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.     

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 effect of porcine somatotropin supplementation in pigs on the lipid profile of subcutaneous and intermuscular adipose tissue and longissimus muscle.

The effect of porcine somatotropin (pST) on the lipid profiles of adipose tissue and muscle was investigated. Sixteen crossbred barrows were injected daily with either 3 mg of pST or a placebo. After slaughter, total lipid and fatty acid composition of raw subcutaneous (SC) adipose and intermuscular (IM) adipose tissue and longissimus muscle were determined. The SC adipose tissue from pST-treated pigs had a 7.5% decrease in total lipid content; specific fatty acids 16:0, 18:0, and 18:1(n-9)c decreased most. The IM fat from pST-treated pigs had lower levels of 16:0 and 20:0. There was no effect of pST treatment on the lipid profile of the longissimus muscle. The data suggest that pST treatment produces small but significant changes in the saturated fatty acid content of adipose tissue in pigs.     

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.     

Effects of an intravenous injection of NPY on leptin and NPY-Y1 receptor mRNA expression in ovine adipose tissue

Neuropeptide Y (NPY) is highly expressed in hypothalami of undernourished and genetically obese animals, and is a potent regulator of food intake and reproduction. Leptin, a protein expressed by adipocytes, has been reported to reduce hypothalamic NPY expression. We recently detected (by ribonuclease protection assay [RPA]) expression of the NPY receptor subtype Y1 (but not Y2) mRNA in adipose tissue. Based on these observations we hypothesized that NPY-Y1 receptors in adipose may represent a peripheral mechanism by which NPY can regulate leptin expression in a direct and rapid manner. To test this hypothesis, adipose samples were biopsied from the tailhead region of 48 ± 3 kg wether lambs immediately before and 30 min after a single intravenous injection of 50 μg porcine NPY (“treated” animals, n = 5), or vehicle (“control” animals, n = 4). Injection of NPY resulted in an increase in expression (P = 0.013; as measured by RPA) of both leptin and NPY-Y1 mRNA. In treated animals, negative correlations were found between response in leptin expression and body weight (r = −0.82, P = 0.092), and between leptin response and initial leptin mRNA levels (r = −0.81, P = 0.097). These data provide evidence of a peripheral mechanism by which NPY may regulate adipocyte expression of both leptin and NPY-Y1 receptor mRNA.     

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.     

Hormonal regulation of leptin receptor expression in primary cultures of porcine hepatocytes

A study was conducted to elucidate hormonal control of leptin receptor gene expression in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (52 kg) and seeded into collagen-coated T-25 flasks. Monolayer cultures were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the 3-day culture period. To establish basal conditions hepatocytes were maintained in serum-free William's E medium containing 10 nM dexamethasone and 1 ng/ml insulin. For the final 24 h, insulin (1 or 100 ng/ml) or glucagon (100 ng/ml), were added in the presence or absence of 100 nM triiodothyronine (T3). RNA was extracted and quantitative RT-PCR was performed with primers specific for the long form and total porcine leptin receptors. Leptin receptor expression was calculated relative to co-amplified 18S rRNA. Expression of the long form of the leptin receptor was confirmed under basal conditions. Insulin, glucagon and synthetic human proteins (ghrelin and GLP-1) at 100 ng/ml had no influence on leptin receptor expression; the addition of T3 was associated with a marked increase (P < 0.001) in expression of total and long forms of the leptin receptor by 1.6 and 2.4-fold, respectively. Addition of leptin to cells which were pre-treated with T3 for 24 h (to up-regulate leptin receptor expression), confirmed the lack of a direct effect of leptin on glucagon-induced glycogen turnover and cAMP production. These data suggest that porcine hepatocytes may be insensitive to leptin stimulation even when leptin receptor expression is enhanced by T3.     

Postnatal development of glucose transporter proteins in bovine skeletal muscle and adipose tissue.

Facilitated diffusion of glucose across the plasma membrane is mediated by a family of glucose transporter (GLUT). GLUT1 is ubiquitously present in all tissues and involved in cellular glucose uptake, while GLUT4 plays a key role in cellular glucose uptake stimulated by insulin in skeletal muscles and adipose tissue. To examine the postnatal change in the GLUTs of ruminants, the protein levels of GLUT1 and GLUT4 were measured by Western blot analysis of skeletal muscles, adipose tissue and brain of Holstein male calves aged from 0 to 12 months. Analysis of rumen short chain volatile fatty acids revealed that rumen fermentation increased around 2-3 months old. The GLUT1 level did not change in all tissues examined during the postnatal period, while the GLUT4 levels in skeletal muscle and subcutaneous adipose tissue decreased gradually, and at 12 month old, it was about 40% of those seen at 0 month old. These results are contrast to those in non-ruminant species, in which GLUT4 increases during postnatal development, and may be related to the insulin-resistance seen in adult ruminants.     

Characterization of somatotropin binding sites in pig skeletal muscle.

The present study was conducted to determine whether somatotropin (ST) binding sites are present in crude membrane preparations containing sarcolemma of pig skeletal muscle. Initial characterization experiments indicated that binding of bovine ST (bST) was time- and temperature-dependent and that binding was reversible. At 23 degrees C, binding was maximized between 36 and 48 h, whereas at 4 degrees C binding had not reached a maximum by 96 h. Somatotropin binding was stable between pH 5.5 and 8.5 and increased linearly between 100 and 600 micrograms of membrane protein. Addition of unlabeled bST decreased specific binding of [125I]bST in a dose-dependent manner (ED50: 1 to 1.6 ng/mL). The binding sites for bST were specific because porcine prolactin poorly inhibited bST binding. Scatchard analysis revealed the presence of a single class of binding sites (Ka: 9 to 15 x 10(9)M-1; Bmax: 5 to 6 fmol/mg of protein). In summary, the present report is the first to demonstrate that specific ST receptors are present in pig skeletal muscle. The role that ST plays in directly stimulating muscle growth and(or) muscle synthesis of insulin-like growth factor I (IGF-I) in pST-treated pigs as opposed to changes that occur as the result of an increase in plasma IGF-I concentration remains to be resolved.     

Effect of somatotropin on adipose tissue metabolism: Ontogeny of the enhanced response to adrenergic challenge in the lactating cow

Seven multiparous Holstein cows (>150 d postpartum) were used to evaluate the time course of the chronic adaptation in lipolytic response to adrenergic challenge with bovine somatotropin (bST) treatment. Cows received daily bST (sometribove; 40 mg/d) or excipient injections for 7 d (single reversal design) with a 7-d interim between periods. Epinephrine challenges (1.4 μg/kg body weight intravenously) were administered on Days −2, −1, 1, 2, 3, 5, and 7 of treatment at 10:00 a.m. (15 hr after bST or excipient injection). Frequent blood samples were collected, and concentrations of plasma glycerol (GLY) and nonesterified fatty acids (NEFA) were determined. Treatment with bST increased milk yield 23% (P < 0.05) and milk fat content 33% (P < 0.001) compared with controls. Somatotropin-treated cows entered negative energy balance by Day 3 and had higher basal plasma concentrations of GLY and NEFA than did controls by Day 2 and Day 3, respectively. Response to epinephrine, expressed as area under the response curve corrected for basal, was enhanced by bST treatment, regardless of energy balance. GLY response was greater than control by Day 1 of bST treatment (P < 0.01), and had plateaued by Day 2 (P < 0.001). The NEFA response area was higher than control and had plateaued by Day 1 of bST treatment (P < 0.001). Day 1 represented 15 hr after the first bST injection. Results illustrate that bST treatment results in enhanced in vivo lipolytic response to catecholamine challenge, and the metabolic adaptation is in place by 15 hr after the first bST injection.     

Plasma leptin and mRNA expression of lipogenesis and lipolysis-related factors in bovine adipose tissue around parturition

The objective was to study changes in plasma leptin concentration parallel to changes in the gene expression of lipogenic- and lipolytic-related genes in adipose tissue of dairy cows around parturition. Subcutaneous fat biopsies were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. Blood samples were assayed for concentrations of leptin and non-esterified fatty acids (NEFA). Subcutaneous adipose tissue was analysed for mRNA abundance by real-time qRT-PCR encoding for leptin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), hormones-sensitive lipase (HSL), perilipin (PLIN), lipoprotein lipase (LPL), acyl-CoA synthase long-chain family member 1 (ACSL1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN) and glycerol-3-phosphate dehydrogenase 2 (GPD2). Body weight and body condition score of the cows were lower after parturition than before parturition. The calculated energy balance was negative in week 1 and 5 p.p., with higher negative energy balance in week 1 p.p. compared with that in week 5 p.p. On day 1 p.p., highest concentrations of NEFA (353.3 μmol/l) were detected compared with the other biopsy time-points (210.6 and 107.7 μmol/l, in week 8 a.p., and week 5 p.p. respectively). Reduced plasma concentrations of leptin during p.p. when compared with a.p. would favour increasing metabolic efficiency and energy conservation for mammary function and reconstitution of body reserves. Lower mRNA abundance of ACC and FASN expression on day 1 p.p. compared with other biopsy time-points suggests an attenuation of fatty acid synthesis in subcutaneous adipose tissue shortly after parturition. Gene expression of AdipoR1, AdipoR2, HSL, PLIN, LPL, ACSL1 and GPD2 was unchanged over time.     

Intercellular signaling between adipose tissue and muscle tissue

Adipose and muscle tissues undergo regulated growth and differentiation processes that are modulated by a wide range of factors. The interactions between myogenic cells and adipocytes play a significant role in growth and development, including the rate and extent of myogenesis, muscle growth, adipogenesis, lipogenesis/lipolysis, and in the utilization of energy substrates. Important hormones and growth factors involved in the regulation of these processes include glucocorticoids, insulin-like growth factors, various cytokines, insulin, and leptin. Interactions among these axes have important implications in their influence on relative fat and lean deposition and the efficiency of energy utilization in growth and development. As research progresses to better clarify the interactions among adipose tissue depots and muscle of different fiber types, pathways will become better understood, ultimately leading to the optimized management of fat and lean growth in domestic livestock species. This review will focus on elements of intercellular signaling, using data from cell culture studies to illustrate specific examples of signaling pathways between cells.     

Leptin in ruminants. Gene expression in adipose tissue and mammary gland, and regulation of plasma concentration

This paper reviews data on leptin gene expression in adipose tissue (AT) and mammary gland of adult ruminants, as well as on plasma leptin variations, according to genetic, physiological, nutritional and environmental factors. AT leptin mRNA level was higher in sheep and goat subcutaneous than visceral tissues, and the opposite was observed in cattle; it was higher in fat than in lean selection line in sheep; it was decreased by undernutrition and increased by refeeding in cattle and sheep, and not changed by adding soybeans to the diet of lactating goats; it was increased by injection of NPY to sheep, and by GH treatment of growing sheep and cattle. Insulin and glucocorticoids in vitro increased AT leptin mRNA in cattle, and leptin production in sheep. Long daylength increased AT lipogenic activities and leptin mRNA, as well as plasma leptin in sheep. Mammary tissue leptin mRNA level was high during early pregnancy and was lower but still expressed during late pregnancy and lactation in sheep. Leptin was present in sheep mammary adipocytes, epithelial and myoepithelial cells during early pregnancy, late pregnancy and lactation, respectively. Plasma leptin in cattle and sheep was first studied thanks to a commercial “multi-species” kit. It was positively related to body fatness and energy balance or feeding level, and decreased by β-agonist injection. The recent development of specific RIA for ruminant leptin enabled more quantitative study of changes in plasma leptin concentration, which were explained for 35–50% by body fatness and for 15–20% by feeding level. The response of plasma leptin to meal intake was related positively to glycemia, and negatively to plasma 3-hydroxybutyrate. The putative physiological roles of changes in leptin gene expression are discussed in relation with published data on leptin receptors in several body tissues, and on in vivo or in vitro effects of leptin treatment.     

Growth and carcass quality of offspring in response to porcine somatotropin (pST) treatment of sows during early pregnancy

The effects of maternal treatment with porcine somatotropin (pST) during early gestation on offspring growth, carcass quality, and immunological characteristics were determined. Thirty-two sows received daily injections of either a placebo (n=16) or 6 mg of pST (n=16) from day 10 to 27 of gestation with gradual withdrawal until day 37. In neonatal piglets, a birth weight group (BWG) by treatment interaction (P<0.01) revealed decreases in the 25% heavy and 50% middle weight groups and an increase in the 25% low weight group within litters. Similar interactions were observed for muscle tissue (P=0.03) and, in opposite direction, for fat (P=0.05) percentages. The average percentage of muscle tissue was reduced (P=0.03) by pST treatment, whereas percentages of internal organs (P=0.05) and skin (P=0.04) were enhanced. The susceptibility of piglets to infections at 2 days after weaning (day 30 of age) was not altered by pST treatment. Carcasses of slaughter pigs (day 182 of age) from pST-treated sows tended to deposit less meat than controls (P=0.10) and to exhibit the same BWG by treatment interaction found at birth (P=0.08). Meat quality at slaughter was changed towards higher intramuscular fat content (P=0.01) and drip loss (P=0.01). The results suggest that pST treatment during early gestation results in more balanced litters, but, on average, is not of advantage for carcass quality.