Functional and phylogenetic analyses of a melanocortin-4 receptor mutation in domestic pigs

The melanocortin-4 receptor (MC4R), a G protein-coupled seven-transmembrane receptor, which is expressed in the brain, plays an important role in the control of mammalian energy homeostasis. A missense mutation (Asp298Asn) was identified in the porcine MC4R gene, which is associated with growth and food intake traits. The Asn298 mutation occurs within a highly conserved motif, NPLIY, of all members of G protein-coupled receptors; whereas, Asp298 is conserved in all five melanocortin receptor subtypes. Functional analysis of the porcine MC4R variant was performed with an in vitro gene expression system in 293 cells. Ligand binding (NDP-alphaMSH) did not differ between Asp298 and Asn298 MC4R proteins. However, the Asn298 MC4R variant was unable to stimulate cAMP production in response to NDP-alphaMSH stimulation; whereas, the Asp298 variant could stimulate cAMP accumulation. These results demonstrate that the Asp298 is required for normal MC4R signaling to the adenylyl cyclase. Sequencing of the MC4R gene of seven diverse genera within the Suiformes that include Hippopotamidae (hippos), Tayassuidae (peccaries) and Suidae (pigs), revealed 62 nucleotide variations in MC4R. Phylogenetic relationships of MC4R variations are consistent with those previously described from morphological and physiological data among the subfamilies of the Suiformes. These findings revealed that a single missense mutation (Asp298Asn) of aspartic acid (Asp) to asparagine (Asn) in MC4R gene decreased cAMP content and MC4R signaling, but with no difference in the ligand binding was associated with growth and feed intake traits in domestic pigs.     

Pharmacological analyses of two naturally occurring porcine melanocortin-4 receptor mutations in domestic pigs

The melanocortin-4 receptor (MC4R) is critical in regulating mammalian food intake and energy expenditure. Numerous mutations in the MC4R gene have been identified from obese humans. So far two naturally occurring porcine MC4R (pMC4R) mutations, D298N and R236H, have been identified from various strains of pigs and D298N is being utilized as a genetic marker to screen performance traits of pigs. In this study, we performed functional analyses of pMC4R D298N and R236H, including their ligand binding and signaling properties in transiently transfected HEK293T cells. Ligand binding assays showed that both D298N and R236H pMC4Rs had similar binding capacities and affinities for the natural agonist -MSH and the natural antagonist Agouti-related protein as wild-type pMC4R. In signaling assays, both mutants had normal EC₅₀ and maximal signaling to -MSH. In summary, pMC4R mutants D298N and R236H do not have any overt functional defects; therefore we suggest caution using these mutations as selection markers in breeding programs.     

Genetic variants of the unsaturated fatty acid receptor GPR120 relating to obesity in dogs

G protein-coupled receptor (GPR) 120 is an unsaturated fatty acid receptor, which is associated with various physiological functions. It is reported that the genetic variant of GPR120, p.Arg270His, is detected more in obese people, and this genetic variation functionally relates to obesity in humans. Obesity is a common nutritional disorder also in dogs, but the genetic factors have not ever been identified in dogs. In this study, we investigated the molecular structure of canine GPR120 and searched for candidate genetic variants which may relate to obesity in dogs. Canine GPR120 was highly homologous to those of other species, and seven transmembrane domains and two N-glycosylation sites were conserved. GPR120 mRNA was expressed in lung, jejunum, ileum, colon, hypothalamus, hippocampus, spinal cord, bone marrow, dermis and white adipose tissues in dogs, as those in mice and humans. Genetic variants of GPR120 were explored in client-owned 141 dogs, resulting in that 5 synonymous and 4 non-synonymous variants were found. The variant c.595C>A (p.Pro199Thr) was found in 40 dogs, and the gene frequency was significantly higher in dogs with higher body condition scores, i.e. 0.320 in BCS4–5 dogs, 0.175 in BCS3 dogs and 0.000 in BCS2 dogs. We conclude that c.595C>A (p.Pro199Thr) is a candidate variant relating to obesity, which may be helpful for nutritional management of dogs.     

Relationship between the melanocortin-1 receptor (MC1R) variant R306ter and physiological responses to mechanical or thermal stimuli in Labrador Retriever dogs

Objective

Variants in the MC1R gene have been associated with red hair color and sensitivity to pain in humans. The study objective was to determine if a relationship exists between MC1R genotype and physiological thermal or mechanical nociceptive thresholds in Labrador Retriever dogs.

Expression of CC chemokine receptor 4 (CCR4) mRNA in canine atopic skin lesion

CC chemokine receptor 4 (CCR4) is a G protein-coupled seven transmembrane receptor that is selectively expressed on Th2 cells and plays an important role in the trafficking of Th2 cells into inflammatory sites. In this study, a full-length canine CCR4 cDNA was cloned and characterized in order to examine the potential role of CCR4 in allergic responses that produce skin lesions in canine atopic dermatitis (AD). The canine CCR4 cDNA reported in this study contained an open reading frame of 1083 nucleotides encoding 360 amino acids. The predicted amino acid sequence of canine CCR4 showed 91.9, 85.3 and 84.5% similarity with those of the human, mouse and guinea pig counterparts, respectively. Expression of CCR4 mRNA was detected in various tissues including thymus, spleen, heart, small intestine and lymph node. Furthermore, it was found that CCR4 mRNA was preferentially expressed in lesional skin of dogs with AD, together with the mRNA of thymus and activation-regulated chemokine (TARC), which is a ligand for CCR4. The present study demonstrates that CCR4 contributes strongly to the immunopathogenesis of canine AD.     

Altered expression of melanocortin-1 receptor (MC1R) in a yellow-coloured wild raccoon dog (Nyctereutes procyonoides)

Background – The melanocortin 1 receptor (MC1R) gene plays a key role in determining coat colour in mammals by controlling the proportion of eumelanin and pheomelanin granules. Wild raccoon dogs have a mixed coat colour, with black to brown and grey hairs. Hypothesis/Objectives – The study was performed to identify the cause of the variant yellow coat colour in a wild raccoon dog. Animals – A wild raccoon dog that showed coat colour change to yellow and four wild‐type raccoon dogs that showed normal coat colour were included. Methods – To identify the cause of the variant yellow coat colour, we examined the sequence of the MC1R gene and its expression at the mRNA and protein levels. Results – The coding region of the MC1R gene of this raccoon dog comprised 954 bp, the same as for wild‐type raccoon dogs and domestic dogs. By comparing the gene with that in the wild‐type raccoon dog, a 2 bp deletion was detected in the 5′‐untranslated region, positioned 152 bp upstream of the start codon. However, there was no significant difference in the mRNA expression level. The yellow raccoon dog revealed a significantly decreased MC1R protein level compared with the wild‐type raccoon dogs, indicating an increase in pheomelanin synthesis. Conclusions and clinical importance – These results suggest that the variant coat colour in the yellow raccoon dog was associated with decreased MC1R function.     

MC4R基因研究进展

黑素皮质素受体-4(melanocortin-4 receptor,MC4R)是下丘脑腹内侧核分泌的一类肽类物质,为黑素皮质素受体家族5个亚型(MC1-5R)之一。在哺乳动物中,MC4R具有介导瘦蛋白(leptin)的功能,是一个调节能量平衡与能量动态平衡的重要信号分子,可与其内源性配体黑素皮质素激素(melanocortin,MC)或刺鼠色蛋白(agouti protein,agouti蛋白)和agouti相关蛋白(agouti related protein,AGRP)相结合,从而在控制食欲和体重稳态中起关键作用。因此,MC4R在人类肥胖研究中作为重要的调节因子倍受关注。最近有研究表明:MC4R的1232位点的G→A的突变与绵羊背膘厚度间存在关联,AG和AA型较GG型具有较高的背膘厚度。     

Melanocortin 2 receptor antagonists in canine pituitary-dependent hypercortisolism: in vitro studies

Canine hypercortisolism is most often caused by an ACTH-secreting pituitary adenoma (pituitary-dependent hypercortisolism; PDH). An interesting target for a selective medical treatment of PDH would be the receptor for ACTH: the melanocortin 2 receptor (MC2R). In this study we investigated whether two peptide compounds, BIM-22776 (#776) and BIM-22A299 (#299), are effective MC2R antagonists in vitro. Their effects on cortisol production and mRNA expression of steroidogenic enzymes, MC2R and melanocortin 2 receptor accessory protein (MRAP) were evaluated in primary adrenocortical cell cultures (n?=?8) of normal canine adrenal glands. Cortisol production stimulated by 50 nM ACTH was dose-dependently inhibited by #299 (inhibition 90.7?±?2.3% at 5 μM) and by #776 (inhibition 38.0?±?5.2% at 5 μM). The ACTH-stimulated mRNA expression of steroidogenic enzymes, MC2R and MRAP was significantly inhibited by both compounds, but most potently by #299. These results indicate that canine primary cell culture is a valuable in vitro system to test MC2R antagonists, and that these compounds, but especially #299, are effective MC2R antagonists in vitro. To determine its efficacy in vivo, further studies are warranted. Antagonism of the MC2R is a promising potential treatment approach in canine PDH.     

Expression of thymic stromal lymphopoietin in canine atopic dermatitis

Background – In humans, thymic stromal lymphopoietin (TSLP) plays a central role in the development of allergic inflammation, such as atopic dermatitis (AD), but it is unknown whether it is involved in the pathogenesis of canine AD (CAD). Hypothesis/Objectives – Our aim was to characterize canine TSLP and to assess its expression in CAD. Methods – Canine TSLP was identified based on sequence homology with human TSLP and the complementary DNA (cDNA) cloned by RT‐PCR. Real‐time quantitative RT‐PCR was established to assess the expression of canine TSLP in cultured canine keratinocytes and in skin biopsy specimens from lesional and nonlesional skin of 12 dogs with CAD and eight healthy control dogs. Results – Partial canine TSLP cDNA was cloned and characterized. It contained four exons that shared 70 and 73% nucleotide identity with human and equine TSLP, respectively, encoding the signal peptide and full‐length secreted protein. We found significantly increased TSLP expression in lesional and nonlesional skin of dogs with CAD compared with healthy control dogs (P < 0.05), whereas no difference was measured between lesional and nonlesional samples. In cultured primary canine keratinocytes, we found increased TSLP expression after stimulation with house dust mite allergen extract or Toll‐like receptor ligands lipopolysaccharide and poly I:C. Conclusions and clinical importance – Increased TSLP expression in the skin of dogs with CAD supports an involvement of TSLP in the pathogenesis of CAD similar to that in humans. Further studies should elucidate the function and therapeutic potential of TSLP in CAD.     

FC‐33 Expression of IL‐12 receptor β2 gene in atopic dogs

Atopic dermatitis (AD) is very common in dogs, but its pathogenesis is not yet fully understood. It has been suggested that a Th2‐dominant status may be associated with the occurrence of canine AD. IL‐12 is thought to be important for the differentiation of Th1 cells. The IL‐12 receptor β2 (IL‐12Rβ2) gene is considered to play a critical role in signal transduction and is attracting attention as one of the causative genes of AD in humans. The purpose of this study was to investigate the relationship between IL‐12Rβ2 gene expression and canine AD. The canine IL‐12Rβ2 gene was cloned by RT‐PCR and its nucleotide sequences were determined. Canine IL‐12Rβ2 showed 76.8% homology at the amino acid level with human IL‐12Rβ2, and its structural motifs were well conserved. cDNA with a 91 bp deletion including the transmembrane region was also cloned, which consequently produced a frame shift and an early stop codon. The deletion region corresponded to exon 14 of the human IL‐12Rβ2 gene on chromosome 1. The expression of deleted canine IL‐12Rβ2 mRNA in phytohemagglutinin‐stimulated peripheral blood mononuclear cells was examined in seven healthy dogs and 11 AD dogs. Both deleted and intact mRNAs were expressed at constant ratios in healthy and AD dogs. The results indicate that the deletion of the transmembrane region is not associated with the occurrence of AD, and that the expression of the deleted mRNA may be constitutive and produced by alternative splicing. Funding: Self‐funded.     

Obesity,expression of adipocytokines,and macrophage infiltration in canine mammary tumors

Obesity influences the development, progression and prognosis of human breast cancer and canine mammary cancer (MC) but the precise underlying mechanism is not well-documented in the fields of either human or veterinary oncology. In the present study, the expression of major adipocytokines, including leptin, adiponectin, and leptin receptor (ObR) in benign (n = 28) and malignant (n = 70) canine mammary tumors was investigated by immunohistochemistry and on the basis of the subject's body condition score (BCS). To evaluate the relationship between obesity and chronic inflammation of the mammary gland, macrophages infiltrating within and around tumoral areas were counted.The mean age of MC development was lower in overweight or obese dogs (9.0 ± 1.8 years) than in lean dogs or optimal bodyweight (10.2 ± 2.9 years), and the evidence of lymphatic invasion of carcinoma cells was found more frequently in overweight or obese group than in lean or optimal groups. Decreased adiponectin expression and increased macrophage numbers in overweight or obese subjects were significantly correlated with factors related to a poor prognosis, such as high histological grade and lymphatic invasion. Leptin expression was correlated with progesterone receptor status, and ObR expression was correlated with estrogen receptor status of MCs, regardless of BCS. Macrophage infiltration within and around the tumor may play an important role in tumor progression and metastasis in obese female dogs and may represent a prognostic factor for canine MCs.     

Molecular cloning of canine thymus and activation-regulated chemokine (TARC) gene and its expression in various tissues

Thymus and activation-regulated chemokine (TARC) is known as a functional ligand for CC chemokine receptor 4 (CCR4), which is selectively expressed on Th2 lymphocytes and induces selective migration of the cells to allergic lesions. In this study, we cloned canine TARC cDNA from canine thymus by RT-PCR with rapid amplification of cDNA ends (RACE) method. The canine TARC clone contained a full-length open reading frame encoding 99 amino acids and included four cysteine residues characteristic to CC chemokine family. The canine TARC cDNA showed 77.5%, 67.4%, and 68.5% amino acid sequence similarity with human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lymph node, lung and heart of the various normal dog tissues examined. TARC cDNA clone obtained in this study will be useful for further investigation on allergic diseases in dogs.     

水貂黑素皮质素受体-4基因部分序列的克隆测序

对水貂黑素皮质素受体-4(MC4R)基因进行了克隆和序列分析,以期为进一步开展水貂MC4R基因与其肥胖性状的相关分析及基因定位等研究提供理论基础。首先采用特定引物对水貂MC4R基因进行PCR扩增、克隆和测序,然后用DNAMAN软件拼接MC4R基因全序列,并进行序列的同源性分析。试验获得水貂MC4R基因序列816 bp。水貂MC4R基因序列与同一种属的水獭的同源性最高,达96%,而与犬、狐、大熊猫、河马、野猪、山羊、人及小鼠这些哺乳动物的同源性在89%～95%之间,另外,与贝加尔湖海豹和加州海狮的同源性也较高,均为94%。本研究成功克隆了水貂的MC4R基因,结果表明,其在物种间具有较高的保守性。     

Validation of a species-optimized enzyme-linked immunosorbent assay for determination of serum concentrations of insulin in dogs

Background: Measurement of canine serum insulin has relied on methods developed to measure human insulin. A species‐optimized test for measurement of serum insulin in dogs is now commercially available. Objective: The purpose of this study was to validate the canine ELISA for determination of serum insulin concentration in dogs. Methods: Precision was determined by evaluating intra‐ and interassay coefficient of variation (CV), and accuracy was determined by dilution and spike recovery studies. A method comparison study with samples from 34 clinically healthy dogs and 73 dogs examined for various illnesses and disorders (“patients”) was performed using the canine ELISA and an ELISA for human insulin. Biologic relevance of the canine assay was evaluated by measuring insulin in samples collected from 8 healthy dogs after administration of glucagon. A stability study was preformed with 6 samples stored at 20°C, 4–8°C, and ?20°C. Results: For the canine ELISA, intra‐ and interassay CVs were 4.3–7.8% and 4.4–7.7%, respectively. Mean recovery after dilution was 99% and recovery after spiking with porcine insulin was 116%. The canine and human ELISAs correlated well (r²=.94 for healthy dogs, r²=.88 for patient samples). After glucagon injection serum insulin concentrations increased significantly in 8 dogs. Insulin was stable for 30 days in 6 serum samples stored at ?20°C and in most samples for 8 days at 4–8°C. Insulin was stable for <3 days at room temperature (20°C). Conclusions: The new canine serum insulin ELISA had good precision and accuracy and correlated well with the previously used assay.     

R132 mutations in canine isocitrate dehydrogenase 1 (IDH1) lead to functional changes

Glioma is the second most common intracranial neoplasia in dogs, but the pathogenic mechanisms remain unclear. In humans, isocitrate dehydrogenase 1 (IDH1) is frequently mutated in gliomas. Although almost all human IDH1 mutations have been identified as involving the Arg132 codon, few studies have reported structural, functional, and mutational information for canine IDH1. Therefore, in this study, we cloned the canine IDH1 homologue and used PCR mutagenesis to substitute the wildtype (WT) Arg132 with His (R132H) or Ser (R132S). WT and mutated IDH1 were overexpressed in HeLa cells, and their presence was confirmed by immunoblotting and immunocytochemistry using mutation-specific antibodies. The IDH1 activity between WT, R132H, and R132S transfectants was compared by measuring the production of NADH and NADPH. NADPH production in R132H and R132S transfectants was lower than that in WT, but NADH levels were not significantly different. Finally, we detected increased expression of hypoxia inducible factor 1 alpha (HIF-1α) in the R132H and R132S transfectants. These results indicated that the canine IDH1 Arg132 mutation has the potential to induce carcinogenesis in canine somatic cells.     

Association of single‐nucleotide polymorphism of melanocortin gene with feed intake in rabbit (Oryctolagus cuniculus)

The aim of the study was to investigate the association of two parts of melanocortin gene (MC4R‐1, MC4R‐2) and feed intake for V‐line rabbits. V‐line rabbits were grouped into high and low daily feed intake during the period from 30 to 63 days of age in order to identify MC4R SNPs useful for association study with feed intake. DNA from blood samples of each group was extracted to amplify the MC4R gene. The purified PCR products were sequenced in those had the highest and lowest feed intake. Alignment of sequence data from each group revealed that there is a variation detected in MC4R‐1 at nucleotide 35 (T‐G) (sense mutation) and another variation was detected in MC4R‐2 gene at nucleotide 19 (T‐C) (sense mutation) for high feed intake rabbits. These sense mutations lead to transform some amino acids and cause a significant change of the MC4R function. The results of average daily feed intake (ADFI) indicated that group (1) had significantly higher feed intake than group (2) of V‐line rabbits. The detected mutations and the analysis of daily feed intake means revealed a significant association between MC4R polymorphism and feed intake in rabbits.     

陆川猪黑皮质激素受体4基因克隆及序列分析

试验旨在对陆川猪黑皮质激素受体4（melanocortin-4 receptor,MC4R）基因进行克隆及相关信息学分析。通过提取陆川猪背最长肌总RNA,采用RT-PCR、克隆等方法获得含目的基因MC4R的质粒pMD18-T-MC4R,经菌落PCR和测序鉴定正确后,应用相关生物信息学软件对陆川猪MC4R基因的理化性质、蛋白质的结构、修饰结构和亚细胞定位等进行预测分析。结果表明,MC4R基因CDS区长999 bp,编码332个氨基酸,与NCBI上公布的野猪MC4R基因序列中的CDS区存在4个碱基差异,其中175和906 bp处为同义突变,110和278 bp处为错义突变,分别引起第37位谷氨酸变为甘氨酸和第93位缬氨酸变为丙氨酸。同源性比对结果发现,MC4R基因在不同物种及进化的过程中具有较高的保守性。陆川猪MC4R蛋白有明显的疏水区域,不存在信号肽,但有7个跨膜结构域,其编码蛋白的二级结构元件有α-螺旋、延伸链、β-转角和无规则卷曲。修饰结构预测表明,MC4R蛋白存在多处N糖基化位点,但无O糖基化位点,可能主要分布于内质网和囊泡。本研究成功克隆了陆川猪MC4R基因,为更好地开发利用地方品种陆川猪及其繁育奠定理论基础。     

Mutations in the melanocortin 1 receptor, β-defensin103 and agouti signaling protein genes, and their association with coat color phenotypes in Akita-inu dogs

To identify factors that control coat color in Akita-inu dogs, we sequenced all the exons of the melanocortin 1 receptor (MC1R), β-defensin103 (CBD103) and agouti signaling protein (ASIP) genes of dogs with four distinct coat colors, namely, brindle, sesame, red and white. Then we examined correlations among specific alleles and coat color. In the case of the MC1R gene, all white dogs were homozygous for a nonsense mutation, R306ter, while brindle, sesame, and red dogs had at least one R306 allele. In the case of the CBD103 gene, all brindle dogs were heterozygous for the G23del mutation (deletion of codon 23, encoding glycine), while all sesame and red dogs were homozygous for G23. In the case of the ASIP gene, all dogs, regardless of coat color, had at least one S82 H83 allele. A missense mutation in the ASIP gene, P87L, was identified for the first time in some Akita-inu dogs but was not associated with any specific coloration. Our results indicate that the 2 key mutations, R306ter in the MC1R gene and G23del in the CBD103 gene, are associated with the phenotypic discriminations among brindle, red/sesame, and white coats, while no mutation that might potentially be associated with the discrimination of a sesame coat from a red coat is present in the coding sequences of these three genes.     

犬细小病毒病原分离及分型研究

为查明4份疑为患细小病毒病军犬的病原及其特性,为进一步的免疫研究奠定基础,本试验将4份送检的犬肠道内容物过滤后分别接种猫肾细胞(F81),培养5 d后,未出现细胞病变的带毒盲传。同时提取病料的总DNA,用犬细小病毒的VP2特异性引物进行PCR扩增,PCR阳性产物克隆至pMD18-T载体测序,并与已知参考毒株序列进行比对及系统发育分析。测序结果表明,用F81细胞分离到4株细小病毒;经VP2基因比对分型结果表明,4株细小病毒毒株均属CPV-2a,分别命名为CPV-JQ、CPV-CM、CPV-M和CPV-KM。