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. I. Assay conditions for homogenates

Previous studies on glycerolipid biosynthesis in swine adipose tissue in vitro resulted in synthesis of primarily phospholipid, whereas triacylglycerol represents the vast majority of adipose tissue lipids. The objectives of this research were to maximize synthesis of triacylglycerol in vitro using the 700 x g infranatant fraction of a swine adipose tissue homogenate as the enzyme source. The capacity for total lipid synthesis was increased by greater than 50%, and the proportion of lipids synthesized as triacylglycerols was increased by increasing the length of incubation time from 20 to 60 min and the concentration of enzyme in the incubation from that obtained from 33 to that obtained from 120 mg adipose tissue. It is recommended that glycerolipid biosynthesis be assessed using two assays. An assay of up to 10 min was linear with incubation time and measured the initial incorporation of glycerol-3-phosphate into the pathway (GPAT); this incorporation was mostly into phospholipids. An assay of about 60 min was not linear with incubation time, but incorporation into total lipids (LSC) was predominantly into the triacylglycerol fraction. Although the LSC assay was not linear with time, it represents steady-state conditions that more closely typify conditions in situ. Oleate at .6 mM was inhibitory with enzyme extracted from 33 or 75 mg adipose tissue, whereas palmitate was not. Palmitoyl-CoA was not a suitable substrate because it produced low LSC and little triacylglycerol. Fluoride increased LSC but inhibited conversion of phospholipids into triacylglycerols, so its presence is not recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitotic activity in fetal and early postnatal porcine adipose tissue

Tritiated thymidine autoradiography and histochemistry were used to study the development of subcutaneous adipose tissue of lean and obese fetuses and postnatal pigs. A pattern of tritiated thymidine uptake by pre-adipocytes and adipocyte lipid accumulation was demonstrated during the growth of the fetal pig. In the youngest fetuses there was a period of intense stromal cell mitotic activity before any adipocyte lipid accumulation. During subsequent fetal development, clusters of tightly arranged stromal cells were formed. Lipid accumulation occurred only in these cell clusters. During this time of cell cluster formation and lipid accumulation, mitotic activity was minimal. In obese fetuses, stromal cell mitotic activity overlapped temporarily with the cell cluster formation and lipid accumulation period. In early postnatal pigs, fat cell clusters increased in size until they "physically filled" the adipose tissue. In pigs 3 d and older, there was extensive mitotic activity of cells within the fat cell clusters. The synthesis of this second bed of pre-adipocytes and the altered developmental pattern in the obese fetuses is suggested to be due to the influence of a high fat diet. The significance of these findings in terms of plausible links between pre-adipocyte mitosis and lipid accumulation is discussed.     

Porcine somatotropin decreases acetyl-CoA carboxylase gene expression in porcine adipose tissue

Crossbred barrows were treated daily with porcine somatotropin (pST; 4 mg/d) from 79 to 127 kg BW to determine whether pST regulates the activity and gene expression of adipose tissue acetyl-CoA carboxylase (ACC), the rate limiting enzyme in de novo fatty acid synthesis. Administration of pST reduced ACC enzyme activity, protein content, and mRNA abundance in adipose tissue by 40 to 50%. When comparisons were made among all pigs, ACC enzyme activity and mRNA abundance were closely associated (r² = .94). In summary, our results indicate that pST decreases ACC enzyme activity and that this is associated with a significant reduction in ACC mRNA abundance. We speculate that decreased ACC enzyme activity results from a reduction in ACC protein and that this occurs because pST reduces the abundance of mRNA available for translation.     

Regulation of uncoupling proteins 2 and 3 in porcine adipose tissue

This study was performed to determine whether or not uncoupling protein 2 (UCP2) and UCP3 expression in porcine subcutaneous adipose tissue are hormonally regulated in vitro and whether their expression is correlated with changes in metabolic activity. Tissue slices (approximately 100 mg) were placed in 12-well plates containing 1 mL of DMEM/F12 with 25 mM Hepes, 0.5% BSA, pH 7.4. Triplicate slices were incubated with basal medium or hormone supplemented media at 37 °C with 95% air/5% CO₂. Parallel cultures were maintained for either 2 or 24 h to evaluate metabolic viability of the tissue. Slices were transferred to test tubes containing 1 mL of DMEM/F12 with 25 mM Hepes, 3% BSA, 5.5 mM glucose, 1 μCi ¹⁴C-U-glucose/mL and incubated for an additional 2 h at 37 °C. Glucose metabolism in 2-h incubations did not differ from 24-h (chronic) incubations, indicating viability was maintained (P > 0.05). Expression of UCP2 and UCP3 was assessed in slices following 24 h of incubation with various combinations of hormones by semi-quantitative RT-PCR. Expression of UCP2 was induced by leptin (100 ng/mL; P < 0.05). Growth hormone (100 ng/mL) inhibited UCP2 expression (P < 0.05). Expression of UCP3 was inhibited by growth hormone (100 ng/mL; P < 0.05), tri-iodothyronine (10 nM; P < 0.05) or leptin (100 ng/mL; P < 0.05). Changes in UCP expression could not be associated with overall changes in glucose metabolism by adipose tissue slices in chronic culture.     

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.     

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.     

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.     

Effect of beta-adrenergic agonists on lipolysis and lipogenesis by porcine adipose tissue in vitro

The effects of the beta-adrenergic agonists isoproterenol, cimaterol, ractopamine and clenbuterol on lipolysis (release of glycerol and free fatty acids) and lipogenesis (incorporation of 14C into fatty acids from [14C]glucose) was examined in porcine adipose tissue explants in vitro. Lipolysis was stimulated by isoproterenol, cimaterol or ractopamine but not by clenbuterol. Insulin reduced the lipolytic effects of the beta-adrenergic agonists (isoproterenol, cimaterol and ractopamine). Lipogenesis was inhibited by all beta-adrenergic agonists tested (isoproterenol, cimaterol, ractopamine and clenbuterol). The antilipogenic effect of the beta-adrenergic agonists was reduced by the presence of insulin in the incubation. Although effects of the different beta-adrenergic agonists varied, all had some direct effects that could be expected to reduce adipose accretion. Effects of beta-adrenergic agonists in the pig are due in part to direct effects on adipose tissue.