Ligand binding to the porcine adipose tissue beta-adrenergic receptor.

We measured ligand binding to the beta-adrenergic receptor from porcine adipocytes using tritiated radioligands, dihydroalprenolol (DHA) and CGP-12177 (CGP), and an iodinated radioligand, cyanopindolol (ICP). Binding was measured in a crude plasma membrane preparation. Equilibrium saturation binding was regular for all three ligands; the Kd were approximately 4,000 pM for DHA, 600 pM for CGP, and 100 pM for ICP. Binding was stereospecific with each radioligand. Association of each radioligand was relatively rapid; dissociation was rapid and complete for DHA, initially rapid but ultimately incomplete for CGP, and minimal for ICP. The Kd estimated from kinetic data were approximately 1,000 pM for DHA and 100 pM for CGP. The receptor did not bind phentolamine, an alpha-adrenergic antagonist, except at concentrations greater than 10(-5) M. Propranolol was bound to the receptor with a Ki of approximately 8 nM regardless of the radioligand used. Metoprolol, a purported beta 1-adrenergic specific antagonist, was bound to the receptor with a Ki of approximately 300 nM when the radioligands were CGP or ICP but with a Ki of approximately 1,000 nM when the radioligand was DHA. The Ki for ICI 118,551, a purported beta 2-adrenergic specific antagonist, were approximately 500 nM when the radioligands were DHA or CGP but 125 nM when the radioligand was ICP. Thus, the choice of radioligand can influence the characterization of the beta-adrenergic receptor being studied.     

The histology of developing porcine adipose tissue

At each of the following days after conception (45, 60, 75, 90 and 105), pig fetuses were removed from sows representing lean and fat stains. From two additional litters, postnatal pigs were sacrificed at 1, 3, 6, 9, 12, 15, 18 and 21 d. Pelikan dye was injected into fetuses and pigs. The whole of the dorsal subcutaneous tissue, including some underlying muscle, was removed. Tissue was fixed into paraffin blocks or was frozen. Paraffin and frozen sections were stained and examined for stromal-vascular and cellular changes during growth. Organized stromal-vascular changes occurred during a period of adipocyte formation from 45 d gestation until 9 d postnatally. At 45 d gestation, the subcutaneous tissue contained many short unorganized connective tissue fibers. Gradually, these fibers became more organized in a ventral to dorsal and caudal to cranial gradient, so that by 1 d postnatally, they formed complete lobules around all existing fat cell clusters. The presumptive adipose space of the complete lobules contained delicate strands of connective tissue and reacted metachromatically for mucin. Connective tissue around lobules became progressively thinner throughout the remaining postnatal ages. Vascularity of the subcutaneous tissue increased as the stromal became organized. Lipid was not present in the subcutaneous tissue at 45 d gestation, but some deposition was apparent in the inner layer at 60 d. Between 60 d gestation and 9 d postnatally, fat cells filled both subcutaneous layers in a ventral to dorsal formation. Presumptive adipose lobules were the source of adipocytes and capillaries of developing fat cell clusters. Adipocytes from fetuses through 1-d postnatal pigs were multilocular, while unilocular fat cells were first observed at 3 d. At 9 d, multilocular adipocytes were found singly or in groups within unilocular fat cell lobules.     

Comparison of plasma free-fatty-acid and blood-glycerol concentrations with measurement of lipolysis in porcine adipose tissue in vitro

Rates of adipose tissue lipid metabolism in vitro are often measured to evaluate the function in vivo of metabolic pathways and thus appraise the accretion or loss of depot fat. This study directly addressed the comparison of degradative metabolism in vitro and in vivo. The concentrations of plasma free-fatty-acids and blood-glycerol as putative representatives of lipolysis in vivo and the lipolytic rate in adipose tissue in vitro obtained at the time of blood sampling were both measured in the same pig. Concentrations of plasma free-fatty-acids and blood-glycerol were increased or decreased by infusion of the norepinephrine analog, isoproterenol or by infusion of the adrenergic antagonist, propranolol, respectively. Although lipolytic rates and sensitivity to isoproterenol in vitro changed during some acute hormonal manipulations of the pig, the modulation in vitro was usually small relative to the large changes observed in plasma free-fatty-acid and blood-glycerol concentrations. Some of the subtle changes in vitro may reflect biological responses to hormone infusion, e.g., desensitization of the response to adrenergic agonists, but the magnitude of rate changes in vitro negates prediction of the rates in vivo from rates in vitro. Extrapolation of lipolytic rates in vitro and several adipose tissue anabolic rates obtained from the literature indicate the improbability for prediction of rates and degree of fat accretion in pigs from metabolic rates in vitro.     

Glycerolipid biosynthesis in porcine adipose tissue in vitro. II. Synthesis by various types of cellular preparations

Glycerolipid biosynthesis by porcine adipose tissue homogenates did not yield the 90+% triacylglycerol observed in situ. Consequently, we compared intact tissue slices and various subcellular fractions to characterize the usefulness of such systems to assess glycerolipid biosynthesis in vitro. Glycerolipid biosynthesis by porcine adipose tissue homogenates was measured in vitro using either [14C]-fatty acid or [14C]-glycerol-3-phosphate (G3P) as a radiolabelled substrate. Removal of residual 14C-labelled fatty acid from lipid extracts was difficult. Because G3P is soluble in water, residual [14C]-G3P separated easily from the glycerolipid-containing organic phase and, thus, was the preferred radiolabelled substrate. With tissue slices, glycerol and G3P were minimally incorporated into lipid so that [14C]-fatty acid was the preferred radiolabelled tracer. A washing procedure followed by thin layer chromatography was devised to separate residual [14C]-fatty acid from glycerolipids, including phospholipids. Fatty acid esterification into glycerolipids in tissue slices yielded about 4% phospholipids, whereas with homogenates, esterification yielded up to 50% phospholipids. Comparison of several subcellular fractions indicated that microsomes contained most of the glycerolipid biosynthetic activity when activity was expressed on a protein basis. However, when activities were expressed on a tissue wet weight basis, the 700 x g infranate and the 10,000 x g supernate had about equal activity that was far greater than the microsomes. The 700 x g infranate was the preferred enzyme preparation for assay of the entrance of G3P into the pathway as well as the capacity to synthesize triacylglycerol. Several methods of freezing and storing tissue or 700 x g infranates were not acceptable. Freezing of the 700 x g infranate in liquid N2 with storage at -80 degrees C may be an acceptable procedure.     

beta-Adrenergic receptor agonists increase apoptosis of adipose tissue in mice

beta-Adrenergic receptor (beta-AR) agonists increase muscle mass and decrease body fat in rodents and livestock. With oral administration, however, the effects of beta1-AR and beta2-AR can be different, depending on the species tested. We tested the effects of clenbuterol, a beta2-AR agonist, and ractopamine, a beta1/beta2-AR agonist, on growth, adiposity and adipose tissue apoptosis in male and female mice by feeding diets containing control, 200 ppm clenbuterol, or 200 or 800 ppm ractopamine. Food intake (FI) was measured daily; body weight (BW) and temperatures (BT) were measured on days 0, 3, 7, 10, 14, 17, and 20. On day 21 mice were sacrificed, body composition was determined using PIXImus densitometry, and muscle and adipose tissues were collected. There were no treatment effects on BT, FI, BW, feed efficiency or body composition. Retroperitoneal (Rp) and epididymal/parametrial (Epi/Par) fat pad masses were reduced in both 800 ppm ractopamine (40+/-3mg and 207+/-20mg, respectively) and clenbuterol (35+/-7 mg and 211+/-22 mg) treated mice compared to control (66+/-8 mg and 319+/-30 mg, P<0.05). Brown adipose tissue (BAT) mass was greater (P<0.05) in clenbuterol treated mice compared to other treatments. Adipose tissue apoptosis (% DNA fragmentation) was increased in Epi/Par fat pads in clenbuterol (5.2+/-1.1%) and 800 ppm ractopamine (4.1+/-0.8%) treated mice compared to control (1.7+/-0.4%, P<0.05). These findings show that WAT apoptosis can be induced by activation of beta-AR in mice, although the mechanism is unknown.     

Effect of chicken egg yolk antibody against adipose tissue plasma membranes on carcass composition and lipogenic hormones and enzymes in pigs

Chicken egg yolk antibody against pig adipose tissue plasma membranes (AIgY) was raised and used in the present experiment to evaluate the effect of dietary AIgY supplementation on pig growth and carcass composition. 160 crossbred (Duroc–Jersey × Landrace·Meishan) pigs, with initial live body weight of 27.5 ± 2.4 kg, were treated with AIgY or non-immunized control egg yolk powder (NIgY) at the inclusion level of 75 mg/kg diet. Following a 104-day trial, the pigs were slaughtered for analyzing the carcass and meat quality traits. The perirenal, mesenteric and subcutaneous fat depots were weighed and the diameter of adipocytes from different fat depots was measured with histological methods. Serum concentrations of insulin and leptin as well as the activities of malic enzyme (ME) and lipoprotein lipase (LPL) in adipose tissue were measured. Dietary supplementation of AIgY enhanced average daily gain and feed efficiency by 13.03% (P < 0.01) and 7.49%, respectively, with no influence on feed consumption. AIgY increased the lean mass by 10.3% (P < 0.01) without affecting the dressing percentage. Backfat thickness at 6th–7th rib and the weights of perirenal, mesenteric and subcutaneous fat depots were reduced by 24.14% (P < 0.01), 27.27% (P < 0.05), 20.42% (P < 0.01) and 29.21% (P < 0.01), respectively. Dietary supplementation of AIgY reduced the size of adipocytes in all the three fat pads (P < 0.05). The meat color was improved whereas the marbling score, the intramuscular fat content, and pH₄₅ of the longissimus muscle remained unaffected. Serum concentration of non-esterified fatty acids (NEFA) was significantly increased (P < 0.01) while urea-N content was reduced (P < 0.05). No alterations were detected for the serum levels of triacylglycerides (TG) and glucose. Serum concentrations of insulin and leptin were decreased by 26.19% (P < 0.05) and 26.53% (P < 0.05), respectively. LPL activity in adipose tissue was depressed significantly (P < 0.05) without affecting ME activity. This study demonstrates that dietary supplementation of AIgY can effectively improve growth and carcass composition of pigs and the changes of serum insulin and leptin levels as well as the tissue LPL activity may be involved in the acting mechanism.     

Effects of adrenergic agonists and insulin on porcine adipose tissue lipid metabolism in vitro

Because this laboratory has been able to demonstrate only a small and somewhat inconsistent stimulation of glucose metabolism by insulin in porcine adipose tissue in vitro, the tissue was preincubated with insulin to attempt to enhance the hormone effect. Preincubation with or without insulin did not increase insulin stimulation. Furthermore, insulin did not stimulate triacylglycerol biosynthesis. Adrenergic hormones stimulated lipolysis in porcine adipose tissue in vitro. Several analogs of norepinephrine incubated with porcine adipose tissue in vitro did not inhibit glucose incorporation into CO2 or total lipids, in contrast to inhibition observed in adipose tissue from other species. Isoproterenol inhibited glycerol-3-phosphate incorporation into lipids; the maximal inhibition was 50% for the initial stages of the pathway. Palmitate incorporation into lipids also was inhibited 50% by isoproterenol but this may have been an artifact. Preincubation of adipose tissue, with no exogenous hormone, might decrease the concentration of endogenous adrenergic hormones and thus make the tissue more responsive to exogenous adrenergic hormones. Preincubation of porcine adipose tissue did not consistently lower the basal lipolytic rate but enhanced the stimulated lipolytic rate; the mechanism is not known. These experiments provide no evidence that preincubation is beneficial to measurement of lipolysis or glucose metabolism in porcine adipose tissue in vitro.     

Dietary protein-induced changes in porcine muscle respiration, protein synthesis and adipose tissue metabolism

Growth rate, physically separable tissues of the ham and loin, heat production, skeletal muscle respiration and protein synthesis, and lipogenesis and lipolysis in s.c. adipose tissue were measured in a single experiment in which pigs were offered a 13 (n = 8), or 21% (n = 6) protein diet from 20 to 100 kg live weight. Pigs that were fed the 13% protein diet gained body weight slower, ate less, converted feed less efficiently and took 31 d longer to reach 100 kg live weight. Fat depth (cm) was greater (P less than .05) and loin eye area (cm2) was less (P less than .01) in pigs fed the 13% protein diet (2.6 vs 2.3 and 29.8 vs 35.3). Pigs that were fed the 13% protein diet had lower (P less than .05) ham and loin separable muscle and greater (P less than .05) ham and loin separable fat. The mean heat production was less (P less than .05) in pigs offered the 13% (22.49) vs 21% (24.63 MJ/d) protein diets. In the intercostal muscle preparation, total and Na+,K+-ATPase-dependent respiration (microliter O2.mg-1.h-1) were lower (P less than .05) in pigs offered the 13% (2.39 and .41) vs the 21% (3.89 and .68) protein diets. The energy used for the support of Na+ transport across membrane accounted for approximately 17% of muscle respiration. Absolute rates of protein synthesis in the muscle preparations were lower (P less than .01) at 13 than at 21% dietary protein. Lipogenesis in s.c. adipose tissue was not affected by dietary protein level. There was no difference in basal and norepinephrine-stimulated lipolysis between the two dietary protein levels.     

Beta-adrenergic receptor activity in ponies with recurrent obstructive pulmonary disease.

Pulmonary function measurements were made in control ponies and in ponies with recurrent obstructive pulmonary disease (principals) during clinical remission and during an attack of acute airway obstruction. The ponies were given beta-adrenergic antagonists and agonists to determine the role of beta receptors in recurrent obstructive pulmonary disease, and to determine the subtypes of beta receptors mediating bronchodilation in ponies. Aerosol administration of the beta antagonists, propranolol (beta 1 and beta 2), atenolol (beta 1), and butoxamine (beta 2) decreased dynamic compliance (Cdyn) and increased pulmonary resistance (RL) in the principal ponies during airway obstruction, but were without effect when the ponies were in clinical remission. Intravenous administration of atropine reversed the effect of atenolol on Cdyn and RL, but was without effect on the decrease in Cdyn and increase in RL observed after butoxamine administration. The beta antagonists did not affect airway function in the control ponies. The effect of beta blockade on Cdyn and RL suggests beta-adrenergic activation in the central and peripheral airways of principal ponies, mediated through both beta 2- and beta 1-adrenergic receptors. The aerosol beta agonists, isoproterenol (beta 1 and beta 2), and clenbuterol (beta 2) attenuated histamine-induced airway obstruction to a similar extent in control ponies that were given histamine IV. In addition, the beta 1 antagonist, atenolol, did not attenuate the bronchodilation observed with isoproterenol. We concluded that, although beta 1- and beta 2-adrenergic receptors exist in pony airways and are activated during acute airway obstruction, bronchodilation in response to beta agonists in ponies seems to be mediated primarily by beta 2-adrenergic receptors.     

Glycerolipid biosynthesis in porcine adipose tissue in vitro: effect of adiposity and depot site

To compare genetic differences in glycerolipid biosynthesis, rates were determined in s.c. adipose tissue of lean and obese pigs at 28, 60 and 110 d of age. To compare depot-specific differences, glycerolipid biosynthetic rates were determined in outer s.c., middle s.c., perirenal and omental adipose tissues obtained from 105-kg contemporary pigs. Rates were determined with a 700 x g infranatant fraction of an adipose tissue homogenate by measuring glycerophosphate incorporation into total lipids (mostly phosphatidic acid) during 4 min. This assay represents entrance of substrates into the glycerolipid synthesis pathway or glycerophosphate acyltransferase (GPAT) activity. Rates measured for 60 min represent maximal synthesis of glycerolipid (more triacylglycerol than phosphatidic acid) or lipid synthesis capacity (LSC). Adipocyte diameter and volume were greater for adipose tissue of obese than of lean pigs both at 60 and 110 d. When expressed per cell, activity of GPAT and LSC were similar for lean and obese pigs at 28 d. At 60 d and 110 d, LSC was greater for obese than for lean pigs; GPAT activity was greater at 60 but not at 110 d in obese than in lean pigs. Expressed on a cell basis, GPAT activity was highest in omental and outer s.c., intermediate in perirenal and lowest in middle s.c. adipose tissue depots. Lipid synthesis capacity was highest in perirenal and lowest in outer and middle s.c. depots. Our results indicate that the LSC assay was more closely related to the accretion of fat in vivo than to GPAT activity.     

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.     

Profile of ligand binding to the porcine beta2-adrenergic receptor

Chinese hamster ovary cells expressing the porcine beta2-adrenergic receptor (betaAR) were used to determine the binding kinetics of agonists and antagonists by competitive displacement of the radioligand [125I]iodocyanopindolol. Several purported agonists, including isoproterenol, epinephrine, norepinephine, dobutamine, salbutamol, and terbutaline, exhibited dual-affinity displacement curves, which is characteristic of agonist binding to betaAR. In each case, the addition of guanosine triphosphate (GTP) eliminated the high-affinity state and resulted in a one-site displacement curve. All of the antagonists modeled to only one site in the presence or absence of GTP. Several ligands, including ones used to promote animal growth (clenbuterol, L-644,969, and ractopamine) and the beta3AR-selective agonist BRL 37344 modeled to only one site, suggesting that these ligands would not be full agonists at the porcine beta2AR (pbeta2AR). Most of the tested ligands exhibited binding affinities that were similar to published values for the beta2AR from other species. However, several exceptions were observed. The BRL 37344 ligand bound the pbeta2AR with a 10-fold higher affinity than the human beta2AR, and the Kd of this was similar to Kd values reported for the human and rat beta3AR. The Kd of the pbeta2AR for ICI 118,551 was 50-fold higher than that for the beta2AR from rats and humans. For both BRL 37344 and ICI 118,551 the subtype-selective character of these ligands was different in the pig compared with the human and rat. These data demonstrate the value of using species-specific betaAR for selection of agonists and antagonists. Further, these data support the growing evidence that few ligands are full agonists for pbetaAR and that binding data may be useful for identifying ligands with full agonist potential.     

脂肪组织细胞膜蛋白卵黄抗体对猪脂肪组织中凋亡相关基因表达的影响

探讨脂肪组织细胞膜蛋白卵黄抗体对猪脂肪组织Bcl-xl、Bax-αmRNA表达的影响。将160头体重27.0 kg左右的杜×(长·梅)三元杂交商品猪,随机分成试验组和对照组,每组设4个重复,每个重复20头,公母各半。在试验组基础日粮中添加75 mg/kg脂肪组织细胞膜蛋白卵黄抗体(简称AIg Y)。饲喂104 d后,从每组选出体重相近的12头猪(公母各半)进行屠宰,分离背部皮下脂肪组织、肾周脂肪组织和肠系膜脂肪组织,并称重,同时采集脂肪组织样,采用RT-PCR检测Bcl-xl、Bax-αmRNA表达水平,并测定各部位脂肪组织DNA、RNA浓度,计算RNA、DNA含量以及RNA/DNA比值。结果显示:试验组皮下、肾周、肠系膜脂肪组织DNA含量分别比对照组下降了38.42%(P0.01)、39.92%(P0.05)和39.16%,而DNA、RNA浓度以及RNA/DNA比值差异不显著;与对照组相比,试验组脂肪组织Bcl-xl mRNA相对表达丰度下降了18.13%(P0.05);Bax-αmRNA相对表达丰度升高了13.79%(P0.05)。提示:日粮中添加脂肪组织细胞膜蛋白卵黄抗体使Bax-α与Bcl-xl基因表达失去平衡,可能诱导猪的脂肪细胞凋亡,上调的Bax-α和下调的Bcl-xl mRNA表达说明AIg Y是通过线粒体途径诱导脂肪细胞凋亡的。     

Effects of ractopamine on adipose tissue metabolism and insulin binding in finishing hogs. Interaction with genotype and slaughter weight

Twenty-four barrows were divided among eight treatments in a 2 x 2 x 2 design to quantify the influence of ractopamine (0 or 20 mg/kg diet) over the final 40 kg of gain on metabolic activity in adipose tissue. Interactions with genotype (Hampshire cross or Landrace cross) and slaughter weight (100 or 127 kg) were investigated also. Backfat was removed at slaughter and rates of lipolysis and fatty acid synthesis (FS), activities of malic enzyme (ME) and fatty acid synthetase (FAS), and insulin binding to adipocytes were assessed. Adipocytes from ractopamine-fed pigs were less sensitive (EC50 increased 90%) and had a lower maximum lipolytic response (40%) to ractopamine stimulation. Rates of basal and insulin-stimulated FS were decreased 40% in ractopamine-fed pigs and were reflected in lower activities of ME (50%) and FAS (15%). Breed and slaughter weight had no consistent influence on the ractopamine response. Landrace-cross pigs had greater insulin binding capacity (30-60%) whether data were expressed on a cell or surface area basis. Ractopamine feeding did not consistently affect insulin binding capacity. Results suggest that ractopamine interacts in vivo with the beta-adrenergic receptor of swine adipocytes, decreasing lipogenic capacity and diminishing responsiveness to beta-adrenergic stimulation.     

A radioimmunoassay for porcine intrauterine folate binding protein.

A RIA was developed for porcine intrauterine folate binding protein (FBP). Displacement of [125I]FBP caused by increasing dilutions of uterine flushings collected from either d-15 pregnant or nonpregnant gilts or media from culture of endometrial tissue from d-15 pregnant or nonpregnant gilts was parallel to the displacement caused by the standard curve. Addition of known amounts of purified allantoic fluid FBP to dilutions of either intrauterine flushings or endometrial culture medium were measured accurately with the RIA. To test specificity, 2-mL samples of uterine flushings collected from d-15 pregnant and nonpregnant gilts were preincubated with 10 microCi of [3H]folic acid and then chromatographed using Sephadex G-100 (Sigma Chemical Co., St. Louis, MO). The fractions were subsequently assayed for radioactivity by liquid scintillation spectrophotometry and for FBP by RIA. The [3H]folic acid and FBP peaks coincided, indicating that the RIA is specific for FBP. Uterine flushings were collected on d 10, 11, 12, 13, 14, and 15 of the cycle or pregnancy from 1) White crossbred, 2) progesterone-treated White crossbred (200 mg of progesterone at 48 and 72 h after estrus), and 3) Meishan gilts and assayed for FBP. Total FBP increased 140-fold from d 10 to 15, and the pattern of change across day did not differ between pregnant and nonpregnant gilts. Progesterone treatment increased intrauterine FBP content on d 10 and 11. No difference in FBP concentrations was detected between White crossbred and Meishan gilts. These results indicate that the RIA for FBP is valid, allowing measurement of this protein in uterine flushings and endometrial culture medium. The onset of FBP secretion by the uterus between d 10 and 15 of the cycle or pregnancy is influenced by the timing of onset of progesterone influence in a manner similar to the endometrial proteins uteroferrin and retinol binding protein. In contrast to these endometrial proteins, FBP concentrations are similar in Meishan and White crossbred gilts.