信号通路调控脂肪沉积的机理，以期为促进维生素A改善肉牛品质和高档牛肉生产提供参考。     

Pathology of congenital generalized lipodystrophy in Agpat2-/- mice

Congenital generalized lipodystrophy (CGL) comprises a heterogeneous group of rare diseases associated with partial or total loss of adipose tissue. Of these, autosomal recessive Berardinelli-Seip congenital lipodystrophy (BSCL) is characterized by the absence of metabolically active subcutaneous and visceral adipose tissues. Metabolic abnormalities associated with lipodystrophy include insulin resistance, hypertriglyceridemia, hepatic steatosis, and diabetes. One form of BSCL has been linked to genetic mutations affecting the lipid biosynthetic enzyme 1-acyl-sn-glycerol 3-phosphate O-acyltransferase 2 (AGPAT2), which is highly expressed in adipose tissue. Precisely how AGPAT2 deficiency causes lipodystrophy remains unresolved, but possible mechanisms include impaired lipogenesis (triglyceride synthesis and storage), blocked adipogenesis (differentiation of preadipocytes to adipocytes), or apoptosis/necrosis of adipocytes. Agpat2(-/-) mice share important pathophysiologic features of CGL previously reported in humans. However, the small white adipose tissue (WAT) depots consisting largely of amoeboid adipocytes with microvesiculated basophilic cytoplasm showed that adipogenesis with deficient lipogenesis was present in all usual locations. Although well-defined lobules of brown adipose tissue (BAT) were present, massive necrosis resulted in early ablation of BAT. Although necrotic or apoptotic adipocytes were not detected in WAT of 10-day-old Agpat2(-/-), the absence of adipocytes in aged mice indicates that these cells must undergo necrosis/apoptosis at some point. Another significant finding in aged lipodystrophic mice was massive pancreatic islet hypertrophy in the face of chronic hyperglycemia, which suggests that glucotoxicity is insufficient by itself to cause β-cell loss and that adipocyte-derived factors help regulate total β-cell mass.     

Adiposity, fatty acid composition, and delta-9 desaturase activity during growth in beef cattle

Oleic acid (18:1n‐9) is the most abundant fatty acid in bovine adipose tissue. Because most of the lipid in bovine muscle is contributed by intramuscular adipocytes, oleic acid also is the predominant fatty acid in beef. In many species, the concentration of oleic acid in adipose tissue is dictated by the average concentration of oleic acid in the diet, but in ruminant species such as beef cattle, oleic acid is hydrogenated largely to stearic acid by ruminal microorganisms. In these species, the concentration of oleic acid in adipose tissue is dependent upon the activity of Δ⁹ desaturase, encoded by the stearoyl coenzyme A desaturase (SCD) gene. Expression of the SCD gene is essential for bovine preadipocyte differentiation, and desaturase gene expression and catalytic activity increase dramatically as adipose tissue mass increases after weaning. Feeding a hay‐based diet to American Wagyu steers to a typical Japanese bodyweight endpoint (650 kg) markedly stimulated desaturase enzyme activity as well as the accumulation of both oleic acid and intramuscular lipid, but the increase in oleic acid and intramuscular lipid was much less in hay‐fed Angus steers. Increasing the concentration of oleic acid improves the palatability and healthiness of beef, and Korean Hanwoo and Japanese Black (and American Wagyu) seem especially well adapted to accumulate oleic acid in their adipose tissue.     

Identification and utilization of genes associated with beef qualities

The fatty acid composition of adipose tissue in beef has been recognized as an important trait because of its relationship with beef quality, including favorable beef flavor and tenderness. Over the last decade, we have tried to identify the genes responsible for the fatty acid composition in cattle, and have found the following. (i) Genetic polymorphism of stearoyl‐CoA desaturase (SCD) is one of the responsible genes associated with fatty acid composition. The average effects of gene substitution of the SCD type A gene on the monounsaturated fatty acid (MUFA) percentage and the melting point of intramuscular fat were approximately +1.0% and ?1.0°C, respectively. (ii) Intron polymorphism of sterol regulatory element binding protein–1 (SREBP‐1) also affected MUFA. (iii) No effect of SCD or SREBP‐1 genotypes on any representative carcass traits of Japanese Black in the field population was observed. (iv) Additional genetic markers adipocytes fatty acid binding protein 4 (FABP4) and liver X receptor α also affected the fatty acid composition. (v) SCD and FABP4 significantly affected fatty acid composition in Holstein steers. These findings will bring new insight into the fat‐related carcass traits of beef cattle and will thus contribute to the beef industry.     

Ground transport stress affects bacteria in the rumen of beef cattle: A real‐time PCR analysis

Transport stress syndrome often appears in beef cattle during ground transportation, leading to changes in their capacity to digest food due to changes in rumen microbiota. The present study aimed to analyze bacteria before and after cattle transport. Eight Xianan beef cattle were transported over 1000 km. Rumen fluid and blood were sampled before and after transport. Real‐time PCR was used to quantify rumen bacteria. Cortisol and adrenocorticotrophic hormone (ACTH) were measured. Cortisol and ACTH were increased on day 1 after transportation and decreased by day 3. Cellulolytic bacteria (Fibrobacter succinogenes and Ruminococcus flavefaciens), Ruminococcus amylophilus and Prevotella albensis were increased at 6 h and declined by 15 days after transport. There was a significant reduction in Succinivibrio dextrinosolvens, Prevotella bryantii, Prevotella ruminicola and Anaerovibrio lipolytica after transport. Rumen concentration of acetic acid increased after transport, while rumen pH and concentrations of propionic and butyric acids were decreased. Body weight decreased by 3 days and increased by 15 days after transportation. Using real‐time PCR analysis, we detected changes in bacteria in the rumen of beef cattle after transport, which might affect the growth of cattle after transport.     

ADCY2基因表达对延边黄牛脂肪前体细胞成脂分化的影响

本试验旨在研究环腺苷酸（3',5'-cyclic adenosine monophosphote,cAMP）环化酶合成酶2（adenylyl cyclase 2,ADCY2）基因对延边黄牛脂肪前体细胞成脂分化的影响。选取3日龄的延边黄牛腹股沟皮下脂肪组织进行脂肪前体细胞的分离、培养和诱导分化;设计ADCY2基因的干扰片段（siADCY2-1、2、3）,构建pEX4过表达载体;分别收集正常诱导（对照组）、干扰和过表达0、5和10 d的脂肪细胞。用实时荧光定量PCR检测过氧化物酶体增殖激活物受体（PPARγ）、CCAAT/增强子结合蛋白（C/EBPα）和ADCY2在细胞分化过程中mRNA的转录水平,并用Western blotting法检测其蛋白的表达;用油红O染色检测脂滴含量的变化;用甘油三酯试剂盒检测甘油三酯含量。试验结果表明,在成脂分化过程中,与未分化相比,ADCY2基因在分化的第5和10天表达量均极显著上升（P<0.01）,而分化第10天与第5天相比水平极显著下降（P<0.01）;siADCY2-1干扰效率最高,过表达ADCY2基因使其mRNA水平升高;与对照组相比,过表达组细胞内ADCY2基因mRNA水平提高了约4 000 000倍,脂肪细胞内脂滴含量和甘油三酯的含量极显著提高（P<0.01）,成脂关键基因C/EBPα和PPARγ表达极显著上调（P<0.01）,而RNA干扰组细胞内ADCY2基因mRNA水平极显著降低（P<0.01）,脂肪细胞内脂滴含量和甘油三酯的含量极显著下降,成脂关键基因C/EBPα和PPARγ表达极显著下调（P<0.01）。因此,ADCY2基因过表达能显著增加成熟脂肪细胞的脂滴含量及甘油三酯含量,促进成脂关键基因C/EBPα和PPARγ的表达,说明ADCY2基因对脂肪细胞分化具有正向调控作用。     

Effect of breed on fatty acid composition and stearoyl-CoA desaturase protein expression in the Semimembranosus muscle and subcutaneous adipose tissue of cattle

The present study investigated (i) the effect of breed on the expression of stearoyl-CoA desaturase (SCD) protein and fatty acid composition in Semimembranosus muscle and subcutaneous adipose tissue of beef cattle and (ii) the relationship between SCD expression, cis-9, trans-11 conjugated linoleic acid (CLA) content, and monounsaturated fatty acid (MUFA) level. The study was conducted on the following breeds: Longhorn (L), Charolais cross with Holstein–Friesian (CX), Hereford (H), Belted Galloway (BG) and Beef Shorthorn (BS). Significant breed differences in the total fatty acid content, saturated fatty acid (SFA) level, MUFA and n−3 PUFA content were observed in subcutaneous adipose tissue but not in muscle. In the case of CLA, the breed differences were observed in both muscle and subcutaneous adipose tissue, with the highest level in L and the lowest level in H. In the case of subcutaneous adipose tissue, the breed with the highest CLA content (L) also had the highest SCD protein expression. The breed-specific pattern of SCD expression in subcutaneous adipose tissue was similar to the breed-specific pattern of one of the products of an SCD-catalysed reaction, C16:1 (BS < BG < H < CX < L). It has been concluded that (i) the mechanisms regulating SCD protein expression and CLA level in beef cattle are tissue-specific; (ii) breed-specific variations in SCD expression might contribute to breed variations in MUFA and CLA content in subcutaneous adipose tissue but not in Semimembranosus muscle.     

DNA甲基化与去甲基化调控脂肪沉积的研究进展

脂肪沉积是一个复杂的生物学过程,受遗传和表观遗传的调控作用。DNA甲基化和去甲基化是表观遗传修饰的重要方式,可通过与转录因子的相互作用或改变染色质的结构调控基因的表达,进而参与机体生长发育和细胞分化等重要的生命过程。动物脂肪沉积是脂肪细胞增殖分化和肥大的结果,脂肪细胞分化是由多能干细胞经前体脂肪细胞向成熟脂肪细胞转化的过程。相关研究表明,转录因子过氧化物酶体增殖物激活受体γ(peroxi-some proliferator activiated receptorγ,PPARγ)和CCAAT增强子结合蛋白家族(CCAAT enchancer binding proteinfamily,CEBPs)在脂肪沉积过程中起关键调控作用。近期研究发现,DNA甲基化可以通过调控脂肪形成过程中相关基因的表达而参与脂肪细胞的分化和脂肪组织的生长发育。去甲基化也可影响动物脂肪沉积过程,但其具体机制目前尚不清楚。作者主要介绍了DNA甲基化和去甲基化的定义、发生位点、生物学功能、参与DNA甲基化和去甲基化过程中的酶及其作用机制,概述了脂肪沉积过程及PPARγ、C/EBPα等转录因子在脂肪沉积过程中的调控作用,重点阐述了DNA甲基化和去甲基化对脂肪形成相关基因的表达和对脂肪细胞分化的影响,旨在为阐明脂肪沉积机制及改善动物肉质品质提供参考。     

饲粮淀粉水平对生长期肉牛肌内脂肪细胞增殖和分化的影响及其机制

肉牛生长期是肌内脂肪细胞发育的重要时期,此阶段脂肪细胞的增殖和分化受到营养物质特别是淀粉供给的重要影响。但饲粮中淀粉水平过高会产生亚急性瘤胃酸中毒,出现一系列健康问题。因此,本文总结生长期肉牛肌内脂肪细胞发育特点,从肌内脂肪细胞脂肪酸底物利用以及脂肪酶和转录因子等方面阐述了饲粮淀粉水平对生长期肉牛肌内脂肪细胞增殖和分化的调控作用和分子机制,为开展生长期肉牛肌内脂肪代谢调控研究和促进肌内脂肪沉积的应用提供理论依据和实践指导。