山本 一郎

G protein-coupled receptor 84 (GPR84) was cloned as an orphan receptor, and medium-chain fatty acids were then revealed as endogenous ligands. GPR84 is expressed in immune cells and is believed to protect liver function from lipotoxicity caused by overeating and high-fat diet intake. This study aimed to present the molecular characterization of GPR84 in domestic cats. The deduced amino acid sequence of the feline GPR84 shows high sequence homology (83-89 %) with the orthologues from other mammalians by cDNA cloning of feline GPR84. Remarkably high mRNA expression was observed in the bone marrow by Q-PCR analysis. The inhibition of intracellular cAMP concentration was observed in cells transfected with feline GPR84 and treated with medium-chain fatty acids. Immunostaining of GPR84 and free fatty acid receptor 2 (FFAR2)/GPR43 in the bone marrow, where high mRNA expression was observed, showed reactions in macrophages and myeloid cells. To clarify whether the receptor formed homo/hetero-merization, GPR84 and FFARs were analyzed using Nano-Luc binary technology and NanoLuc bioluminescence resonance energy transfer technologies, which revealed that GPR84 formed more heteromers with FFAR2 than homomers with each other. In addition, when GPR84 and FFAR2/GPR43 were cotransfected in the cell, their localization on the cell membrane was reduced compared with that when single receptors were transfected. These results indicated that GPR84 is a functional receptor protein that is expressed in cat tissues and may have a protein-protein interaction with FFAR2/GPR43 on the cell membrane.

G protein-coupled receptors 41 and 43 were identified and characterized as free fatty acid receptors (FFAR) 3 and 2, respectively. FFAR2 and FFAR3 mediate short-chain fatty acids (SCFAs) as signalling molecules. The present study aimed to give molecular characterization of FFAR2 and FFAR3 in the domestic cat. High homology with that in other mammals was revealed by cDNA cloning of cat FFAR2 FFAR3. We analyzed the tissue distribution of cat FFAR2 and FFAR3 mRNA using quantitative polymerase chain reaction. The inhibition of intracellular cAMP concentrations was observed in cells transfected with cat FFAR2 or FFAR3 and treated with SCFAs. The activation of nuclear factor of activated T cells-luciferase reporter was only observed in cat FFAR2 transfected cells but not in FFAR3. Split luciferase assay (NanoLuc Binary Technology; NanoBiT) for FFAR2 or FFAR3 and Arrestin-3/β-arrestin-2 revealed acetate-/propionate-induced recruitment to cat FFAR2 or FFAR3 in CHO-K1 cells. Our results indicate that FFAR2 and FFAR3 are functional receptor proteins that are expressed in cat tissues and show differential distribution patterns.

Melanocortin 4 receptor (MC4R), which is a member of the G protein-coupled receptor (GPCR) family, mediates regulation of energy homeostasis upon the binding of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system (CNS). Melanocortin 2 receptor accessory protein 2 (MRAP2) modulates the function of MC4R. We performed cDNA cloning of cat MC4R and MRAP2 and characterized their amino acid sequences, mRNA expression patterns in cat tissues, protein–protein interactions, and functions. We found high sequence homology (&gt 88%) with other mammalian MC4R and MRAP2 encoding 332 and 206 amino acid residues, respectively. Reverse transcription-polymerase chain reaction analysis revealed that cat MC4R and MRAP2 mRNA were expressed highly in the CNS. In CHO-K1 cells transfected with cat MC4R, stimulation with α-MSH increased intracellular cyclic adenosine monophosphate (cAMP) concentration in a dose-dependent manner. Furthermore, the presence of MRAP2 enhanced the cat MC4R-mediated cAMP production. These results suggested that cat MC4R acts as a neuronal mediator in the CNS and that its function is modulated by MRAP2. In addition, our NanoBiT study showed the dynamics of their interactions in living cells stimulation with α-MSH slightly affected the interaction between MC4R and MRAP2, and did not affect MC4R homodimerization, suggesting that they interact in the basal state and that structural change of MC4R by activation may affect the interaction between MC4R and MRAP2.

Back ground: Aging is generally associated with alterations in physical activity, weight status and energy metabolism, which predisposes aged individuals to metabolic syndrome. In this manuscript, age effects on energy metabolic indicators of similar physical activity and weight status but of varying ages were investigated. Materials and Methods: Energy metabolic indicators, such as plasma adiponectin, leukocytic AMP-activated protein kinase, plasma malate dehydrogenase and lactate dehydrogenase along with common plasma metabolites, were measured in healthy young (AV = 7.1 years) and aged (AV = 14.1 years) riding horses of similar physical activity, diet and weight status. Malate dehydrogenase and lactate dehydrogenase ratio was also calculated as the indicator of energy metabolism. Results: Plasma adiponectin concentration and leukocytic AMP-activated protein kinase activity in aged horses were significantly lower than those in young horses (p&lt 0.05, Mann-Whitney U test). Although not significant, energy metabolism indicators, malate dehydrogenase, lactate dehydrogenase and their ratio were lower in aged group when compared to those of young group. Conclusion: The present results indicate the decline in energy metabolism with aging in healthy horses even without any visible changes in adiposity. Such changes reflect dysfunction of energy metabolism and predispose the aged individuals to the development of metabolic syndrome.

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.

Background: Obesity and overweight have been frequently observed in dogs and cats in recent years as in humans. The compositions of fatty acids (FAs) in the accumulated lipids in tissues of obese animals may have important roles in the process and mechanisms related to the onset of metabolic disorders. The purpose of this study was to evaluate the effects of a high fat (HF) diet, which contained a higher proportion of saturated FAs, on FA metabolism and distribution in obese cats. Cats (N = 12) were divided into control diet group (crude fat; 16.0 %) (n = 4) or a high fat (HF) diet group (crude fat; 23.9 %) (n = 8). The HF diet contained up to 60 % of calories from fat and was rich in stearic acid. Blood samples were collected at 0, 2, 4 and 6 weeks after the feeding. Adipose and liver tissues were collected at the 6th week after feeding. We performed analysis of histological findings and fatty acid composition in serum and tissues. Results: Body weights of the cats significantly increased in the HF group. The increased activities of hepatic enzymes and the accumulation of lipid droplets were found in hepatocytes in the HF group at the 6th week after feeding. In this study, the stearic acid (C18:0)-rich HF diet contained less oleic acid (C18:1n-9) and more linoleic acid (C18:2n-6) than the control. However, the composition of oleic acid in the liver was higher, and those of stearic acid and linoleic acid were lower in the HF group at the 6th week after feeding. The higher oleic acid: stearic acid ratio suggests an increase in the conversion from saturated FA to mono-unsaturated FAs, which may reflect the hepatic storage of FAs as a relatively harmless form. Conclusion: The stearic acid-rich HF diet increased hepatic lipid accumulation accompanied by the increased of hepatic oleic acid, increased serum oleic acid and activation of hepatic enzymes. These findings could be an important sign of early stages of dyslipidemia and hepatic damage. Also, the higher oleic acid: stearic acid ratio might be related to the increased activity of SCD-1, which suggests that the stearic acid-rich HF diet evoked hepatic lipogenesis in the feline liver.

Nuclear factor kappa B (NF-kappa B) plays an important role in the immune system. The p65 subunit is an important part of NF-kappa B unit, and studies of dog and cat p65 subunits of NF-kappa B (dp65 and cp65) are important in understanding their immune function. In this study, we described the molecular characterization of dp65 and cp65. The dp65 and cp65 complementary DNA encoded 542 and 555 amino acids, respectively, showing a high sequence homology with the mammalian p65 subunit (>87.5%). Quantitative polymerase chain reaction revealed that the p65 messenger RNA is highly expressed in the dog stomach and cat heart and adipose tissue. Functional NF-kappa B promoter-luciferase reporter vectors revealed that our isolated dp65 and cp65 cDNA encodes a functionally active protein. Transiently expressed dp65 and cp65 up-regulated pro-inflammatory cytokine expression levels in dog and cat, respectively. These findings suggest that dp65 and cp65 play important roles in regulating immune function. (C) 2015 Elsevier Ltd. All rights reserved.

Obesity is associated with the metabolic syndrome, diabetes, hypertension and chronic inflammation and early detection of weight gain and prompt intervention are the keys to promoting increased quality of life and longevity in veterinary animals. We evaluated the changes in Malate Dehydrogenase (MDH), Lactate Dehydrogenase (LDH) and MDH/LDH ratio as energy metabolism markers in dogs before and after the 4-week overfeeding trial. The acute weight gain was attained by overfeeding of 2x Daily Energy Requirement (DER) separated into 3 meals/day (overfed group). The experimentally overfed dogs showed about 28.2% increase in the Body Weight (BW), the increase of Body Condition Score (BCS) from 1.9-3.4 and significant elevations were noted in Triglyceride (TG), total cholesterol (TC), glucose (GLU) alkaline phosphatase (ALB), Blood Urea Nitrogen (BUN), leukocyte MDH and LDH. Although not significant, both the plasma MDH and LDH activities decreased, whereas leukocytic MDH and LDH activities increased in the overfed group after the feeding trial. Both the resultant plasma and leucocytic M/L ratios showed mild increase in the over-fed group after the feeding trial. In conclusion, assays of MDH, LDH and M/L ratio on plasma and leukocytes are not sensitive as diagnostic tools for detecting acute weight gain. The diagnostic significance of the above mentioned parameters should be further examined on various types of weight gain and target tissues.

G-protein-coupled receptors (GPRs) 40 and 120 are members of the Free Fatty Acid (FFA) receptor group and are termed FFAR1 and FFAR4, respectively. The aim of this study was to clone cat GPR40 and GPR120 cDNAs in several tissues. There was high sequence homology to other mammalian GPR40 and GPR120, with encoding 320 and 361 amino acid residues, respectively. Cat GPR40 encoded extra 21 amino acid residues in the C-terminal cytoplasmic region. Quantitative RT-PCR revealed expression of GPR40 mRNA in the duodenum, liver and pancreas. The GPR120 mRNA was expressed in adipose tissues, cerebral cortex and colon. In conclusion, GPR40 and GPR120 were well conserved and were expressed in cat tissues with different distribution patterns.

Background: The aim of this study is to compare metabolic parameters, malondialdehyde as a lipid oxidation marker, and lipid profiles between dogs with untreated hyperlipidemia and hyperlipidemia with treatment, in order to examine the usefulness of malondialdehyde and lipid profiles as diagnostic parameters at early stages of hyperlipidemia. Results: Dog samples were collected from four different veterinary clinics across Japan from March to June 2013. They were separated into three groups: control, untreated hyperlipidemia based on temporally screening, and hyperlipidemia with current anti-hyperlipidemic (statins and fibrates) treatment. Triglyceride levels of untreated hyperlipidemia dogs were significantly higher than those of control dogs. ALT levels of hyperlipidemic dogs with treatment were the highest among three groups. VLDL and LDL of both cholesterol and triglyceride of untreated hyperlipidemia dogs were the highest among three groups. HDL1 levels in triglyceride of hyperlipidemia dogs with treatment were significantly higher than those of control and untreated hyperlipidemia dog. Malondialdehyde concentrations of untreated hyperlipidemia dogs were significantly higher than those of control and hyperlipidemic dogs with treatment. Conclusions: In this study, dogs with untreated hyperlipidemia clearly showed abnormal lipid status, whereas hyperlipidemic dogs under anti-hyperlipidemia treatment showed more normal lipid status suggesting the effectiveness of the therapy. Anti-hyperlipidemics (statins and fibrates) for dogs are also effective in relieving elevated levels of lipids and lipid oxidation. Plasma lipid (triglyceride and cholesterol) profiles and malondialdehyde are useful diagnostic tools for identifying early stages of untreatment hyperlipidemia in dogs.

Background: In dogs, occurrence of lipid metabolism disorders such as obesity and diabetes mellitus has increased markedly in recent years. Hyperlipidemia has been regarded as a common characteristic for obese animals and hyperlipidemic condition may be associated with inflammation, oxidative stress and lipid composition changes. In this study, we investigated the changes in plasma cholesterol and triglyceride (TG) profiles and metabolite concentrations in 24 dogs (young group: 0-7 years old, n = 12, aged group: 8-13 years old, n = 12). Results: Plasma adiponectin (ADN) concentrations were significantly lower in aged dogs than those in young dogs (mean +/- SD, 17.2 +/- 10.0 mu g mL(-1) vs 29.3 +/- 12.5 mu g mL(-1), respectively; P < 0.05). Although there were no significant differences statistically, aged dogs showed significantly higher plasma alpha1-acid glycoprotein (alpah1-AG) levels compared to those in young dogs. Plasma cholesterol lipoprotein and TG lipoprotein were divided into four fractions by biphasic agarose gel electrophoresis technique. The levels of the third TG-lipoprotein fraction from the positive pole (TG Fraction 3) were significantly higher in aged dogs than in young dogs (mean +/- SD, 143.0 +/- 109.3 mg dL(-1) vs 55.2 +/- 31.3 mg dL(-1), respectively; P < 0.05). On the correlation coefficient analysis by Peason's method, moderate positive correlations were seen between the age and TG (r = 0.446, P = 0.029), TG Fraction 3 (r = 0.516, P = 0.010), malondialdehyde (r = 0.146, P = 0.043), alpha-1 AG (r = 0.448, P = 0.028) levels, respectively. Moderate negative correlations were seen the age and total cholesterol (TC) Fraction 2 (r = -0.446, P = 0.029), glucose (r = -0.637, P = 0.001), ADN (r = -0.408, P = 0.048), respectively. Conclusions: Present data suggest biochemical characteristics of lipid metabolism disorder may be affected by aging in dogs.

The objective of this study was to determine whether Peripheral Blood Leukocytes (PBL) are sensitive enough to detect early signs of diet induced obesity related changes occurring in insulin sensitive tissues, such as abdominal omental and subcutaneous adipose, liver and skeletal muscle, by comparing transcriptome profiles of insulin signaling (IRS-1, IRS-2 and PI3-K p85 α), adiponectin signaling (AdipoR1 and AdipoR2), energy homoeostasis (G6PDH and MDH) and sterol metabolism (FASN) genes as determined by RT-PCR in cats fed on High Fat (HF) diet. Regarding PBL concordance, using a HF diet induced obesity cat model, out of seven genes examined, concordance was observed with ~60% (5 out of 8) of them (IRS-1, IRS-2, Adipo-R1, Adipo-R2 and MDH) between PBL and tissue transcriptomes. HF diet cat PBL IRS-1 and IRS-2 mRNA expression were both reduced, when compared to control diet which was in concordance with reduced IRS-1 and IRS-2 mRNA expression in both abdominal and subcutaneous adipose of HF diet cats. Similar to IRS-1 and IRS-2, AdipoR1 and AdipoR2 mRNA expression in HF diet cats was also reduced, when compared to control diet which was in concordance with reduced AdipoR1 and AdipoR2 in liver and skeletal muscle, respectively of HF diet cats. Lastly, PBL MDH mRNA expression was reduced and was concordant with reduced mRNA expression in liver and skeletal muscle. Overall, our results demonstrate that PBL are sufficiently sensitive to high fat diet induced alterations to transcriptomes of insulin sensitive tissues and can serve as surrogate tissue for various insulin sensitive tissues. © 2014 Academic Journals Inc.

The efficiency of licorice flavonoid supplement as anti-obesity substance was investigated in 14 client-owned dogs. This flavonoid supplement reduced whole and visceral fat in overweight humans. All experimental dogs were given the supplement at a dose of 10 mg kg-1 day-1 in the evening with food for 8 weeks. After 8 weeks post-administration, the body weight was decreased in 11 animals (78.6%). The decreasing rate was 2.51±0.6% (Mean±SE). Serum glucose (GLU), Triglyceride (TG), Malondialdehyde (MDA) concentrations and aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Glutathione peroxidase (GSHPx) activities were decreased but the changes were not statistically significant. On the contrary, serum adiponectin (ADN) concentrations, though statically significance was not seen, tended to increase after 8 weeks post-administration of the supplement compared to those in baseline. In conclusion, the licorice flavonoid supplement was suggested to show anti-obesity effect accompanied with improvement of lipid metabolism in tissues of dogs.

The prevalence of obese cats has increased because of over calorie diet and physical inactivity. Obesity has been found to be associated with oxidative stress and Reactive Oxygen Species (ROS). Unfortunately oxidative stress status at the early phase of obesity in high fat fed cats is not well understood. The objectives of this study were (1) To evaluate lipid and glucose metabolism using enzymatic, hormonal and oxidative stress biomarkers at the early obese phase of cats fed on a high-fat diet and (2) To identify rapidly changing variables to use as a diagnostic marker for lipid metabolic disorders in cats. Total 13 domestic female cats were divided into two groups which were fed on control and high-fat diet for eight weeks, respectively. After the feeding period, they were compared in metabolic variables and oxidative stress markers in plasma and tissues. As results, High-fat diet including much long chain fatty acids promoted rapid changes in lipid metabolism, particularly accelerated β-oxidation of fatty acids and oxidative stress in the liver of the cats. G6PD, GPx and SOD were increased in the liver. Insulin resistance was not apparent at the early phase of obesity in cats. Plasma activities of SOD also increased at the early phase of obesity in cats. Remarkable alternation for oxidative stress in liver was observed at the early phase of obesity in cats fed on high fat diet and SOD may be a potential marker of the early phase of obesity in cats. © 2014 Academic Journals Inc.

Neurotensin is a tridecapeptide that has multiple functions as a neurotransmitter and as a circulating hormone. Neurotensin and its related peptide, LANT6, have been isolated in the chicken, but the mRNA encoding these peptides has not been identified. In this study, we first cloned the cDNA for the chicken neurotensin precursor mRNA from the duodenum and characterized its primary structure and then investigated tissue expression patterns of neurotensin precursor and receptor mRNA. The cDNA encoded a protein of 495 amino acids that contains the sequences of chicken neurotensin and LANT6 in the C-terminal region. Real-time PCR analysis showed that the neurotensin precursor mRNA is preferentially expressed in intestinal tissues, such as the duodenum, jejunum, ileum, and colon/rectum, with temporal increases during the hatching period. The expression levels of neurotensin receptor 1 mRNA were relatively higher during the late embryonic period compared with the posthatching period in the duodenum and jejunum, whereas the expression levels were higher in the colon/rectum during the posthatching period. In the liver, the expression levels of neurotensin receptor 1 were markedly increased during the early posthatching period. These results suggest that chicken neurotensin is involved in the regulation of gastrointestinal and hepatic functions, especially during the hatching period. © 2013 Poultry Science Association Inc.

Background: Mammalian sirtuins are homologs to the yeast silent information regulator 2 (Sir2), which is an NAD-dependent deacetylase. Sirtuins are comprised of 7 proteins, and each has different target proteins. Sirtuin 1 (SIRT1) plays important roles in maintaining metabolic functions and immune responses, and SIRT3 protects cells from oxidative stress-induced cell death. Both SIRT1 and SIRT3 are regulated by metabolic status and aging. Hence, SIRT1 and SIRT3 have been researched in metabolic diseases, such as type 2 diabetes mellitus (DM), fatty liver, and heart diseases. Although these diseases have been increasing, there is little information about relation between the diseases and SIRT1 and SIRT3 in cats. Therefore we cloned SIRT1 and SIRT3 cDNA, examined mRNA expression in cat tissues, and investigated the changes in SIRT1 and SIRT3 mRNA expression in peripheral blood leukocyte of cats fed on HFD for 6 weeks. Results: Cat SIRT1 and SIRT3 contained a catalytic core region and showed high sequence homology with other vertebrate SIRT1 (&gt 61.3%) and SIRT3 (&gt 65.9%) amino acids. Real-time polymerase chain reaction analyses revealed that high expression levels were observed in the liver and skeletal muscle for SIRT1 and in the heart for SIRT3 in cats. In addition, both cat SIRT1 and SIRT3 expression levels in the pancreas were different between individuals. Cat SIRT1 mRNA expression in peripheral blood leukocytes was significantly elevated in obese cats fed on HFD (P &lt 0.05). Conclusions: Cat SIRT1 and SIRT3 genes are highly conserved among vertebrates, and HFD feeding may be related to SIRT1 mRNA expression mechanisms in cat peripheral blood leukocytes. © 2013 Ishikawa et al. licensee BioMed Central Ltd.

Insulin is a critical hormone in the regulation of blood glucose levels and is produced exclusively by pancreatic islet beta-cells. Insulin deficiency due to reduced pancreatic islet beta-cell number underlies the progression of diabetes mellitus, prompting efforts to develop beta-cell replacement therapies. However, precise information on beta-cell replacement and differentiation in canines is limited. In this study, we established insulin-producing cells from bone marrow derived mesenchymal stem cells transiently expressing canine pancreatic and duodenal homeobox 1 (Pdx1), beta cell transactivator 2 (Beta2) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) using a gene transfer technique. Real-time PCR analysis revealed an increase in insulin mRNA expression of transfected cells. And ELISA revealed that insulin protein expressed was detected in cytoplasmic fraction. Insulin immunostaining analysis was performed and observed in cytoplasmic fraction. These results suggest that co-transfection of Pdx1, Beta2 and Mafa induce insulin production in canine BMSCs. Our findings provide a clue to basic research into the mechanisms underlying insulin production in the canines. (C) 2013 Elsevier Inc. All rights reserved.

Australian Burmese cats are predisposed to diabetes mellitus and, compared to other breeds, have delayed triglyceride clearance that may result in subtle changes within cells and tissues that trigger specific alterations in gene expression within peripheral blood leucocytes (PBLs). Expression of genes involved in energy metabolism (glucose-6-phosphate dehydrogenase and malate dehydrogenase), lipogenesis (ATP citrate lyase [ACL], fatty acid synthase [FAS] and sterol regulatory binding protein-1c [SREBP-1c]), and insulin signalling (insulin receptor substrates 1 and 2, and phosphatidylinositol-3 kinase), as well as cholesterol lipoprotein subfraction profiling were carried out on PBLs from lean Burmese cats and compared with similar profiles of age and gender matched lean and obese Australian domestic shorthaired cats (DSHs) in an attempt to identify possible biomarkers for assessing obesity.For the majority of the genes examined, the lean Burmese cats demonstrated similar PBL gene expression patterns as age and gender matched obese Australian DSH cats. Lean Burmese had increased expression of ACL and FAS, but not SREBP-1c, a main upstream regulator of lipid synthesis, suggesting possible aberrations in lipogenesis. Moreover, lean Burmese displayed a 3- to 4-fold increase in the very low density cholesterol fraction percentage, which was double that for obese DSH cats, indicating an increased degree of lipid dysregulation especially in relation to triglycerides. The findings suggest that Burmese cats may have a particular propensity for dysregulation in lipid metabolism. © 2012 Elsevier Ltd.

Peripheral Blood Leukocytes (PBL) continually interact with virtually every organ and tissue in the whole body. A remarkable concordance (80%) of gene expression profiles between peripheral blood mononuclear cells and different tissues has been previously demonstrated in humans. As such, gene expression responses of circulating PBL can therefore, potentially provide early warning of any abnormalities they discover. Weight alterations (increase or decrease) when associated with obesity has been reported to lead to alterations to PBL gene expression, especially those related to insulin and adiponectin signaling genes and even genes involved with energy homeostasis. As such, a pilot study involving PBL profiles of the following genes involved in energy homoestasis (5' Adenosine Monophosphate-Activated Protein Kinase (AMPK)-alpha 1 and 2, -beta 1 and 2 and -gamma 1 and 2); Glucose-6-Phosphate Dehydrogenase (G6PDH) and Malate Dehydrogenase (MDH), lipogenesis (Fatty Acid Synthase (FAS) and insulin signaling Adiponectin Receptor (ADIPOR) (-1 and 2); Insulin. Receptor Substrates (IRS) (-1 and 2); Phosphatidylitositol-3 Kinase (PI3-K) were evaluated between lean and overweight dogs in an attempt to identify possible PBL biomarkers for assessing obesity in dogs. As compared to lean dogs, overweight dog PBL demonstrated reduced mRNA expression of IRS-1, IRS-2, FAS, G6PDH and AMPK beta 1 genes. Overall, these findings suggest that dysregulation of energy metabolism, associated with obesity in overweight dogs may carry over with alterations in PBL gene expression of genes involved in energy homeostasis and sterol metabolism. As such, PBL gene expression profiles may aid in early detection of PBL biomarkers for assessing obesity in dogs.

Hyperlipidemia refers to increase of triglyceride (TG) and/or total cholesterol (T-cho) in blood. Fatty Acids (FAs) have important roles in the lipid metabolism. The aim of this study was to determine the FA composition of plasma lipid fractions in dogs with hyperlipidemia and to evaluate the FA composition as a new diagnostic marker for obesity at early stage. Thirty-nine dogs were classified into healthy or hyperlipidemia based on the criteria to diagnose hyperlipidemia. The blood biochemical values, such as TG, T-cho, glucose, insulin, adiponectin and Non-Esterified Fatty Acid (NEFA) were measured. FA composition profile was performed on GC/MS system. The values of plasma TG, insulin and NEFA of the hyperlipidemia group were significantly higher than that of control group. Hyperlipidemia group tended to show lower concentration of adiponectin. It was found that only the levels of TG and NEFA, but not T-cho increased significantly in early stage of hyperlipidemia. In hyperlipidemia group, percentages of myristic acid (C14:0), parmitoleic acid (C16:1) and oleic acid (C18:1) increased in total FAs. And the percentage of C18:1 increased in NEFA. Indeed, the higher level of insulin and lower adiponectin concentration were seen in hyperlipidemia group. These results suggest that appearance of insulin resistance may be the result of increases of certain FAs in early stage of insulin resistance. © 2013 Academic Journals Inc.

Pancreatic and duodenal homeobox 1 (Pdx-1) is a critical insulin transcription factor expressed by pancreatic beta-cells, and is crucial in the early stage of pancreas development. Unfortunately, nothing concerning Pdx-1 in canine has been elucidated yet. In this study, full length canine Pdx-1 cDNA was cloned and it was 1498 bp in length, consisting of a 99 bp 5'-untranslated region (UTR), a 849 bp coding region, and a 550 bp 3'-UTR region. A deduced 282 amino acid sequence of canine PDX-1 displayed high overall sequence identity with human, bovine, and mouse PDX-1. qRT-PCR analysis revealed that a high level of Pdx1 mRNA expression is exists in the duodenum and pancreas of canines. In addition, functional canine insulin promoter-luciferase reporter constructs with various canine insulin promoter region fragments revealed that our Pdx-1 isolated cDNA sequence encodes for a functionally active PDX-1 protein. Significant promoter activity was observed within the 583 bp 5'-upstream region of canine insulin gene with Chinese hamster ovary cells. In addition, Pdx-1 appears to have a synergistic effect with beta cell transactivator 2 (BETA2) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), which also have important roles in the activation of the insulin gene promoter. Our results confirm that similar to humans, Pdx1 plays an important role in expression of insulin gene in canines. (c) 2011 Elsevier Ltd. All rights reserved.

Background: Bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis. Results: Samples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM-and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM-and AT-MSCs. Conclusion: Our results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.

Continuous high intensity training may induce alterations to enzyme activities related to glucose and lipid metabolism in horses. In our study, five Thoroughbred race horses (3 male and 2 female, avg age = 5 yrs old) were compared against five riding horses (1 male, 1 female, 3 gelding, avg age = 13 yrs old) in terms of energy metabolism, by examining plasma malate (MDH) and lactate (LDH) dehydrogenase activities and M/L ratio. MDH is involved in NADH and ATP generation, whereas LDH can convert NADH back into NAD(+) for ATP generation. An increase in plasma M/L ratio can reflect heightened energy metabolism in the liver and skeletal muscle of horses adapted to continuous intensive exercise. Moreover, plasma lipid metabolism analytes (adiponectin, NEFA, total cholesterol (T-Cho), and triglycerides (TG)) can reflect changes to lipolysis rate, which can also indicate a change in energy metabolism. Overall, race horses demonstrated increased MDH and LDH activity in plasma (4x and 2x greater, respectively), in addition to a plasma M/L ratio twice as high as that of riding horses (2.0 vs 1.0). In addition, race horses also demonstrated significantly higher levels of plasma NEFA (50% greater), TG (2x greater), and T-Cho (20% greater) as compared to riding horses. Therefore, race horse muscles may have adapted to prolonged high intensity endurance exercise by gaining a higher oxidative capacity and an increased capacity for fat utilization as an energy source, resulting in heightened energy metabolism and increased rate of lipid mobilization.