Insulin‐independent actions of glucagon‐like peptide‐1 in wethers

Insulin‐independent actions of glucagon‐like peptide‐1 (GLP‐1) are not yet clear in ruminants. Four Suffolk mature wethers (60.0 ± 6.7 kg body weight (BW)) were intravenously infused with insulin (0.5 mU/kg BW/min; from 0 to 90 min) and GLP‐1 (0.5 μg/kg BW/min; from 60 to 150 min) with both hormones co‐administered from 60 to 90 min, in a repeated‐measure design under euglycemic clamp for 150 min, to investigate whether GLP‐1 has insulin‐independent actions. Jugular blood samples were taken at 15‐min intervals for plasma hormones and metabolites analysis. Compared to baseline concentrations (at 0 min), insulin infusion decreased (P < 0.05) plasma concentrations of glucagon, non‐esterified fatty acids (NEFA), lactate, nonessential amino acids (NEAA), branched‐chain amino acids (BCAA), total amino acids (TAA) and urea nitrogen (UN). Insulin plus GLP‐1 infusion induced a greater increase (P < 0.05) in plasma concentrations of insulin and triglyceride (TG), but decreased (P < 0.05) glucagon, total cholesterol (T‐Cho), NEAA and UN plasma concentrations. GLP‐1 infusion increased (P < 0.05) NEFA, β‐hydroxybutyrate and TG, but decreased (P < 0.05) glucagon, T‐Cho, NEAA, BCAA and UN plasma concentrations. In conclusion, GLP‐1 exerts extrapancreatic roles in ruminants not only insulin‐independent but probably, in contrast to non‐ruminants, antagonistic to insulin effects.     

The canine prostate cancer cell line CHP‐1 shows over‐expression of the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α

Although androgen therapy resistance and poor clinical outcomes are seen in most canine prostate cancer cases, there are only a few tools for analysing canine prostate cancer by using a cell biological approach. Therefore, to evaluate androgen‐independent neoplastic cell growth, a new canine prostate cancer cell line (CHP‐1) was established in this study. CHP‐1 over‐expressed the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α (SGTA), which is over‐expressed in human androgen‐independent prostate cancer. The CHP‐1 xenograft also showed SGTA over‐expression. Although CHP‐1 shows poor androgen receptor (AR) signalling upon dihydrotestosterone stimulation, forced expression of AR enabled evaluation of AR signalling. Taken together, these results suggest that CHP‐1 will be a useful model for investigating the pathogenesis of androgen‐dependent and androgen‐independent canine prostate cancer.     

Association of Sphingosine‐1‐phosphate (S1P)/S1P Receptor‐1 Pathway with Cell Proliferation and Survival in Canine Hemangiosarcoma

Background

Sphingosine‐1‐phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs.

Hypothesis/Objectives

S1P/S1P₁ signaling will contribute to the progression of hemangiosarcoma (HSA).

Animals

Thirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 endothelial cell line derived from a dog with splenic hematoma were used.

Methods

This was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT‐PCR, immunohistochemistry, and immunoblotting were performed to examine S1P₁ expression. S1P concentrations were measured by high‐performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca²⁺ mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining.

Results

Canine HSA cells expressed higher levels of S1P₁ mRNA than nonmalignant endothelial cells. S1P₁ protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P₁, decreased S1P₁ protein expression and induced apoptosis of HSA cells.

Conclusions and clinical importance

S1P/S1P₁ signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P₁ or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA.     

The Influence of Restricted Feeding on Glucagon‐Like Peptide‐1 (GLP‐1)‐Containing Cells in the Chicken Small Intestine

The influence of restricted feeding on the distribution of glucagon‐like peptide‐1 (GLP‐1)‐containing endocrine cells in the chicken small intestine was investigated using immunohistochemical and morphometrical techniques. This study demonstrated that the restricted feeding had an influence on the activity of GLP‐1‐immunoreactive cells in the chicken small intestine. There were differences in the localization and the frequency of occurrence of GLP‐1‐immunoreactive cells in the small intestine between control and restricted groups, especially 25% feed supply group provided with 25% of the intake during the adapting period. GLP‐1‐immunoreactive cells in the control chickens were mainly located in epithelium from crypts to the lower part of intestinal villi. Those in restricted groups, however, tended to be located from crypts to the middle part of intestinal villi. The frequency of occurrence of GLP‐1‐immunoreactive cells was lowest in the control group, medium in 50% feed supply group and highest in 25% feed supply group at each intestinal region examined in this study, that is, increased with the advancement of restricting the amount of feed supply. These data show that the quantity of food intake is one of signals that have an influence on the secretion of GLP‐1 from L cells in the chicken small intestine.     

PD‐1 expression by canine T cells and functional effects of PD‐1 blockade

The co‐inhibitory checkpoint molecule programmed death receptor 1 (PD‐1) can trigger T cell functional exhaustion upon binding to its ligand PD‐L1 expressed on tumour cells or macrophages. PD‐1 blocking antibodies have generated remarkable results in human cancer patients, including inducing durable responses in a number of advanced cancers. Therefore, monoclonal antibodies specific for canine PD‐1 were assessed for T cell binding and induction of functional activation. A total of 5–10% of CD4 T cells and 20–25% of CD8 T cells from healthy dogs expressed PD‐1, and PD‐1 expression was upregulated on T cells from dogs with cancer. Functionally, PD‐1 antibodies significantly enhanced T‐cell activation, as assessed by proliferation and interferon‐gamma (IFN‐γ) production. PD‐1 antibodies also reversed T‐cell suppression induced by canine soluble PD‐L1 and by tumour cells and tumour explant fragments. These findings indicate that PD‐1 antibodies have potential for use in cancer immunotherapy in dogs.     

Mitochondrial biogenesis and PGC‐1α gene expression in male broilers from ascites‐susceptible and ‐resistant lines

Ascites is a cardiovascular metabolic disease characterized by accumulation of fluid around the heart and in the abdominal cavity that eventually leads to death. This syndrome is the end‐point result of a series of metabolic incidents that are generally caused by impaired oxygen availability. Mitochondria are the major sites of oxygen consumption, therefore major contributors to oxidative stress. Genetic, metabolic and dietary factors can influence variations in mitochondrial biogenesis (mitochondrial size, number and mass) that might have an effect on oxygen consumption and reactive oxygen species production. This study evaluated the effect of genotype on PGC‐1α mRNA gene expression and mitochondrial biogenesis. These parameters were examined in male broiler chickens at 22 weeks of age from the SUS and RES lines divergently selected for ascites phenotype. From each line, five birds were sampled for right ventricle and breast muscle. Gene expression and mtDNA copy number were assessed by quantitative PCR. Results showed that birds from SUS had significantly higher PGC‐1α mRNA gene (p = .033) and mitochondrial DNA copy number (p = .038) in breast muscle. There was no difference in right ventricle PGC‐1α expression or mitochondrial DNA copy number between the two lines. These findings indicate that mitochondrial biogenesis and PGC‐1α mRNA gene expression differ between male broiler chickens from RES and SUS lines in a tissue‐specific manner.     

Effect of genistein on IGF‐1 and IGFBP‐1 in young and aged female rat ovary

The objective of this study was to assess the effects of genistein (GEN) on expression of insulin‐like growth factor 1 (IGF‐1) and insulin‐like growth factor binding protein 1 (IGFBP‐1) in young and aged rat ovary. Forty young female Sprague Dawley (SD) rats (200 ± 20 g) and forty aged female SD rats (490 ± 20 g) were selected and according to weight, they were divided into the following five groups with eight animals in each: negative control group (NC), low‐dose group (L), middle‐dose group (M), high‐dose group (H) and positive control group (PC). GEN group received GEN of 15, 30, 60 mg/kg respectively. It lasted 30 days. Concentrations of serum hormones, IGF‐1 and IGFBP‐1 were determined by enzyme‐linked immunosorbent assay (ELISA). Gene and protein expressions of IGF‐1 and IGFBP‐1 were determined by real‐time PCR and Western blot respectively. Compared with NC, GEN significantly increased oestradiol‐17β(E₂) level in aged rat, reduced luteinizing hormone (LH) level in young and aged rat. Serum levels of IGFBP‐1 in young rats were significantly higher in GEN groups (p < 0.05). mRNA and protein expression levels of IGF‐1 and IGFBP‐1 were positively correlated with GEN dose. GEN could significantly reduce the ratio of IGF‐1/IGFBP‐1 of aged rats. Multivariate Cox regression analysis result showed IGF‐1 and IGFBP‐1 levels significantly correlated with GEN dose. We speculate that there is an association between the addition of GEN and expression of IGF‐1 and IGFBP‐1, and the relationship between them is different in young and aged rat.     

Three‐dimensional T1‐weighted gradient echo is a suitable alternative to two‐dimensional T1‐weighted spin echo for imaging the canine brain

Volumetric imaging (VOL), a three‐dimensional magnetic resonance imaging (MRI) technique, has been described in the literature for evaluation of the human brain. It offers several advantages over conventional two‐dimensional (2D) spin echo (SE), allowing rapid, whole‐brain, isotropic imaging with submillimeter voxels. This retrospective, observational study compares the use of 2D T1‐weighted SE (T1W SE), with T1W VOL, for the evaluation of dogs with clinical signs of intracranial disease. Brain MRI images from 160 dogs who had T1W SE and T1W VOL sequences acquired pre‐ and postcontrast, were reviewed for presence and characteristics of intracranial lesions. Twenty‐nine of 160 patients were found to have intracranial lesions, all visible on both sequences. Significantly better grey‐white matter (GWM) differentiation was identified with T1W VOL (P < .001), with fair agreement between the two sequences (weighted κ = 0.35). Excluding a mild reduction in lesion intensity in three dogs precontrast on the T1W VOL images compared to T1W SE, and meningeal enhancement noted on the T1W VOL images in one dog, not identified on T1W SE, there was otherwise complete agreement between the two sequences. The T1W VOL sequence provided equivalent lesion evaluation and significantly improved GWM differentiation. Images acquired were of comparable diagnostic quality to those produced using a conventional T1W SE technique, for assessment of lesion appearance, number, location, mass effect, and postcontrast enhancement. T1W VOL, therefore, provides a suitable alternative T1W sequence for canine brain evaluation and can facilitate a reduction in total image acquisition time.     

Tumor necrosis factor‐α‐induced inflammatory responses in cattle

Tumor necrosis factor‐alpha (TNF‐α) is recognized as a cytokine because of its involvement in inflammation‐mediated biological defense functions. Although TNF‐α is primarily produced by macrophages, it is also produced by other cells, including lymphocytes, Kupffer cells, natural killer cells and adipocytes. While TNF‐α has diverse immune system functions, including antitumor activity, antimicrobial activity and mediation of inflammation, it also regulates a number of physiological functions, including appetite, fever, energy metabolism and endocrine activity. Factors such as viruses, parasites, other cytokines, and endotoxins induce TNF‐α production. In combination with other cytokines, TNF‐α plays a clinically important role in cattle by mediating immune inflammatory responses such as mastitis and endotoxic shock. It has been reported that cytokines such as TNF‐α are involved in metabolic disease such as acidosis. On the other hand, several data suggest that lactoferrin (LF) acts to prevent the release of a number of inflammatory mediators from various activated cells, and further suggest that the prophylactic effect of LF involves inhibition of cytokine production, including TNF‐α, that are principal mediators of the inflammatory response leading to death from toxic shock. This review discusses the role of TNF‐α in pathological conditions in cattle, including infections and metabolic diseases caused by perturbation of metabolism and endocrine functions.     

B‐cell intestinal lymphoma with Mott cell differentiation in a 1‐year‐old miniature Dachshund

Abstract: A 1‐year‐old intact female miniature Dachshund was presented with hematochezia, vomiting, and diarrhea of more than 1‐week duration. An abdominal mass was palpated, which at exploratory surgery was found to be a 7‐cm‐long thickened section of ileum. The thickened ileum was resected. Impression smears revealed numerous small‐ to medium‐sized lymphocytes, with a smaller number of cells resembling Mott cells. The Mott‐like cells contained multiple pale vacuoles that were positive for periodic acid‐Schiff (PAS) in wet‐fixed smears, consistent with Russell bodies. Histologic evaluation of the surgically excised ileum revealed 2 populations of neoplastic lymphoid cells. The majority were uniform medium‐sized lymphocytes with hyperchromatic oval or round nuclei and inconspicuous nucleoli. The remaining cells resembled Mott cells, which contained several PAS‐positive eosinophilic globules in the cytoplasm, occasionally compressing the nucleus. The majority of neoplastic cells stained positively for vimentin, CD20, CD79a, and Pax‐5, but were negative for CD3 and lysozyme; 43.5% of cells stained positively for Ki‐67. The Mott cells were strongly positive for immunoglobulin but were negative for Pax‐5. Using electron microscopy, a homogenous substance of intermediate electron density was observed frequently in the cisternae of rough endoplasmic reticulum in the cytoplasm of the Mott cells, and rarely in the perinuclear cisternae of the lymphoid cells, corresponding to the site of immunoglobulin staining. Monoclonal rearrangement of immunoglobulin heavy‐chain (IgH) gene was observed by PCR testing for lymphocyte–antigen receptor rearrangement. The morphologic features, immunophenotype, and IgH gene rearrangement verified the lymphoid cells were neoplastic (mature cell type) and had a B‐cell phenotype, with evidence of immunoglobulin production and differentiation into Mott cells. This case was unusual because of the age of the dog and because most intestinal lymphomas are T‐cell phenotype. The Mott cell morphology also differed from typical mature B‐cell lymphoma types and may be a unique B‐cell lymphoma variant.     

Expression analysis of an α‐1, 3‐galactosyltransferase,an enzyme that creates xenotransplantation‐related α–Gal epitope,in pig preimplantation embryos

α‐1,3‐Galactosyltransferase (α‐GalT), an enzyme creating Galα1‐3Gal (α‐Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference‐based suppression of endogenous α‐GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi‐quantitative RT‐PCR (qRT‐PCR) and the cytochemical method using a fluorescence‐labeled Bandeiraea simplicifolia Isolectin B₄ (BS‐I‐B₄). Staining with BS‐I‐B₄ demonstrated that α‐Gal epitope expression was first recognized at the 8‐cell stage, and increased up to the hatched blastocyst stage. Single embryo‐based qRT‐PCR also confirmed this pattern. These results indicate that creation of α‐Gal epitope is proceeded by de novo synthesis of α‐GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.     

Orexin‐B‐like immunoreactivity in pituitary αMSH‐producing cells and median eminence GnRH‐containing fibres of the flat‐tailed house gecko

We examined the distribution of the orexin‐like peptides in the pituitary and median eminence of the flat‐tailed house gecko (Hemidactylus platyurus) using immunohistochemistry. Orexin‐B‐like, but not orexin‐A‐like, immunoreactivity was detected in the pituitary, specifically in the pars intermedia, and these cells corresponded to alpha‐melanocyte‐stimulating hormone (αMSH)‐producing cells. Orexin‐B and αMSH secreted from pars intermedia may modulate secretion of adenohypophyseal cells in the pars distalis. In the median eminence, orexin‐B‐immunoreactive puncta and fibres were observed, and these structures corresponded to gonadotropin‐releasing hormone (GnRH)‐immunoreactive puncta and fibres. Orexin‐B secreted from GnRH‐containing neurons in the hypothalamus may affect thyrotropin‐releasing hormone‐containing neurons resulting in modulation of αMSH secretion of melanotrophs in the pars intermedia.     

Recent Outbreaks of Foot‐and‐Mouth Disease Type Asia 1 in China

Foot‐and‐mouth disease (FMD) virus is the aetiological agent of an important disease of livestock and has a great global impact. The outbreak of FMD type Asia 1 in China was first confirmed at the beginning of May 2005. Subsequent outbreaks occurred in 10 areas in different provinces of China.     

Oral bioavailability of P‐glycoprotein substrate drugs do not differ between ABCB1‐1Δ and ABCB1 wild type dogs

Mealey, K.L., Waiting, D., Raunig, D.L., Schmidt, K.R., Nelson, F.R. Oral bioavailability of P‐glycoprotein substrate drugs do not differ between ABCB1‐1Δ and ABCB1 wild type dogs. J. vet. Pharmacol. Therap. 33 , 453–460. Previous studies have indicated that intestinal P‐glycoprotein (P‐gp) limits the oral bioavailability of substrate drugs and alters systemic pharmacokinetics. In this study, dogs lacking functional P‐gp were used to determine the contribution of P‐gp to the oral bioavailability and systemic pharmacokinetics of several P‐gp substrate drugs. The P‐gp substrates quinidine, loperamide, nelfinavir, cyclosporin and the control (non P‐gp substrate) drug diazepam were individually administered intravenously and per os to ABCB1‐1Δ dogs, which have a P‐gp null phenotype and ABCB1 wildtype dogs. ABCB1‐1Δ dogs have been shown to have greater brain penetration of P‐gp substrates, but limited information is available regarding oral bioavailability of P‐gp substrate drugs in this animal model. Plasma drug concentration vs. time curves were generated and pharmacokinetic parameters were calculated for each drug. There were no differences in oral bioavailability between ABCB1‐1Δ dogs and ABCB1 wildtype dogs for any of the drugs studied, suggesting that intestinal P‐gp does not significantly affect intestinal absorption of these particular substrate drugs in ABCB1‐1Δ dogs. However, small sample sizes and individual variability in CYP enzyme activity may have affected the power of the study to detect the impact of P‐gp on oral bioavailability.     

PLAG1 and NCAPG‐LCORL in livestock

A recent progress on stature genetics has revealed simple genetic architecture in livestock animals in contrast to that in humans. PLAG1 and/or NCAPG‐LCORL, both of which are known as a locus for adult human height, have been detected for association with body weight/height in cattle and horses, and for selective sweep in dogs and pigs. The findings indicate a significant impact of these loci on mammalian growth or body size and usefulness of the natural variants for selective breeding. However, association with an unfavorable trait, such as late puberty or risk for a neuropathic disease, was also reported for the respective loci, indicating an importance to discriminate between causality and association. Here I review the recent findings on quantitative trait loci (QTL) for stature in livestock animals, mainly focusing on the PLAG1 and NCAPG‐LCORL loci. I also describe our recent efforts to identify the causative variation for the third major locus for carcass weight in Japanese Black cattle.