Comparison of insulin signaling gene expression in insulin sensitive tissues between cats and dogs

Diabetes mellitus (DM) is a common endocrine disease in cats and dogs with increasing prevalence. Type 1 DM appears to be the most common form of diabetes in dogs whereas Type 2 DM prevails for cats. Since insulin resistance is more frequently encountered in cats than dogs, our laboratory was interested in determining whether differences at the insulin signaling pathway level and differences in glucose and lipid metabolism could be observed between cats and dogs. Insulin resistance has been positively correlated to insulin signaling pathway abnormalities. As such, this study measured insulin receptor substrate-1 (IRS-1), insulin receptor substrate-2 (IRS-2), and phosphatidylinositol 3-kinase (PI3-K) P-85α mRNA expression levels in classical insulin-responsive sensitive tissues (liver, skeletal muscle, and abdominal fat) and peripheral leukocytes between cats and dogs by qRT-PCR. Different tissues were sampled because it is currently unknown where insulin-resistance arises from. In addition, enzymes involved in glucose and lipid metabolism, malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH) and fatty acid synthase (FAS) were also assessed since glucose and lipid metabolism differs between cats and dogs. Overall, IRS-1, IRS-2, PI3-K, MDH, G6DPH, and FAS mRNA tissue expression profiles demonstrated different levels of expression, in various tissues for both canines and felines, which was expected. No distinct expression pattern emerged; however, differences were noted between canines and felines. In addition, IRS-1, IRS-2, PI3-K, MDH, G6DPH, and FAS mRNA expression was significantly higher in canine versus feline tissues, including peripheral leukocytes. Remarkable differences in insulin signaling gene expression between felines and canines indicate that cats may have an underlying low insulin sensitivity level due to low IRS-1, IRS-2, and PI3-K P-85α mRNA expression levels which would predispose cats to develop insulin resistance. Moreover, differences in glucose and lipid metabolism related gene expression (MDH, G6DPH, and FAS) demonstrate that felines have an overall lower metabolic rate in various tissues which may be attributed to overall lower insulin signaling gene expression and a lack of physical activity as compared to canines. Therefore, a combination of genetic and environmental factors appears to make felines more prone to suffer from insulin resistance and type 2 DM than canines.     

Prevalence,risk factor analysis,and hematological findings of hemoplasma infection in domestic cats from Valdivia,Southern Chile

Four distinct cat hemoplasma species are recognized worldwide. However, this is the first study to investigate the prevalence, risk factors, and hematological findings of hemoplasmas in cats from Chile. Complete blood count and 16S rRNA real-time PCR for cat hemoplasma species were performed in 384 blood samples from domestic cats in Valdivia, Chile. Among the 384 samples the species-specific prevalence was as follows: ‘Candidatus Mycoplasma haemominutum’ (7.8%), Mycoplasma haemofelis (4.4%), ‘Candidatus Mycoplasma turicensis’ (1%), ‘Ca. M. haemominutum’ + M. haemofelis (0.78%), ‘Ca. M. haemominutum’ + ‘Ca. M. turicensis’ (0.52%), ‘Ca. M. haemominutum’ + ‘Candidatus Mycoplasma haematoparvum’ (0.26%) and ‘Ca. M. haemominutum’ + M. haemofelis + ‘Ca. M. haematoparvum’ (0.26%). Male sex, older age, outdoor access, and FIV status were risk factors for hemoplasmosis. Mycoplasma haemofelis-positive cats had higher mean corpuscular volume and monocyte count. Four hemoplasma species circulate in the cat population of Valdivia. ‘Candidatus M. turicensis’ and ‘Ca. M. haematoparvum’ have been reported for the first time in Chilean cats.     

A validation of 10 feline reference genes for gene expression measurements in snap-frozen tissues

For a proper determination of relative mRNA expression levels with real-time quantitative PCR (Q-PCR) internal standards, such as the expression of reference genes, are of utmost importance. For cats, in contrast to dogs, no validation of reference genes has been published. Our goal was to evaluate frequently used reference genes for the analysis of relative mRNA levels from feline tissues in a SYBR Green-based Q-PCR protocol. First, primers were optimized on mRNA-derived cDNA from liver and kidney tissues of randomly chosen (healthy and diseased) cats. Then, the expression variation and stability of each reference gene within a specific tissue was determined. Dental roots and crowns, heart (left ventricle), renal, liver, lung, and mammary gland tissues from 3 to 11 cats of different breeds, sexes, ages, and disease status were included in this study. Averaging relative stabilities over these six tissues revealed the usefulness of each tested gene as reference gene. In order to compensate for the expression variation of a reference gene within a specific tissue, as much as six reference genes (e.g. RPL17, RPL30, RPS7, YWHAZ, and HPRT) were required to obtain highly reliable data in cat tissues. The optimal set of reference genes depended on the tissue analyzed and should, ideally, be selected and evaluated at the start of each experimental condition. A comparison with a similar evaluation in dogs revealed three issues: (i) most ribosomal genes are suitable in both species; (ii) good non-ribosomal reference genes differ; (iii) more feline than canine reference genes are required for proper analysis.     

Comparison of nested PCR and real-time PCR for the detection of Toxoplasma gondii in biological samples from naturally infected cats

There are different protocols of molecular diagnosis methods available including DNA extraction methods to diagnose of Toxoplasma gondii, being necessary to perform comparative studies in biological samples. The aim of this study is to compare real-time PCR (rtPCR) and nested PCR (nPCR) to evaluate the detection of T. gondii in naturally infected cats. Biological samples of Toxoplasma-seropositive cats were assayed for detection of T. gondii DNA - extracted by both the lysis buffer and proteinase K (LB proteinase K) method and the acid guanidinium thiocyanate (GuSCN) method - using rtPCR and nPCR. T. gondii DNA was detected by nPCR in 43.6% and 40.8% of the samples from which it was extracted by the LB proteinase K and the GuSCN method, respectively. With rtPCR these figures fell significantly to 33.8% and 14.1%. Despite of nPCR showed higher sensitivity, the agreement observed between two PCRs was good; this agreement, however, was affected by the DNA extraction method used, LB proteinase K method showed better results.     

Oral glucose leads to a differential response in glucose,insulin, and GLP-1 in lean versus obese cats

The response to oral glucose was examined in 10 obese and 9 lean age-matched, neutered cats. In all cats, oral administration of 2 g/kg glucose was followed by a prompt increase in glucose, insulin, and glucagon-like peptide (GLP)-1. There were significant differences between lean and obese cats in the areas under the curve for glucose, insulin, and GLP-1. However, the responses were variable, and a clear distinction between individual lean and obese cats was not possible. Therefore, this test cannot be recommended as a routine test to examine insulin resistance in individual cats as it is used in people. A further disadvantage for routine use is also the fact that this test requires gastric tubing for the correct administration of the glucose and associated tranquilization to minimize stress and that it was associated with development of diarrhea in 25% of the cats. GLP-1 concentrations were much lower in obese than lean cats. The low GLP-1 concentrations in obese cats might indicate a contribution of GLP-1 to the lower insulin sensitivity of obese cats, but this hypothesis needs to be further investigated.     

High prevalence of Giardia detected in cats by PCR

Microscopy, PCR and a Giardia CELISA test were used to determine the prevalence of Giardia in 40 faecal samples obtained from domestic cats in the Perth metropolitan area. A prevalence of 5, 80 and 60% was found by the tests, respectively. The results show that more sensitive techniques such as PCR may be necessary, and may yield more reliable results, in the detection of low levels of Giardia in domestic cats.     

Investigation of Bartonella henselae in cats in Ankara, Turkey

The purpose of this study was to determine Bartonella henselae prevalance in cats in Ankara. Whole bloods and sera collected from 256 cats were investigated for the presence feline Bartonella species by culture and sera were tested for the presence of antibodies against B. henselae IgG using immunofluorescence assay. Bartonella species were isolated by blood culture from 24 (9.4%) cats. Bartonella isolates were subjected to restriction fragment length polymorphism (RFLP) by using TaqI and HhaI endonucleases to identify species. Twenty-one isolates were determined as B. henselae and three of 24 isolates were determined as Bartonella clarridgeiae with RFLP. The bacteraemia prevalence and seroprevalence of B. henselae IgG antibodies in cats was detected as 8.2% and 18.6% respectively. This is the first report on B. henselea and B. clarridgeiae in cats in Turkey.     

骨形态发生蛋白IB型受体(BMPR-IB)基因在蒙古羊卵巢和子宫组织中的表达分析

BMPR-IB基因是发现最早且对绵羊排卵率影响机理已经阐明的多胎主效基因,主要在与繁殖有关的组织和器官中表达,并且对绵羊排卵起重要的调控作用。本研究采用实时荧光定量PCR(Real-time Quantitative PCR,RQ-PCR)技术的相对定量方法对BMPR-IB基因在不同生理时期蒙古羊的卵巢和子宫组织进行了差异表达的研究。定量结果得到-βActin基因的扩增曲线回归方程为y=-3.358x+35.708,回归系数R2=0.990,BMPR-IB基因的扩增曲线回归方程为y=-2.119x+31.424,回归系数R2=0.992。以非发情期蒙古羊的卵巢组织定量结果为对照计算得到BMPR-IB基因在发情期蒙古羊的卵巢组织中表达量最高,在发情期的子宫组织中表达量最低,在发情期卵巢组织中的表达量是非发情期的2.65倍。     

Lipogenic gene expression in abdominal adipose and liver tissues of diet-induced overweight cats

The effect of overweight status on the expression of SREBP-1c and downstream lipogenic genes, such as ATP citrate lyase (ACL) and fatty acid synthase (FAS), in abdominal adipose and liver tissues was determined in cats using a diet-induced weight gain model. ACL and SREBP-1c mRNA expression was significantly reduced (～65% and 20%, respectively) in liver tissue, whereas FAS and SREBP-1c expression was significantly increased (～80% and 45%, respectively) in abdominal omental adipose tissue of overweight animals as compared to healthy animals. Additionally, ACL, FAS, and SREBP-1c expression was significantly reduced by ～50%, 75%, and 70%, respectively, in abdominal subcutaneous adipose tissue of overweight animals. Omental adipose tissue appeared to foster, whereas subcutaneous adipose and liver tissues appeared to defer lipid storage based on differences in SREBP-1c mRNA expression. Overall, reduced lipogenic gene mRNA expression patterns support the hypothesis that SREBP-1c expression is reduced in overweight and possibly obese cats, reflecting down-regulation of the lipogenic pathway to prevent further fat accumulation and weight gain.     

催乳素受体基因mRNA表达与山羊绒毛生长关系的研究

为探讨绒山羊皮肤组织中催乳素受体(prolactin receptor,PRLR)基因mRNA表达水平与绒山羊绒毛生长的关系,通过埋植褪黑激素(me-latonin,MT)刺激绒毛提前生长,利用实时荧光定量聚合酶链式反应(Real-time PCR)技术检测绒山羊从绒毛开始萌发到绒毛快速生长期间皮肤组织中PRLR基因mRNA表达量的变化。结果显示:在内蒙古白绒山羊皮肤中总的催乳素受体(T-PRLR)基因mRNA表达量从6至11月逐渐降低,而MT埋植组的短型催乳素受体(S-PRLR)mRNA表达水平在绒毛快速生长的7、8、9月份显著高于对照组(P<0.05)。结果表明,山羊绒毛生长可能与S-PRLR mRNA表达水平升高有直接的关系。     

Influence of glucose dosage on interpretation of intravenous glucose tolerance tests in lean and obese cats

Intravenous glucose tolerance tests (IVGTTs) are used in cats and other species to assess insulin sensitivity. Several dosages have been reported but the dosage that maximally stimulates insulin secretion in cats has not been determined nor has it been compared in lean and obese animals. IVGTTs were performed in 4 lean and 4 obese spayed female cats with 5 glucose dosages: 0.3 (A), 0.5 (B), 0.8 (C), 1.0 (D). and 1.3 (E) g/kg body weight (BW). Each cat received each dosage in a random design. The glucose disposal rate was significantly different only between lean and obese cats at the highest glucose dosage. The area under the curve for insulin increased significantly among A, B, C, and D in lean and among A, B, and C in obese cats but not between D and E in lean and among C, D, and E in obese cats. Baseline insulin secretion was significantly higher (P = .03) and 1st peak insulin secretion was approximately 50% lower in obese as compared to lean cats (P = .03). Lean but not obese cats reached baseline insulin concentrations at all dosages at 120 minutes. We conclude that the glucose dosage for maximal insulin secretion is 1.0 g/ kg BW in lean and 0.8 g/kg BW in obese cats, supporting routine use of 1 g/kg BW to maximally stimulate insulin secretion regardless of body composition. Obese cats showed an abnormal insulin secretion pattern, indicating a defect in insulin secretion with obesity and insulin resistance.     

蒙古马不同组织器官TLR1、TLR2、TLR4和TLR6 mRNA转录水平研究

根据GenBank中马Toll样受体基因序列设计特异性引物,建立检测马Toll样受体（TLRs）mRNA转录水平的SYBR GreenⅠ实时荧光定量PCR方法,检测TLR4、TLR2、TLR1和TLR6在蒙古马不同组织器官中的转录水平。4种TLRs在心脏、肝脏、脾脏、肺脏、肾脏、胃、十二指肠、空肠、盲肠和骨髓中均有转录。其中,TLR4mRNA除在空肠和肝脏外,在其他组织器官中的表达水平均高于TLR2、TLR1、TLR6。免疫器官中,TLR4、TLR1mRNA在骨髓中表达量高于脾脏,而TLR2、TLR6mRNA在脾脏中表达量高于骨髓。各肠段,TLR4、TLR2、TLR1、TLR6mRNA表达水平之间在空肠的差异不是很大,而在十二指肠和盲肠中差异很大。结果表明,TLRs mRNA在马各组织器官转录水平差异较大,可能与其对病原体的识别和抵抗能力有关。     

捻转血矛线虫ES24抗原基因的克隆与表达特性分析

根据捻转血矛线虫ES24抗原基因序列(U64793.1)设计1对特异性引物,用RT-PCR方法扩增出大小约为670 bp的DNA片段。将该DNA片段克隆到pMD18-T载体后进行序列测定和分析,结果发现该基因与GenBank中已知的捻转血矛线虫24 ku ES抗原基因的相似性达96%～98%。将该基因的开放阅读框插入pET28a(+)载体中,获得原核表达质粒pET28/ES24,并转化大肠杆菌BL21。重组细菌用IPTG诱导,经SDS-PAGE分析,结果表明该基因获得了表达,重组蛋白分子量大小约为25 ku。用实时荧光定量PCR技术对该基因在捻转血矛线虫的虫卵、第3期幼虫、雌虫和雄虫等不同发育阶段、不同性别虫体内的表达情况进行了定量分析,结果显示ES24基因在雄性成虫中表达量最高,雌虫和虫卵其次,在第3期幼虫中表达最低。     

IGF-1和IGF-1R基因在辽宁绒山羊皮肤组织中的表达

为了研究类胰岛素生长因子1(IGF-1)和类胰岛素生长因子1受体(IGF-1R)基因在绒山羊绒毛生长不同阶段皮肤组织中的表达差异,试验采用实时荧光定量PCR技术对非产绒期和产绒期辽宁绒山羊皮肤中IGF-1和IGF-1R基因的表达进行了测定。结果表明:辽宁绒山羊皮肤中IGF-1和IGF-1R mRNA的表达量在产绒期均极显著高于非产绒期(P<0.01),产绒期IGF-1、IGF-1RmRNA的表达水平分别是非产绒期的8.30倍和7.49倍。说明IGF-1和IGF-1R基因可能与山羊绒周期性生长有关。     

β-defensins gene expression in tissues of the crossbred and Tibetan pigs

Delayed resumption of postpartum oestrous cycles in dairy cows is linked to low plasma insulin status and associated changes in ovarian function. Previous studies showed that plasma insulin can be raised by increasing dietary starch concentration, but it is not known if rumen digestible starch and bypass starch would be equally effective. The objective of the two experiments reported here was to investigate whether site of dietary starch digestion influences metabolic hormones and ovarian function. Cows were fed on a standard diet from calving until 40 days postpartum and then fed on treatment diets until 70 days postpartum. In Experiment 1, six cows were transferred to each of five diets with a starch content of 190 g/kg DM, of which 0.40, 0.45, 0.50, 0.55 or 0.60 was bypass starch. In Experiment 2, eight cows were transferred to each of four diets containing concentrates with low (L) or high (H) starch content and predominantly grass (G) or maize (M) silages in a factorial design. Between 60 and 70 days postpartum, plasma insulin was not influenced by site of starch digestion in Experiment 1, but insulin was increased by H (P = 0.020) and M (P = 0.048) in Experiment 2. Dietary bypass starch content negatively influenced plasma glucagon concentration (P = 0.046) and the insulin to glucagon ratio (P = 0.005) in Experiment 1. Growth hormone, insulin-like growth factor-I and leptin did not vary among diets in either experiment. The number of small (< 5 mm) ovarian follicles before ovulation tended (P = 0.056) to decrease with increasing dietary bypass starch content and was negatively related (P = 0.046) to plasma glucagon in Experiment 1, but there was no other dietary effect on ovarian function in either experiment. The main conclusion from these experiments is that rumen digestible starch and rumen bypass starch can be equally effective for maintaining plasma insulin and ovarian function of high-yielding dairy cows in early lactation.     

Evaluation of endogenous control genes for gene expression studies across multiple tissues and in the specific sets of fat- and muscle-type samples of the pig

To normalize a set of quantitative real-time PCR (q-PCR) data, it is essential to determine an optimal number/set of housekeeping genes, as the abundance of housekeeping genes can vary across tissues or cells during different developmental stages, or even under certain environmental conditions. In this study, of the 20 commonly used endogenous control genes, 13, 18 and 17 genes exhibited credible stability in 56 different tissues, 10 types of adipose tissue and five types of muscle tissue, respectively. Our analysis clearly showed that three optimal housekeeping genes are adequate for an accurate normalization, which correlated well with the theoretical optimal number (r ≥ 0.94). In terms of economical and experimental feasibility, we recommend the use of the three most stable housekeeping genes for calculating the normalization factor. Based on our results, the three most stable housekeeping genes in all analysed samples (TOP2B, HSPCB and YWHAZ) are recommended for accurate normalization of q-PCR data. We also suggest that two different sets of housekeeping genes are appropriate for 10 types of adipose tissue (the HSPCB, ALDOA and GAPDH genes) and five types of muscle tissue (the TOP2B, HSPCB and YWHAZ genes), respectively. Our report will serve as a valuable reference for other studies aimed at measuring tissue-specific mRNA abundance in porcine samples.     

Mutations in the 3c and 7b genes of feline coronavirus in spontaneously affected FIP cats

Feline infectious peritonitis (FIP) is the most frequent lethal infectious disease in cats. However, understanding of FIP pathogenesis is still incomplete. Mutations in the ORF 3c/ORF 7b genes are proposed to play a role in the occurrence of the fatal FIPV biotype. Here, we investigated 282 tissue specimens from 28 cats that succumbed to FIP. Within one cat, viral sequences from different organs were similar or identical, whereas greater discrepancies were found comparing sequences from various cats. Eleven of the cats exhibited deletions in the 3c gene, resulting in truncated amino acid sequences. The 7b gene was affected by deletions only in one cat. In three of the FIP cats, coronavirus isolates with both intact 3c genes as well as 7b genes of full length could also be detected. Thus, deletions or stop codons in the 3c sequence seem to be a frequent but not compelling feature of FIPVs.