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Chinese hamster ovary cell constructs expressing either the β 1-, β 2- or β 3-adrenergic receptor (AR) were used to determine whether a novel β-AR modulator, lubabegron fumarate (LUB; Experior, Elanco Animal Health) might exert greater potency for a specific β-AR subtype. EC50 values calculated based on cAMP accumulation in dose response curves indicate that LUB is highly selective for the β 3-AR subtype, with an EC50 of 6 × 10-9 M, with no detectible agonistic activity at the β 2-AR. We hypothesized that the accumulation of lipolytic markers would reflect the agonist activity at each of the β-receptor subtypes of the specific ligand; additionally, there would be differences in receptor subtype expression in subcutaneous (s.c.) and intrmuscular (i.m.) adipose tissues. Total RNA was extracted from adipose tissue samples and relative mRNA levels for β 1-, β2-, and β 3-AR were measured using real-time quantitative polymerase chain reaction. Fresh s.c. and i.m. adipose tissue explants were incubated with isoproterenol hydrochloride (ISO; β-AR pan-agonist), dobutamine hydrochloride (DOB; specific β 1-AA), salbutamol sulfate (SAL; specific β 2-AA), ractopamine hydrochloride (RAC), zilpaterol hydrochloride (ZIL), BRL-37344 (specific β 3-agonist), or LUB for 30 min following preincubation with theophylline (inhibitor of phosphodiesterase). Relative mRNA amounts for β 1-, β 2-, and β 3-AR were greater (P < 0.05) in s.c. than in i.m. adipose tissue. The most abundant β-AR mRNA in both adipose tissues was the β 2-AR (P < 0.05), with the β 1- and β 3-AR subtypes being minimally expressed in i.m. adipose tissue. ISO, RH, and ZH stimulated the release of glycerol and nonesterified fatty acid (NEFA) from s.c. adipose tissue, but these β-AR ligands did not alter concentrations of these lipolytic markers in i.m. adipose tissue. LUB did not affect glycerol or NEFA concentrations in s.c. or i.m. adipose tissue, but attenuated (P < 0.05) the accumulation of cAMP mediated by the β 1- and β 2-AR ligands DOB and SAL in s.c. adipose tissue. Collectively, these data indicate that bovine i.m. adipose tissue is less responsive than s.c. adipose tissue to β-adrenergic ligands, especially those that are agonists at the β 1- and β3-receptor subtypes. The minimal mRNA expression of the β 1- and β 3 subtypes in i.m. adipose tissue likely limits the response potential to agonists for these β-AR subtypes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science.)

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., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Fat randomization and interesterification change triacylglycerol (TAG) structure and its solid fat content profile. It has not been thoroughly investigated whether these changes affect lipid metabolism., Methods: Two experiments were conducted to investigate the effects of TAG structure and solid fat content on feed intake, body weight change, and serum metabolite concentrations in mice. An experiment used two fats rich in 1,2-dipalmitoyl-3-oleoylglycerol (PPO) and 1,3-dipalmitoyl-2-oleoylglycerol (POP) as comparative pair of fats to assess the effect of TAG structure since PPO and POP have the same fatty acid composition and solid fat content at 37 °C. Another experiment used a fat rich in 1-palmitoyl-2,3-dioleoylglycerol (POO) with solid fat content of zero at 37 °C and a mixture of fats that had the same general fatty acid composition and palmitic acid positional distribution, but with solid fat content of 22 % at 37 °C. This pair of fats was used to examine the effect of solid fat content on blood lipid profile., Results: After 6-week feeding, the pair of fats with different solid fat contents did not significantly affect the concentrations of total serum cholesterol, HDL cholesterol, TAG, non-esterified fatty acid (NEFA), or blood glucose. However, the PPO fat significantly reduced feed intake, body weight, and serum glucose concentration as compared to POP., Conclusion: These results suggest that the presence of solid fat at the level examined does not affect lipid metabolism and lipemia, but PPO diet significantly affects NEFA and glucose concentrations. Palmitic acid at the sn-2 position of the TAG may have significant effect on appetite, which may be mediated via the gut receptors.

Fatty acids have distinct cellular effects related to inflammation and insulin sensitivity. Dietary saturated fat activates toll-like receptor 4, which in turn can lead to chronic inflammation, insulin resistance, and adipose tissue macrophage infiltration. Conversely, n3 fatty acids are generally antiinflammatory and promote insulin sensitivity, in part via peroxisome proliferator-activated receptor γ. Ossabaw swine are a useful biomedical model of obesity. We fed Ossabaw pigs either a low-fat control diet or a diet containing high-fat palm oil with or without additional n3 fatty acids for 30 wk to investigate the effect of saturated fats and n3 fatty acids on obesity-linked inflammatory markers. The diet did not influence the inflammatory markers C-reactive protein, TNFα, IL6, or IL12. In addition, n3 fatty acids attenuated the increase in inflammatory adipose tissue CD16(-)CD14(+) macrophages induced by high palm oil. High-fat diets with and without n3 fatty acids both induced hyperglycemia without hyperinsulinemia. The high-fat only group but not the high-fat group with n3 fatty acids showed reduced insulin sensitivity in response to insulin challenge. This effect was not mediated by decreased phosphorylation of protein kinase B. Therefore, in obese Ossabaw swine, n3 fatty acids partially attenuate insulin resistance but only marginally change inflammatory status and macrophage phenotype in adipose tissue.

Adiponectin is an antiinflammatory and insulin-sensitizing hormone that is decreased in obesity. Although controversial, it has been suggested that decreased adiponectin contributes to colorectal cancer risk in obesity. To further investigate the role of adiponectin in obesity-linked colorectal carcinogenesis, we used male and female adiponectin knockout (KO) and wild-type (Wt) C57BL/6J mice. Tumorigenesis was induced in all mice with the combined treatment of azoxymethane (AOM) and dextran sodium sulfate (DSS). Following AOM/DSS treatment, mice were fed a low-fat control (LFC), or high-fat lard (HFL) diet for 7 1/2 wk. KO mice developed fewer total lesions than Wt mice, males developed fewer lesions than females, and mice fed the HFL diet developed fewer lesions than those fed the LFC diet. Early lesion multiplicity was influenced by genotype, whereas advanced lesion development was influenced by sex and diet. Moreover, lesion types were differentially correlated with serum adipokines and colon gene expression of adiponectin receptors, insulin receptor, and toll-like receptor 4. These data suggest that in the AOM/DSS model of carcinogenesis, adiponectin functions to promote early lesion development whereas sex and diet are important regulators of advanced lesion development through pathways involved in inflammation and insulin signaling.

Obesity is associated with a decrease in the antiinflammatory hormone, adiponectin, and increases in the circulating concentrations of multiple proinflammatory cytokines. These changes contribute to colon tumorigenesis. Resveratrol increases adiponectin production in adipocytes and attenuates the development of colon cancer. Thus, we hypothesized that adiponectin is an integral component of the mechanism by which resveratrol antagonizes colorectal tumorigenesis. To investigate this, we induced tumorigenesis in adiponectin knockout (KO) and wild-type (Wt) C57BL/6 mice through combined azoxymethane and dextran sodium sulfate treatment during which mice were fed a high-fat, lard-based diet, or the same diet containing 20 mg/kg resveratrol. After 14 wk on diet, Wt mice gained more weight and, on a percentage basis, had higher fat mass and lower lean mass than KO mice. Resveratrol tended to attenuate this response in male Wt mice. Resveratrol also tended to reduce aberrant crypt foci development and decrease circulating interleukin 6 and insulin concentrations in male but not female Wt mice. Taken together, resveratrol improved overall health of obese Wt but not KO mice as hypothesized with a differential sex response.

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.

The objective of the present study was to determine whether a mitochondria-targeted vitamin E derivative (MitoVit E) would affect certain mitochondrial parameters, as well as systemic oxidative stress. A total of sixty-four mice were fed a high-fat (HF) diet for 5 weeks. They were then switched to either a low-fat (LF) or a medium-fat (MF) diet, and administered orally with MitoVit E (40 mg MitoVit E/kg body weight) or drug vehicle (10 % (v/v) ethanol in 0·9 % (w/v) NaCl solution), every other day for 5 weeks. Mitochondrial ATP and H(2)O(2) production rates in both the liver and the gastrocnemius were not affected by MitoVit E administration in either LF or MF diet-fed mice. However, the number and average size of the subsarcolemmal mitochondria, but not the intermyofibrillar mitochondria, from the soleus muscle were significantly higher in the MF group receiving MitoVit E (MF-E) than in the MF group receiving vehicle only (MF-C). After the mice were switched from the HF diet to the four dietary treatments (LF-C, LF-E, MF-C and MF-E), the decrease in urinary isoprostane concentration was significantly greater in the LF-E group than in the other three groups during the whole study (weeks 6-10). In addition, MitoVit E significantly increased plasma superoxide dismutase (SOD) activity in the MF diet-fed group without affecting plasma glutathione peroxidase activity or H(2)O(2) levels. Overall, these data suggest that MitoVit E affects subsarcolemmal mitochondrial density and systemic oxidative stress parameters such as plasma SOD activity and urinary isoprostane concentration.

Reperfusion injury (RI) remains an important limitation of myocardial revascularization. The aim of the present study was to evaluate the influence of the intracoronary injection of adiponectin on RI and cardiomyocyte death in a porcine myocardial infarction model. Acute infarction in 14 Polish domestic pigs was induced by inflation of an over the wire balloon (OTW) catheter in the medial left anterior descending artery for 60 min. The study group consisted of 7 pigs in which intracoronary adiponectin (50 µg) was infused through the OTW catheter immediately before reperfusion. The control group (n=7) was administered placebo. Animals were sacrificed after two days of follow-up. The infarct area (IA) was stained with tetrazoline and the area at risk (AAR) with intracoronary administration of Evans Blue dye before euthanasia. Hearts in each group had similar AARs (46.2±9.9% vs. 48.4±6.2% of the whole myocardium, p=ns). The IA/AAR% and IA were smaller in the study group when compared to the control (24.7±4.0% vs. 45.3±22.5%, p=0.005; and 11.7±4.9% vs. 20.5±5.6%, p=0.01, respectively). These outcomes corresponded well with the peak troponin levels after 12 h (109.9±60.9 ng/ml vs. 185.5±39.4 ng/ml, p=0.017). After two days there was a significantly higher LVEF in the study group (51.4±8.5% vs. 33.9±8.6%, p=0.002). There was also a trend toward lower apoptosis enhancement in the viable myocardium in the study group (3.11±2.3 vs. 8.92±6.3; p=0.07). The administration of adiponectin into the infarct- related artery is safe and feasible. The treatment significantly reduced the infarct size.

We have previously shown that toll-like receptor-4 (Tlr4) is involved in obesity-induced inflammation in adipose tissue (AT). However, less is known about the role of Tlr2 in this process. To determine the involvement of this receptor in obesity-induced inflammation, we utilized male Tlr2(-/-) mice that were backcrossed onto a mouse model of diet-induced obesity (DIO). Mice were fed either low-fat control (LFD) or high-fat diet (HFD) ad libitum for 16 weeks. Despite negligible differences in body weight or energy intake, Tlr2(-/-) mice were protected from HFD-induced adiposity as was evident by reduced epididymal fat pad weight and carcass lipid content. Corresponding with these effects was a blunted accumulation of F4/80-positive macrophages in AT of Tlr2(-/-) mice. Furthermore, transcript abundance of proinflammatory mediators, including monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNFα) and nitric oxide synthase-2 (NOS2) in AT of Tlr2(-/-) mice, was lower or less responsive to DIO. There were no significant differences in serum markers of insulin sensitivity (data not shown). However, adipocytes derived from stromal vascular cells (SVCs) isolated from AT of Tlr2(-/-) mice had considerably greater basal and insulin-stimulated glucose uptake as compared with those obtained from Tlr2(+/+) mice. Furthermore, the absence of Tlr2(-/-) precluded the induction of insulin resistance by zymosan A (ZymA) but not by palmitate. These data indicate that Tlr2 may be directly involved in HFD-induced inflammation and may also regulate basal and insulin-stimulated glucose uptake in adipocytes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

Our objective was to determine sensitivity of myostatin null (MN) mice to obesity induction by dietary or genetic means. To induce dietary obesity, 3-week-old wild type (WT) and MN mice were fed diets with 60% calories (HF) or 10% calories from fat (LF) for 4 weeks. MN mice did gain body fat on the HF diet but to a lesser extent than WT mice. Body weight and fat content was similar in MN mice fed HF and LF diets. To induce genetic obesity, the MN mutation was incorporated into leptin db/db (DB) mice generating mice homozygous for each mutation (MNDB). Nine-week-old MNDB mice were obese, similar to DB mice. Body weight, body fat content, fat pad weight and adipocyte size were all increased in MNDB mice compared to MN and WT mice and were quite similar to DB mice. However, fasting blood glucose, an indicator of insulin resistance and diabetes, was reduced in MNDB mice compared to DB mice. These results indicate that MN mice gain less body fat than WT on a HF diet, but the MN mutation does not alter fat accumulation caused by DB mutation. Thus, MN mice are not always resistant to obesity development., (© 2010 The Authors; Journal compilation © 2010 Japanese Society of Animal Science.)

Our objective in this study was to determine whether a mitochondria-targeted vitamin E derivative (MitoVit E) would decrease oxidative stress and associated obesity by preventing a previously proposed aconitase inhibition cascade. Sixty-four mice were fed a high-fat (HF) diet for 5 wk. They were then switched to either a low-fat (LF) or a medium-fat (MF) diet and gavaged with MitoVit E (40 mg MitoVit E x kg body weight(-1)) or drug vehicle (10% ethanol in 0.9% NaCl solution) every other day for 5 wk. Epididymal fat weight, as well as liver lipid and remaining carcass lipid, were significantly lower in the MF group receiving MitoVit E (MF-E) than in the MF group receiving vehicle only (MF-C). Liver mitochondrial H(2)O(2) production and the protein carbonyl level were also significantly lower in MF-E than in MF-C mice. In contrast, none of the biochemical variables (aconitase activity, ATP and H(2)O(2) production, and protein carbonyl level) in the muscle mitochondria were modified by MitoVit E in either MF or LF groups. Expression of acetyl-CoA carboxylase and fatty acid synthase in both liver and adipose tissue of MF groups was not affected by MitoVit E. However, expression of carnitine palmitoyltransferase 1a in the liver and uncoupling protein 2 in adipose tissue were significantly enhanced by MitoVit E in both LF and MF groups. In conclusion, MitoVit E attenuates hepatic oxidative stress and inhibits fat deposition in mice but not through alleviation of the aconitase inhibition cascade.

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.

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.

The current study utilized Ussing chambers to examine the impact of supplementing maternal gestation and/or lactation diets with n-3 polyunsaturated fatty acids (PUFA) provided via a protected fish oil (PFO) product on intestinal fatty acid profiles and ex vivo glucose uptake in the jejunum of weanling piglets. Jejunum tissues were enriched with n-3 PUFA as a result of feeding the sows the PFO during gestation and/or lactation (P<.05). Glucose uptake improved by twofold (P<.042) in intestinal preparations obtained from the offspring of sows fed PFO during gestation or throughout gestation/lactation versus lactation alone. This was also reflected in the jejunum protein expressions of glucose transporter 2 (GLUT2) and sodium-dependent glucose transporter 1 (SGLT1). Furthermore, adding docosahexaenoic acid (DHA) or an AMP-activated protein kinase (AMPK) agonist to the chamber buffer improved glucose uptake (P<.05) in intestinal preparations obtained from the offspring fed the control diet, devoid of the PFO product and containing minimal concentrations of n-3 PUFA. Collectively, these data indicate two important points. First, long-term exposure to n-3 PUFA via the maternal gestation diet effectively enhances glucose uptake in the weanling piglet, and the underlying mechanism may be associated with changes in the intestinal fatty acid profile. Secondly, there is an apparent direct and acute effect of DHA that is achieved within a time frame that precludes substantial changes in the intestinal fatty acid profile. Additionally, both mechanisms may involve activation of AMPK. Thus, n-3 PUFA delivered in utero and postnatally via the maternal diet may help the offspring adapt quickly to rapidly changing diets early in life and allow optimal nutrient uptake.

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.

Toll-like receptor-4 (Tlr-4), a key pattern recognition receptor involved in innate immune response, is activated by saturated fatty acids (SFAs). To investigate the involvement of this receptor in obesity caused by consumption of diets high in fat, we utilized male Tlr-4-deficient 10ScN mice and 10J controls. Mice were fed either low fat (low-fat control (LFC)), high unsaturated fat (high-fat control (HFC)), or high saturated fat + palmitate (HFP) diets ad libitum for 16 weeks. Relative to the LFC diet, the HFC diet resulted in greater epididymal fat pad weights and adipocyte hypertrophy in both Tlr-4-deficient and normal mice. However, the 10ScN mice were completely protected against the obesigenic effects of the HFP diet. Moreover, macrophage infiltration and monocyte chemotactic protein-1 (MCP-1) transcript abundance were lower in adipose tissue of 10ScN mice fed the HFP diet, and the hyperinsulinemic response was negated. Tlr-4-deficient mice also had markedly lower circulating concentrations of MCP-1 and much less nuclear factor-kappaB (NFkappaB) protein in nuclear extracts prepared from adipose tissue, irrespective of diet. In contrast, Tlr-4 deficiency did not attenuate the induction of tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6) expression in adipose tissue. These data indicate that Tlr-4 deficiency selectively protects against the obesigenic effects of SFA and alters obesity-related inflammatory responses in adipose tissue.

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.

Despite aggressive research aimed at understanding the myriad biochemical factors that are integrated to balance energy intake and expenditure to maintain normal body weight, obesity is increasing at an alarming rate, and the long-term success of prevention and intervention strategies is minimal. Because much of the scientific literature addressing obesity has originated with rodent models, there is considerable interest among researchers and funding agencies in the development of comparative animal models. Furthermore, numerous disparate results between rodent models and humans (i.e., adipsin, leptin, resistin, tumor necrosis factor-alpha, and other adipokines) have hindered the translation of rodent data into actionable technologies for humans. The pig is an exceptional restenosis model, and is emerging rapidly as a biomedical model for energy metabolism and obesity in humans because it is devoid of brown fat postnatally and because of their similar metabolic features, cardiovascular systems, and proportional organ sizes. This article highlights the current literature devoted to the development of porcine models for obesity and the metabolic syndrome, with a particular emphasis on the role of adipose tissue and adipokines in the regulation of energy balance and the inflammation associated with obesity.

The objective of this study was to test the hypothesis that the inflammatory response to lipopolysaccharide (LPS) in vivo is accompanied by down-regulation of toll-like receptor (TLR) 4 in adipose tissue, and a source of protected n-3 polyunsaturated fatty acid (PUFA) attenuates this response. Seventy-two castrated male pigs were individually fed either a control (CONT) diet, or the CONT diet containing 1.87% (LF) or 7.50% (HF) protected n-3 PUFA on a weight basis for 7 weeks. Adipose and muscle tissue biopsy samples were taken at Weeks 1, 2, 3, 4 and 7 to assess gene expression and/or confirm tissue enrichment with eicosapentaenoic acid and docosahexaenoic acid and reflected the n-3 PUFA contained in the diet. The LPS challenge was performed at week 7 and consisted of sequential injections of 10 and 2.5 mug LPS per kilogram of body weight 23 h apart. The LPS challenge resulted in a marked down-regulation (P=.004) of TLR4 at the protein level in the adipose tissue of challenged vs. control pigs, but LF and HF clearly blocked this response at the mRNA level. Although LF and HF also attenuated (P<.001) the LPS-induced acute febrile response and lowered (P<.002) serum concentrations of tumour necrosis factor alpha. Cyclooxygenase 2 and 12-lipoxygenase were readily expressed in porcine adipose tissue, but there was no effect of LF, HF or LPS on expression levels of these inflammatory mediators, or that of TNF and interleukin 6, at the conclusion of the challenge period. These findings indicate that adipose tissue responds to LPS administration in vivo by reducing TLR4 mRNA and protein abundance and that the anti-inflammatory effects of n-3 PUFA do not include down-regulation of TLR4 in adipose tissue.

The aim of this research was to determine whether feeding gestating and lactating sows (n-3) PUFA [eicosapentaenoic acid (EPA) and/or docosahexenoic acid (DHA)] or coconut fat (saturated fat) influences ex vivo glucose absorption in the proximal jejunum and glucose and glycogen concentration of liver and muscle of their offspring at weaning. Sows were fed 1 of 4 diets for 150 d, which included the entire gestation and lactation periods. The diets consisted of basal corn/soybean meal (CONT), CONT + protected EPA and DHA-rich fish oil (PFO), CONT + DHA Gold fat (DHAGF), and CONT + coconut fat (COCO). All tissues were collected from piglets (n = 4 per treatment) following a 24-h period of food deprivation, which was initiated at weaning. Proximal jejunum samples were mounted in modified Ussing chambers for transport determinations. Relative to the CONT (7 muA/cm(2)), active glucose transport was greater (P = 0.013) in piglets from sows fed the PFO (30 microA/cm(2)) and DHAGF (40 microA/cm(2)) diets, but not the COCO diet (19 microA/cm(2); pooled SEM = 5). Likewise, jejunum expression of glucose transporter 2 and sodium glucose transporter 1 protein tended (P < 0.10) to be greater in piglets from dams fed the PFO and DHAGF diets, as did AMP-activated protein kinase activity. Piglets' muscle glycogen was greater than in CONT (34 +/- 5.2 mg/g wet tissue) only in piglets from dams fed the DHAGF (46 +/- 5.2 mg/g wet tissue; P < 0.05). These results indicate that (n-3) PUFA, particularly DHA, improves intestinal glucose absorption and muscle glycogen concentrations in newly weaned pigs. These findings may also have important implications for human mothers and infants.

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.

Fatty acids and their metabolites regulate gene expression and immunological pathways. Furthermore, obese individuals frequently have increased circulating fatty acid concentrations, and localized inflammation in adipose tissue may facilitate the systemic inflammation associated with the insulin resistance of obesity. Although palmitate induces inflammation (i.e., activates proinflammatory pathways) in myotubes, the effects of fatty acids on inflammatory processes in adipocytes have not been established. Therefore, we examined the potential for palmitate, laurate, and docosahexaenoic acid (DHA) to modulate inflammation in 3T3-L1 adipocytes. Palmitate, but not DHA or laurate, induced nuclear factor kappaB (NF-kappaB)-driven luciferase activity and interleukin-6 (IL-6) expression (P < 0.05). Inhibition of fatty acyl Co-A synthase (FACS) with triacsin C suppressed palmitate-induced NF-kappaB activation (P < 0.05), but caused an additive increase in palmitate-induced IL-6 expression (P < 0.05). Disrupting mitogen-activated protein kinase/Erk kinase (MEK) and protein kinase C (PKC) activity with U0126 and Bisindolylmaleimide (Bis), respectively, suppressed palmitate-induced IL-6 expression (P < 0.05), but had no effect on NF-kappaB reporter gene activity (P > 0.05). However, the phosphoinositide-3 kinase (PI3K) inhibitor, wortmannin, alone and additively with palmitate, activated the NF-kappaB reporter gene and induced IL-6 expression (P < 0.05). Palmitate also induced the mRNA expression of tumor necrosis factor alpha (TNFalpha) (P < 0.05), but the increase in mRNA abundance was not reflected in a greater protein concentration in the media (P > 0.05). These data indicate that palmitate induces inflammation in adipocytes, and that this is not a generalized effect of all SFA. Furthermore, PI3K may act constitutively to suppress inflammation. Consequently, inhibition of this enzyme may promote and exacerbate the inflammation in adipose tissue that is associated with obesity and insulin resistance.

Obesity and insulin resistance are often associated with lower circulating adiponectin concentrations and elevated serum interleukin-6 (IL-6) and/or tumor necrosis factor-alpha (TNF-alpha). Adiponectin suppresses activation of nuclear factor-kappaB (NF-kappaB) in aortic endothelial cells and porcine macrophages. Accordingly, we hypothesized that adiponectin is an anti-inflammatory hormone and suppresses activation of NF-kappaB in adipocytes. Because peroxisome proliferator-activated receptor gamma2 (PPARgamma2) antagonizes the transcriptional activity of NF-kappaB, we determined whether adiponectin alters PPARgamma2 expression in pig adipocytes. In addition, we determined whether interferon-gamma alters the expression of PPARgamma2 in the presence or absence of adiponectin. Primary adipocytes from pig subcutaneous adipose tissue were treated with or without lipopolysaccharide (LPS; 10 microg/ml) and adiponectin (30 microg/ml), and nuclear extracts were obtained for gel shift assays to assess nuclear localization of NF-kappaB. Whereas LPS induced an increase in NF-kappaB activation, adiponectin suppressed both NF-kappaB activation and the induction of IL-6 expression by LPS (P<0.05). Similar results were obtained in 3T3-L1 adipocytes. In addition, adiponectin antagonized LPS-induced increase in TNF-alpha mRNA expression (P<0.05) and tended (P<0.065) to diminish its accumulation in the culture media in 3T3-L1 adipocytes. Adiponectin also induced an upregulation of PPARgamma2 mRNA (P<0.05). Although IFN-gamma did not reduce the basal expression of PPARgamma2, it suppressed PPARgamma2 induction by adiponectin (P<0.05). These findings indicate that adiponectin may be a local regulator of inflammation in the adipocyte and adipose tissue via its regulation of the NF-kappaB and PPARgamma2 transcription factors.

We recently provided evidence that interleukin-15 (IL-15) is expressed lowly in the pig adipocyte and that interferon-gamma (IFN-gamma) markedly increases this expression through a pathway regulated in part by protein kinase C. In the present study, we tested the hypothesis that IL-15 acts directly on the adipocyte to regulate lipid accretion by enhancing lipolysis or suppressing lipogenesis. Using recombinant porcine IL-15, we determined that this cytokine stimulates lipolysis in a dose-dependent manner (P < 0.001). Furthermore, comparative studies with other cytokines showed that IL-15 is more potent in its acute stimulation of lipolysis than either TNF-alpha, IL-6, or LPS (P < 0.001). When specific inhibitors of protein kinase A or Janus kinase are present, the lipolytic effect of IL-15 is attenuated (P < 0.01). These data indicate that, in addition to its regulation of muscle protein accretion and T-cell growth and development, IL-15 also targets the adipocyte directly to alter stimulate lipolysis. Thus, when induced by IFN-gamma or other inflammatory mediators, IL-15 may be a significant homeorhetic factor that mobilizes and directs energy away from the adipocyte to other cells during the acute phase of the inflammatory response.

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.

Adiponectin, an adipocyte-derived hormone, attenuates the production of TNFalpha by activated human macrophages. In the present study, we used porcine blood-derived macrophages to test the hypothesis that the anti-inflammatory action of adiponectin includes suppression of IL6 and an induction of IL10. Adiponectin suppressed both TNFalpha and IL6 production in macrophages activated with lipopolysaccharide (P<0.01). In contrast, adiponectin increased IL10 expression (P<0.05) and augmented (P<0.05) the induction of this cytokine by lipopolysaccharide (LPS). Mechanistically, the attenuation of proinflammatory cytokine production by adiponectin was associated with an attenuation of the translocation of NFkappaB to the nucleus. Either adiponectin or inhibition of ERK1/2 with U0126 diminished the induction of IL6 by LPS (P<0.05), but the combination of adiponectin and the inhibitor did not further reduce IL6 production. In contrast, the inhibitory actions of adiponectin and a p38 MAPK inhibitor (SB203580) were additive (P<0.05). These data indicate that the anti-inflammatory actions of adiponectin include suppression of IL6 and induction of IL10. In addition, we provide evidence that some of the anti-inflammatory actions of adiponectin are mediated in part by suppression of NFkappaB signaling and ERK1/2 activity.

3T3-L1 adipocytes express the lipopolysaccharide (LPS) receptor and respond to direct stimulation with the antigen by increasing the expression of inflammatory mediators. Activation of this receptor by its ligand in the macrophage causes the activation and translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus where it regulates the expression of proinflammatory cytokines and other target genes. We investigated whether LPS could stimulate NF-kappaB translocation in primary pig adipocytes and regulate the expression and secretion of TNF-alpha and IL-6. LPS clearly induced the nuclear translocation of NF-kappaB and also upregulated (P < 0.05) the mRNA expression and secretion of IL-6 into the culture medium. An induction of TNF-alpha expression by LPS was not detected, but with extended incubation (8 h), there was a modest increase (P < 0.09) in the media concentration of this cytokine. Inhibition of either ERK1/2, PKC, or the inhibitory G protein (Gi) with U-0126, bisindolylmaleimide HCl, and pertussis toxin, respectively, blocked (P < 0.05) the increase in IL-6 expression caused by LPS. Because LPS administration in vivo increases circulating concentrations of IFN-gamma, and because this cytokine also regulates multiple immune modulators in the adipocyte, we also determined whether IFN-gamma regulates cytokine expression in primary adipocytes. Although the expression of IL-6 and TNF-alpha was unresponsive to IFN-gamma, the expression of IL-15 was markedly upregulated (P < 0.01). Furthermore, the induction of IL-15 expression by IFN-gamma was blocked by inhibition of PKC. These data indicate that NF-kappaB is responsive to LPS in the adipocyte and also identify key mediators of LPS-induced IL-6 expression. In addition, we provide novel evidence that IFN-gamma targets the adipocyte to induce IL-15 expression, thus indicating a possible role for the adipocyte in the regulation of T-cell function and muscle metabolism during the innate immune response.

Direct effects of leptin on gluconeogenesis in rat hepatocytes are equivocal, and model systems from other species have not been extensively explored in assessing the regulation of glucose metabolism by leptin. Therefore, the goal of the present study was to compare the effects of leptin on gluconeogenesis in pig and rat hepatocyte cultures as well as to investigate an underlying mechanism of action at the level of phosphoenolpyruvate carboxykinase (PEPCK). In rat hepatocytes, leptin exposure (3 h, 50 and 100 nM) attenuated glucagon-stimulated hepatic gluconeogenesis by 35 and 38% (P < 0.05), respectively. However, leptin did not produce any significant acute effect in pig hepatocytes. Leptin exposure for 24 h failed to produce any significant effect on gluconeogenesis in either rat or pig hepatocytes cultured in the presence of glucagon or dexamethasone. Mechanistically, there was a 25-35% decrease (P < 0.05) in glucagon-induced PEPCK mRNA levels in rat but not pig hepatocytes cultured with leptin. This effect on PEPCK mRNA was not due to an alteration in the relative abundance of the leptin receptor or the ability of PEPCK to respond to cAMP. The nonuniformity of the effects of leptin on gluconeogenesis in pig and rat hepatocytes indicates differences in leptin action between species. Furthermore, the unique action of leptin in porcine hepatocytes points to the utility of this model system for biomedical research and also underscores the value of comparative studies.

Two in vivo studies were conducted with pigs to determine the effects of exogenous leptin on the expression of peroxisome proliferator activated receptors (PPAR), and on serum concentrations of selected metabolites and hormones. Initially, leptin was administered i.m. to young pigs for 15 days at 0 (control), 0.003 (low), 0.01 (medium) and 0.03 (high) mg. kg(-1). day(-1). There was no leptin effect on serum glucose (P > 0.84), triglycerides (P > 0.69), non-esterified fatty acids (NEFA, P > 0.53), or glycerol (P > 0.33). Leptin at the intermediate and high doses depressed adipose expression of both PPARgamma1 (P < 0.06) and PPARgamma2 (P < 0.01). In a second study, we used a paired-feeding experimental design to determine the effects of a higher dose of leptin (0.05 mg. kg(-1). day(-1)) on serum metabolites and PPAR expression in selected tissues. At this dose, leptin increased (P < 0.0001) serum NEFA concentrations relative to both the ad libitum and pair-fed control groups. However, in this study, there was no difference in the expression of PPARgamma1 in adipose tissue, but PPARgamma2 mRNA was upregulated by leptin (P < 0.08). In contrast, leptin had no impact on the expression of PPARalpha in liver, skeletal muscle or adipose tissue. Adipose tissue explants were also incubated with leptin to assess the effect on PPARgamma expression, in vitro. The abundance of PPARgamma1 mRNA (P < 0.05) was increased after 24 hr of exposure, but the effect of leptin on gamma2 was not significant (P > 0.24). The lipolytic effect of leptin was also evaluated in vitro using isolated adipocytes. In keeping with the increase in serum NEFA concentrations in vivo, leptin stimulated lipolysis in vitro, increasing glycerol concentrations in the medium to about 219% of that in basal (non-treated) culture medium after 8 hr of incubation. Collectively, the data presented herein indicate that leptin modulates lipid metabolism in the pig, but that PPARalpha expression is not a parallel target of leptin as it is in rodent models. The regulation of PPARgamma by leptin seems complex in that it varied in relation to dose in vivo, and may be impacted by in vitro vs. in vivo circumstances.

A combination of in vivo and in vitro experiments were performed to determine the extent to which exogenous leptin regulates serum growth hormone (GH) and insulin-like growth factor I (IGF-1) concentrations, and the abundance of IGF-1 mRNA in major peripheral tissues. Initially (Experiment 1), a recombinant human leptin analog was administered i.m. to young growing pigs (approximately 27 kg body weight) for 15 days at 0 (control), 0.003, 0.01 and 0.03 mg. kg(-1). day(-1). Although there was no sustained effect of leptin on serum GH, there was a reduction (P < 0.02) in serum IGF-1 at the intermediate dose that paralleled a decrease (P < 0.09) in hepatic IGF-1 expression. Leptin, at these doses, did not reduce feed intake (P > 0.57), nor was there an effect of leptin on dietary nitrogen retention (P > 0.97). In a second experiment, pigs were injected with vehicle or a higher dose of leptin (0.05 mg. kg(-1). day(-1)) for 14 days. A third treatment group was injected with vehicle and pair-fed to the intake of the group treated with leptin. In this study, exogenous leptin resulted in a sustained increase in serum leptin (P < 0.0001) and reduction in feed intake of approximately 30% (P < 0.0001). Serum IGF-1 was depressed in both the leptin-treated and pair-fed groups, relative to the group allowed ad-libitum intake (P < 0.01). Furthermore, there was no difference among treatments in the relative abundance of IGF-1 mRNA in skeletal muscle (P > 0.42) or adipose tissue (P > 0.26), and liver mRNA abundance was actually increased (P < 0.01) by leptin, despite the lower feed intake. Finally, to determine whether leptin altered the secretion of IGF-1 by isolated pig hepatocytes, primary cultures were incubated with leptin for 24 to 48 hr (Experiment 3). Leptin (100 nM) caused a sharp reduction (P < 0.0001) in dexamethasone-induced IGF-1 secretion at 24 hr (47% reduction) and at 48 hr (40% reduction). Collectively, these data indicate that leptin may regulate hepatic IGF-1 production in the pig, independent of GH, but that hepatocyte sensitivity to leptin may be depend on dose and in vitro vs. in vivo conditions.

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.

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.

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., (Copyright 2001 Academic Press.)

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.

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).

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 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.

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.

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.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the PPAR subfamily of nuclear hormone receptors. In rodents and humans, expression of PPARgamma is predominantly found in adipose tissue where it regulates adipocyte differentiation and the expression of multiple adipocyte genes. The primary aim of this work was to clone the porcine PPARgamma cDNA and examine the regulation of gene expression in porcine subcutaneous adipose tissue. The porcine PPARgamma gene encodes a 1.8-kb mRNA transcript and shares 99, 96 and 97% amino acid sequence identity to the human, mouse and cow PPARgamma molecules, respectively. Both isoforms of PPARgamma (gamma1 and gamma2) are highly expressed in porcine adipose tissue. The gamma2 isoform is expressed in low abundance in porcine spleen, whereas the gamma1 isoform is highly expressed in spleen and lung and at a low abundance in several other tissues. Western blot analysis confirmed a high level of PPARgamma protein expression in porcine adipose tissue compared to other tissues. Both caloric restriction and fasting significantly reduced PPARgamma2 but not gamma1 mRNA and PPARgamma protein abundance in subcutaneous adipose tissue compared to ad-libitum fed controls. We provide the first evidence that PPARgamma is abundantly expressed in porcine subcutaneous adipose tissue, and that expression is regulated by caloric intake. Thus, PPARgamma may play an important role in adipogenesis and hormone action in porcine adipocytes.

The physiologic response to infection includes reductions in tissue concentrations of anabolic growth factors as a means of reducing growth and conserving nutrients for immunologic processes. This repartitioning of nutrients is accompanied by anorexia, which has been linked to increased leptin expression. Furthermore, leptin and growth hormone (GH) concentrations are inversely related, with leptin being required for normal GH release. The objective of this study was to determine if pretreatment with GH would influence endotoxin-induced changes in leptin expression or attenuate endotoxin-induced reductions in serum insulin-like growth factor-1 (IGF-1) and IGF-1 expression in liver and longissimus muscle. In experiment 1, 40 pigs were assigned to four treatments (n = 10 per treatment) arranged as a 2x2 factorial with GH (s.c. injection, 2 mg 1 h before challenge and 2 mg 2 h after challenge) and endotoxin (single i.m. injection, 25 microg/kg body weight) as main effect variables. Pretreatment with GH resulted in a marked increase (p<0.001) in serum GH within 1 h that was sustained throughout the study. Endotoxin challenge reduced (p<0.003) serum IGF-1 independent of GH (GH x endotoxin, p>0.682), and reduced (p<0.05) IGF-1 expression in longissimus muscle but not liver. Leptin mRNA abundance was reduced 56% (p<0.005) by GH but was not affected by endotoxin (p>0.81). In experiment 2, 36 pigs (n = 12 per treatment) were either allowed ad libitum feed consumption with no injection or deprived of feed and injected twice with either saline or endotoxin 24 h apart. Feed deprivation reduced leptin expression (p<0.05). However, endotoxin did not change leptin expression but markedly increased (p<0.05) serum haptoglobin. These data indicate that changes in IGF-1 status in endotoxin-challenged pigs are independent of serum GH and that leptin expression is not increased by endotoxin challenge in the pig. These data also indicate a regulatory linkage between GH and leptin in vivo.

The objective of this study was to establish the developmental pattern and tissue specificity of porcine myostatin expression and to evaluate expression in skeletal muscle during circumstances in which muscle growth was altered. Northern blot analysis revealed two transcripts (1.5 and 0.8 kb). Myostatin mRNA was detected in whole fetuses at 21 and 35 days and was markedly increased (P < 0.05) by 49 days. At birth, mRNA abundance in longissimus muscle had declined significantly (P < 0.05) from that at day 105 of gestation and continued to decrease (P < 0.05) to its lowest level 2 wk postnatally (4 kg body wt). Myostatin expression was higher (P < 0. 05) at 55, 107, and 162 kg body wt than at 4 kg body wt. Postnatally, myostatin mRNA was detected in skeletal muscle and mammary gland. Expression at birth was 65% higher (P < 0.04) in longissimus muscle of low-birth-weight piglets (0.57 +/- 0.052 kg body wt) vs. normal (1.37 +/- 0.077 kg body wt) littermates, irrespective of gender. However, suppression of longissimus muscle growth by food deprivation (3 days) did not alter (P > 0.15) myostatin expression in either 4- or 7-wk-old piglets. Additionally, myostatin mRNA abundance was not changed by porcine growth hormone administration in growing animals. These data indicate that myostatin expression in skeletal muscle peaks prenatally and that greater expression is associated with low birth weight. Expression in mammary gland indicates a possible role for myostatin in mammary gland development and/or lactation.

The objective of the present study was to evaluate the effect of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-alpha (IL-1a), on myoblast proliferation and fusion and on myocyte protein metabolism and stress protein expression. Proliferation was suppressed (p < 0.05) by both cytokines, alone and in combination, and at lower concentrations, the suppression was additive. Likewise, fusion was retarded (p < 0.05) by these cytokines alone and in combination. Myosin synthesis was not altered acutely or chronically by TNF-alpha alone or by the combination of this cytokine with IL-1alpha. Chronic exposure to TNF-alpha did not alter total cellular protein synthesis, but exposure to IL-1alpha and the cytokine combination resulted in an increase (14% to 19%, p < 0.05) in synthesis. Neither total cellular protein nor myosin degradation were influenced by either cytokine alone or by the combination. There was no detectable induction, acutely or chronically, of any of the stress proteins evaluated (HSC70, HSP70, or HSP60). These data suggest that cytokines may alter muscle growth and development prenatally and postnatally and that the changes in muscle protein metabolism during periods of immune challenge are not direct effects of TNF-alpha or IL-1alpha.

The objective of this study was to determine whether the improvements in growth and efficiency of gain achieved by recombinant porcine somatotropin (pST) are associated with altered expression of the p94, calpastatin, or alpha-actin genes in porcine longissimus (LD) muscle. Forty-eight barrows (initial 64.2 to 67.4 kg BW) were assigned to four treatments (n = 12) arranged as a 2 x 2 factorial in a randomized complete block design. Factors were duration of treatment (3 or 6 wk) and pST administration (0 or 3 mg x pig(-1) x d(-1)). Plasma samples were obtained 24 h after the first pST injection and at the end of the each treatment period for assays of selected variables. The LD samples were obtained at 3 and 6 wk of pST treatment. Northern blot analysis of calpastatin expression in LD muscle revealed three distinct transcription products of approximately 8.5 (CPST I), 5.5 (CPST II), and 2.5 (CPST III) kb; CPST II was reduced (P < .02) 33 and 61% by pST at 3 and 6 wk, respectively, whereas CPST I and III were not influenced (P > .12). Neither alpha-actin nor p94 was responsive to pST injection. As expected, pST resulted in higher (50%, P < .02) plasma insulin within 24 h and one- and twofold higher (P < .01) concentrations at 3 and 6 wk, respectively. Glucose was increased (P < .01) at 3 (15%) and 6 (10%) wk, whereas urea nitrogen was reduced (32 to 36%, P < .01). The efficacy of pST was evident in that ADG was improved (P < .01) 11 to 13% independent of time. Likewise, feed intake was reduced (P < .01) 10 to 11% and gain: feed improved (P < .01) approximately 26% for pigs receiving pST independent of time. These data indicate that the enhanced muscle growth achieved by pST is not associated with altered expression of p94 or alpha-actin, or an increase in the abundance of any calpastatin transcription product.

Leptin, a 16-kDa protein secreted from white adipocytes, has been implicated in the regulation of food intake, energy expenditure, and whole-body energy balance in rodents and humans. The gene encoding leptin was identified by positional cloning and is the mutation leading to the profound obese phenotype of the ob/ob mouse. Exogenous administration of leptin to ob/ob mice leads to a significant improvement in reproductive and endocrine status as well as reduced food intake and weight loss. The expression and secretion of leptin is highly correlated with body fat mass and adipocyte size. Cortisol and insulin are potent stimulators of leptin expression, and expression is attenuated by beta-adrenergic agonists, cAMP, and thiazolidinediones. The role of other hormones and growth factors in the regulation of leptin expression and secretion is emerging. Leptin circulates specifically bound to proteins in serum, which may regulate its half-life and biological activity. Isoforms of the leptin receptor, members of the interleukin-6 cytokine family of receptors, are found in multiple tissues, including the brain. Many of leptin's effects on food intake and energy expenditure are thought to be mediated centrally via neurotransmitters such as neuropeptide Y. Multiple peripheral effects of leptin have also been recently described, including the regulation of insulin secretion by pancreatic beta cells and regulation of insulin action and energy metabolism in adipocytes and skeletal muscle. Leptin is thought to be a metabolic signal that regulates nutritional status effects on reproductive function. Leptin also plays a major role in hematopoeisis and in the anorexia accompanying an acute cytokine challenge. The profound effects of leptin on regulating body energy balance make it a prime candidate for drug therapies for humans and animals.