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.     

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.     

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.     

Beta-adrenergic receptor binding in crude porcine adipose tissue plasma membranes.

Methods have been detailed to prepare a crude membrane fraction from isolated porcine adipose tissue cells. Adipocytes were obtained after incubation of 5 g of adipose tissue slices with 4,500 units of a selected lot of collagenase in a total volume of 15 mL at 37 degrees C for 90 min. There was no bovine serum albumin present during cell isolation because albumin did not enhance cell yield or yield of lipolytic activity. Isolated cells were lysed by exposure to hypotonic conditions in the presence of 7.5 mM ethylene glycol tetraacetic acid (EGTA) and .8 mM phenylmethylsulfonyl fluoride (PMSF). A 30,000 x g centrifugal pellet was used as the crude membrane preparation. Binding of tritiated dihydroalprenolol (DHA), a beta-adrenergic antagonist, was measured in the presence of 7.5 mM EGTA and .2 mM PMSF, because these protease inhibitors improved specific binding by approximately 50% to greater than 150 fmol/mg of protein and decreased non-specific binding to less than 10% at 2.5 nM DHA.     

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.     

Hormonal control of porcine adipose tissue fatty acid release and cyclic AMP concentration

Compared with adipose tissue from other mammals, porcine adipose tissue has stringent specificity for stimulation of lipolysis by analogs of norepinephrine. This study was to ascertain whether the specificity for control was reflected in the concentration of tissue cyclic AMP (cAMP). Adipose tissue slices were incubated and concentrations of tissue cAMP and free fatty acids (FA) released to the medium were measured. It was necessary to include theophylline in the incubation medium to be able to measure changes in cAMP concentration. Fatty acid release and cAMP production were increased most effectively by the beta-adrenergic agonists; isoproterenol, fenoterol, dobutamine and the mixed alpha- + beta-adrenergic agonist, epinephrine. Isoproterenol-stimulated FA and cAMP production both inhibited by the beta-adrenergic antagonist, propranolol. There was no evidence for alpha 2-adrenergic inhibition of lipolysis in porcine adipose tissue because clonidine (alpha 2-adrenergic agonist) did not lower isoproterenol-induced FA or cAMP levels and phentolamine, an alpha-adrenergic antagonist, did not increase epinephrine-stimulated FA release or cAMP generation. These results imply that the stringent specificity observed for stimulation of swine adipose tissue lipolysis resides in the beta-adrenergic receptor coupled to cAMP production.     

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.     

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.     

Stimulation of lipogenesis by insulin in swine adipose tissue: antagonism by porcine growth hormone

The effects of physiological (1, 10 ng/ml) and pharmacological (1,000 ng/ml) concentrations of insulin (INS) and porcine growth hormone (pGH) on lipid metabolism were determined in short-term (2 h) and long-term (26, 50 h) incubations of swine adipose tissue. The short-term effects of three different commercial sources of bovine serum albumin (BSA) on adipose tissue metabolism were also evaluated. Two of the three BSA preparations were found to be unsuitable for inclusion in the short-term incubation buffer because they caused a stimulation of lipid synthesis in adipose tissue and masked the stimulatory effects of insulin. Physiological concentrations of insulin stimulated glucose metabolism in 2-h incubations by 100% in adipose tissue from 80-kg swine. After a 26-h incubation period, INS maintained rates of glucose metabolism at levels comparable to maximally stimulated rates in fresh tissue. Insulin also enhanced glucose metabolism following 50-h incubations; however, rates were less than for 2- or 26-h incubations. Glucose metabolism was also stimulated in adipose tissue from 127-kg swine when incubated for 2 h with INS; however, INS responsiveness declined with increasing body weight. Lipogenesis and glucose oxidation were partially maintained by INS using tissue from the heavier swine. A pharmacological but not physiological concentration of pGH stimulated glucose metabolism in short-term incubations by 50% in adipose tissue from 80-kg swine, and by 10% in adipose tissue from 127-kg swine. Long-term culture of adipose tissue in the presence of pGH had no effect on glucose metabolism. Physiological levels of pGH directly antagonized the stimulation of glucose metabolism by INS in short- and long-term incubations. In summary, these results are the first to establish that swine adipose tissue is quite sensitive to insulin and that pGH directly antagonizes insulin action.     

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.     

Gene cloning of porcine adiponectin gene from adipose tissue and construction of its eukaryotic expression vector

To clone adiponectin (ADPN) gene from Shaziling porcine adipocyte and construct its eukaryotic expression vector, total RNA was extracted from subcutaneous fatty tissue. One pair of specific primers was designed by Primer 5.0 software according to the sequence of ADPN gene of porcine available in GenBank. The ADPN gene was amplified by PCR from cDNA and cloned into pMD18‐T vector to construct recombinant clonal vector pMD‐ADPN, sequenced and analysed. A recombinant expression plasmid pPICZaA‐ADPN was constructed by subcloning the cloned ADPN gene into the linearized pPICZaA vector. Then, the plasmid pPICZaA‐ADPN was expressed in Pichia pastoris (GS115) by electrotransformation. Western blot and Bradford analysis were used to determine the target protein induced by methanol. Results showed that the genome size of ADPN was 732 bp and encoded 244 amino acid, the nucleotide sequence of ADPN shared 100% identity with that of porcine available in GenBank. Western blot and Bradford analysis showed that the recombinant ADPN was expressed in GS115 correctly and has certain immune activity. The expression level of ADPN was 28.5 μg/ml. In conclusion, the recombinant ADPN could express in eukaryotic expression vector pPICZaA‐ADPN constructed in this study effectively.     

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)     

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.