Acute effects of beta-adrenergic agonists on porcine adipocyte metabolism in vitro

Backfat was obtained at slaughter from market weight hogs to study the acute effects of clenbuterol (CB), ractopamine (RAC) or epinephrine (EPI), in the presence and absence of theophylline (THEO) or adenosine deaminase (ADA), on rates of lipolysis and fatty acid synthesis in vitro. Only EPI increased lipolytic rate in the absence of THEO or ADA. In the presence of THEO or ADA, RAC and CB were lipolytic, although CB had a lower maximal response. With THEO present, RAC and EPI increased lipolysis with a similar potency and responsiveness. Lipolytic responses from all agonists were prevented by propranolol. Insulin stimulated glucose incorporation into fatty acids 50 to 100%; stimulated rates were not influenced by any agonist, either alone or in the presence of ADA. When THEO was present, EPI and RAC inhibited fatty acid synthesis approximately 50%. Clenbuterol was not inhibitory under any conditions. Results indicate that, under appropriate conditions, beta-adrenergic agents increase lipolysis and decrease lipogenesis in porcine adipocytes. Combined evidence suggests that lipolysis is more sensitive to beta-adrenergic stimulation than is insulin-stimulated lipogenesis. Finally, RAC and CB possess only partial agonist activity relative to EPI, CB being least active.     

Regulation of protein metabolism by insulin: value of different approaches and animal models

Insulin induces protein accretion by stimulating protein synthesis and inhibiting proteolysis. However, the mechanisms of regulation of protein metabolism by insulin are complex and still not completely understood. The use of approaches combining hyperinsulinemic clamp and isotopic methods, or measurement of the activation of intracellular kinases involved in insulin signaling, in addition to the use of different animal models in a comparative physiology process, provide better understanding of the potential regulation of protein metabolism by insulin. Studies using the clamp technique in lactating goats have shown a clear inhibitory effect of insulin on proteolysis, with an interaction between the effects of insulin and amino acids. Such studies revealed that the insulin-inhibited proteolysis is improved in lactating goats, this adaptative process limiting the mobilization of body protein under the conditions of amino acid deficit which occurs during early lactation. Insulin signaling studies in growing chickens have also provided some interesting features of insulin regulation compared to mammals. Refeeding or insulin injection leads to the activation of the early steps of insulin receptor signaling in the liver but not in the muscle. Muscle p70 S6 kinase, a kinase involved in the insulin activation of protein synthesis, was found to be markedly activated in response to insulin and to refeeding, suggesting that other signaling pathways than those classically described in mammalian muscles may be involved in signal transduction. Finally, although the role of insulin has been doubtful and has long been considered to be minor in ruminants and in avian species, this hormone clearly regulates protein metabolism in both species.     

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.     

Fatty acid metabolism by the porcine placenta

Two experiments were performed to determine whether esterification is a major pathway of fatty acid utilization within porcine placenta and to determine what metabolic parameters may limit fatty acid transfer to the fetal pig. Maternal (endometrium) and fetal (chorioallantois) placenta were obtained by Caesarean section at d 110 of gestation in both experiments. Eight gilts were used in the first experiment. Tissue sections were incubated with palmitate at concentrations ranging from .25 to 2.0 mM. Maternal placenta metabolized palmitate at a higher rate than fetal placenta, although fetal placenta was more efficient in esterifying palmitate. Esterification composed the majority of palmitate utilization within fetal and maternal placenta. The second experiment evaluated the effect of dietary lipid on placental fatty acid metabolism and evaluated the ability of placenta to mobilize lipids. Fourteen gilts were divided into two groups of seven and fed a diet containing 15% tallow diet or a diet not supplemented with tallow (control) from d 90 to 110 of gestation. Dietary lipid had no detectable effects on lipoprotein lipase activity, [14C]palmitate metabolism, or lipolysis by the maternal or fetal placenta. Lipolytic activity of placental tissues was minimally affected by incubation with various proposed lipolytic activity of placental tissues was minimally affected by incubation with various proposed lipolytic agents. The data indicate that supply of fatty acids to the fetal pig may be limited by transfer of plasma fatty acids into the cytoplasm of placental cells or by regulatory enzymes for intermediate esterification; both types of limitations have been proposed to be influenced by fatty-acid binding proteins.     

The effect of adenosine on pro-inflammatory cytokine production by porcine T cells

Adenosine is a well described anti-inflammatory modulator of immune responses. The aim of the present study was to describe the role of common adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) in cytokine production by main porcine T cell subpopulations. TNF-α, IFN-γ, IL-2 and IL-10 were detected by multicolor flow cytometry together with cell surface markers CD3, CD4 and CD8. It was found that NECA inhibits (in a dose-dependent manner) production of pro-inflammatory TNF-α and Th1-associated cytokines IFN-γ, IL-2 in all concanavalin A-stimulated T cell subpopulations. Moreover, production of IL-10 was potentiated in all T cell subpopulations tested. These corresponded well with the fact that all T cell subsets expressed mRNA for adenosine receptor (AR) subtypes to comparable extents. Contrary to concanavalin A-stimulated cells, NECA had a moderate effect on PMA-stimulated T cells, suggesting that AR in pigs acts via signaling pathways not associated with protein-kinase C. Non-selective antagonist CGS15943 as well as allosteric modulator SCH202676 failed to reverse the effect of NECA in pigs. In conclusion, NECA has an anti-inflammatory effect on porcine T cell subpopulations.     

Porcine leptin alters isolated adipocyte glucose and fatty acid metabolism

This study examined if leptin can acutely affect glucose or fatty acid metabolism in pig adipocytes and whether leptin's actions on lipogenesis are manifested through interaction with insulin or growth hormone. Subcutaneous adipose tissue was obtained from approximately 55 kg crossbred barrows at the USDA abattoir. Isolated adipocytes were prepared using a collagenase procedure. Experiments assessed U-14C-glucose or 1-14C-palmitate metabolism in isolated adipocytes exposed to: basal medium (control), 100 nM insulin, 100 ng/ml porcine growth hormone, 100 ng/ml recombinant porcine leptin, and combinations of these hormones. Treatments were performed in triplicate and the experiment was repeated with adipocytes isolated from five different animals. Cell aliquots (250 microl) were added to 1 ml of incubation medium, then incubated for 2h at 37 degrees C for measurement of glucose and palmitate oxidation or incorporation into lipid. Incubation of isolated adipocytes with insulin increased glucose oxidation rate by 18% (P<0.05), while neither growth hormone nor leptin affected glucose oxidation (P>0.5). Total lipid synthesis from glucose was increased by approximately 25% by 100 nM insulin or insulin+growth hormone (P<0.05). Insulin+leptin reduced the insulin response by 37% (P<0.05). The combination of all three hormones increased total lipid synthesis by 35%, relative to controls (P<0.05), a rate similar to insulin alone. Fatty acid synthesis was elevated by insulin (32%, P<0.05) or growth hormone (13%, P<0.05). Leptin had no effect on fatty acid synthesis (P>0.05). Leptin reduced the esterification rate by 10% (P<0.05). Growth hormone and insulin could overcome leptin's inhibition of palmitate esterification (P>0.05).     

Short-term regulation of hepatic triacylglycerol metabolism by insulin and glucagon

This paper presents a review of our current knowledge of short-term regulation of triacylglycerol metabolism by insulin and glucagon in isolated rat hepatocytes. Insulin is devoted to rapidly improve the lipogenic climate of the hepatocyte. The hormone stimulates glycolysis, the conversion of pyruvate into acetyl-CoA, the synthesis of fatty acids and their esterification. The opposite pathways, hydrolysis of triacylglycerols and fatty acid oxidation, are depressed by insulin. Glucagon does all but deteriorate the lipogenic climate within the liver cell as the hormone inhibits triacylglycerol synthesis and stimulates the breakdown of triacylglycerols.     

过氧化物酶体增殖物激活受体对脂肪代谢的调控

过氧化物酶体增殖物激活受体(PPARs)是一个由3种核受体组成的家族。其作为脂肪感受器,调节相关基因表达。PPARs具有多种生物学功能,如调控体内能量平衡、脂质和脂蛋白代谢、葡萄糖平衡等,其在脂肪细胞分化、生成和代谢等多方面起到重要作用。本文论述了PPARs的结构和调控基因转录的分子机制,综述了PPARs在脂肪细胞分化和脂肪代谢中的作用,并讨论了一些脂肪因子对PPARs和脂肪代谢的影响。     

Heart fatty acid binding protein is upregulated during porcine adipocyte development

Heart fatty acid binding protein (H-FABP) has been associated with intramuscular fat content in pigs. In the current study, we showed that expression of H-FABP mRNA in adipose tissue of adult pigs was 8.5% of that in heart and 30% of that in skeletal muscle, and that H-FABP mRNA level was more than 10% of that of adipocyte fatty acid binding protein mRNA in adipose tissue. Levels of H-FABP mRNA reached a maximum in adipose tissue from 7-d neonates, with no further increase in the adult. Also, H-FABP mRNA was induced during adipogenic differentiation of stromal-vascular cells derived from adipose tissue and skeletal muscle. In conclusion, H-FABP may play a role in adipose tissue development and function in the pig.     

Regulation of adipose tissue metabolism during pregnancy and lactation in the ewe: the role of insulin

This study was conducted to examine the effect of insulin on lipid metabolism of adipocytes during pregnancy and lactation in ewes. During the first 3 mo of pregnancy, metabolism of adipocytes from omental adipose tissue was characterized by a high rate of de novo lipogenesis (90 to 125 nmol of acetate incorporated into lipids.2 h-1.10(6) cells-1) and a 38% reduction in response to beta-lipolytic stimulus (isoproterenol 10(-6) M). Simultaneously, there was a rise in the number of high-affinity insulin receptors (Kd = .2 nM), and insulin binding characteristics showed a decrease in the negative cooperativity phenomenon. Moreover, lipogenesis stimulated by insulin (1 mU/ml) increased in comparison with observations in nonpregnant ewes. The last third of pregnancy and early lactation were characterized by a marked fall in lipogenesis and a simultaneous increase in isoproterenol-stimulated lipolysis. During lactation, the number of total insulin receptors was decreased by 62% and insulin stimulation of lipogenesis became inefficient. Results suggest that insulin plays a direct role in adipose tissue metabolism during pregnancy.     

Regulation of bovine satellite cell proliferation and differentiation by insulin and triiodothyronine

Satellite cells activity contributes to postnatal muscle growth. Herein, we have studied the respective influence of insulin and triiodothyronine (T3) on the proliferation and differentiation of primary bovine satellite cells isolated from Semitendinosus muscle of Montbéliard steers. Under basal conditions, satellite cells proliferated until the fifth day of culture, began to fuse into myotubes and expressed differentiation markers such as connectin, myogenin, and myosin heavy chain (MHC) isoforms. Insulin behaved as an effective mitogen. Moreover, it promoted extensive myotube formation and enhanced differentiation as shown by an increase in the accumulation of differentiation markers. Maximal differentiation occurred with insulin physiological range concentrations. A delay in the stimulation of differentiation was registered with a high dose that promoted maximal proliferation. Conversely, T3 decreased cell proliferation in a dose-dependent manner. In addition, fusion and biochemical differentiation (accumulation of connectin, MyoD1, myogenin, and myosin heavy chain isoforms) were also enhanced. Bovine satellite cells seemed to respond differentially to insulin and T3 for proliferation. Interestingly, both hormones displayed a myogenic influence. Our observations suggest that both hormones could influence bovine satellite cells in vivo and contribute to the regulation of postnatal muscle growth.     

Decreased insulin binding to porcine adipocytes in vitro by beta-adrenergic agonists

The present study was conducted to determine the influence of dibutyryl-cAMP (dbcAMP), epinephrine, ractopamine and clenbuterol on insulin binding to porcine adipocytes. Dibutyryl-cAMP decreased insulin binding to swine adipocytes by 40 and 20% at 1.8 and 25.8 ng insulin/ml, respectively. Ractopamine and clenbuterol directly reduced insulin binding at the low insulin concentration and decreased binding at high insulin concentrations in the presence of adenosine deaminase. Scatchard analysis suggested that the reduction of insulin binding was due to a decrease in receptor number. Epinephrine alone did not influence insulin binding. In the presence of theophylline, epinephrine decreased binding at both low and high insulin concentrations; however, ractopamine plus theophylline decreased binding only at the low insulin concentration. Clenbuterol did not affect insulin binding in the presence of theophylline. Propranolol blocked the inhibitory effect of epinephrine on insulin binding. These beta-adrenergic agonists can inhibit insulin binding and, thus, antagonize insulin action in swine adipocytes.     

Determination of the affinity of ractopamine and clenbuterol for the beta-adrenoceptor of the porcine adipocyte

Dissociation constants (Kd) of ractopamine and clenbuterol for the swine adipocyte beta-adrenergic receptor were estimated from competition studies with epinephrine for the stimulation of lipolysis. Both compounds competitively inhibited epinephrine-stimulated lipolysis in the absence of adenosine deaminase. Three methods for estimating Kd values were used and similar estimates were obtained with each method. Ractopamine and clenbuterol showed greater affinity for the beta-receptor than did epinephrine and had similar Kd values of 1 to 2 x 10(-7) M. The low capacity of ractopamine and clenbuterol to stimulate lipolysis in vitro does not result from poor coupling to the beta-receptor. Ractopamine and clenbuterol may be considered partial agonists, possessing high affinity for the beta-adrenoceptor but exhibiting a relative ineffectiveness for adenylate cyclase activation.     

Regulation of protein and energy metabolism by the somatotropic axis.

The somatotropic axis plays a key role in the co-ordination of protein and energy metabolism during postnatal growth. This review discusses the complexity of the regulation of protein and energy metabolism by the somatotropic axis using three main examples: reduced nutrition, growth hormone (GH) treatment and insulin-like growth factor-1 (IGF-1) treatment. Decreased nutrition leads to elevated GH secretion, but it reduces hepatic GH receptor (GHR) number and plasma levels of IGF-1; it also changes the relative concentrations of IGF binding proteins (IGFBPs) in plasma. GH treatment improves the partitioning of nutrients by increasing protein synthesis and decreasing protein degradation and by modifying carbohydrate and lipid metabolism. However, these well-established metabolic responses to GH can change markedly in conditions of reduced nutritional supply or metabolic stress. Short-term infusion of IGF-1 in lambs reduces protein breakdown and increases protein synthesis. However, long-term IGF-1 administration in yearling sheep does not alter body weight gain or carcass composition. The lack of effect of IGF-1 treatment can be explained by activation of feedback mechanisms within the somatotropic axis, which lead to a reduction in GH secretion and hepatic GHR levels. The somatotropic axis has multiple levels of hormone action, with complex feedback and control mechanisms, from gene expression to regulation of mature peptide action. Given that GH has a much wider range of biologic functions than previously recognized, advances in research of the somatotropic axis will improve our understanding of the normal growth process and metabolic disorders.     

羊抗猪脂肪细胞膜抗血清对猪生长发育与脂质代谢的影响

选取24头刚断奶(5周龄)的上海大白猪,随机分成3大组(6小组),即对照组C(C1和C2小组)、试验组Ⅰ(Ⅰ1和Ⅰ 2小组)、试验组Ⅱ(Ⅱ 1和Ⅱ2小组),每小组4头猪.试验期为11周和17周.42日龄时,试验组Ⅰ和Ⅱ,分别腹腔和皮下注射山羊抗猪脂肪细胞膜抗血清,连续以每日2.5 mL/kg荆量注射.时照组以相同剂量注射山羊正常血清.分别在免疫后11、17周末屠宰.结果显示,山羊抗猪脂肪细胞膜被动免疫猪后,各试验组(腹腔免疫组与皮下免疫组)的饲料报酬均明显高于时照组(P＜0.05);体重和日增重也高于时照组.在免疫后7～17 d各试验组血清FFA浓度与对照组相比差异显著(P＜0.05),65 d后各试验组均无明显差异;血清尿素氮在处理后各试验组均明显低于对照组(P＜0.05),35 d时皮下免疫组无明显差异;在免疫后7～17 d各试验组血清总蛋白与对照组相比均明显升高(P＜0.05),65 d时各试验组均不存在显著性差异;免疫后7～17 d各试验组血清胆固醇均显著低于对照组(P＜0.05).     

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.