Fatty acid elongation and desaturation enzyme activities of bovine liver and subcutaneous adipose tissue microsomes

Assay conditions were established for the fatty acid elongation and the delta 9 desaturase enzyme systems of bovine liver and adipose tissue microsomes; rat liver microsomes were used as a reference. Overall fatty acid elongation was determined by measuring the incorporation of [2-14C]malonyl-coenzyme A (CoA) to 14C-labeled stearate. Rat liver elongation activity was .50 +/- .02 nmol.min-1.mg protein-1; bovine liver microsomal elongation activity was substantially lower (P less than .05), with a mean value of .15 +/- .02 nmol.min-1.mg protein-1. The elongation activity of bovine s.c. adipose tissue microsomes (.42 +/- .10 nmol.min-1.mg protein-1) was not different (P greater than .05) from the activity observed in rat liver microsomes. To determine the fatty acid delta 9 desaturase activity, microsomes were incubated in the presence of [1-14C]stearoyl-CoA and nicotinamide adenine dinucleotide (reduced form) (NADH), and the production of radioactively labeled oleate was quantified. Microsomal delta 9 desaturase activity was similar in rat liver and bovine s.c. adipose tissue microsomes with rates of .15 +/- .04 and .21 +/- .05 nmol.min-1.mg protein-1, respectively. However, no desaturase activity was detected in bovine liver microsomes, indicating that the liver is not a major site of oleate synthesis in this species. To investigate differences in fatty acid metabolism relative to breed type, eight Angus and seven Braford heifers were slaughtered at approximately 12 mo of age. Subcutaneous fat thickness over the 12th-13th thoracic vertebrae was greater in the Angus heifers than in the Braford heifers. However, no differences (P greater than .05) were observed in mean adipocyte size or number of cells per gram of adipose tissue between the Angus and Braford heifers. Similarly, there were no significant differences between the Angus and Braford s.c. adipose tissues for microsomal fatty acid elongation or delta 9 desaturation, or for nicotinamide adenine dinucleotide phosphate (NADP)-malate dehydrogenase, fatty acid synthetase, or the pentose cycle reductases. The inability of bovine liver to convert stearate to oleate was in agreement with the fatty acid composition of the liver lipid, which had a smaller percentage of oleate and a higher percentage of stearate than s.c. adipose tissue.     

Lipogenesis in acute and 48-hour cultures of bovine intramuscular and subcutaneous adipose tissue explants

In this study, the interactions among breed of cattle, adipose tissue site and specific incubation conditions were investigated. Subcutaneous and i.m. adipose tissues were obtained from 10 Angus and 9 Santa Gertrudis steers immediately postmortem. Adipose tissue explants were incubated acutely for 2 h immediately at slaughter or after being cultured 48 h with or without 1 mU/ml insulin and 30 mg/ml bovine serum albumin; the incorporation of 14C-labeled acetate and glucose (5 mM, plus 5 mM unlabeled lactate) into lipid fractions was measured. AT the same chronological age, Angus steers had a more youthful lean maturity score, higher USDA marbling score and higher USDA quality grade (P less than .05) than did carcasses from Santa Gertrudis steers. The lower marbling score of the Santa Gertrudis steers was paralleled by smaller i.m. adipocytes (P less than .05) relative to Angus steers. Pentose cycle reductase and NADP-malate dehydrogenase activities were greater in Angus i.m. adipose tissue than in Santa Gertrudis i.m. adipose tissue, which would provide more reducing equivalents (NADPH) and glycerol for fatty acid biosynthesis and triacylglycerol esterification. Correspondingly, Angus i.m. adipose tissue exhibited a greater rate of lipogenesis from acetate and glucose (P less than .05) than did Santa Gertrudis i.m. adipose tissue in acute incubations. The presence of insulin resulted in higher rates of lipogenesis from acetate in Angus s.c. adipose tissue than in Santa Gertrudis s.c. adipose tissue after 48 h of explant culture. These data indicate that i.m. and s.c. adipose tissues exhibit aspects of lipid metabolism unique to each tissue and suggest that breed-related differences in adipose tissues may explain the divergent responses to insulin observed in different laboratories.     

Postnatal development of stearoyl coenzyme A desaturase gene expression and adiposity in bovine subcutaneous adipose tissue

This investigation addressed the hypothesis that stearoyl coenzyme A desaturase (SCD) gene expression would serve as a postnatal marker of adipocyte differentiation in bovine s.c. adipose tissue. Samples of tailhead s.c. adipose tissue were obtained by biopsy from preweaning steer calves 2.5 wk, 5 mo, and 7.5 mo of age and from yearling steers 12 mo of age. Samples also were obtained at slaughter when the steers were 18 mo of age. The steers sampled as yearlings were fed native pasture from weaning until 12 mo of age, and the steers sampled at slaughter were fed a high-concentrate diet from 12 to 18 mo of age. Major peak adipocyte volumes for the 2.5-wk-, 5-mo-, and 7.5-mo-old steers were 14, 270, and 700 pL, respectively (P < .001). The steers did not gain weight during pasture feeding, and at 12 mo of age peak adipocyte volume had decreased (P = .009) to 270 pL. At this time, a second, smaller population of adipocytes had appeared with a peak volume of 115 pL. At slaughter, adjusted fat thickness of the steers was 1.60 +/- .13 cm, the USDA yield grade of the carcasses was 3.51 +/- .31, and peak adipocyte volume had increased (P = .01) to over 2,500 pL. The number of adipocytes per 100 mg of adipose tissue doubled (P = .006) between 2.5 wk and 5 mo of age, concurrent with the nearly 20-fold increase in peak adipocyte volume, indicating that this was a period of apparent adipocyte hyperplasia. Uncoupling protein mRNA was undetectable at all stages of postnatal growth, indicating that differentiating tailhead s.c. adipocytes do not acquire brown adipocyte characteristics postnatally. Lipogenesis expressed on a cellular basis was low in all preweaning samples and increased significantly above preweaning values only in the 18-mo-old steers. Stearoyl coenzyme A desaturase mRNA concentration also was low in all preweaning samples, but it peaked (P = .07) at 12 mo of age. Because the peak in SCD mRNA concentration preceded a significant rise in lipogenesis and lipid filling, we conclude that the level SCD gene expression may be indicative of the extent of terminal differentiation in bovine tailhead s.c. adipose tissue.     

Effect of nutritional status on swine adipose tissue lipolytic activities

Young swine (28 days of age) were fed an isocaloric and isonitrogenous diet with either a high fat or a low fat content for 3 to 4 weeks. The adipose tissue lipolytic rate was higher in the group fed the high fat diet. However, there was no effect of diet on the activities of several of the enzymes controlling the lipolytic process, i.e., adenylate cyclase, phosphodiesterase and hormone-sensitive lipase. No effect of diet on the activity of lipoprotein lipase was detected. Fasting for 72 hr, but not for 24 or 48 hr, caused an increase in the lipolytic rate. There was also a decrease in cell size after a 72-hr fast (P greater than .05) such that the increased rate was not significant when the data were expressed on a cell basis. Inexplicable transient changes in adenylate cyclase activity, as well as a decrease in the activity of the low affinity phosphodiesterase (doubtful physiological significance), were detected during starvation. Starvation depressed the adipose tissue lipoprotein lipase activity but had no effect on the hormone-sensitive lipase activity.     

Alterations with age in composition and lipolytic activity of adipose tissue from male and female chickens

The composition and lipolytic activity of adipose tissue from male and female chickens between 4 weeks of age and maturity was studied. The triglyceride to DNA ratio doubled in male adipose tissue during this time but increased seven‐fold in females, suggesting that the latter had larger adipocytes. The protein to DNA ratios were constant throughout.

The lipolytic sensitivity of adipocytes to glucagon did not alter with age but the concentration of glucagon producing a maximal response decreased. Noradrena‐line sensitivity, never very great, was lost after 16 weeks but re‐appeared in the adult male. The total lipolytic response of the tissue decreased, in terms of the dry weight (representing triglyceride content), but was constant in terms of the DNA content of the tissue, suggesting that the cell protoplasm was equally active at all ages from 12 weeks to maturity.     

Lipogenic and lipolytic activities in isolated adipocytes from cattle with fat necrosis

The metabolic activity and cellularity of adipocytes isolated from the abdominal adipose tissue of normal heifers and heifers with fat necrosis were compared. The basal rate of U-¹⁴C glucose incorporation into total lipids in adipocytes from the periphery of the necrotic mass was higher than that in the colonic mesentery of both the affected and normal heifers. In the affected animals, adipocytes from the mesentery of the spiral colon and adipocytes from the periphery of the necrotic mass failed significantly to increase the incorporation of labelled acetate and glucose, respectively, in response to insulin. In the presence of adrenalin, adipocytes from the colonic mesentery and the periphery of the necrotic mass of the affected heifers released more glycerol than adipocytes from the colonic mesentery of normal animals. In addition, the mean diameters of adipocytes from the colonic mesentery and the periphery of the necrotic mass of the affected heifers were significantly greater than those from the colonic mesentery of normal animals. These results indicate that excessive fattiness in abdominal adipose tissue may predispose cattle to fat necrosis.     

Factors affecting measurements of glucose metabolism and lipolytic rates in porcine adipose tissue slices in vitro

Porcine adipose tissue glucose metabolism and lipolytic rates have been measured for many years by numerous investigators. However, there is little or no documented indication of the effects of variation in tissue handling procedures or variations in incubation medium components on metabolic rates. We have systematically varied conditions to provide such documentation for these much used techniques. The temperature (18 to 38 C) of tissue during transport had little effect. The medium for tissue transport probably should be buffered. Use of Hepes buffer at greater than 10 or 25 mM in incubation media inhibited glucose metabolism and lipolysis. Calcium ion effects on glucose metabolism or lipolysis could not be demonstrated. Dimethyl sulfoxide should not be used routinely. Ascorbate at .56 mM did not inhibit glucose metabolism or lipolysis. Glucose metabolism was increased by glucose concentration to about 5 mM and not inhibited at higher concentrations; we recommend 10 or 20 mM glucose to ensure maximal rates. Insulin stimulated glucose metabolism but effects were slight, not related to insulin concentration and not consistently observed. Addition of some albumin preparations did not allow expression of insulin stimulation; we recommend albumin be omitted or, if included, carefully monitored. Lipolytic rates were dependent on albumin concentration, but rates were similar with all albumin preparations. Insulin markedly inhibited hormone-stimulated but not basal lipolysis. Adenosine, an inhibitor of lipolysis, did not affect glucose metabolism rates. An artificial oxygen carrier did not increase anabolic activity. Incubation in serum increased rates of glucose metabolism relative to lipolysis so that refinement of the incubation might lead to greater anabolic than catabolic rates in vitro to reflect the status of adipose tissue in growing pigs in vivo. Tissue handling and incubation conditions can markedly affect metabolic rates, and should be understood and controlled.     

Influence of abomasal carbohydrates on subcutaneous, omental, and mesenteric adipose lipogenic and lipolytic rates in growing beef steers

To determine the response to alteration in site and form of carbohydrate delivery to the digestive tract, in vitro rates of lipogenesis and lipolysis in mesenteric (MESA), omental (OMA), and subcutaneous (SQA) adipose depots were compared. Forty crossbred beef steers (243 +/- 2 kg of BW) were fed 161 (LI) or 214 (HI) kcal of ME/(kg of BW(0.75) x d) or they were fed LI and infused for 35 d into the rumen (R) or abomasum (A) with starch hydrolysate (SH) or into the abomasum with glucose (G). Jugular blood samples were collected, steers were slaughtered, and adipose depots were sampled and prepared for assessment of lipogenesis and lipolysis in vitro. Blood concentrations of glucagon were increased (P = 0.04) in HI-H2O compared with LI-H2O steers, whereas A-SH tended to increase (P = 0.08) circulating IGF-I relative to R-SH, and A-G tended to have elevated (P = 0.09) T3 compared with A-SH. Lipolysis, as assessed by NEFA release, was unaffected by treatment. Glycerol release by the MESA and SQA was increased or tended to be increased (P < or = 0.08) in HI-H2O compared with LI-H2O steers. In A-G compared with A-SH steers, glycerol release from OMA increased (P = 0.008) and from SQA tended to be increased (P = 0.08). Acetate incorporation into total neutral lipids (TNL) increased or tended to increase with ME intake and SH infusion (P < or = 0.09) across all depots. Rates of acetate incorporation into fatty acids (FA) also increased or tended to be increased (P < or = 0.1) by SH infusion across all depots, but only that of SQA was increased with ME intake (HI-H2O vs. LI-H2O; P = 0.02). Rates of acetate incorporation into FA and TNL in MESA were increased (P < or = 0.03) by A-SH compared with R-SH, but site of SH infusion did not affect the rates in SQA or OMA. Glucose incorporation into TNL for MESA and SQA increased or tended to be increased (P < or = 0.1) by dietary and infused energy, whereas for OMA they tended to be increased (P = 0.1) only by SH infusion. In contrast, glucose incorporation into FA was unaffected by energy supply but tended to be increased (P = 0.07) by SH in MESA and tended to be greater (P = 0.08) for A-G than A-SH in OMA. The general across-depot pattern of acetate incorporation rate into FA and TNL was SQA > OMA > MESA, whereas, for glucose incorporation, rates across depots were equivalent. These data provide evidence that the postruminal supply of energy, specifically carbohydrate, stimulates lipogenesis from acetate and glucose and is more pronounced in abdominal depots relative to the subcutaneous depot.     

Effects of in vitro ronnel on metabolic activity in subcutaneous adipose tissue and skeletal muscle from steers

Ronnel [0,0-dimethyl 0-(2,4,5-trichlorophenyl) phosphorothioate] is an organophosphate pesticide with growth-promoting properties. Experiments were conducted to determine effects of ronnel on oxidation of and fatty acid synthesis from acetate and glucose as indices of metabolic activity in subcutaneous adipose tissue and skeletal muscle from 6-, 12- and 18-mo-old steers. Ronnel depressed metabolic activity in adipose tissue from 6- and 12-mo-old steers without concomitantly decreasing metabolic activity in skeletal muscle. Production of CO2 and fatty acids from acetate and glucose in tissues from 18-mo-old steers was influenced less by ronnel than in tissues from younger steers. Interactions of ronnel with thyroxine or growth hormone on acetate oxidation and conversion to fatty acids in adipose tissue also were investigated. Thyroxine increased acetate oxidation and decreased fatty acid synthesis. Ronnel interfered with the metabolic effects of thyroxine. Growth hormone, with or without ronnel, did not affect metabolic activity of adipose tissue. Ronnel seemingly alters the partitioning of acetate and glucose between major metabolic processes in adipose tissue and skeletal muscle.     

Human acylation-stimulating protein and lipid biosynthesis in bovine adipose tissue explants

Human acylation-stimulating protein (hASP) up-regulates triacylglycerol synthesis in human adipocytes. The objectives of this research were 1) to determine the effect of hASP on triacylglycerol synthesis in bovine adipose explants and 2) to determine whether nutritional status influences the sensitivity of adipose tissue to hASP. Fresh s.c. adipose tissue was sectioned into 20- to 30-mg explants and incubated for 1 to 6 h in M199 media containing 3% BSA and either 0.75 mM [1-14C]palmitate, 0.75 mM [9, 10-3H]oleate, or 2.5 mM [1-14C] acetate, as well as hASP and(or) insulin. The explants were extracted, and lipid fractions were separated by TLC and quantified by liquid scintillation. Acetate incorporation into lipids increased 15 to 30%, and palmitate or oleate incorporation increased 10 to 25%, when explants were exposed to hASP, although this response was not significant in every experiment. Insulin increased triacylglycerol synthesis in some experiments, but not in others. Our interpretation is that acylation-stimulating protein (ASP) can mildly enhance triacylglycerol synthesis in bovine adipose tissue. To fulfill the second objective, nine 9-mo-old steers were housed individually for two periods of 3 wk each. During the first period, four of the nine steers were fed to 50% of NEm requirement and the other five consumed the same diet ad libitum. After the first period, all steers consumed feed ad libitum for 2 wk and were assigned the opposite ration for the second period. Steers gained 40.5 kg BW when allowed ad libitum access to feed but lost 30.2 kg BW when feed intake was restricted (SE = 7.84; P < 0.01). At the end of each period, s.c. adipose tissue was sectioned into explants and incubated as described above. Four explants per steer per period were used to test effects of insulin (0 and 1 nM) and hASP (0, 0.01, 0.1, and 1 microM). Insulin did not influence incorporation of acetate or oleate. Acetate incorporation (P < 0.32) was 0.99, 1.03, 1.04, and 1.10 nmol x mg(-1) h(-1) (SE = 0.13) and oleate incorporation (P < 0.01) was 0.347, 0.357, 0.353, and 0.420 nmol x mg(-1)h(-1) (SE = 0.022) for 0, 0.01, 0.1, and 1 microM hASP, respectively. Feed restriction reduced (P < 0.01) acetate and oleate incorporation by 95 and 40%, respectively. No interactions among feed intake, insulin, and hASP were detected. In conclusion, the effect of hASP on fatty acid esterification is not influenced by feed restriction.     

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.     

五指山猪皮下脂肪组织lncRNA和mRNA的鉴定与分析

为了鉴定和分析五指山猪背部和腹部皮下脂肪组织中差异表达的长链非编码RNA(long noncoding RNA, lncRNA)和信使RNA(messenger RNA, mRNA),采用RNA-Seq和生物信息学方法对五指山猪皮下脂肪组织中的lncRNA和mRNA进行分析筛选,运用DESeq鉴定背部和腹部皮下脂肪组织中差异表达的lncRNA和mRNA,并对差异表达的lncRNA进行靶基因预测分析。结果显示:在五指山猪皮下脂肪组织中共鉴定出12 875个lncRNA,其中正义型10 155个、反义型278个、内含子型246个、基因间型2 196个;在背部与腹部皮下脂肪间,存在184个差异表达的mRNA,其中前十位分别是ZIC1、ZIC4、HAND2、CCBE1、RPH3A、ISM1、ANXA8、SLITRK4、DSG2、EVPL,存在45个差异表达的lncRNA,其中6个只在背部皮下脂肪中表达、18个只在腹部皮下脂肪中表达;获得差异表达lncRNA的靶基因共109个,包括顺式作用的靶基因和反式作用的靶基因。本试验为进一步研究lncRNA和mRNA调控猪皮下脂肪发育的分子机制提供科学依据。     

Effects of pregnancy and lactation on the metabolism of bovine adipose tissue

The incorporation of [2-14C] acetate into the triglycerides of adipocytes from bovine omental adipose tissue was at a minimum during peak lactation but increased again in late lactation. Similar but somewhat greater changes were observed for adipocytes from subcutaneous adipose tissue. In both tissues the activities of four enzymes concerned with fatty acid synthesis were lowest during peak lactation. In omental tissue lipoprotein lipase activity was lowest during peak lactation but there was a marked increase in its activity in the udder during lactation. In both omental and subcutaneous adipocytes the rate of glycerol release at peak lactation was higher than at all other stages of pregnancy and lactation. At peak lactation plasma insulin levels were low but growth hormone levels were higher than at all other stages of pregnancy and lactation. These results suggest that lipogenic precursors were diverted away from adipose tissue in early lactation and stored triglycerides were made available for use elsewhere in the body.     

Evidence that phosphofructokinase limits glucose utilization in bovine adipose tissue

Bovine subcutaneous adipose tissue slices were incubated with 10 mM [U--14C] acetate in the absence and presence of 2 mM glucose, 10 mM lactate and 33 mU insulin/ml. The incorporation of acetate into fatty acids was stimulated significantly by glucose and lactate, but not by insulin. The concentration of glycolytic intermediates was measured in tissue slices incubated in vitro with the same substrate combinations. Glucose significantly increased the cellular content of glucose 6-phosphate and fructose 6-phosphate, but had no effect on any other glycolytic intermediate. Under certain conditions, acetate and lactate tended to decrease the monophosphorylated hexoses and increase certain triose phosphates, indicating increased flux through phosphofructokinase. Insulin generally had no effect on metabolite levels. The data indicate that phosphofructokinase has a key regulatory role in controlling glycolytic flux in bovine adipose tissue incubated in vitro. The data did not indicate regulatory roles for hexokinase or insulin.     

牛皮下注射爱普菌素的组织药代动力学试验

牛皮下单次注射爱普菌素注射剂,剂量为0.5 mg/kg,给药后在不同时间点采取肌肉、肝脏、肾脏和脂肪等组织样品检测爱普菌素残留,采用3P97软件对组织残留-停药时间数据进行分析.结果表明,注射部位、肝脏中爱普菌素残留浓度变化符合二室开放模型,肌肉、肾脏、脂肪中EPR残留浓度变化符合一室开放模型.爱普菌素经皮下注射后Tmax均小于1 d(0.17～0.76 d),Cmax范围在37.32～1453.79 ng/g之间.MRT范围在7.54～14.79 d之间,与T1/2el范围2.91～19.50 d相一致,说明药物在动物体内消除缓慢.