Cloning, expression and chromosome localization of porcine adiponectin and adiponectin receptors genes

Adiponectin is a cytokine secreted specifically by adipocytes that has been proposed to enhance insulin sensitivity and prevent atherosclerosis. Adiponectin receptors (adipoR1 and adipoR2) are recently found in mice which act as receptors for globular and full-length adiponectin to mediate the fatty-acid oxidation and glucose uptake in muscle and liver. The primary goal of this study was to examine chromosome localization of porcine adiponectin and adiponectin receptors and the gene expression pattern in various tissues of pigs of the three genes. Radiation hybrid mapping demonstrated that porcine adiponectin, adipoR1 and adipoR2 were located to chromosome13q36-41, 10p11 and 5q25, in the regions that were syntenic to the homologs of human genes, respectively. Semi-quantitative RT-PCR showed that porcine adiponectin mRNA was specifically expressed in adipose tissue and porcine adipoR1 and adipoR2 mRNA were ubiquitously expressed in many tissues except brain. Comparison to adipoR2 mRNA which was highly expressed in liver, heart, kidney, adipose tissues and lung, adipoR1 mRNA was expressed at relatively high levels in porcine muscle, leukocytes and epididymis. Our data provide basic molecular information useful for the further investigation on the function of the three genes.     

Fasting regulates the expression of adiponectin receptors in young growing pigs

Adiponectin is an adipocyte-derived hormone that can improve insulin sensitivity. Its functions in regulating glucose utilization and fatty acid metabolism in mammals are mediated by 2 subtypes of adiponectin receptors (AdipoR1 and AdipoR2). This study was conducted to determine the effect of fasting on the expression of adiponectin and its receptors. The expression of adiponectin was not affected in s.c. adipose tissue, but adiponectin expression increased in visceral adipose tissue after fasting. In contrast, expression of both AdipoR mRNA was increased in the liver and s.c. adipose tissue of 24-h-fasted pigs compared with fed pigs, but the mRNA in muscle and visceral adipose tissue was not affected by fasting. A third putative adiponectin receptor, T-cadherin, was cloned and the mRNA expression was determined. T-Cadherin has been recognized to act as a vascular adiponectin receptor in vascular endothelial and smooth muscle cells. Our data showed that the expression of T-cadherin was decreased in the muscle of fasted pigs, suggesting that the expression of T-cadherin can be regulated by feeding status. In summary, in young pigs, adiponectin mRNA was up-regulated by fasting in visceral, but not s.c., adipose tissue, whereas AdipoR1 and AdipoR2 mRNA were increased in s.c., but not visceral, adipose tissue. The adiponectin receptor, T-cadherin, was expressed in s.c. and visceral adipose tissue and in muscle, but only muscle mRNA expression was decreased by fasting.     

Transition period-related changes in the abundance of the mRNAs of adiponectin and its receptors,of visfatin,and of fatty acid binding receptors in adipose tissue of high-yielding dairy cows

Adipose tissue expresses adipokines, which are involved in regulation of energy expenditure, lipid metabolism, and insulin sensitivity. To adapt for the transition from pregnancy to lactation, particularly in high-yielding dairy cows, adipokines, their receptors, and particular G-protein coupled receptors (GPRs) are of potential importance. Signaling by GPR 41 stimulates leptin release via activation by short-chain fatty acids; GPR 43/109A inhibits lipolysis, and GPR 109A thereby mediates the lipid-lowering effects of nicotinic acid and β–hydroxybutyrate. The aim of this study was to compare the mRNA expression of adiponectin and visfatin, adiponectin receptors 1 and 2 (AdipoR1/2), leptin receptor (obRb), insulin receptor as of the aforementioned GPRs during the transition period in high-yielding dairy cows. Biopsies from subcutaneous fat and blood samples were obtained from 10 dairy cows 1 week before and 3 weeks after calving. For AdipoR1 and AdipoR2 mRNA abundance as well as for leptin concentrations in plasma, a reduction (P ≤ .05) was observed postpartum; for visfatin and putative GPR 109A mRNA abundance in adipose tissue, there was a trend (P < .1) for analogous changes. In contrast, the mRNA content of obRb and GPR 41 in adipose tissue was higher (P ≤ .05) in samples from early lactation than in those from late gestation. Our results indicate decreasing adiponectin sensitivity in adipose tissue after calving, which might be involved in the reduced insulin sensitivity of adipose tissue during early lactation. In addition, visfatin, GPR 41, and GPR 109A may further modulate insulin sensitivity.     

Distinct adiponectin profiles might contribute to differences in susceptibility to type 2 diabetes in dogs and humans

Dogs develop obesity-associated insulin resistance but not type 2 diabetes mellitus. Low adiponectin is associated with progression to type 2 diabetes in obese humans. The aims of this study were to compare total and high molecular weight (HMW) adiponectin and the ratio of HMW to total adiponectin (S_A) between dogs and humans and to examine whether total or HMW adiponectin or both are associated with insulin resistance in naturally occurring obese dogs. We compared adiponectin profiles between 10 lean dogs and 10 lean humans and between 6 lean dogs and 6 age- and sex-matched, client-owned obese dogs. Total adiponectin was measured with assays validated in each species. We measured S_A with velocity centrifugation on sucrose gradients. The effect of total and HMW adiponectin concentrations on MINMOD-estimated insulin sensitivity was assessed with linear regression. Lean dogs had total and HMW adiponectin concentrations three to four times higher than lean humans (total: dogs 32 ± 5.6 mg/L, humans 10 ± 1.3 mg/L, P<0.001; HMW: dogs 25 ± 4.5 mg/L, humans 6 ± 1.3 mg/L, P<0.001) and a higher S_A (dogs: 0.78 ± 0.05; humans: 0.54 ± 0.08, P = 0.002). Adiponectin concentrations and S_A were not lower in obese dogs (0.76 ± 0.05 in both groups; P=1). Total adiponectin, HMW adiponectin, and S_A were not associated with insulin sensitivity in dogs. We propose that differences in adiponectin profiles between humans and dogs might contribute to the propensity of humans but not dogs to develop type 2 diabetes. Dogs with chronic, naturally occurring obesity do not have selectively reduced HMW adiponectin, and adiponectin does not appear to be important in the development of canine obesity-associated insulin resistance.     

Enhanced protective response and immuno-adjuvant effects of porcine GM-CSF on DNA vaccination of pigs against Aujeszky’s disease virus

This study was conducted to investigate whether the co-delivery of DNA encoding porcine cytokines would enhance a protective immune response in pigs to a Pseudorabies virus (PRV; or Aujeszky’s disease virus) DNA vaccine. Aujeszky’s disease in pigs results in respiratory and nervous symptoms with important economic losses. To evaluate cytokine effects, eukaryotic expression vectors were constructed for porcine GM-CSF, IL-2 and IFN-γ. cDNA for each of these cytokines was inserted under the control of a CMV promoter in the pcDNA3 plasmid and cytokine expression was confirmed after DNA transfection in various mammalian cell cultures by bioassays (GM-CSF and IL2) and ELISA (IFN-γ). Pigs were vaccinated by single intramuscular injection with plasmid DNA encoding PRV gB and gD along with various combinations of cytokine plasmid constructs. Pig serum was tested for the production of antibody by isotype specific anti-PRV ELISA. Pigs were then challenged with the highly virulent PRV strain NIA3 on day 21 after vaccination. The survival and growth rate of pigs were monitored for seven days after the viral challenge. The co-administration of GM-CSF plasmid increased the immune response induced by gB and gD PRV DNA vaccine. This immune response was characterized by an earlier appearance of anti-PRV IgG2, a significantly enhanced anti-PRV IgG1 and IgG2 antibody response, a significantly decreased and shortened viral excretion in nasal swabs and an improved protection to the viral challenge. In contrast, the co-administration of porcine IL-2 or IFN-γ had no adjuvant effects. Our results thus demonstrate for the first time that the application of porcine GM-CSF gene in a DNA vaccine formulation can exert immuno-adjuvant and protective effects with single vaccination in the natural host pig against Aujeszky’s disease.     

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

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

Effects of leptin and adiponectin on the growth of porcine myoblasts are associated with changes in p44/42 MAPK signaling

We hypothesized that both adiponectin and leptin affect the growth of porcine skeletal muscle cells, with fatty acids acting as modifiers in adipokine action and that both adipokines influence the gene expression of their receptors. Therefore, the objective of this study was to investigate the effects of recombinant adiponectin and leptin on cell number (DNA) and DNA synthesis rate with and without oleic acid supplementation, on cell death, and on key intracellular signaling molecules of proliferating porcine myoblasts in vitro. Moreover, the mRNA expression of genes encoding for the leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) as affected by leptin or adiponectin was examined. Recombinant porcine adiponectin (40 μg/mL) and leptin (20 ng/mL) increased DNA synthesis rate, measured as [³H]-thymidine incorporation (P < 0.01), reduced cell viability in terms of lactate dehydrogenase release (P < 0.05), or lowered DNA content after 24 h (P < 0.05). In adiponectin-treated cultures, oleic acid supplementation increased DNA synthesis rate and reduced cell number in a dose-dependent manner (P < 0.05). Both adiponectin (P = 0.07) and leptin (P < 0.05) induced a transient activation of p44/42 mitogen-activated protein kinase (MAPK) after 15 min, followed by decreases after 60 and 180 min (P < 0.05). Adiponectin tended to increase c-fos activation (P = 0.08) and decreased p53 activation at 180 min (P = 0.03). Both adiponectin and leptin down-regulated the abundance of ADIPOR2 mRNA and, transiently, of LEPR mRNA (P < 0.05). In conclusion, adiponectin and leptin may adversely affect the growth of porcine myoblasts, which is related to p44/42 MAPK signaling and associated with changes in ligand receptor gene expression.     

Canine adiponectin: cDNA structure, mRNA expression in adipose tissues and reduced plasma levels in obesity

Adiponectin is a protein synthesized and secreted by adipocytes. Decreased adiponectin is responsible for insulin resistance and atherosclerosis associated with human obesity. We obtained a cDNA clone corresponding to canine adiponectin, whose nucleotide and deduced amino acid sequences were highly identical to those of other species. Adiponectin mRNA was detected in adipose tissues, but not in other tissues, of dogs. When 22 adult beagles were given a high-energy diet for 14 weeks, they became obese, showing heavier body weights, higher plasma leptin concentrations, but lower plasma adiponectin concentrations. The adiponectin concentrations of plasma samples collected from 71 dogs visiting veterinary practices were negatively correlated to plasma leptin concentrations, being lower in obese than non-obese dogs. These results are compatible with those reported in other species, and suggest that adiponectin is an index of adiposity and a target molecule for studies on diseases associated with obesity in dogs.     

Immunohistochemical expression of progesterone receptors, growth hormone and insulin growth factor-I in feline fibroadenomatous change

The immunohistochemical expression, tissue-specific and cell-specific distribution patterns of progesterone receptors (PR), growth hormone (GH) and insulin growth factor-I (IGF-I) have been studied in 22 cases of feline fibroadenomatous change (FFAC). PR and GH were detected in all cases and were distributed homogeneously throughout the lesion, while IGF-I was detected in 77% of the cases at the site of ductal budding. The simultaneous expression of PR, GH and IGF-I was detected in epithelial cells in 14 of 22 cases while PR and GH expression only was detected in epithelial cells in 11 cases. Cases that expressed GH and IGF-I without PR expression in the stroma were the most numerous. Double immunohistochemical staining showed the co-localisation of PR and GH in a subset of ductal epithelial cells located between basal/myoepithelial and luminal cells (probably undifferentiated stem cells). These results suggest that ligand-activated progesterone receptors may induce the local synthesis of GH which in turn may exert its proliferative action directly and also indirectly through the production of other growth factors, such as IGF-I, in an autocrine/paracrine manner.     

Effects of feeding pattern on ghrelin and insulin secretion in pigs

Ghrelin is a peptide hormone that has been implicated in the regulation of feed intake, but little is known about its secretion in pigs. Hence, the effect of feeding pattern on the regulation of ghrelin secretion was tested. In experiment 1, barrows were allotted randomly into 1 of 2 groups, (1) ad libitum fed (CONT) and (2) limited access to feed (once per day, MEAL). Blood samples were taken through jugular catheters every 15 min for 6 h after 7 d on the experimental feeding regimen. Plasma concentrations of ghrelin and insulin were determined by radioimmunoassay. Ghrelin concentrations in the MEAL pigs were elevated before feeding and declined after feeding (P < 0.01). No pattern in plasma ghrelin concentrations was observed in the CONT pigs, but ghrelin concentrations were lower than in the MEAL group. Insulin concentrations were greater in CONT pigs (P < 0.01) during most of the sampling and increased after feeding in the MEAL pigs (P < 0.01). In experiment 2, the treatments were the same as in experiment 1; however, the amount of feed was increased in the MEAL group so that their daily intake was similar to the CONT pigs. Ghrelin concentrations in the MEAL group were again elevated before the meal and declined afterward (P < 0.01). Insulin but not glucose concentrations were negatively correlated with ghrelin. Once-per-day feeding resulted in increased plasma concentrations of ghrelin, which decreased after feeding. Ghrelin may be involved in the regulation of feed intake in pigs.     

Adiponectin enhances in vitro development of swine embryos

Recent studies have shown that factors from adipose tissue influence and regulate the reproductive system. Hormones such as leptin and resistin are now known to regulate several reproductive processes. Adiponectin is the most abundant protein secreted by adipose tissue, and its circulating concentration is inversely related to adiposity and body mass index. Little is known about the involvement of adiponectin in reproduction. In the present study, the effect of recombinant adiponectin on the meiotic maturation and early embryo development in vitro was investigated, using porcine oocytes. Adiponectin receptors, AdipoR1 and AdipoR2, were found to be expressed in porcine oocytes and cumulus cells of both small and large follicles. Both AdipoR1 and AdipoR2 were immunolocalized to cumulus-oocyte complexes (COCs), oocytes, and early developing embryos. When included in oocyte maturation medium for 46 h, adiponectin significantly decreased the frequency of meiotic immature oocytes derived from large follicles (3-6 mm) but not from small follicles (<3mm). From studies of oocytes matured in the presence of adiponectin and mitogen-activated protein kinase (MAPK) pathway inhibitors MEK1 (PD98059), MEK1/2 (U0126), and p38MAPK (SB203580) it was concluded that adiponectin enhances oocyte maturation thought the p38MAPK pathway. Finally, a superior rate of embryo development to the blastocyst stage was achieved by embryos cultured in the presence of adiponectin. These results indicate that adiponectin has a positive effect on the meiotic maturation and in vitro embryo development of porcine oocytes and suggests a physiological role for this adipokine in early development in mammals.     

重组脂联素对皖南花猪脂肪细胞脂联素及其受体mRNA表达的影响

脂联素（Adp）是主要由脂肪组织分泌的细胞因子,有重要的生理作用。本试验旨在研究重组脂联素（rAdp）对皖南花猪脂肪细胞脂联素及其受体2,AMP激活蛋白激酶（AMPK）、过氧化物增殖剂活化受体α（PPARα）mR-NA表达的影响。选择10d皖南花猪皮下脂肪组织分离前体脂肪细胞,增殖培养至80%融合后换分化培养基培养,细胞分化后用0、2和10mg/L rAdp分别处理12和48h。油红O染色法鉴定脂肪细胞,MTT方法检测细胞活力;酶法测定培养液中甘油释放量,荧光定量RT-PCR方法检测脂肪细胞脂联素（Adp）、脂联素受体1（AdpR1）、脂联素受体2（AdpR2）、PPARα和AMPK mRNA表达。结果显示,rAdp处理后,脂肪细胞活力总体有降低趋势,10mg/L处理48h达到显著水平（P〈0.05）;rAdp处理对甘油释放的抑制作用未达到差异水平。rAdp处理12h后,脂肪细胞AdpR1和AdpR2mRNA表达显著升高（P〈0.01）,但无剂量依赖性;rAdp处理48h后,脂肪细胞AdpmRNA表达显著下降（P〈0.05）。rAdp处理12h后,脂肪细胞PPARαmRNA表达显著升高（P〈0.01）,且有剂量效应性;而AMP AMPK mRNA表达均无显著性变化。结果提示,重组脂联素处理猪原代脂肪细胞有降低细胞活力和抑制脂肪细胞甘油释放量的趋势,能显著上调AdpR1、AdpR2和PPARα基因的表达,从而刺激脂肪酸氧化和甘油三酯的水解作用。     

Obesity induced changes to plasma adiponectin concentration and cholesterol lipoprotein composition profile in cats

Feline obesity generally results in aberrations to plasma metabolite levels, such as lipid concentrations and lipoprotein composition. This study sought to investigate the resultant effect of obesity on cholesterol lipoprotein composition and circulating adiponectin concentrations in cats. Plasma glucose, lipids (triglyceride, cholesterol and free fatty acid), insulin and adiponectin concentrations, and cholesterol lipoprotein composition were measured and compared between body condition score (BCS) determined normal healthy control and obese cats. Although the obese group demonstrated higher levels of plasma cholesterol, glucose, and triglycerides, as compared to healthy controls, the difference was insignificant thus indicating that the BCS determined obese cats may have been overweight and not morbidly obese. Plasma insulin levels were significantly higher (25–30%) versus healthy control animals thereby possibly hinting at the ensuing emergence of obesity induced insulin resistance. However, the BCS determined obese cat demonstrated a significant reduction (p < 0.05) in plasma adiponectin concentration and a significant increase (p < 0.05) in LDL-cholesterol % as compared to age matched healthy control animals. This would indicate that changes in plasma adiponectin concentration and cholesterol lipoprotein composition may be good early indicators of obesity in cats.     

Detection and molecular characterization of porcine group C rotaviruses in South Korea

Group C rotaviruses (GCRVs) cause acute diarrhea in humans and animals worldwide and the evidence for a possible zoonotic role of GCRVs has been recently provided. However, there is little evidence of porcine GCRV infections or of their genetic diversity in South Korea. We examined 137 diarrheic fecal specimens from 55 farms collected from six provinces. RT-PCR utilizing primer pairs specific for the GCRV VP6 gene detected GCRV-positive reactions in 36 (26.2%) diarrheic fecal samples. Of these, 17 samples (12.4%) tested positive for porcine GCRVs alone and 19 samples (13.8%) were also positive for other pathogens. Other enteric pathogens except for GCRV were detected in 64 feces samples (46.7%) and no enteric pathogens were evident in 37 feces samples (27.0%). Phylogenetic and sequence homology analyses of GCRV partial VP6 gene between 23 Korean and other known porcine GCRVs demonstrated that Korean strains belonged to the porcine lineage. Furthermore, one Korean porcine strain shared the highest nucleotide (89.7–89.0%) and deduced amino acid sequence (92.9–93.9%) identities with bovine GCRV strains and was placed in the bovine GCRV lineage indicative of bovine origin. In conclusion, porcine GCRV infections are widespread in piglets with diarrhea in South Korea. The infecting porcine GCRVs mostly belong to the porcine lineage with the exception of one bovine-like GCRV, which possibly originated from bovine GCRV due to interspecies transmission.     

Adiposity and adiponectin in dogs: investigation of causes of discrepant results between two studies

Although one study showed lower adiponectin concentrations in obese dogs, other recent studies indicate that adiponectin might not be decreased in obese dogs, raising the possibility that the physiology of adiponectin is different in dogs than in humans. The aim of this study was to investigate possible causes of the discrepancy between the two largest studies to date that assessed the association between adiposity and adiponectin concentration in dogs, including the validity of the assay, laboratory error, and the effects of breed, sex, and neuter status on the relationship between adiposity and adiponectin concentrations. Adiponectin concentrations measured with a previously validated adiponectin ELISA were compared with those estimated by Western blotting analysis of reduced and denatured plasma samples. The possibility of laboratory error and the effect of EDTA anticoagulant and aprotinin were tested. Adiponectin concentration was measured by ELISA in 20 lean dogs (10 male and 10 female, 5 neutered in each sex). There was close correlation between adiponectin concentrations measured by ELISA and those estimated by Western blotting analysis (r = 0.90; P < 0.001). There was no substantial effect of EDTA, aprotinin, or laboratory error on the results. There was confounding by neuter status of the relationship between adiposity and adiponectin concentrations, but adiponectin concentrations were not significantly lower in male than in female lean dogs (females, 36 mg/L; males, 26 mg/L; P > 0.20) and were not significantly lower in intact than in neutered lean male dogs (intact, 28 mg/L; neutered, 23 mg/L; P = 0.49). We conclude that the adiponectin ELISA previously validated for use in dogs appears to be suitable for determination of canine adiponectin concentrations and that testosterone does not appear to have a strong effect on plasma adiponectin concentrations in dogs. Obesity might decrease adiponectin concentrations in intact but not in neutered dogs.     

Expression of adiponectin and its receptors in swine

Adiponectin is an adipocyte-derived hormone that plays an important role in lipid metabolism and glucose homeostasis. Objectives of this study were 1) to determine the presence and distribution of adiponectin and its receptors 1 and 2 (adipoR1 and adipoR2) in porcine tissues; 2) to characterize pig adiponectin, adipoR1, and adipoR2 mRNA levels in various fat depots from three different breeds of pigs; and 3) to study, in stromal-vascular cell culture, the effects of leptin and tumor necrosis factor-alpha (TNFalpha) on pig adiponectin, adipoR1, and adipoR2 gene expression. To this end, fat Chinese Upton Meishan (UM, n = 10), lean Ham Line (HL, n = 10), and Large White (LW, n = 10) gilts were used. We report the isolation of partial cDNA sequences of pig adipoR1 and adipoR2. Porcine-deduced AA sequences share 97 to 100% homology with human and murine sequences. Pig adipoR1 mRNA is abundant in skeletal muscle, visceral fat, and s.c. fat tissues, whereas adipoR2 mRNA is predominantly expressed in liver, heart, skeletal muscle, and visceral and s.c. fat tissues. Pig adiponectin mRNA levels in s.c. and visceral fat tissues were not associated with plasma insulin and glucose in fasting animals. Subcutaneous (r = -0.44, P < 0.05), visceral (r = -0.43, P < 0.05), and total body fat (r = -0.42, P < 0.05) weights were negatively correlated with adiponectin mRNA levels measured in visceral, but not s.c., fat. Pig adipoR1 and adipoR2 mRNA levels, in visceral fat, were less expressed in fat UM gilts than in the lean HL gilts (P < 0.05). Inverse associations were found between s.c. (r = -0.57, P < 0.01), visceral (r = -0.46, P < 0.05), and total body fat (r = -0.56, P < 0.01) weights and adipoR2 mRNA levels in visceral fat only. We were unable to find such associations for adipoR1 mRNA levels in the overall gilt population. The current study demonstrated that TNFalpha downregulates adiponectin and adipoR2, but not adi-poR1, mRNA levels in stromal-vascular cell culture. Moreover, leptin significantly decreased adiponectin mRNA levels, whereas there was no effect on adiponectin receptors. We conclude that adiponectin and adi-poR2 mRNA levels, but not adipoR1, are modulated in pig visceral fat tissues. Furthermore, our results indicate that TNFalpha interferes with adiponectin function by downregulation of adipoR2 but not of adipoR1 mRNA levels in pigs.