Adipose triglyceride lipase protein abundance and translocation to the lipid droplet increase during leptin-induced lipolysis in bovine adipocytes.

Proper regulation of lipid metabolism is critical for preventing the development of metabolic diseases. It is clear that leptin plays a critical role in the regulation of energy homeostasis by regulating energy intake. However, leptin can also regulate energy homeostasis by inducing lipolysis in adipocytes, but it is unclear how the major lipases are involved in leptin-stimulated lipolysis. Therefore, the objectives of this study were to determine if (1) leptin acts directly to induce lipolysis in bovine adipocytes, (2) the potential lipases involved in leptin-induced lipolysis in bovine adipocytes, and (3) increases translocation of adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) during leptin-stimulated lipolysis in bovine stromal vascular cell-derived adipocytes. As hypothesized, leptin induced a lipolytic response (P = 0.02) in isolated adipocytes which was accompanied by an increase in phosphorylation of signal transducer and activator of transcription (STAT)3 (P = 0.03), a well-documented secondary messenger of leptin, and ATGL protein abundance (P < 0.01). Protein abundance of STAT3, perilipin, HSL, and phosphorylation of HSL by PKA and AMPK were not altered during leptin-stimulated lipolysis (P > 0.05). Immunostaining techniques were employed to determine the location of HSL and ATGL. Both lipases translocated to the lipid droplet after 2 h of exposure to isoproterenol (P < 0.02). However, only ATGL was translocated to the lipid droplet during leptin-stimulated lipolysis (P = 0.04), indicating ATGL may be the active lipase in leptin-stimulated lipolysis. In summary, leptin stimulates lipolysis in bovine adipocytes. The lack of phosphorylated HSL and translocation of HSL to the lipid droplet during leptin-stimulated lipolysis suggest minimal activity by PKA. Interestingly, leptin-stimulated lipolysis is accompanied by an increase in ATGL protein abundance and translocation to the lipid droplet, indicating its involvement in leptin-stimulated lipolysis either due to an increase in protein abundance or through a novel lipolytic cascade.

Effects of ad libitum and restricted feed intake on growth performance and body composition of Yorkshire pigs selected for reduced residual feed intake.

Residual feed intake (RFI), defined as the difference in the observed and expected feed intake while accounting for growth and backfat, has gained much attention, but little is known about why pigs selected for reduced RFI are more efficient. To this end, a line of Yorkshire pigs selected for reduced RFI was developed. The objective of this study was to evaluate the 5th generation of this select line against a randomly selected control line for performance, carcass and chemical carcass composition, and overall efficiency toward the later part of the growth phase. Eighty barrows, 40 from each line, were paired by age (~132 d, P < 0.60) and BW (74.8 ± 9.9 kg, P < 0.49) and randomly assigned to 1 of 4 feeding treatments in 10 replicates: 1) ad libitum, 2) 75% of ad libitum, 55% of ad libitum, and BW stasis, with weekly adjustments in intake to keep BW constant for each pig. Pigs were individually penned (group housing was used for selection) and on treatment for 6 wk. Initial BW did not differ between the lines (P < 0.49). The ad libitum select pigs consumed 10% less feed (P < 0.09) than the ad libitum control with no significant difference in BW (P < 0.80) and slight differences in carcass fat composition (P < 0.20) and backfat (P < 0.11), which resulted in significantly less carcass energy (P < 0.03). Under restricted feeding, the select line had an increase in BW (P = 0.10) while consuming the same ration of feed as the control line with no significant difference in chemical carcass composition and lighter visceral weights, which was significant for the 75% of ad libitum treatment (P < 0.01). Under BW stasis feeding the select line consumed 7.6% less feed overall (P = 0.21) and 18% less feed at the end of the 6 wk (P < 0.08), to maintain static BW with no significant difference in chemical carcass composition compared with the control line. Overall, the select line had lighter visceral weight (P < 0.02) and a greater dressing percentage (P < 0.03) compared with the control line. Using regression, the select line had reduced energy retention (P < 0.04) and feed energy utilization (P < 0.34); however, the select line appeared to have reduced maintenance requirements (P < 0.13). In conclusion, selection for reduced RFI decreases feed intake with no significant difference (P > 0.05) in growth performance, reduced backfat, increased dressing percentage, and reduced maintenance requirements. All of these traits are appealing to the producer and result in increased profits in the production setting.

Effects of ad libitum and restricted feeding on early production performance and body composition of Yorkshire pigs selected for reduced residual feed intake.

Residual feed intake (RFI), defined as the difference between observed and expected feed intake based on growth and backfat, has been used to investigate genetic variation in feed efficiency in cattle, poultry and pigs. However, little is known about the biological basis of differences in RFI in pigs. To this end, the objective of this study was to evaluate the fifth generation of a line of pigs selected for reduced RFI against a randomly selected Control line for performance, carcass and chemical carcass composition and overall efficiency. Here, emphasis was on the early grower phase. A total of 100 barrows, 50 from each line, were paired by age and weight (22.6 ± 3.9 kg) and randomly assigned to one of four feeding treatments in 11 replicates: ad libitum (Ad), 75% of Ad (Ad75), 55% of Ad (Ad55) and weight stasis (WS), which involved weekly adjustments in intake to keep body weight (BW) constant for each pig. Pigs were individually penned (group housing was used for selection) and were on treatment for 6 weeks. Initial BW did not significantly differ between the lines (P > 0.17). Under Ad feeding, the low RFI pigs consumed 8% less feed compared with Control line pigs (P < 0.06), had less carcass fat (P < 0.05), but with no significant difference in growth rate (P > 0.85). Under restricted feeding, low RFI pigs under the Ad75 treatment had a greater rate of gain while consuming the same amount of feed as Control pigs. Despite the greater gain, no significant line differences in carcass composition or carcass traits were observed. For the WS treatment, low RFI pigs had similar BW (P > 0.37) with no significant difference in feed consumption (P > 0.32). Overall, selection for reduced RFI has decreased feed intake, with limited differences in growth rate but reduced carcass fat, as seen under Ad feeding. Collectively, results indicate that the effects of selection for low RFI are evident during the early grower stage, which allows for greater savings to the producer.

Chronic activation of 5'-AMP-activated protein kinase changes myosin heavy chain expression in growing pigs.

The purpose of this study was to determine the effect of 5'-AMP-activated protein kinase (AMPK) on energy metabolism and myosin heavy chain (MyHC) isoform expression in growing pigs using chronic treatment with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) as a model. Four-week-old pigs were given daily injections of AICAR or 0.9% saline for 10 d. Treatment with AICAR increased (P < 0.05) AMPK activity in semitendinosus muscles (STM). Expression of skeletal muscle specific glucose transporter 4 (GLUT4) was also enhanced (P < 0.05) by AICAR treatment. Using real-time PCR, electrophoresis, and Western blot analyses, we confirmed that AICAR treatment caused a decrease (P < 0.05) in type IIa MyHC isoform mRNA and protein levels and a concomitant increase (P < 0.05) in type IIx MyHC containing fibers. Consistent with a MyHC isoform shift from IIa to IIx, muscles from pigs treated with AICAR had greater (P < 0.05) lactate dehydrogenase (LDH) activity. Moreover, muscle of treated pigs expressed greater (P < 0.05) message for LDH. Administration of AICAR, however, did not alter expression of PPAR-gamma coactivator-1alpha, fatty acid translocase, citrate synthase, or the activity of cytochrome c oxidase. Overall, these results indicate that activation of AMPK by AICAR causes muscle to assume a faster-contracting, more glycolytic nature. These data are in direct contrast to documented effects in rodent models, but these effects may be dependent on the time of administration and the overall growth status of the animal.

The c-Jun N-terminal kinase mediates the induction of oxidative stress and insulin resistance by palmitate and toll-like receptor 2 and 4 ligands in 3T3-L1 adipocytes.

Saturated fatty acids (SFAs) are known to induce inflammation and insulin resistance in adipocytes through toll-like receptor-4 (Tlr4) signaling, but the mechanisms are not well delineated. Furthermore, the potential roles of Tlr2 and the c-Jun N-terminal kinase (JNK) in inflammation in adipocytes have not been investigated. We demonstrated that palmitate, lipopolysaccharide (LPS), and the toll-like receptor-2 (Tlr2) agonist, zymosan A (ZymA), induced insulin resistance in a time- and dose-dependent manner in 3T3-L1 adipocytes. Corresponding with the reduction of insulin sensitivity was an increased expression of IL-6, as well as activation of the proinflammatory transcription factors, nuclear factor kappa B, and activator protein-1. Reactive oxygen species (ROS) accumulation was also observed in palmitate and Tlr agonist treated adipocytes. The JNK inhibitor, SP600125, attenuated insulin resistance mediated by SFA and Tlr agonists, which corresponded with a diminished proinflammatory response and reduced ROS accumulation. Collectively, these results demonstrated Tlr2 involvement in adipocyte inflammation and therefore implicated the receptor as a potential target for SFA. Moreover, activation of JNK also appeared to be essential to Tlr2-, as well as Tlr4-induced insulin resistance and oxidative stress.

Regulation of hepatic peroxisome proliferator-activated receptor alpha expression but not adiponectin by dietary protein in finishing pigs.

Soy protein regulates adiponectin and peroxisome proliferator-activated receptor alpha (PPARalpha) in some species, but the effect of dietary soy protein on adiponectin and PPARalpha in the pig has not been studied. Therefore, the objective of this study was to determine whether soya bean meal reduction or replacement influences serum adiponectin, adiponectin mRNA, serum metabolites and the expression of PPARalpha and other genes involved in lipid deposition. Thirty-three pigs (11 pigs per treatment) were subjected to one of three dietary treatments: (i) reduced crude protein (CP) diet containing soya bean meal (RCP-Soy), (ii) high CP diet containing soya bean meal (HCP-Soy) or (iii) high CP diet with corn gluten meal replacing soya bean meal (HCP-CGM) for 35 days. Dietary treatment had no effect on overall growth performance, feed intake or measures of body composition. There was no effect of dietary treatment on serum adiponectin or leptin. Dietary treatment did not affect the abundance of the mRNAs for adiponectin, PPARalpha, PPARgamma2, lipoprotein lipase or fatty acid synthase in adipose tissue. The mRNA expression of PPARalpha, PPARgamma2, lipoprotein lipase or fatty acid synthetase in loin muscle was not affected by dietary treatment. In liver tissue, the relative abundance of PPARalpha mRNA was greater (p < 0.05) in pigs fed the HCP-Soy diets when compared to pigs fed RCP-Soy or HCP-CGM diets. Hepatic mRNA expression of acyl-CoA oxidase or fatty acid synthase was not affected by dietary treatment. Western blot analysis indicated that hepatic PPARalpha protein levels were decreased (p < 0.05) in pigs fed the RCP-Soy diets when compared to pigs fed the HCP-Soy diets. These data suggest that increasing the soy protein content of swine diets increases hepatic expression of PPARalpha without associated changes in body composition.

Integrating the immune system with the regulation of growth and efficiency.

Muscle growth in meat animals is a complex process governed by integrated signals emanating from multiple endocrine and immune cells. A generalized phenomenon among meat animal industries is that animals commonly fail to meet their genetic potential for growth in commercial production settings. Recent evidence indicates that adipocytes and myofibers are equipped with functional pattern recognition receptors and are capable of responding directly to the corresponding pathogens and other receptor ligands. Thus, these cells are active participants in the innate immune response and, as such, produce a number of immune and metabolic regulators, including proinflammatory cytokines and adiponectin. Specifically, the transcription factor, nuclear factor kappa B, is activated in adipocytes and muscle cells by bacterial lipopolysaccharide and certain saturated fatty acids, which are potent agonists for the Toll-like receptor-4 pattern recognition receptor. Receptor activation results in the local production of interleukin-6 and tumor necrosis factor-alpha, and creates a local environment by which these cytokines regulate both metabolic and immunological pathways. However, adipocytes are also the predominant source of the antiinflammatory hormone, adiponectin, which suppresses the activation of nuclear factor kappa B and the production of proinflammatory cytokines. The molecular ability to recognize antigens and produce regulatory molecules strategically positions adipocytes and myofibers to regulate growth locally and to reciprocally regulate metabolism in peripheral tissues.

Adipocytes, myofibers, and cytokine biology: new horizons in the regulation of growth and body composition.

Muscle growth in meat animals is a complex process governed by integrated signals emanating from multiple endocrine and immune cells. A generalized phenomenon among meat animal industries is that animals commonly fail to meet their genetic potential for growth in commercial production settings. Therefore, understanding the impact of stress and disease on muscle growth is essential to improving production efficiency. The adipocyte in particular seems to be well positioned as an interface between energy status and immune function, and may thus influence nutrient partitioning and growth through a combination of signals that influence fat metabolism, glucose uptake, and insulin sensitivity. Adipocytes and myofibers are active participants in the innate immune response, and as such, produce a number of metabolic regulators, including leptin, adiponectin, and proinflammatory cytokines. Specifically, adipocytes and muscle cells respond directly to bacterial lipopolysaccharide (LPS) by producing interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFalpha). However, adipocytes are also the predominant source of the antiinflammatory hormone adiponectin, which regulates the nuclear factor kappa-B transcription factor. The ability to recognize antigens and produce regulatory molecules strategically positions adipocytes and myofibers to regulate growth locally, and to reciprocally regulate metabolism peripherally.

Cloning and expression of porcine adiponectin, and its relationship to adiposity, lipogenesis and the acute phase response.

Adiponectin is an adipocyte-derived hormone that has been implicated recently in the regulation of inflammation in immunocytes, and in lipid metabolism and glucose homeostasis in liver, skeletal muscle and adipocytes. However, information in non-rodent models is limited. We have cloned and sequenced the porcine adiponectin open reading frame and evaluated the regulation of adiponectin in vivo following lipopolysaccharide (LPS) or E. coli administration. The porcine sequence shares approximately 88, 86, 85 and 83% homology with the dog, human, cow and mouse adiponectin respectively, and 79-83% similarity with dog, human, cow and mouse proteins at the amino acid level, based on the translated porcine sequence and GenBank submissions for the other species. Relative serum adiponectin concentrations were not altered in pigs infused with E. coli, and mRNA expression in adipose tissue was not responsive to LPS. However, analysis of serum from very lean vs a substantially fatter genotype of pig indicated that relative circulating adiponectin concentrations are higher (P<0.01) in the lean pigs than in the fatter genotype, and that the difference is established relatively early in the growth curve. Also, incubating pig adipocytes for 6 h with recombinant pig adiponectin resulted in an approximately 30% reduction (P<0.05) in lipogenesis compared with adipocytes under basal conditions and with those incubated in the presence of insulin. This is the first report in any species that adiponectin antagonizes the incorporation of glucose carbon into lipid in the adipocyte, and provides additional evidence that adiponectin acts as an autocrine regulatory factor to regulate energy metabolism.

Leptin alters antibody isotype in the pig in vivo, but does not regulate cytokine expression or stimulate STAT3 signaling in peripheral blood monocytes in vitro.

Although leptin modulates immunological pathways in some species, the role of leptin as a regulator of immunocyte function in the pig has not been studied. Therefore, the primary objective of this study was to determine whether leptin influences specific immunocyte response variables in the pig in vivo or in vitro. Fifteen pigs (five pigs per treatment) were 1) injected with recombinant human leptin and allowed to consume feed ad libitum, 2) injected with vehicle and allowed to consume feed ad libitum, or 3) injected with vehicle and limit-fed to the intake of the leptin-injected group. All the pigs also were injected with the antigen, Limulus hemocyanin, on d 0 and 15 of the experiment. Exogenous leptin decreased (P < 0.05) daily feed intake and antigen-specific immunoglobulin (Ig) G1, but had no effect on lymphocyte proliferation or antigen-specific IgG2. In a second series of experiments, peripheral blood mononuclear cells (PBMC) were isolated from venous blood to determine the effect of stimulation with the polyclonal mitogen, concanavalin A (ConA), on the long form of the leptin receptor (Ob-Rl) mRNA abundance, and to determine whether leptin altered mitogen-induced proliferation, cytokine production, or signal transducer and activator of transcription 3 (STAT3) activation. Leptin had no effect on the proliferation of PBMC or on cytokine mRNA abundance or secretion. The abundance of Ob-Rl mRNA was decreased (P < 0.05) in response to stimulation with ConA. Constitutive STAT3 DNA binding was evident in mobility shift assays, but was not altered by either leptin or serum deprivation. These data indicate that leptin modifies antibody isotypes in the pig, and that Ob-Rl expression is downregulated in response to polyclonal mitogens in porcine PBMC. The constitutive activation of STAT3, coupled with the absence of leptin-inducible binding, indicates an alternative signaling pathway for leptin in pig PBMC.

Beta-adrenergic receptor subtypes that mediate ractopamine stimulation of lipolysis.

Ractopamine HCl is an beta-adrenergic receptor (betaAR) ligand that was recently approved for use in swine to enhance carcass leanness. The RR stereoisomer of ractopamine is the most active of the four stereoisomers exhibiting the highest affinity and signaling response. The RR isomer exhibits selective activation of the porcine beta2AR, which might limit the lipolytic response to ractopamine because the betaAR is the predominant subtype in swine adipocytes and may mediate most of the lipolytic response. Therefore, we determined the betaAR subtypes that mediate the lipolytic response to ractopamine in swine adipocytes. In order to confirm the predominant role of the beta1AR in porcine adipocytes, isoproterenol-stimulated lipolysis was inhibited by increasing doses of subtype-selective antagonists. Inhibition curves were biphasic using beta1AR antagonists (CGP 20712A and bisoprolol) and curve analysis indicated that both beta1AR an beta2AR contributed to lipolysis with 50 to 60% of the response coming from the beta1AR. Inhibition with the beta2AR antagonist clenbuterol revealed only one class of betaAR that closely approximated the kinetics of the beta1AR. When the RR isomer of ractopamine was the lipolytic agent, similar results to isoproterenol were observed, except that the estimated contribution of the beta1AR was 38%. That beta2AR antagonists did not detect a contribution of the beta2AR to lipolysis may indicate that the beta1AR masked the response to the beta2AR. Dose titration with the RR isomer in the presence of a saturating concentration of beta1AR or beta2AR antagonists indicated that each subtype was present in sufficient quantities to stimulate lipolysis near maximally. Data indicate that both the beta1AR and beta2AR are functionally linked to lipolysis in swine adipocytes and that ractopamine activates each subtype. The RR isomer of ractopamine stimulated adenosine 3',5'-cyclic phosphate accumulation with equal efficacy to isoproterenol through the cloned porcine beta2AR, but was only 35% as efficacious through the cloned porcine beta1AR. These data confirm the beta2AR selectivity of the RR stereoisomer, but suggest the partial agonism through the beta1AR is sufficient to activate lipolysis through both subtypes in swine adipocytes.

Nutritionally induced adipose hypertrophy in young pigs is transient and independent of changes in the expression of the obese and peroxisome proliferator activated receptor genes.

Previous studies have shown that piglets weaned to a liquid milk replacer (MR), rather than a typical dry diet (DD) regimen, have improved growth rates and deposit more energy as body fat. In the present study, we used this model to determine whether changes in the expression of genes linked to the regulation of adiposity were related to the accelerated fat accretion. We also determined whether the increase in body fat was sustained throughout a substantial proportion of the growth curve. At weaning (19 plus minus 2 days of age), 96 piglets were placed in 12 replicate pens per diet (4 pigs per pen, 2 barrows and 2 gilts), and fed a liquid MR or conventional DD regimen for 5 weeks. Thereafter, 6 barrows and 6 gilts pigs from each diet were killed for determination of whole body chemical composition (less gastrointestinal contents). The remaining pigs were assigned randomly to weight target groups (60, 85, and 110 kg), placed in individual pens, and fed a conventional dietary regimen until killed at their respective weight targets for tissue sampling and determination of whole body chemical composition. Over the 5-week period in which the MR was fed, the growth rate of the pigs consuming the MR exceeded that of the pigs fed the DD by 36% (P <.05). Fat gain in these pigs was increased to 1.8 times that of the pigs fed the DD, and percentage body fat was 45% greater (P <.05). Acetyl Co-A carboxylase (ACC) activity (per mg of adipose extract protein) was not different between the two diet groups at the conclusion of the 5-week period, or at 110 kg body weight. During the MR period, actual protein gain was increased (P <.05) 22% in the pigs fed the MR as well. By 110 kg of body weight, body fat was reduced (P <.05) by 7.7% (total fat mass) and 8.3% (percentage of body weight basis) in the pigs fed MR vs. the DD group. The expression of the peroxisome proliferator activated receptors (PPAR) alpha and gamma was not influenced by diet or by body weight. Expression of the obese gene was independent of diet, but was greater (P <.09) in pigs at 110 kg body weight than at 60 kg. These data provide additional evidence that piglets weaned to liquid diets have greater rates of growth and deposit more body fat, but that this difference subsides quickly when a typical dry dietary regimen is imposed. Furthermore, the biochemical changes responsible for the increased adiposity are independent of changes in the expression of the obese or PPAR genes, at least at the mRNA level.

Expression and complement d activity of porcine adipsin.

To learn how signals from adipocytes might be involved in regulation of energy intake and storage, we have begun to characterize the porcine complement protein, adipsin. Adipsin was originally identified as a protein that is produced rather specifically by adipocytes, is secreted, and is nearly absent in several obese rodent models. We now report that porcine adipsin mRNA sequence is 74% identical to rat and predicts a protein that has 82 and 68% identity to human and rat forms, respectively. Porcine adipsin has none of the asparagine glycosylation consensus sites which make recombinant expression of mouse adipsin in Escherichia coli impractical. We present a method for engineering the porcine cDNA to facilitate expression by E. coli and provide a protocol for refolding and purifying porcine adipsin protein and for immunoassay. We have found that in addition to adipose tissue, adipsin mRNA is present in gut tissues. Coupled with the fact that adipsin is required for processing of complement C3a-desArg, and that C3a-desArg is a potent stimulant of fatty acid acylation in adipocytes, the production of adipsin in the gut may be related to a mechanism for adipocyte removal of lipid from chylomicrons.

Changes in the expression of uncoupling proteins and lipases in porcine adipose tissue and skeletal muscle during feed deprivation*(1).

The hormone-sensitive and lipoprotein lipases are critical determinants of the metabolic adaptation to starvation. Additionally, the uncoupling proteins have emerged with potential roles in the metabolic adaptations required by energy deficiency. The objective of this study was to evaluate the expression (mRNA abundance) of uncoupling proteins 2 and 3 and that of hormone-sensitive and lipoprotein lipase in the adipose tissue and skeletal muscle of the pig in relationship to feed deprivation. Thirty-two male castrates (87 kg +/- 5%) were assigned at random to fed and feed-deprived treatment groups. After 96 hr, the pigs were euthanized and adipose and skeletal muscle tissue obtained for total RNA extraction and nuclease protection assays. Feed deprivation increased uncoupling protein 3 mRNA abundance 103-237% (P < 0.01) in longissimus and red and white semitendinosus muscle. In contrast, the increase in uncoupling protein 3 mRNA in adipose tissue was only 23% (P < 0.06), and adipose uncoupling protein 2 mRNA was not influenced (P > 0.66) by feed deprivation. The increased abundance of uncoupling protein 2 mRNA in the longissimus muscle of feed-deprived pigs was small (22%), but significant (P < 0.04). The expression of hormone-sensitive lipase was increased 46% and 64% (P < 0.04) in adipose tissue and longissimus muscle, respectively, by feed deprivation, whereas adipose lipoprotein lipase expression was reduced (P < 0.01) to 20% of that of the fed group. Longissimus lipoprotein lipase expression in the feed-deprived group was 37% of that of the fed group (P < 0.01), and similar reductions were detected in red and white semitendinosus muscle. Overall, these findings indicate that uncoupling protein 3 expression in skeletal muscle is quite sensitive to starvation in the pig, whereas uncoupling protein 2 changes are minimal. Furthermore, we conclude that hormone-sensitive lipase is upregulated at the mRNA level with prolonged feed deprivation, whereas lipoprotein lipase is downregulated.

Regulation of PPARgamma but not obese gene expression by dietary fat supplementation.

Leptin, the product of the obese gene, and peroxisome proliferator activated receptor gamma (PPARgamma) are important regulators of energy metabolism, adipogenesis, and immune function. In rodent models, both genes seem to respond at the mRNA and/or protein levels to dietary fat consumption. To determine the effect(s) of dietary saturated and polyunsaturated fatty acids on the expression (mRNA abundance) of these genes, adipose tissue was obtained from pigs fed three different dietary fat sources. Corn-soybean meal diets containing no added fat (NO, control) or 10% beef tallow (BT), safflower oil (SO), or fish oil (FO) were fed ad libitum (n = 12) for 12 weeks. The abundance of obese, PPARgamma1, and PPARgamma2 mRNA was quantified relative to 18S rRNA using ribonuclease protection assays. The gain:feed ratio was improved (P < 0.05) 21% by all fats with a corresponding reduction (P < 0.05) in feed intake. Relative to pigs fed NO, serum total cholesterol was increased (P < 0.01) in pigs fed BT and triglyceride and nonesterified fatty acid concentrations were increased (P < 0.01) by all supplemental fats. Serum insulin was increased (P < 0.10) only by SO. Neither obese nor PPARgamma1 mRNA abundance were responsive to added fat (P > 0.15). However, the abundance of PPARgamma2 mRNA was increased fourfold by SO compared with the NO diet. These data indicate that the abundance of obese mRNA is independent of dietary fat consumption per se, whether saturated or unsaturated, when feed consumption is reduced due to greater dietary caloric density. Furthermore, we provide evidence that expression of the PPARgamma2 gene in porcine adipose tissue is selectively responsive to SO (presumably linoleic acid, 18:2n-6).

Physiological response to acute endotoxemia in swine: effect of genotype on energy metabolites and leptin.

Certain high lean gain swine genotypes have greater sensitivity to pathogen and nonpathogen stressors evident by reduced productivity and increased mortality during disease stress or in suboptimal production environments. Saline (control) and an immunologic challenge (LPS; 25 microg lipopolysaccharide/kg BW) were administered to three genetic populations (each pig used as its own control): high lean (H), moderate lean terminal cross (MT), and moderate lean maternal cross (MM). LPS induced anorexia, and significantly increased body temperature and circulating TNF-alpha, cortisol, and NEFA in all genotypes (P < 0.0004). LPS reduced circulating glucose, insulin, and IGF-1 in all genotypes (P < 0.05). The LPS-induced hypoglycemia was significantly greater in MM versus MT and H pigs (P < 0.03). The hypoinsulinemia was significantly greater in MM versus H pigs (P < 0.02). MM pigs recovered from hypoinsulinemia slower than MT pigs (P < 0.03). Control insulin was higher in H versus MT pigs (P < 0.08), but relative to basal, the insulin response to LPS was similar. Plasma haptoglobin response to LPS was lower for MM versus MT and H pigs (P < 0.02), and tended to be lower in MT versus H pigs (P < 0.09). LPS treatment caused similar decreases in plasma IGF-1 concentrations among genotypes. Ten hours after LPS treatment, leptin mRNA abundance in adipose tissue was significantly reduced (relative to control) in MM and H pigs (P < 0.02) but not in MT pigs (P > 0.05). Physiological differences in leptin, a potent regulator of food intake and energy metabolism, may be important factors in the genetic variation in sensitivity to environmental stress.

Leptin expression is reduced with acute endotoxemia in the pig: correlation with glucose, insulin, and insulin-like growth factor-1 (IGF-1).

Leptin has been implicated in the regulation of anorexia associated with cachexia in rodents and humans. Regulation of leptin expression is under complex endocrine and metabolic control. To determine if leptin expression is regulated by acute inflammation and to define the endocrine and metabolic factor(s) that regulates leptin expression during acute inflammation, castrate male pigs (ad libitum fed, used as their own controls) were treated with saline (control period) and endotoxin (lipopolysaccharide [LPS] period). Frequent blood samples were collected to identify dynamic changes in hormones and metabolites that are known to regulate leptin expression. LPS caused fever and elevated plasma cortisol (p < 0.0004), tumor necrosis factor-alpha (TNF-alpha) (p < 0.0001), and plasma nonesterified fatty acids (NEFA) (p < 0.001) compared with control. Circulating insulin (p < 0.01), glucose (p < 0.003), and insulin-like growth factor-1 (IGF-1) (p < 0.0001), as well as adipose leptin mRNA abundance (p < 0.01), were profoundly reduced following LPS treatment compared with control. Our data indicate that during acute endotoxemia (1-10 h after injection), leptin gene expression is decreased compared with ad libitum fed animals and is more closely related to energy homeostasis than cytokine profiles in plasma.

Growth hormone regulates leptin gene expression in bovine adipose tissue: correlation with adipose IGF-1 expression.

Leptin, the product of the ob gene, is secreted from white adipocytes and regulates food intake and whole-body energy metabolism. In rodents and humans, leptin gene expression is under complex endocrine and metabolic control, and is strongly influenced by energy balance. Growth hormone (GH) has myriad effects on adipose tissue metabolism. The primary aim of this study was to determine the ability of GH to regulate leptin mRNA expression in bovine adipose tissue in vitro and in vivo. Incubation of subcutaneous adipose tissue explants for 24 h with GH alone had no effect on bovine leptin gene expression, whereas high concentrations of insulin or dexamethasone (DEX) potently stimulated bovine leptin mRNA abundance. GH, in combination with high concentrations of insulin, DEX, or both, attenuated the ability of insulin or DEX to stimulate leptin expression in vitro. These data indicate that GH can indirectly regulate leptin expression in vitro by altering the adipose tissue response to insulin or DEX. We extended these studies to examine the ability of GH to regulate leptin expression in vivo, using young castrate male cattle treated with no hormone (control) or GH (200 micrograms/kg body weight per day) for 3 days. GH increased plasma GH and insulin concentrations, but not those of cortisol or non-esterified fatty acid (NEFA) concentrations. GH treatment increased adipose tissue leptin and IGF-1 mRNA concentrations (n=9, P>0.001). In addition, leptin abundance was highly correlated with adipose tissue IGF-1 mRNA in GH-treated animals (P>0.001). The timing of GH-induced changes in leptin gene expression preceded measurable GH effects on adiposity.

Soybean isoflavones, genistein and genistin, inhibit rat myoblast proliferation, fusion and myotube protein synthesis.

The isoflavones, genistein and genistin, are cytotoxic in vitro (e.g. , inhibition of cell proliferation), due in part to inhibition of protein tyrosine kinase and DNA topoisomerase activities. Normal cell functions associated with these enzymatic activities could potentially be impaired in animals through ingestion of soybean products. In this study, cultured rat myogenic cells (L8) were used to determine whether genistein or genistin influences myoblast proliferation and fusion, and myotube protein synthesis and degradation. Genistein or genistin was dissolved in dimethylsulfoxide and included in the culture medium at 0, 1, 10 or 100 micromol/L. Myoblast proliferation was measured by methyl-3H-thymidine incorporation over 48 h. Myoblast differentiation was evaluated by the number of nuclei in multinucleated myotubes. Myotube protein synthesis was measured by 2-h 3H-amino acid incorporation into the myosin and total protein pools after acute (2 h) or chronic (24 h) exposure to similar treatments; protein degradation was measured by measuring radioactivity in protein pools following a time course of protein breakdown after myotube proteins were prelabeled with 3H-amino acids. Genistein or genistin strongly inhibited in vitro myoblast proliferation (P < 0.001) and fusion (P < 0.001) in a dose-dependent manner with effective genistein concentration as low as 1 micromol/L. Genistein or genistin inhibited protein accretion in myotubes (P < 0.001). Decreased protein accretion is largely a result of inhibition on cellular (myofibrillar) protein synthesis rate. No adverse effect on protein degradation was observed. Results suggest that if sufficient circulating concentrations are reached in tissues of animals consuming soy products, genistein/genistin can potentially affect normal muscle growth and development.

Weaning anorexia may contribute to local inflammation in the piglet small intestine.

Compromising alterations in villus-crypt structure are common in pigs postweaning. Possible contributions of local inflammatory reactions to villus-crypt alterations during the weaning transition have not been described. This study evaluated local inflammatory responses and their relationship with morphological changes in the intestine in 21-d-old pigs (n = 112) killed either at weaning (Day 0) or 0.5, 1, 2, 4 or 7 d after weaning to either milk- or soy-based pelleted diets. Cumulative intake averaged <100 g during the first 2 d postweaning, regardless of diet. During this period of weaning anorexia, inflammatory T-cell numbers and local expression of the matrix metalloproteinase stromelysin increased while jejunal villus height, crypt depth and major histocompatibility complex (MHC) class I RNA expression decreased. Upon resumption of feed intake by the fourth d postweaning, villus height and crypt depth, CD8(+) T cell numbers, MHC class I RNA expression and local expression of stromelysin returned to Day 0 values. Together the results indicate that inadequate feed intake during the immediate postweaning period may contribute to intestinal inflammation and thereby compromise villus-crypt structure and function.