RESUMEN

Kalirin (gene: KALRN) is a Rho-GEF kinase linked to neurodegenerative diseases in humans. Unexpectedly, various polymorphisms in KALRN gene were previously associated with resistance to bacterial infections in ruminants. In this study, we evaluated the effect of the rs384223075 (RS-075) deletion in KALRN intron 5 on the occurrence of Mycobacterium bovis and Brucella abortus infections in cattle. We performed two separate case-control association analyses: one for bovine tuberculosis (bTB) using 308 Holstein and Jersey cows from three herds with prevalence between 5 and 15% for this infection; and another for brucellosis using 140 Holstein and beef crossbred cows from two herds with high prevalence for brucellosis (> 30%). In the bTB analysis, the RS-075 deletion frequency was higher among cases than controls (p = 0.0001), and the absence of the RS-075 deletion allele was associated with negative PPD-skin test results (p = 0.0009) at genotype level. On the contrary, RS-075 was not associated with Brucella spp. serological status (p = 0.72) but, unexpectedly, the deletion allele was more frequent among controls than cases in the beef crossbred herd (0.31 vs. 0.14, p = 0.02). In concordance with this observation, in vitro assays showed that the RS-075 deletion could be linked to an enhanced cellular response to bacterial antigens and unspecific stimulation in mononuclear cells derived from beef crossbred cows, specifically the reactive nitrogen species production (p = 0.008) and proliferation capacity (p = 0.018). This study is consistent with other reports that support an important role of the KALRN gene and its polymorphisms in the host response to intracellular pathogens.

Asunto(s)

Brucelosis Bovina , Brucelosis , Enfermedades de los Bovinos , Tuberculosis Bovina , Humanos , Femenino , Bovinos , Animales , Tuberculosis Bovina/genética , Tuberculosis Bovina/epidemiología , Intrones , Brucelosis/epidemiología , Brucelosis/veterinaria , Brucelosis Bovina/genética , Brucelosis Bovina/epidemiología , Rumiantes , Fenotipo

RESUMEN

Brucellosis is an important zoonotic disease caused by infection with Brucella spp. It generates major economic losses in livestock production worldwide. Goats are the principal hosts of B. melitensis, the main infection agent of caprine and human brucellosis. The selection of resistance-related genes is considered one of the best long-term means to improve control to bacterial infection in domestic ruminants. We performed a candidate gene association study to test if six short insertion/deletion polymorphisms (InDels) at bacterial-infection related genes influence the resistance to Brucella infection in female creole goats. InDels (IRF3-540: rs660531540, FKBP5-294: rs448529294, TIRAP-561: rs657494561, PTPRT-588: rs667380588, KALRN-989: rs667660989 and RAB5a-016: rs661537016) were resolved by PCR-capillary electrophoresis in samples from 64 cases and 64 controls for brucellosis. Allelic frequencies were significantly different between cases and controls at IRF3-540 and KALRN-989 (p = 0.001 and 0.005). Indeed, the minor alleles (a and k) at InDels IRF3-540 and KALRN-989 were more frequent among controls than cases, providing evidence that these alleles confer protection against Brucella infection. Moreover, IRF3-540 a-containing genotypes (Aa and aa) were associated with absence of Brucella-specific antibodies in goats (p = 0.003; OR = 3.52; 95% CI = 1.55-7.96), and more specifically, a-allele was associated with resistance to Brucella infection in a dose-dependent manner. Also, we observed that the IRF3-540 deletion (a-allele) extends a conserved upstream ORF by 75 nucleotides to the main ORF, and thus it may decrease gene expression by reducing translation efficiency from the main ORF. These results suggest a potential functional role of IRF3-540 deletion in genetic resistance to Brucella infection in goats.

Asunto(s)

Brucelosis/genética , Cabras/genética , Factor 3 Regulador del Interferón/genética , Polimorfismo Genético/genética , Alelos , Animales , Brucella/patogenicidad , Femenino , Frecuencia de los Genes/genética , Genotipo , Sistemas de Lectura Abierta/genética

RESUMEN

BACKGROUND: Bovine leukemia virus (BLV) infection is omnipresent in dairy herds causing direct economic losses due to trade restrictions and lymphosarcoma-related deaths. Milk production drops and increase in the culling rate are also relevant and usually neglected. The BLV provirus persists throughout a lifetime and an inter-individual variation is observed in the level of infection (LI) in vivo. High LI is strongly correlated with disease progression and BLV transmission among herd mates. In a context of high prevalence, classical control strategies are economically prohibitive. Alternatively, host genomics studies aiming to dissect loci associated with LI are potentially useful tools for genetic selection programs tending to abrogate the viral spreading. The LI was measured through the proviral load (PVL) set-point and white blood cells (WBC) counts. The goals of this work were to gain insight into the contribution of SNPs (bovine 50KSNP panel) on LI variability and to identify genomics regions underlying this trait. RESULTS: We quantified anti-p24 response and total leukocytes count in peripheral blood from 1800 cows and used these to select 800 individuals with extreme phenotypes in WBCs and PVL. Two case-control genomic association studies using linear mixed models (LMMs) considering population stratification were performed. The proportion of the variance captured by all QC-passed SNPs represented 0.63 (SE ± 0.14) of the phenotypic variance for PVL and 0.56 (SE ± 0.15) for WBCs. Overall, significant associations (Bonferroni's corrected -log10p > 5.94) were shared for both phenotypes by 24 SNPs within the Bovine MHC. Founder haplotypes were used to measure the linkage disequilibrium (LD) extent (r2 = 0.22 ± 0.27 at inter-SNP distance of 25-50 kb). The SNPs and LD blocks indicated genes potentially associated with LI in infected cows: i.e. relevant immune response related genes (DQA1, DRB3, BOLA-A, LTA, LTB, TNF, IER3, GRP111, CRISP1), several genes involved in cell cytoskeletal reorganization (CD2AP, PKHD1, FLOT1, TUBB5) and modelling of the extracellular matrix (TRAM2, TNXB). Host transcription factors (TFs) were also highlighted (TFAP2D; ABT1, GCM1, PRRC2A). CONCLUSIONS: Data obtained represent a step forward to understand the biology of BLV-bovine interaction, and provide genetic information potentially applicable to selective breeding programs.

Asunto(s)

Enfermedades de los Bovinos/genética , Leucosis Bovina Enzoótica/genética , Genómica/métodos , Polimorfismo de Nucleótido Simple , Animales , Bovinos , Enfermedades de los Bovinos/virología , Leucosis Bovina Enzoótica/virología , Femenino , Haplotipos , Virus de la Leucemia Bovina/fisiología , Leucocitos/metabolismo , Leucocitos/virología , Desequilibrio de Ligamiento , Provirus/fisiología , Factores de Transcripción/genética , Carga Viral

RESUMEN

The prevention and control of bovine mastitis by enhancing natural defenses in animals is important to improve the quality of dairy products. Mastitis resistance is a complex trait which depends on genetic components, as well as environmental and physiological factors. The limitations of classical control measures have led to the search for alternative approaches to minimize the use of antibiotics by selecting naturally resistant animals. Polymorphisms in genes associated with the innate immune system are strong candidates to be evaluated as genetic markers. In this work, we evaluated a set of single nucleotide polymorphisms (SNPs) in candidate genes for health and production traits, and determined their association with the somatic cell score (SCS) as an indicator of mastitis in Argentinean dairy cattle. We evaluated 941 cows: Holstein (n = 677) and Holstein × Jersey (n = 264) crossbred, daughters from 22 bulls from 14 dairy farms located in the central dairy area of Argentina. Two of the 21 successfully genotyped markers were found to be significantly associated (p < 0.05) with the SCS: GHR_140 and OPN_8514C-T. The heterozygote genotype for GHR_140 showed a favorable effect in reducing the SCS. On the other hand, heterozygote genotypes for OPN8514C-T caused an increase in the SCS; moreover, combined genotypes for OPN SNPs showed an even larger effect. These findings can contribute to the design of effective marker-assisted selection programs.

Asunto(s)

Resistencia a la Enfermedad/genética , Mastitis Bovina/genética , Leche/citología , Polimorfismo de Nucleótido Simple , Animales , Bovinos , Femenino , Estudios de Asociación Genética , Marcadores Genéticos , Genotipo , Heterocigoto , Masculino , Análisis de Secuencia de ADN

RESUMEN

BACKGROUND: Islet NADPH oxidase activity is modulated by glucose and other insulin secretagogues and it might be part of the regulatory mechanism of insulin secretion. We studied its modulatory role of islet NADPH oxidase upon ß-cell function in rats with fructose-induced oxidative stress. METHODS: Normal rats were fed for 3weeks with a standard diet, a fructose-rich diet or both diets plus apocynin. We measured plasma glucose, insulin, triacylglycerol and lipid peroxidation levels and the homeostasis model assessment-insulin resistance (HOMA-IR) and HOMA-ß indexes, and performed an oral glucose tolerance test. ß-cell volume density and the number of islets per mm(2) were determined by immunomorphometric analysis of the pancreas. Insulin secretion, glucose metabolism, glucokinase and NADPH oxidase activities were studied in islets isolated from each experimental group. RESULTS: Fructose-fed rats had increased plasma triacylglycerol, insulin and lipid peroxidation levels associated with an insulin resistance state; the reactive higher secretion was unable to cope with the increased demand of insulin, leading to an impaired glucose tolerance. They also have a lower number of islets per area unit with a decreased ß-cell volume density. All these alterations were prevented by blocking NADPH oxidase activity with apocynin. CONCLUSION: Fructose-induced changes are partly mediated by modulation of NADPH oxidase activity. GENERAL SIGNIFICANCE: The metabolic dysfunctions and enhanced oxidative stress measured in fructose-fed rats resemble those recorded in human prediabetes; thus, successful strategies employed in this model could be later used to prevent the progression of this state towards type 2 diabetes in human beings.

RESUMEN

OBJECTIVES: This study aimed to determine the cellular distribution of islet cannabinoid receptors (CBs) and their involvement in the development of metabolic and hormonal changes in rats fed a fructose-rich diet (F). METHODS: In normal rat islets, we determined CBs (immunofluorescence and retrotranscription-polymerase chain reaction) and glucose-stimulated insulin secretion (GSIS) of isolated islets incubated with the CB1 antagonist rimonabant (R) and/or different CBs agonists. In 3-week F-fed rats, we determined the in vivo effect of R on serum glucose, triglyceride, and insulin levels; homeostasis model assessment for insulin resistance, GSIS, and CBs and insulin receptor substrate gene expression levels (real-time polymerase chain reaction). RESULTS: Cannabinoid receptors appeared exclusively in islet α cells. Whereas different CB agonists enhanced GSIS in normal rat islets, R did not affect it. F rats had higher serum triglyceride and insulin levels and homeostasis model assessment for insulin resistance than control rats; these alterations were prevented by R coadministration. Although R did not correct the increased GSIS observed in F islets, it modulated CBs and insulin receptor substrate gene expression. CONCLUSIONS: Islet CBs would exert an important modulatory role in metabolic homeostasis. Administration of R and F affected islet CB expression and prevented the development of F-induced metabolic impairment. Selective islet CB1 blockers could be useful to prevent/treat the alterations induced by the intake of unbalanced/unhealthy diets.

Asunto(s)

Islotes Pancreáticos/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Animales , Agonistas de Receptores de Cannabinoides/farmacología , Antagonistas de Receptores de Cannabinoides/farmacología , Expresión Génica , Células Secretoras de Glucagón/efectos de los fármacos , Células Secretoras de Glucagón/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Masculino , Piperidinas/farmacología , Pirazoles/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB2/genética , Rimonabant , Distribución Tisular

RESUMEN

The aim of the present study was to test the effect of sitagliptin and exendin-4 upon metabolic alterations, ß-cell mass decrease and hepatic steatosis induced by F (fructose) in rats. Normal adult male Wistar rats received a standard commercial diet without (C) or with 10% (w/v) F in the drinking water (F) for 3 weeks; animals from each group were randomly divided into three subgroups: untreated (C and F) and simultaneously receiving either sitagliptin (CS and FS; 115.2 mg/day per rat) or exendin-4 (CE and FE; 0.35 nmol/kg of body weight, intraperitoneally). Water and food intake, oral glucose tolerance, plasma glucose, triacylglycerol (triglyceride), insulin and fructosamine concentration, HOMA-IR [HOMA (homoeostasis model assessment) for insulin resistance], HOMA-ß (HOMA for ß-cell function) and liver triacylglycerol content were measured. Pancreas immunomorphometric analyses were also performed. IGT (impaired glucose tolerance), plasma triacylglycerol, fructosamine and insulin levels, HOMA-IR and HOMA-ß indexes, and liver triacylglycerol content were significantly higher in F rats. Islet ß-cell mass was significantly lower in these rats, due to an increase in the percentage of apoptosis. The administration of exendin-4 and sitagliptin to F animals prevented the development of all the metabolic disturbances and the changes in ß-cell mass and fatty liver. Thus these compounds, useful in treating Type 2 diabetes, would also prevent/delay the progression of early metabolic and tissue markers of this disease.

Asunto(s)

Inhibidores de la Dipeptidil-Peptidasa IV/uso terapéutico , Hígado Graso/prevención & control , Células Secretoras de Insulina/efectos de los fármacos , Síndrome Metabólico/prevención & control , Pirazinas/uso terapéutico , Triazoles/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Dieta , Ingestión de Líquidos/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Ingestión de Alimentos/efectos de los fármacos , Exenatida , Hígado Graso/etiología , Hígado Graso/patología , Fructosa/administración & dosificación , Prueba de Tolerancia a la Glucosa/métodos , Hipoglucemiantes/uso terapéutico , Células Secretoras de Insulina/patología , Masculino , Síndrome Metabólico/patología , Péptidos/uso terapéutico , Ratas , Ratas Wistar , Fosfato de Sitagliptina , Ponzoñas/uso terapéutico

RESUMEN

NADPH oxidase expression and activity have been measured in pancreatic islets under normal conditions, but its potential modulatory role upon insulin secretion remains unclear. We have currently studied NADPH oxidase activity in islets isolated from normal rats as well as the effect of its inhibition upon insulin secretion stimulated by different secretagogues. Glucose, arginine, fatty acids and KCl increased islet NADPH oxidase activity in a stimulus-dependent manner. DPI inhibited such increase in different proportions and affected unevenly insulin secretion, namely, it decreased the effect of high glucose, increased that of oleic acid and KCl, without changing the one induced by palmitic acid. Our data provide evidence that the contribution of NADPH activity to reactive oxygen species production in normal rat islets as well as its effect upon insulin secretion is uneven and highly stimulus-dependent.

Asunto(s)

Glucosa/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Insulina/metabolismo , NADPH Oxidasas/metabolismo , Animales , Arginina/farmacología , Activación Enzimática/efectos de los fármacos , Ácidos Grasos/farmacología , Técnicas In Vitro , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasa 4 , Cloruro de Potasio/farmacología , Ratas , Ratas Wistar

RESUMEN

AIMS: The effect of a three-week fructose-rich diet (FRD) upon gene expression, protein and activity levels of liver antioxidant system and carbohydrate metabolism was studied. MAIN METHODS: Serum glucose (fasting and after a glucose load), triglyceride and insulin levels of normal male Wistar rats were measured. In liver, we measured gene/protein expression and enzyme activity of catalase (CAT), copper-zinc-superoxide dismutase (CuZnSOD) and glutathione peroxidase (GSHPx); reduced glutathione (GSH); protein carbonyl content; thiobarbituric acid reactive substances (TBARS) content and microsomal membrane susceptibility to lipid peroxidation; glucokinase (GK), glucose-6-phosphatase (G-6-Pase) and glucose-6-phosphate dehydrogenase (G-6-PDH) activity; and glycogen, pyruvate, lactate and triglyceride content. KEY FINDINGS: Similar body weights and caloric intake were recorded in both groups. FRD rats had higher serum glucose, insulin and triglyceride levels, molar insulin:glucose ratio, HOMA-IR values and impaired glucose tolerance, whereas CAT, CuZnSOD and GSHPx relative gene expression levels were significantly lower. CAT and CuZnSOD protein expression, CAT activity and GSH content were also lower, while protein carbonyl content was higher. No differences were recorded in CuZnSOD, MnSOD and GSHPx activity, TBARS content and membrane susceptibility to lipid peroxidation. Glycogen, lactate and triglyceride content and GK, G-6-Pase and G-6-PDH activity were significantly higher in FRD rats. SIGNIFICANCE: In the presence of oxidative stress, the liver exhibits changes in the carbohydrate and lipid metabolic pathways that would decrease reactive oxygen species production and their deleterious effect, thus inducing little impact on specific antioxidant mechanisms. This knowledge could facilitate the design and implementation of strategies to prevent oxidative stress-induced liver damage.

Asunto(s)

Antioxidantes/metabolismo , Dieta , Carbohidratos de la Dieta/farmacología , Fructosa/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Conducta de Ingestión de Líquido/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Glutatión/metabolismo , Insulina/sangre , Peroxidación de Lípido/efectos de los fármacos , Hígado/enzimología , Masculino , Carbonilación Proteica/efectos de los fármacos , Ratas , Ratas Wistar , Triglicéridos/sangre

RESUMEN

We studied the effect of insulin resistance (IR) induced by administration of a fructose-rich diet (FRD) to normal Wistar rats for 21days, upon islet plasma membrane calcium ATPases (PMCAs) and insulin secretion. FRD rats showed significantly higher triglyceride and insulin levels, insulin:glucose ratio and HOMA-IR index than controls. FRD islets released significantly more insulin in response to glucose and showed (a) marked changes in PMCA isoform protein content (decreased PMCA 2 and increased PMCA 3), (b) a decrease in total PMCAs activity, and (c) higher levels of cytosolic calcium [Ca(2+)](i). The lower PMCAs activity with the resultant increase in [Ca(2+)](i) would favor the compensatory greater release of insulin necessary to cope with the IR state present in FRD rats and to maintain normal glucose homeostasis. Thus, changes in PMCAs activity and isoform expression play a modulatory role upon insulin secretion during long-term adaptation to an increased hormone demand.

Asunto(s)

Resistencia a la Insulina , Islotes Pancreáticos/enzimología , Isoenzimas/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Animales , Calcio/metabolismo , Citosol/metabolismo , Carbohidratos de la Dieta/metabolismo , Fructosa/metabolismo , Glucosa/farmacología , Inmunohistoquímica , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Masculino , ARN Mensajero/genética , Ratas , Ratas Wistar , Triglicéridos/sangre , Triglicéridos/metabolismo

RESUMEN

The effect of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) administration to normal male hamsters upon serum glucose and triglyceride levels, beta-cell mass and function was studied. INGAP-PP (500 mug) or saline was injected twice daily during 10 days. Both groups showed comparable body weight, serum glucose and triglyceride levels. INGAP-PP treated animals had significantly higher HOMA-IR and HOMA-beta and their islets released more insulin in response to glucose; they had lower islet DNA content, significantly increased number of islets/unit area, beta-cell replication rate and mass, cells co-expressing Pdx-1/INGAP and islets in contact with ducts, and decreased beta-cell apoptosis rate. The percentage of cells expressing Pdx-1 alone or together with INGAP or insulin increased significantly in ducts. These animals also showed a significantly higher concentration of Pdx-1 and Ngn-3 mRNA and a lower number of INGAP-positive cells. In conclusion, INGAP-PP promoted a controlled and functionally active increase of beta-cell mass; our data demonstrate for the first time the mechanism responsible for such changes; that Ngn-3 would be involved in INGAP-PP-induced neogenesis; and the existence of a negative feedback loop with endogenous INGAP-producing cells. Accordingly, INGAP-PP could be used to induce these effects in people with or at risk of developing diabetes.

Asunto(s)

Antígenos de Neoplasias/farmacología , Biomarcadores de Tumor/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Fragmentos de Péptidos/farmacología , Animales , Antígenos de Neoplasias/administración & dosificación , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Biomarcadores de Tumor/administración & dosificación , Glucemia/análisis , Peso Corporal/efectos de los fármacos , Cricetinae , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Lectinas Tipo C/administración & dosificación , Masculino , Mesocricetus , Proteínas del Tejido Nervioso/genética , Proteínas Asociadas a Pancreatitis , Fragmentos de Péptidos/administración & dosificación , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genética , Triglicéridos/análisis